Note: Descriptions are shown in the official language in which they were submitted.
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ALTERATION AND MODULATION OF PROTEIN ACTIVITY
BY VARYING POST-TRANSLATIONAL MODIFICATION
PRIORITY CLAIM
This application claims the benefit of the filing date of United States Patent
Application Serial No.
12/655,993, filed January 12, 2010, for "ALTERATION AND MODULATION OF PROTEIN
ACTIVITY
BY VARYING POST-TRANSLATIONAL MODIFICATION," which application claims priority
as a
continuation-in-part under 35 U.S.C. 119 to United States Patent Application
Serial No. 12/380,450, filed
February 26, 2009, and to PCT International Patent Application Serial No.
PCT/US2009/035307, filed
February 26, 2009.
GOVERNMENT RIGHTS
This invention was made with government support under Contract Numbers DE-AC07-
99ID13727
and DE-AC07-05ID14517 awarded by the United States Department of Energy. The
government has certain
rights in the invention.
TECHNICAL FIELD
The present invention relates generally to the field of biotechnology. More
specifically,
embodiments of the present invention relate to post-translational modification
of proteins.
BACKGROUND
It has been believed until only very recently that bacteria in general do not
glycosylate their proteins.
While there have been some instances reported, these were dismissed as unusual
anomalies (Borman 2006).
It is now becoming more accepted that bacteria do glycosylate their proteins
in perhaps more ways than
eukaryotes do, although this belief is not yet widespread (Schaffer et al.,
2001). In a recent review article, it
was stated that glycosylation has been shown to assist in protein stability,
modulate physical properties such
as solubility, protect against proteolysis, modify activity profiles, and
target for externalization (Upreti et al.,
2003). In 1994, a group purified an amylase from Alicyclobacillus
acidocaldarius and showed that the
amylase was cell-bound during active growth (Schwermann et al., 1994). As the
culture entered stationary
phase, the cells released an active soluble glycosylated version of the
amylase into the medium (Schwermann
et al., 1994). No attempt was made to compare the activities of the various
forms of the amylases.
DISCLOSURE
Embodiments of the invention include methods of altering the enzymatic
activity of an extremophilic
enzyme or other protein via means of chemical glycosylation and/or isolated or
partially purified
glycosyltransferases and/or post-translational modification proteins, extracts
of cells comprising
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glycosyltransferases and/or post-translational modification proteins, and/or
in cells comprising one or more
glycosyltransferases and/or post-translational modification proteins.
Embodiments of the invention include
methods of post-translationally modifying proteins. In some embodiments, the
post-translational
modification may occur using means of glycosylation (including chemical
glycosylation), pegylation,
phosphorylation, methylation or other forms of post-translational modification
and/or be isolated or partially
purified glycosyltransferases and/or post-translational modification proteins,
extracts of cells comprising
glycosyltransferases and/or post-translational modification proteins, and/or
in cells comprising one or more
glycosyltransferases and/or post-translational modification proteins.
Embodiments of the invention include
post-translationally modified proteins including, but not limited to, SEQ ID
NOS:307 (celB), 331 (an
Endoglucanase C), 333 (a Peptidoglycan N-acetylglucosamine deacetylase), 335
(a Beta-galactosidase), 337
(an arabinofuranosidase), and 338 (an alpha-xylosidase). Embodiments thus
include glycosylated versions of
the aforementioned proteins.
A first aspect of the present invention relates to an enzyme isolated from an
extremophilic
microbe that displays optimum enzymatic activity at a temperature of greater
than about 80 C, and a pH
of less than about 2.
Another aspect of the present invention relates to a hemicellulase that was
derived from
Alicyclobacillus acidocaldarius (ATCC 27009).
Another aspect of the present invention relates to an enzyme that is useful in
the degradation of
complex biomolecules.
Still further, another aspect of the present invention relates to an enzyme
that may be useful in a
simultaneous saccharification and fermentation process to convert a biomass
sugar into an end product.
Yet another aspect of the present invention relates to a method for the
treatment of a biomass that
includes the steps of providing a source of a biomass having a biomass sugar;
pretreating the biomass
with a water soluble hemicellulase derived from Alicyclobacillus
acidocaldarius (ATCC 27009) to
produce an end product.
Another aspect of the present invention relates to a method for the
preparation of a hemicellulase
that includes the steps of providing a source of Alicyclobacillus
acidocaldarius (ATCC 27009);
cultivating the Alicyclobacillus acidocaldarius (ATCC 27009) in a microbial
nutrient medium having a
supernatant; separating the cells of the Alicyclobacillus acidocaldarius from
the nutrient medium
supernatant; and recovering and purifying the hemicellulase derived from the
Alicyclobacillus
acidocaldarius (ATCC 27009) from the nutrient medium supernatant.
Moreover, another aspect of present invention relates to a method for
hydrolyzing a
polysaccharide that includes the steps of providing a water soluble
hemicellulase derived from a microbe;
and conducting hydrolysis of a polysaccharide with the water soluble
hemicellulase at a pH of less than
about 2.
These and other aspects of the present invention will be described in greater
detail hereinafter.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a sequence alignment between SEQ ID NO:I (RAA000164) and
reflYP_001223775.11, reflYP_729290.11, reflZP_01084440.11, reflZP01079150.11,
and
reflZP_01471594.1 I (SEQ ID NOS:3-7) respectively, which all have the function
assigned to SEQ ID
NO:1 in Table 1. Amino acids conserved among all sequences are indicted by a
"*" and generally
conserved amino acids are indicated by a ":".
FIG. 2 depicts a sequence alignment between SEQ ID NO:18 (RAA000517) and
reflZP_00589533.11, reflZP_01386435.11, reflYP_378533.1l, reflZP_00513158.1I,
and reflYP_374173.1I
(SEQ ID NOS:20-24) respectively, which all have the function assigned to SEQ
ID NO: 18 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIG. 3 depicts a sequence alignment between SEQ ID NO:35 (RAA000650) and
reflYP_001127183.11, reflZP_02038504.11, ref7YP_001647987.11,
reflYP_001377114.11, and
reflNP_835081.1l (SEQ ID NOS:37-41) respectively, which all have the function
assigned to SEQ ID
NO:35 in Table 1. Amino acids conserved among all sequences are indicted by a
"*" and generally
conserved amino acids are indicated by a ":".
FIG. 4 depicts a sequence alignment between SEQ ID NO:52 (RAA000991) and
reflZP_02327412.11, reflYP001487207.11, reflZP_01172765.11, reW_831314.11, and
reflNP_844008.11
(SEQ ID NOS:54-58) respectively, which all have the function assigned to SEQ
ID NO:52 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a
FIGs. 5A and 513 depict a sequence alignment between SEQ ID NO:69 (RAAC01110)
and
reflYP_001519856.11, reflYP_711688.11, reflZP_01331931.11,
reflYP_001076955.11, and
reflYP_336440.11 (SEQ ID NOS:71-75) respectively, which all have the function
assigned to SEQ ID
NO:69 in Table 1. Amino acids conserved among all sequences are indicted by a
"*" and generally
conserved amino acids are indicated by a ":".
FIGs. 6A and 6B depict a sequence alignment between SEQ ID NO:86 (RAAC01166)
and
gblAAR99615.1I, gbIABM68334.21, re f ZP_01372248.11, reflYP_519555.11, and
reflZP_02234077.11
(SEQ ID NOS:88-92) respectively, which all have the function assigned to SEQ
ID NO:86 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIG. 7 depicts a sequence alignment between SEQ ID NO:103 (RAAC01167) and
re f ZP_01515212.11, reflYP_001277643.11, reflZP_02291400.11,
reflYP_001633727.11, and
re f YP_001434357.11 (SEQ ID NOS:105-109) respectively, which all have the
function assigned to SEQ
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ID NO: 103 in Table 1. Amino acids conserved among all sequences are indicted
by a "*" and generally
conserved amino acids are indicated by a
FIGs. 8A and 8B depict a sequence alignment between SEQ ID NO:120 (RAAC01170)
and
reflYP_001324592.11, reflYP_342776.11, reflNP_780975.11, reflYP_001636830.11,
and
reflYP_001299026.11 (SEQ ID NOS:122-126) respectively, which all have the
function assigned to SEQ
ID NO: 120 in Table 1. Amino acids conserved among all sequences are indicted
by a "*" and generally
conserved amino acids are indicated by a ":".
FIG. 9 depicts a sequence alignment between SEQ ID NO:137 (RAAC01248) and
reflZP_02170160.11, reflZP01171895.11, reflYP_076646.11, reff YP_590910.11,
and reflZP_02175410.1
(SEQ ID NOS: 139-143) respectively, which all have the function assigned to
SEQ ID NO: 137 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a
FIGs. 10A and l0B depict a sequence alignment between SEQ ID NO:154
(RAAC01348) and
reflZP_01665289.11, reflZP_01643350.11, gbIAAW77167.1I, reflYP 452722.11, and
reflZP_02241787.11
(SEQ ID NOS: 156-160) respectively, which all have the function assigned to
SEQ ID NO:154 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIG. 11 depicts a sequence alignment between SEQ ID NO:171 (RAAC01377) and
reflYP_147952.11, reflYP_520670.11, reflYP_001395809.11, reflYP_001309701.1I,
and
reflYP_001643660.11 (SEQ ID NOS: 173-177) respectively, which all have the
function assigned to SEQ
ID NO: 171 in Table 1. Amino acids conserved among all sequences are indicted
by a "*" and generally
conserved amino acids are indicated by a ":".
FIG. 12 depicts a sequence alignment between SEQ ID NO:188 (RAAC01611) and
reflYP_146214.11, reflYP001124463.1 1, reflNP_865262.11, reflYP_426013.1!, and
reflZP_01885526.11
(SEQ ID NOS: 190-194) respectively, which all have the function assigned to
SEQ ID NO: 188 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIGs. 13A and 13B depict a sequence alignment between SEQ ID NO:205
(RAAC01612) and
reflYP_146215.11, reflYP001124464.11, reflYP_074948.1!, reflYP_001039503.1I,
and refNP_621770.1
(SEQ ID NOS:207-21 1) respectively, which all have the function assigned to
SEQ IDNO:205 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIGs. 14A and 14B depict a sequence alignment between SEQ ID NO:222
(RAAC01926) and
reflYP_001038202.11, refZP_01667587.11, refZP_01575301.11,
reflYP_001211020.11, and
reflYP_516465.11 (SEQ ID NOS:224-228) respectively, which all have the
function assigned to SEQ ID
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NO:222 in Table 1. Amino acids conserved among all sequences are indicted by a
"*" and generally
conserved amino acids are indicated by a ":".
FIG. 15 depicts a sequence alignment between SEQ ID NO:239 (RAAC01998) and
reflNP_348940.11, refNP_721244.11, dbj JBAC75700.11, reflZP_00605123.11, and
reflYP_015329.11 (SEQ
ID NOS:241-245) respectively, which all have the function assigned to SEQ ID
NO:239 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIG. 16 depicts a sequence alignment between SEQ ID NO:256 (RAAC02011) and
reflYP_754819.11, reflYP_184322.11, reflNP_577787.11, reflNP142068.11, and
reflNP_125751.11 (SEQ
ID NOS:258-262) respectively, which all have the function assigned to SEQ ID
NO:256 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a ":".
FIGs. 17A and 17B depict a sequence alignment between SEQ ID NO:273
(RAAC02381) and
reflNP_622177.11, reflYP_848858.1I, reflYP_001374688.11, reflNP_470039.11, and
ref7ZP_01929325.1
(SEQ ID NOS:275-279) respectively, which all have the function assigned to SEQ
ID NO:273 in Table 1.
Amino acids conserved among all sequences are indicted by a "*" and generally
conserved amino acids
are indicated by a
FIG. 18 depicts a sequence alignment between SEQ ID NO:290 (RAAC02421) and
reflZP_01721811.11, reflNP_241897.11, refYP001486101.11, reflZP-O 11705 32.
11, and
reflZP_02327994.1I (SEQ ID NOS:292-296) respectively, which all have the
function assigned to SEQ ID
NO:290 in Table 1. Amino acids conserved among all sequences are indicted by a
"*" and generally
conserved amino acids are indicated by a ":".
FIG. 19 is a graph depicting an effect of temperature on xylanase activity, as
provided by the
present invention.
FIG. 20 is a graph depicting an effect of temperature on cellulase activity,
as provided by the
present invention at a pH of 4Ø
FIG. 21 is a graph depicting an effect of pH on cellulase activity of the
present invention at a
temperature of 60 C.
FIG. 22 is a graph depicting an effect of pH on xylanase activity of the
present invention at a
temperature of 60 C.
FIG. 23 is a graph depicting the xylanase activity of SEQ ID NO:307 as
determined using wheat
arabinoxylan (WAX) at various pH and temperature levels. The enzyme was
isolated from
Alicyclobacillus acidocaldarius (black bars) or produced in E. coli (white
bars). There is no available
data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (60 C
and 80 C).
FIG. 24 is a graph depicting cellulase activity of SEQ ID NO:307 as determined
using
carboxymethyl cellulose (CMC) at various pH and temperature levels. The enzyme
was isolated from
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Alicyclobacillus acidocaldarius (black bars) or produced in E. coli (white
bars). There is no available
data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (60 C
and 80 C).
FIG. 25 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID
NO:307 as
determined from FIGs. 23 and 24 at various pH and temperature levels. The
enzyme was isolated from
Alicyclobacillus acidocaldarius (black bars) or produced in E. coli (white
bars). There is no available
data for enzyme isolated from Alicyclobacillus acidocaldarius at pH 5.5 (60 C
and 80 C). The tops of the
bars at pH 5.5 for the enzyme produced in E. coli are left open to indicate
that the ratio was greater than
10.
FIG. 26 is a graph depicting xylanase activity of SEQ ID NO:307 as determined
using wheat
arabinoxylan (WAX) at various pH and temperature levels. The enzyme was
produced in Pichia pastoris
(black bars) or produced in E. coli (white bars).
FIG. 27 is a graph depicting cellulase activity of SEQ ID NO:307 as determined
using
carboxymethyl cellulose (CMC) at various pH and temperature levels. The enzyme
was produced in
Pichia pastoris (black bars) or produced in E. coli (white bars).
FIG. 28 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID
NO:307 as
determined from FIGs. 26 and 27 at various pH and temperature levels. The
enzyme was produced in
Pichia pastoris (black bars) or produced in E. coli (white bars). The tops of
the bars at pH 5.5 for the
enzyme produced in E. coli are left open to indicate that the ratio was
greater than 10.
FIG. 29 is a graph depicting xylanase activity of SEQ ID NO:307 as determined
using wheat
arabinoxylan (WAX) at various pH and temperature levels. The enzyme was
isolated from
Alicyclobacillus acidocaldarius (black bars) or produced in Pichia pastoris
(white bars). There is no
available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH
5.5 (60 C and 80 C).
FIG. 30 is a graph depicting cellulase activity of SEQ ID NO:307 as determined
using
carboxymethyl cellulose (CMC) at various pH and temperature levels. The enzyme
was isolated from
Alicyclobacillus acidocaldarius (black bars) or produced in Pichia pastoris
(white bars). There is no
available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH
5.5 (60 C and 80 C).
FIG. 31 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID
NO:307 as
determined from FIGs. 29 and 30 at various pH and temperature levels. The
enzyme was isolated from
Alicyclobacillus acidocaldarius (black bars) or produced in Pichia pastoris
(white bars). There is no
available data for enzyme isolated from Alicyclobacillus acidocaldarius at pH
5.5 (60 C and 80 C).
FIG. 32 is a graph depicting xylanase activity of SEQ ID NO:307 lacking the C-
terminal 203
amino acids as determined using wheat arabinoxylan (WAX) at various pH and
temperature levels. The
enzyme was produced in Pichia pastoris (black bars) or produced in E. coli
(white bars).
FIG. 33 is a graph depicting cellulase activity of SEQ ID NO:307 lacking the C-
terminal 203
amino acids as determined using carboxymethyl cellulose (CMC) at various pH
and temperature levels.
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The enzyme was produced in Pichia pastoris (black bars) or produced in E. coli
with an N-terminal His
tag (white bars).
FIG. 34 is a graph depicting a ratio of cellulose/xylanase activity of SEQ ID
NO:307 lacking the
C-terminal 203 amino acids as determined from FIGs. 32 and 33 at various pH
and temperature levels.
The enzyme was produced in Pichia pastoris (black bars) or produced in E. coli
(white bars).
FIG. 35 is a graph depicting a ratio of arabinofuranosidase activity of
RAAC00307 (SEQ ID
NO:337) produced in E. coli. Activity at 50 C (diamonds), 60 C (squares), 70 C
(triangles), 80 C ("x"s),
and 90 C ("*"s) at various pH levels are shown. The enzyme had no activity at
pH 2.
FIG. 36 is a graph depicting a ratio of beta xylosidase activity of RAAO00307
(SEQ IDNO:337)
produced in E. coli. Activity at 50 C (diamonds), 60 C (squares), 70 C
(triangles), 80 C ("x"s), and 90 C
("*"s) at various pH levels are shown. The enzyme had no activity at pH 2.
FIG. 37 is a graph depicting a ratio of beta xylosidase activity of RAAC00307
(SEQ ID NO:337)
produced in Pichia pastoris. Activity at 60 C (diamonds) and 80 C (squares),
at various pH levels are
shown.
MODE(S) FOR CARRYING OUT THE INVENTION
One aspect of the present invention, as described hereinafter, relates, in
part, to enzymes isolated
from an extremophilic microbe that display optimum enzymatic activity at a
temperature of greater than
about 80 C, and an optimum pH of less than about 2. In further aspects of the
present invention, the
enzyme may be a hemicellulase and/or xylanase that was derived from
Alicyclobacillus acidocaldarius,
where the organism is further identified as ATCC 27009. The enzyme, as
discussed hereinafter, appears
to display enzymatic activity at a pH of about 1. Still further, this same
enzyme has a molecular weight
of at least about 120 kDa. In the present invention, the enzyme, as disclosed,
may be useful in a
simultaneous saccharification and fermentation process and/or a sequential
hydrolysis and fermentation
process to convert a biomass sugar into an end product. Still further, the
enzyme, as described herein,
may be useful in the pretreatment of a biomass slurry to degrade a water-
soluble or water-insoluble
oligomer and/or polysaccharide that is present in the biomass slurry to
produce an end product.
As used hereinafter, the term "extremophilic microbe" means an organism that
can live and thrive
under conditions that humans would consider extreme, such as boiling water,
ice, battery acid or at the
bottom of the ocean. Examples of such microbes include, but are not limited
to, Pyrolobusfumarii that
grows at temperatures up to 235 F (113 C), Psychrobacter cryopegella that
survives at temperatures of
-20 C (-4 F), Deinococcus radiodurans that can survive in a nuclear reactor,
Photobacterium profundum
that thrives at pressures 300 times the atmospheric pressure at sea level, and
Picrophilus torridus that
lives at a pH of 0, the same as battery acid. Environments in which these
microbes can be found include
boiling hot springs, deep ocean thermal vents, glaciers, salt flats, and
nuclear reactors. The microbes used
in the present invention can be obtained from natural and artificial sources
or commercially from culture
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depositories. In the present invention, the cultivation is preferably
conducted at temperatures above 40 C
and a pH below about 5, and more preferably above 50 C and below a pH of 4,
and most preferably
above 55 C and below a pH of 3.5, and under anaerobic, aerobic, and/or micro-
aerophilic conditions.
While the cultivation period varies depending upon the pH, temperature and
nutrient medium used, a
period of 12 hours to several days will generally give favorable results. As
used herein, Alicyclobacillus
acidocaldarius is defined as a microorganism that can be obtained from the
American Type Culture
Collection (ATCC), Manassas, Virginia, and that is identified as
Alicyclobacillus acidocaldarius (ATCC
27009).
As used hereinafter, the phrase "enzymatic activity" means the reaction an
enzyme causes to
occur. Enzymes are proteins produced by all living organisms that mediate,
cause and/or promote
reactions that change a chemical into another type of chemical without
themselves being altered or
destroyed. In the context of the present application, the word "optimum," when
used in combination with
the term "enzymatic activity," means the most favorable conditions that allow
the enzyme to work the
best and the fastest for a given end result. The optimum enzymatic activity
may be affected by conditions
that include temperature, pH, and salt concentrations.
As used hereinafter, the word "xylanase" means an enzyme that breaks apart
hemicellulose by
breaking the chemical bonds between the xylose sugars that make up the
backbone of the hemicellulose
molecule, or by breaking bonds between xylose sugars in the hemicellulose side
chains.
The word "polysaccharide" as used hereinafter shall mean a chain of sugars
(can be the same
sugars or different sugars) that are linked together by chemical bonds.
Polysaccharides can consist of
straight chains of these sugars with or without side chains. Examples of
polysaccharides include starch,
pectin, cellulose, and hemicellulose.
The word "hydrolysis" in the context of this present application shall mean a
chemical reaction in
which water reacts with a molecule and breaks it into at least two pieces.
As used hereinafter, the phrase "biomass sugar" shall mean sugars that have
come from the
breakdown of biomass components, such as cellulose and hemicellulose. Examples
of biomass sugars
include, but are not limited to, saccharides, glucose, xylose, galactose,
mannose, arabinose, as well as
combinations, oligomers, and/or modified or substituted forms thereof.
The phrase "simultaneous saccharification and fermentation process" shall mean
hereinafter a
process for making a fuel or chemical such as ethanol from a biomass that may
or may not have been
pretreated by chemical means, and where cellulase and/or hemicellulase
enzyme(s) are used to break
down biomass polysaccharides into sugars (saccharification); and the sugars
are fermented by source(s) of
microorganism(s) into the product fuel or chemical (fermentation). These two
processes occur at the
same time, in the same reaction vessel (simultaneous).
The phrase "end product" as used in the present application shall mean
hereinafter the
chemical(s) that is/are produced by a chemical or enzymatic reaction. Examples
of end products
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contemplated by the present invention include simpler saccharides and sugars
(e.g., monomers, dimers,
trimers, oligomers, etc.), alcohols, fuels, and/or other products of an
enzymatic reaction.
The word "biomass" in the context of the present invention shall mean plant
and other
lignocellulosic material such as corn stalks, wheat straw, and wood by-
products, such as sawdust and the
like.
The phrase "pretreatment of a biomass slurry" shall mean, in the context of
the present
application, the preparation of a biomass for its subsequent conversion to
fuels, such as ethanol. This
pretreatment includes the steps of grinding the biomass to a powder or small
particles, and adding water
(this constitutes a slurry). This slurry is then treated by a number of
methods designed to partially or
completely remove the lignin from the biomass, and convert the hemicellulose
and cellulose into a form
that can be more easily degraded into their component sugars using enzymes
such as cellulases and
hemicellulases. Some pretreatments degrade hemicellulose to its component
sugars while leaving the
cellulose as part of the solid residue. This treatment step is called a
"pretreatment" because it occurs
before both the enzymatic degradation step and before the fermentation step
that converts the sugars into
ethanol.
The phrase "water-soluble" in the context of the present invention shall mean
a chemical or other
substance that can be dissolved completely in water without leaving any solid
residue.
The word "hemicellulose" in the context of the present invention means one
component of a plant
(the other two being cellulose and lignin), that is made of a linear chain of
sugars such as xylose, or
mannose that are connected by a chemical bond. This linear chain also has
branches consisting of sugars
and other chemicals along the chain.
The word "hemicellulase" in the context of the present invention means a class
of enzymes that
can break hemicellulose into its component sugars and other chemical monomers.
Examples of
hemicellulases include, but are not limited to, xylanases, mannanases,
glucuronidases, and
arabinofuranosidases.
The phrase "sequential hydrolysis and fermentation process" in the context of
the present
invention shall mean a process for making a fuel or chemical from the biomass,
such as ethanol, and
where the biomass is treated physically or with a reactive chemical or
solvent, or mixtures thereof, to
remove the lignin, and to convert the cellulose and hemicellulose present in
the biomass into their
component sugars or into a form that can be more easily degraded into their
component sugars using
enzymes such as cellulases and hemicellulases. Examples of these include
grinding, milling, acids,
alkalis, organosolvents, and the like. These treatments can be performed at
temperatures ranging from
ambient to 300 C or more, and at pressures ranging from ambient to 2000 psig
or more. The sugars,
which are dissolved in water, are then cooled, and the pH adjusted to neutral,
and then subsequently
fermented by microorganisms of various types into a product fuel(s) or
chemical(s) (fermentation). These
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two processes occur in separate reaction vessels with the hydrolysis step
conducted first, and the
fermentation step conducted second (e.g., sequential).
The phrase "cultivating Alicyclobacillus acidocaldarius" in the context of the
present invention
shall mean providing the aforementioned microbe with a food source (soluble or
insoluble lignocellulose
or other source of polysaccharides or sugars) and various vitamins and
minerals dissolved in water (this
constitutes the nutrient medium), and giving the microbe the proper conditions
that allow it to grow (a
temperature of 140 F (60 C), a pH of 3.5, and oxygen).
The phrase "separating the cells of the Alicyclobacillus acidocaldarius" in
the context of the
present invention shall include means for removing the bacterial cells from
the nutrient medium by, for
example, centrifugation.
The phrase "recovering and purifying the hemicellulase" in the context of the
present invention
shall mean separating the hemicellulase enzyme from the nutrient medium. In
the present invention, a
process called cation exchange was used to separate hemicellulase from the
nutrient medium. In this
regard, the nutrient medium (with hemicellulase) was pumped through the cation
exchange material.
When brought into contact with the cation exchange material, the hemicellulase
will attach itself to the
cation exchange material, but the nutrient medium will pass through. The
hemicellulase enzyme is then
removed from the cation exchange material and is purified.
The phrase "microbial nutrient medium" in the context of the present
application means a food
source for the microbe (Alicyclobacillus acidocaldarius) and vitamins and
minerals, all dissolved in water
and adjusted to the pH needed by the microbe to grow. More specifically, the
microbial nutrient medium
includes about 1 gram per liter of Xylan; about 10 mM NH4C1; about 5.2 mM
K2HPO4i about 0.8 mM
MgSO4-7 H2O; about 1.74 mM Na2SO4i about 25 mg per liter MgCl2; about 6.6 mg
per liter of CaC12;
about 2.0 mg per liter MnSO4; about 0.5 mg per liter ZnSO4; about 0.5 mg per
liter of boric acid; about 5
mg per liter of FeC13; about 0.15 mg per liter of CuSO4; about 0.025 mg per
liter of NaMoO4; about 0.05
mg per liter of CoNO3i about 0.02 mg per liter of NiC12; about 0.08 mg per
liter of pyridoxine
hydrochloride; about 0.01 mg per liter of folic acid; about 0.1 mg per liter
of thiamine hydrochloride;
about 0.04 mg per liter of riboflavin; about 0.08 mg per liter of
nicotinamide; about 0.08 mg per liter of
p-aminobenzoate; about 0.01 mg per liter of biotin; about 0.0004 mg per liter
cyanocobalamin; about 0.08
mg per liter D-pantothenic acid-Ca; about 0.02 mg per liter of myo-inositol;
about 0.05 mg per liter of
choline bromide; about 0.02 mg per liter of monosodium orotic acid; and about
0.1 mg per liter
spermidine, wherein the resulting nutrient medium is adjusted to a pH of about
3.5.
The word "supernatant" in the context of the present application shall mean
the nutrient medium
that is leftover after the bacterial cells are substantially removed from
same.
The inventors have isolated and characterized temperature and acid stable
endoglucanase and/or
xylanases that demonstrate activity at elevated temperatures, and low pH, and
that show stability when
incubated under these conditions for extended periods of time. The inventors
recognize that heat and acid
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stable hemicellulases and cellulases, as described hereinafter, have
particular value in, or as an accessory
to, processes that would lead, on the one hand, to the reduction in the
severity of pretreatment processes,
earlier described, and/or the elimination of these limitations in various
processes. In this regard, the
inventors screened numerous organisms from Yellowstone National Park and
various culture collections
for microbes that had the ability to produce enzymes that were stable at both
high temperature, and low
pH. In this regard, water and sediment samples were collected from six springs
in the Norris Geyser
Basin of Yellowstone National Park. These samples were inoculated into a
liquid mineral salt medium
having a pH 3.5, and further containing either 0.5 grams per liter of oat
spelt xylan, or 0.5 grams per liter
of ground corn cobs. The subsequent cultures were incubated at 80 C and were
observed daily for
growth, both visually and microscopically. Still further, a search of the
American Type Culture
Collection (ATCC) and the Deutsche Sammlung von Mikroorganismen Und
Zellkulturen (DSMZ)
yielded four possible heterotrophic organisms whose optimal temperatures, and
pH, for growth, were
greater than about 60 C, and less than about a pH of 4. These several
organisms were grown in the media
recommended by ATCC or DSMZ with a carbon source replaced by either oat spelt
xylan, or ground corn
cobs, as described above. These cultures where then later incubated at their
optimum growth temperature
and a pH of 3.5. Subsequent microbial growth was assessed visually by the
appearance of turbidity.
In this investigation, hemicellulase and/or cellulase activities were
presumptively assumed
present if growth occurred in the presence of xylan. Cultures where growth
occurred were harvested after
approximately three days incubation. Cells were removed from the culture by
centrifugation. The culture
supernatant was concentrated at about 1000-2000 fold using an AMICON
ultrafiltration cell with a
10000 MWCO membrane. The subsequent supernatant concentrate was then tested
for hemicellulase and
cellulase activity using arsenomolybdate reducing sugar acid assay (previously
described by Somogyi
(1952), J. Biol. Chem. 195:19-23) with wheat arabinoxylan (commercially
secured from Megazyme), or
carboxyinethylcellulose (secured from Sigma-Aldrich). These were used as
substrates for the
hemicellulase and cellulase activities, respectively. Standard conditions for
the assays were set at 60 C,
and a pH of about 3.5. As will be seen by reference to the drawings, the
hemicellulase and cellulase
activities were measured at temperatures up to about 90 C to determine the
optimum temperature for
enzymatic activity. The reducing sugar assay referenced above was modified by
changing the incubation
temperature of the supernatant concentrate with the substrate. Similarly, the
enzyme activities were
measured at a pH ranging from 1 to about 8 to determine the optimum pH for the
enzyme activity. For
these studies, the reducing sugar assay was modified by preparing the assay
components in the
appropriate pH buffer (pH 1-2, 50 mM sodium maleate or 50 mM glycine; pH 2-6,
50 mM sodium
acetate; pH 6-8, 50 mM sodium phosphate; and pH 8-9, 50 mM Tris).
In addition to the foregoing, stabilities of the hemicellulases and cellulases
as a function of
temperature and pH were examined by incubating the supernatant concentrate at
a temperature of about
70 C, and a pH of 2Ø In this regard, a layer of mineral oil was placed over
the concentrate to limit
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evaporation during this exam. Samples were periodically collected and assayed
for hemicellulase and
cellulase activity at the standard assayed conditions earlier described. With
respect to the hemicellulase
and cellulase reaction kinetics, these were determined using the reducing
sugar assay with varying
amounts of wheat arabinoxylan or carboxymethylcellulose. The reaction kinetics
were determined at
60 C and a pH of 3.5. Michaelis-Menten parameters Vmax and Km were calculated
by nonlinear
analysis using ENZYME KINETICS PROTM that is available through SynexChem.
After the process
noted above, the inventors identified Alicyclobacillus acidocaldarius (ATCC
27009) for further
examination.
Subsequently, a crude enzyme preparation was made by concentrating the cell
free culture
material. A subsequent SDS-page gel showed five major bands and several minor
bands. The
subsequently calculated masses of these bands were consistent with other
reported xylanases and
cellulases. As seen in FIGS. 19, 20, 21, and 22, the inventors discovered that
the enzyme isolated from
the Alicyclobacillus acidocaldarius, which is identified herein as ATCC 27009,
had an optimum
temperature for enzymatic activity (xylanase and cellulase) at about 80 C. As
seen in FIG. 19, the
relative enzymatic activity is contrasted against the enzymatic activity that
is provided by a similar
enzyme that is isolated from another similar microbe T. lanuginosus. Further,
it was found that the
isolated endoglucanase and/or xylanase exhibited enzymatic activity at a pH as
low as 1, with an optimum
pH of 2, while the optimum pH for the cellulase activity was at a pH of about
4, although it did show
some activity at a pH as low as 2. FIG. 22 shows the xylanase activity as a
function of pH as described
above. To the best knowledge of the inventors, the lowest optimum
hemicellulase pH previously reported
was in the reference to Collins (2005, FEMS, Micro. Review, 29(l):3-23). It is
conceivable that the
present water soluble endoglucanase and/or xylanase enzyme that has been
isolated perhaps has activity
at a pH lower than 1, however, presently, the reducing sugar assay reagents
were unstable below a pH of
1. Further investigation revealed that the newly isolated hemicellulase and
cellulase activities showed no
decrease in activity when incubated at 70 C and a pH of 2. The aforementioned
investigation lead the
inventors to conclude that the Alicyclobacillus acidocaldarius (ATCC 27009) is
capable of growth on a
xylan substrate, and further produces extracellular hemicellulase and
cellulase activity, which are both
water-soluble and display significant hemicellulase activity at a pH of about
2, and which further has a
molecular weight of at least about 120 kDa. Again, see FIG. 22.
The prior art discloses that an acid stable xylanase has been purified and
characterized from
Aspergillus kawachii that has a pH optimum of 2.0 and a temperature optimum
between 50 C to 60 C.
(Purification and Properties of Acid Stable Xylanases from Aspergillus
kawachii, K. Ito, H. Ogasawara,
T. Sugomoto, and T. Ishikawa, Bioscience Biotechnology and Biochemistry 56
(4):547-550, April 1992.)
Additionally, several xylanases have been reported with pH optima in the range
of 4 to 5, and numerous
xylanases have been reported that have temperature optima up to 100 C.
However, in the inventors'
knowledge, the enzyme as described hereinafter, is the first enzyme known that
has activities at such a
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low pH, and at such a high temperature, as claimed herein. In addition to the
foregoing, the inventors are
aware that a xylanase has been purified from the same organism, that is,
Alicyclobacillus acidocaldarius
(ATCC 27009), that is reported to have xylanase activity associated with it.
It is reported that this
enzyme had a pH optimum of 4.0, and a temperature optimum of about 80 C. In
this regard, a
thermoacidophilic endoglucanase and/or xylanase (ce1B) from Alicyclobacillus
acidocaldarius (ATCC
27009) displayed high sequence similarity to arabinofuranosidases belonging to
Family 51 of glycoside
hydrolases (K. Eckert and E. Schneider, European Journal of Biochemistry, 270
(17):3593-3602,
September, 2003). The aforementioned cellulase precursor as described in this
prior art reference is best
understood by a study of SEQ ID NO:307, which is shown below:
1 MKRPWSAALA ALIALGTGAS PAWAAAHPSP KVPAGAAGRV RAADVVSTPI
51 SMEIQVIHDA LTVPELAAVQ AAAQAASNLS TSQWLQWLYP NATPTTSAQS
101 QAAQAVANLF NLATYGAVST RGSNAAQILQ TLQSISPLLS PRAVGLFYQS
151 FLTEIGQSSK AILARQASSS IVGNALAQAA SLSPTISAYL RQNGLSPSDL
201 ARTWSSFETQ VDPQGAAQTA LATRICTNAL GFGAPTASAT ITVNTAARLR
251 TVPATAFGLN AAVWDSGLNS QTVISEVQAL HPALIRWPGG SISDVYNWET
301 NTRNDGGYVN PNDTFDNFMQ FVNAVGASPI ITVNYGTGTP QLAADWVKYA
351 DVTHHDNVLY WEIGNEIYGN GYYNGNGWEA DDHAVPNQPQ KGNPGLSPQA
401 YAQNALQFIQ AMRAVDPNIK IGAVLTMPYN WPWGATVNGN DDWNTVVLKA
451 LGPYIDFVDV HWYPETPGQE TDAGLLADTD QIPAMVAELK REINAYAGSN
501 AKNIQIFVTE TNSVSYNPGQ QSTNLPEALF LADDLAGFVQ AGAANVDWWD
551 LLNGAEDNYT SPSLYGQNLF GDYGLLSSGQ ATPKGVQEPP QYTPLPPYYG
601 FQLVSDFARP GDTLLGSASS QSDIDVHAVR EPNGDIALML VNRSPSTIYS
651 ADLNVLGVGP YAITKALVYG EGSSAVSPAL TLPTAHSVKL MPYSGVDLVL
701 HPLIPAPHAA ASVTDTLALS SPTVTAGGSE TVTASFSSDR PVRDATVELE
751 LYDSTGDLVA NHEMTGVDIA PGQPVSESWT FAAPAANGTY TVEAFAFDPA
801 TGATYDADTT GATITVNQPP AAKYGDIVTK NTVITVNGTT YTVPAPDASG
851 HYPSGTNISI APGDTVTIQT TFANVSSTDA LQNGLIDMEV DGQNGAIFQK
901 YWPSTTLLPG QTETVTATWQ VPSSVSAGTY PLNFQAFDTS NWTGNCYFTN
951 GGVVNFVVN
In some embodiments of the invention, SEQ ID NO:307 may be glycosylated. In
further
embodiments, SEQ ID NO: 307 may be glycosylated at least at positions 174,
193, 297, 393, and 404.
With respect to the present invention, the new enzyme that was isolated from
an extremophilic
microbe has an N-terminal sequence comprising SEQ ID NO:326 as shown below:
DVVSTPISMEIQV.
It will be noted, that this N-terminal sequence of the present enzyme
aligns/corresponds to
positions 44-56 of SEQ ID NO:307.
In the present invention, an enzyme as contemplated by the present invention
and that is isolated
from an extremophilic microbe comprises that which is seen in SEQ ID NO:327,
which is provided
below:
QAS SSIVGNALAQAASLSPTISAYLRQNGLSPSDLARTWSSYYCTQFDDPQGAAQTALAT
RICNDQALGGGAPTASATITVNTAAR.
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As should be understood, this SEQ ID NO:327 aligns/corresponds to positions
166-248 of the
ce1B sequence as seen in SEQ ID NO:307. It should be noted, that SEQ ID NO:327
includes changed
amino acids at positions 207, 208, 212, 229, and 231; and added amino acids at
positions 209, 213, and
230, respectively.
In the present invention, the enzyme of the present invention may be further
characterized, and is
best understood by a study of SEQ ID NO:328 below:
GLNAAV WDSGLNSQTVISEV QALHPALIRWPGGSISDMDYNWETNTR
As should be understood SEQ ID NO:328, aligns/corresponds to positions 258-304
of SEQ ID
NO:307. It should be noted that SEQ ID NO:328 has a changed amino acid at
position number 295, and
an additional amino acid at position 296.
In the present invention, the enzyme as contemplated by the present invention
further comprises
the SEQ ID NO:329 as seen below:
EADDHAVPNQPQKGNPGLSPQAYAQNALQFMQSPVVYYR.
SEQ ID NO:329 aligns/corresponds to positions 379-415 of SEQ ID NO:307. It
should be
understood that with respect to the earlier SEQ ID NO:307, the present SEQ ID
NO:329 has changes in
amino acids at positions 409, 411, and 413, respectively. Still further,
additional amino acids are located
at positions 412, 414, and 415, respectively.
In the prior art reference noted above to Eckert and Schneider, it is observed
that work had been
conducted on the Alicyclobacillus acidocaldarius (ATCC 27009) for purposes of
determining the
presence of extracellular thermoacidophilic enzymes with polysaccharide-
degrading activities. The
authors noted that the organism was found to utilize a variety of
polysaccharides including xylan as a sole
source of carbon and energy. However, the authors failed to detect xylanase
activity in the culture
supernatant. The authors assumed a cell-associated enzyme and succeeded in
extracting cellulose
degrading activity with associated xylan degrading activity from the intact
cells with TRITON X-100.
The authors observed that the cellulose degrading activity and its associated
xylanase activity remained
cell bound even after the culture reached the stationary phase of growth. In
contrast, the enzyme isolated
from the extremophilic microbe of the present invention that displays optimum
enzymatic activities at
temperatures equal to or greater than 80 C and at a pH of less than 2, is
considered to be water-soluble,
and further has been isolated from cell supernatant.
The present invention is also directed to a method for the preparation of a
hemicellulase that
includes the steps of providing a source of Alicyclobacillus acidocaldarius
(ATCC 27009); cultivating the
Alicyclobacillus acidocaldarius (ATCC 27009) in a microbial nutrient medium
having a supernatant;
separating the cells of the Alicyclobacillus acidocaldarius from the nutrient
medium supernatant; and
recovering and purifying the hemicellulase derived from the Alicyclobacillus
acidocaldarius (ATCC
27009) from the nutrient medium supernatant. The methodology, as described,
produces a hemicellulase
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that is water-soluble and displays significant enzymatic activity at a pH of
less than about 2, and at
temperatures greater than about 80 C. In the methodology as described, the
hemicellulase comprises the
sequence as depicted in SEQ ID NOS:326, 327, 328, and/or 329. Still further,
and in the methodology as
described, the nutrient medium that is utilized further includes about 1 gram
per liter of Xylan, about 10
mM NH4C1, about 5.2 mM K2HPO4, about 0.8 mM MgSO4-7 H2O, about 1.74 mM Na2SO4,
about 25 mg
per liter MgC12i about 6.6 mg per liter of CaCl2, about 2.0 mg per liter
MnSO4, about 0.5 mg per liter
ZnSO4, about 0.5 mg per liter of boric acid, about 5 mg per liter of FeC13,
about 0.15 mg per liter of
CuSO4, about 0.025 mg per liter of NaMoO4, about 0.05 mg per liter of CoNO3,
about 0.02 mg per liter of
NiCl2, about 0.08 mg per liter of pyridoxine hydrochloride, about 0.01 mg per
liter of folic acid, about 0.1
mg per liter of thiamine hydrochloride, about 0.04 mg per liter of riboflavin,
about 0.08 mg per liter of
nicotinamide, about 0.08 mg per liter of p-aminobenzoate, about 0.01 mg per
liter of biotin, about 0.0004
mg per liter cyanocobalamin, about 0.08 mg per liter D-pantothenic acid-Ca,
about 0.02 mg per liter of
myo-inositol, about 0.05 mg per liter of choline bromide, about 0.02 mg per
liter of monosodium orotic
acid, and about 0.1 mg per liter spermidine, wherein the resulting nutrient
medium is adjusted to a pH of
about 3.5. As discussed earlier in the application, the present enzyme may be
used in various processes.
Therefore, the methodology, as described above, includes the step of supplying
the recovered and purified
hernicellulase to a simultaneous saccharification and fermentation process to
facilitate the conversion of a
biomass polysaccharide into an end product. One process for using the enzyme
as noted above includes a
step of pretreating a biomass slurry with the recovered and purified
hemicellulase or with a crude enzyme
preparation prepared from the organism containing a majority of the protein
comprised of the
hemicellulase to degrade an oligomer and/or polysaccharide that is present in
the biomass slurry to
produce an end product.
Other possible methods for using the enzyme as described above may be
employed. For
example, the enzyme that has been isolated from the extremophilic microbe may
be used in a method for
hydrolyzing a polysaccharide, which includes the step of providing a water-
soluble hemicellulase derived
from an extremophilic microbe; and conducting hydrolysis of a polysaccharide
with the water-soluble
hemicellulase at a pH of less than about 2. As was discussed, earlier, the
water-soluble hemicellulase has
an optimal enzymatic activity at a temperature of about 80 C.
OPERATION
The operation of the described embodiment of the present invention is believed
to be readily
apparent and is briefly summarized at this point.
As described, an enzyme isolated from extremophilic microbe that displays
optimum enzymatic
activity at a temperature of about 80 C and a pH of less than about 2 is best
understood by a study of SEQ
ID NOS:327-329, respectively. The enzyme that has been isolated is useful in a
method for treating a
biomass, which includes the steps of providing a source of a biomass having a
biomass sugar; pretreating
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the biomass with a water-soluble hemicellulase derived from Alicyclobacillus
acidocaldarius (ATCC
27009) to produce an end product. In the present methodology, the biomass
sugar comprises a
polysaccharide, and the hemicellulase hydrolyzes the polysaccharide. As
discussed above, the
hemicellulase displays enzymatic activity at a pH of less than about 2, and at
temperature of greater than
about 80 C.
The hemicellulase, as contemplated by the present invention, has a molecular
weight of about 120
kDa. In the methodology as described above, the methodology includes
additional steps of pretreating the
biomass in the presence or absence of the hemicellulase; providing a
sequential hydrolysis and
fermentation process to convert the biomass sugar into the end product; and
supplying the hemicellulase
to the sequential hydrolysis and fermentation process to facilitate the
conversion of the biomass sugar into
the end product. After the step of pretreating the biomass as discussed above,
which can be performed at
reduced severity in the presence or absence of the hemicellulase, the method
includes a further step of
providing a simultaneous saccharification and fermentation process to convert
the biomass sugar into the
end product; and supplying the hemicellulase to the simultaneous
saccharification and fermentation
process to facilitate the conversion of the biomass sugar into the end
product.
Therefore, it will be seen that the present enzyme and methodology, as
described above, avoids
many of the shortcomings attendant with the prior art enzymes and practices
employed heretofore, and
further provides a convenient means for producing various desirable end
products, while simultaneously
reducing the severity of pretreatment steps that had the propensity for
generating various deleterious
waste products, as well as for increasing the cost of the overall process
through the requirement of high
temperatures, pressures and quantities of acid to attain the high pretreatment
severity.
Embodiments of the invention include methods of post-translationally modifying
proteins. In
some embodiments, the post-translational modification may occur using isolated
or partially purified
glycosyltransferases and/or post-translational modification proteins, extracts
of cells comprising
glycosyltransferases and/or post-translational modification proteins, and/or
in cells comprising one or
more glycosyltransferases and/or post-translational modification proteins.
Glycosyltransferases and/or
post-translational modification proteins may be, without limitation, of the
following classes: UDP
beta-glucosephosphotransferases, Dolichol-phosphate mannosyltransferases, and
Glycosyltransferases. In
some embodiments, the glycosyltransferases and/or post-translational
modification proteins may be those
of a thermoacidophilic organism. Examples of thermoacidophiles, include, but
are not limited to,
Alicyclobacillus acidocaldarius, and those organisms belonging to the genera
Acidianus, Alicyclobacillus,
Desulfurolobus, Stygiolobus, Sulfolobus, Sulfurisphaera, Sulfurococcus,
Thermoplasma, and Picrophilus.
Examples of glycosyltransferases and/or post-translational modification
proteins include, but are not
limited to, those provided by SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137,
154, 171, 188, 205, 222,
239, 256, 273, and 290, as well as those available from the NCBI at accession
numbers XP_002490630.1,
Q541L5.1, ABN66322.2, XP_002493240.1, XP002491463.1, XP002491326.1,
CAY71061.1,
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NP_344219.1, NP_343051.1, ACR41289.1, ACP37471.1, NP_111396.1, NP111382.1,
NP_394039.1,
NP 394183.1, YP023099.1, P_023093.1, AAT43750.1, and AAT42890.1. Embodiments
of the
invention also include proteins glycosylated according to methods of the
invention. Examples of such
proteins include, but are not limited to, glycosylated forms of the proteins
of SEQ ID NOS:307, 331, 333,
335, 337, and 338.
Embodiments of the invention include methods of altering the physical and/or
kinetic properties
of an endoglucanase and/or xylanase from a thermoacidophile. In some
embodiments, the ratio of
cellulase activity to xylanase activity of an endoglucanase and/or xylanase
from a thermoacidophile is
altered by post-translational modification of the endoglucanase and/or
xylanase. In some embodiments,
the post-translational modification may be glycosylation. In further
embodiments, the solubility of an
endoglucanase and/or xylanase from a thermoacidophile is altered by post-
translational modification of
the endoglucanase and/or xylanase. In some embodiments, the endoglucanase
and/or xylanase is an
endoglucanase and/or xylanase of Alicyclobacillus acidocaldarius. In further
embodiments, the
endoglucanase and/or xylanase is celB (SEQ ID NO:307).
Embodiments of the invention include modification of the ratio of cellulase
activity to xylanase
activity of SEQ ID NO:307 through glycosylation of SEQ ID NO:307.
Glycosylation of SEQ ID NO:307
may be performed, by way of non-limiting example, by expression of a sequence
encoding SEQ ID
NO:307 in an organism capable of glycosylating SEQ ID NO:307, by exposure of
SEQ ID NO:307 to an
enzyme having glycosylating activity or a cell producing an enzyme having
glycosylating activity; or by
chemical methods known in the art.
In further embodiments of the invention, the ratio of cellulase activity to
xylanase activity of an
endoglucanase and/or xylanase may be altered based on the level and location
of post-translational
modification. In some embodiments, the level and location of post-
translational modification may be
manipulated, by way of non-limiting examples, through the use of different
enzymes having glycosylating
activity, different cells capable of glycosylating a protein, different
chemical methods of glycosylation,
and/or by varying the amount of glycosylating activity or time the
endoglucanase and/or xylanase is
exposed to. In some embodiments, post-translational modification of the
endoglucanase and/or xylanase
results in increased xylanase activity at an acidic pH. In some embodiments,
the modified form of the
endoglucanase and/or xylanase has increased xylanase activity compared to an
un-modified form of the
endoglucanase and/or xylanase at, by way of non-limiting example, pH of less
than about 5, pH of about
5, pH of about 3.5, and pH of about 2.
In some embodiments, post-translational modification of the endoglucanase
and/or xylanase
results in greater solubility at an acidic pH. In some embodiments, the
modified form of the
endoglucanase and/or xylanase is more soluble that the un-modified form of the
endoglucanase and/or
xylanase at, by way of non-limiting example, pH of less than about 5, pH of
about 5, pH of about 3.5, and
pH of about 2.
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Embodiments of the invention include genes and associated proteins related to
the glycosylation
and/or post-translational modification of proteins of the thermoacidophile
Alicyclobacillus
acidocaldarius. Coding sequences for genes related to these processes were
determined from sequence
information generated from sequencing the genome of Alicyclobacillus
acidocaldarius. These genes and
proteins may represent targets for metabolic engineering of Alicyclobacillus
acidocaldarius or other
organisms. Non-limiting examples of nucleotide sequences found within the
genome of Alicyclobacillus
acidocaldarius, and amino acids coded thereby, associated with glycosylation
and/or post-translational
modification of proteins are listed in Table 1. Glycosyltransferases and/or
post-translational modification
proteins may be, without limitation, of the following classes: UDP beta-
glucosephosphotransferases,
Dolichol-phosphate mannosyltransferases, and Glycosyltransferases; and others.
Embodiments of the invention relate in part to the gene sequences and/or
protein sequences
comprising genes and/or proteins of Alicyclobacillus acidocaldarius. Genes and
proteins included are
those which play a role in glycosylation and/or post-translational
modification of proteins. Intracellular
enzyme activities may be thermophilic and/or acidophilic in nature and general
examples of similar genes
are described in the literature. Classes of genes, sequences, enzymes and
factors include, but are not
limited to, those listed in Table 1.
TABLE 1
Alicyclobacillus acidocaldarius genes and proteins related to glycosylation
Reference Protein Sequence Gene Sequence Function
RAA000164 SEQ ID NO:1 SEQ ID NO:2 Glycosyltransferase
RAA000517 SEQ ID NO:18 SEQ ID NO:19 Glycosyltransferase
RAA000650 SEQ ID NO:35 SEQ ID NO:36 Glycosyltransferase
RAA000991 SEQ ID NO:52 SEQ ID NO:53 Glycosyltransferase
RAAC01110 SEQ ID NO:69 SEQ ID NO:70 Glycosyltransferase
RAACO 1166 SEQ ID NO: 86 SEQ ID NO: 87 UDP beta-glucosephosphotransferase
RAACO1167 SEQ ID NO: 103 SEQ ID NO: 104 Glycosyltransferase
RAACO1170 SEQ ID NO:120 SEQ ID NO: 121 Glycosyltransferase
RAAC01248 SEQ ID NO:137 SEQ ID NO:138 Glycosyltransferase
RAAC01348 SEQ ID NO:154 SEQ ID NO:155 Glycosyltransferase
RAAC01377 SEQ ID NO:171 SEQ ID NO:172 Glycosyltransferase
RAAC01611 SEQ ID NO:188 SEQ ID NO:189 Glycosyltransferase
RAAC01612 SEQ ID NO:205 SEQ ID NO:206 Glycosyltransferase
RAAC01926 SEQ ID NO:222 SEQ ID NO:223 Glycosyltransferase
RAAC01998 SEQ ID NO:239 SEQ ID NO:240 Glycosyltransferase
RAAC02011 SEQ ID NO:256 SEQ ID NO:257 Dolichol-phosphate mannosyltransferase
RAAC02381 SEQ ID NO:273 SEQ ID NO:274 Glycosyltransferase
RAAC02421 SEQ ID NO:290 SEQ ID NO:291 Glycosyltransferase
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The present invention relates to nucleotides sequences comprising isolated
and/or purified
nucleotide sequences of the genome of Alicyclobacillus acidocaldarius selected
from the sequences of
SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,
257, 274, 291, 332, 334,
336, 339, and 340, or one of their fragments.
The present invention likewise relates to isolated and/or purified nucleotide
sequences,
characterized in that they comprise at least one of. a) a nucleotide sequence
of at least one of the
sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189,
206, 223, 240, 257, 274,
291, 332, 334, 336, 339, and 340 or one of their fragments; b) a nucleotide
sequence homologous to a
nucleotide sequence such as defined in a); c) a nucleotide sequence
complementary to a nucleotide
sequence such as defined in a) or b), and a nucleotide sequence of their
corresponding RNA; d) a
nucleotide sequence capable of hybridizing under stringent conditions with a
sequence such as defined in
a), b) or c); e) a nucleotide sequence comprising a sequence such as defined
in a), b), c) or d); and f) a
nucleotide sequence modified by a nucleotide sequence such as defined in a),
b), c), d) or e).
Nucleotide, polynucleotide, or nucleic acid sequence will be understood
according to the present
invention as meaning both a double-stranded or a single-stranded nucleic acid
in the monomeric and
dimeric (so-called in "tandem") forms and the transcription products of said
nucleic acids.
Aspects of the invention relate nucleotide sequences which it has been
possible to isolate, purify
or partially purify, starting from separation methods such as, for example,
ion-exchange chromatography,
by exclusion based on molecular size, or by affinity, or alternatively,
fractionation techniques based on
solubility in different solvents, or starting from methods of genetic
engineering such as amplification,
cloning, and subcloning, it being possible for the sequences of the invention
to be carried by vectors.
Isolated and/or purified nucleotide sequence fragment according to the
invention will be
understood as designating any nucleotide fragment of the genome of
Alicyclobacillus acidocaldarius, and
may include, by way of non-limiting examples, length of at least 8, 12, 20 25,
50, 75, 100, 200, 300, 400,
500, 1000, or more, consecutive nucleotides of the sequence from which it
originates.
Specific fragment of an isolated and/or purified nucleotide sequence according
to the invention
will be understood as designating any nucleotide fragment of the genome of
Alicyclobacillus
acidocaldarius, having, after alignment and comparison with the corresponding
fragments of genomic
sequences of Alicyclobacillus acidocaldarius, at least one nucleotide or base
of different nature.
An homologous isolated and/or purified nucleotide sequence in the sense of the
present invention
is understood as meaning an isolated and/or purified nucleotide sequence
having at least a percentage
identity with the bases of a nucleotide sequence according to the invention of
at least about 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%,
99.5%, 99.6%, or 99.7%, this percentage being purely statistical and it being
possible to distribute the
differences between the two nucleotide sequences at random and over the whole
of their length.
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Specific homologous nucleotide sequence in the sense of the present invention
is understood as
meaning a homologous nucleotide sequence having at least one nucleotide
sequence of a specific
fragment, such as defined above. Said "specific" homologous sequences can
comprise, for example, the
sequences corresponding to the genomic sequence or to the sequences of its
fragments representative of
variants of the genome of Alicyclobacillus acidocaldarius. These specific
homologous sequences can thus
correspond to variations linked to mutations within strains of
Alicyclobacillus acidocaldarius, and
especially correspond to truncations, substitutions, deletions and/or
additions of at least one nucleotide.
Said homologous sequences can likewise correspond to variations linked to the
degeneracy of the genetic
code.
The term "degree or percentage of sequence homology" refers to "degree or
percentage of
sequence identity between two sequences after optimal alignment" as defined in
the present application.
Two amino-acids or nucleotidic sequences are said to be "identical" if the
sequence of
amino-acids or nucleotidic residues, in the two sequences is the same when
aligned for maximum
correspondence, as described below. Sequence comparisons between two (or more)
peptides or
polynucleotides are typically performed by comparing sequences of two
optimally aligned sequences over
a segment or "comparison window" to identify and compare local regions of
sequence similarity. Optimal
alignment of sequences for comparison may be conducted by the local homology
algorithm of Smith and
Waterman, Ad. App. Math 2:482 (1981), by the homology alignment algorithm of
Neddleman and
Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of
Pearson and Lipman, Proc.
Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementation of
these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer
Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection.
"Percentage of sequence identity" (or degree of identity) is determined by
comparing two
optimally aligned sequences over a comparison window, where the portion of the
peptide or
polynucleotide sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as
compared to the reference sequence (which does not comprise additions or
deletions) for optimal
alignment of the two sequences. The percentage is calculated by determining
the number of positions at
which the identical amino-acid residue or nucleic acid base occurs in both
sequences to yield the number
of matched positions, dividing the number of matched positions by the total
number of positions in the
window of comparison and multiplying the result by 100 to yield the percentage
of sequence identity.
The definition of sequence identity given above is the definition that would
be used by one of
skill in the art. The definition by itself does not need the help of any
algorithm, said algorithms being
helpful only to achieve the optimal alignments of sequences, rather than the
calculation of sequence
identity.
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From the definition given above, it follows that there is a well defined and
only one value for the
sequence identity between two compared sequences which value corresponds to
the value obtained for the
best or optimal alignment.
In the BLAST N or BLAST P "BLAST 2 sequence," software which is available at
the web site
worldwideweb.nebi.nlm.nih.gov/gorf/bl2.html, and habitually used by the
inventors and in general by the
skilled person for comparing and determining the identity between two
sequences, gap cost which
depends on the sequence length to be compared is directly selected by the
software (i.e., 11.2 for
substitution matrix BLOSUM-62 for length>85).
Complementary nucleotide sequence of a sequence of the invention is understood
as meaning any
DNA whose nucleotides are complementary to those of the sequence of the
invention, and whose
orientation is reversed (antisense sequence).
Hybridization under conditions of stringency with a nucleotide sequence
according to the
invention is understood as meaning hybridization under conditions of
temperature and ionic strength
chosen in such a way that they allow the maintenance of the hybridization
between two fragments of
complementary DNA.
By way of illustration, conditions of great stringency of the hybridization
step with the aim of
defining the nucleotide fragments described above are advantageously the
following.
The hybridization is carried out at a preferential temperature of 65 C in the
presence of SSC
buffer, 1 x SSC corresponding to 0.15 M NaCl and 0.05 M Na citrate. The
washing steps, for example,
can be the following: 2 x SSC, at ambient temperature followed by two washes
with 2 x SSC, 0.5% SDS
at 65 C.; 2 x 0.5 x SSC, 0.5% SDS; at 65 C for 10 minutes each.
The conditions of intermediate stringency, using, for example, a temperature
of 42 C in the
presence of a 2 x SSC buffer, or of less stringency, for example a temperature
of 37 C in the presence of a
2 x SSC buffer, respectively, require a globally less significant
complementarity for the hybridization
between the two sequences.
The stringent hybridization conditions described above for a polynucleotide
with a size of
approximately 350 bases will be adapted by a person skilled in the art for
oligonucleotides of greater or
smaller size, according to the teachings of Sambrook et al., 1989.
Among the isolated and/or purified nucleotide sequences according to the
invention, are those
that can be used as a primer or probe in methods allowing the homologous
sequences according to the
invention to be obtained. These methods, such as the polymerase chain reaction
(PCR), nucleic acid
cloning, and sequencing, are well known to a person skilled in the art.
Among said isolated and/or purified nucleotide sequences according to the
invention, those are
again preferred which can be used as a primer or probe in methods allowing the
presence of SEQ ID
NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,
274, 291, 332, 334, 336, 339,
and 340, one of their fragments, or one of their variants such as defined
below to be diagnosed.
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The nucleotide sequence fragments according to the invention can be obtained,
for example, by
specific amplification, such as PCR, or after digestion with appropriate
restriction enzymes of nucleotide
sequences according to the invention, these methods in particular being
described in the work of
Sambrook et al., 1989. Such representative fragments can likewise be obtained
by chemical synthesis
according to methods well known to persons of ordinary skill in the art.
Modified nucleotide sequence will be understood as meaning any nucleotide
sequence obtained
by mutagenesis according to techniques well known to the person skilled in the
art, and containing
modifications with respect to the normal sequences according to the invention,
for example mutations in
the regulatory and/or promoter sequences of polypeptide expression, especially
leading to a modification
of the rate of expression of said polypeptide or to a modulation of the
replicative cycle.
Modified nucleotide sequence will likewise be understood as meaning any
nucleotide sequence
coding for a modified polypeptide such as defined below.
The present invention relates to nucleotide sequence comprising isolated
and/or purified
nucleotide sequences of Alicyclobacillus acidocaldarius, characterized in that
they are selected from the
sequences SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206,
223, 240, 257, 274, 291,
332, 334, 336, 339, and 340 or one of their fragments.
Embodiments of the invention likewise relate to isolated and/or purified
nucleotide sequences
characterized in that they comprise a nucleotide sequence selected from: a) at
least one of a nucleotide
sequence of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189,
206, 223, 240, 257, 274,
291, 332, 334, 336, 339, and 340 or one of their fragments; b) a nucleotide
sequence of a specific
fragment of a sequence such as defined in a); c) a homologous nucleotide
sequence having at least 80%
identity with a sequence such as defined in a) or b); d) a complementary
nucleotide sequence or sequence
of RNA corresponding to a sequence such as defined in a), b) or c); and e) a
nucleotide sequence
modified by a sequence such as defined in a), b), c) or d).
Among the isolated and/or purified nucleotide sequences according to the
invention are the
nucleotide sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155,
172, 189, 206, 223, 240,
257, 274, 291, 332, 334, 336, 339, and 340, or fragments thereof and any
isolated and/or purified
nucleotide sequences which have a homology of at least 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or
99.7% identity
with the at least one of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87,
104, 121, 138, 155, 172, 189,
206, 223, 240, 257, 274, 291, 332, 334, 336, 339, and 340 or fragments
thereof. Said homologous
sequences can comprise, for example, sequences corresponding to genomic
sequences Alicyclobacillus
acidocaldarius. In the same manner, these specific homologous sequences can
correspond to variations
linked to mutations within strains of Alicyclobacillus acidocaldarius and
especially correspond to
truncations, substitutions, deletions and/or additions of at least one
nucleotide. As will be apparent to one
of ordinary skill in the art, such homologues are easily created and
identified using standard techniques
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and publicly available computer programs such as BLAST. As such, each
homologue referenced above
should be considered as set forth herein and fully described.
Embodiments of the invention comprise the isolated and/or purified
polypeptides coded for by a
nucleotide sequence according to the invention, or fragments thereof, whose
sequence is represented by a
fragment. Amino acid sequences corresponding to the isolated and/or purified
polypeptides may be coded
by one of the three possible reading frames of at least one of the sequences
of SEQ ID NOS:2, 19, 36, 53,
70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 332, 334,
336, 339, and 340.
Embodiments of the invention likewise relate to the isolated and/or purified
polypeptides,
characterized in that they comprise a polypeptide selected from at least one
of the amino acid sequences
of SEQ ID NOS:I, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,
239, 256, 273, 290, 307,
331, 333, 335, 337, and 338 or one of their fragments.
Among the isolated and/or purified polypeptides, according to embodiments of
the invention, are
the isolated and/or purified polypeptides of amino acid sequence SEQ ID NOS:1,
18, 35, 52, 69, 86, 103,
120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335,
337, and 338, or fragments
thereof or any other isolated and/or purified polypeptides which have a
homology of at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%,
99.5%, 99.6%, or 99.7% identity with at least one of the sequences of SEQ ID
NOS:1, 18, 35, 52, 69, 86,
103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333,
335, 337, and 338 or fragments
thereof. As will be apparent to one of ordinary skill in the art, such
homologues are easily created and
identified using standard techniques and publicly available computer programs
such as BLAST. As such,
each homologue referenced above should be considered as set forth herein and
fully described.
Embodiments of the invention also relate to the polypeptides, characterized in
that they comprise
a polypeptide selected from: a) a specific fragment of at least five amino
acids of a polypeptide of an
amino acid sequence according to the invention; b) a polypeptide homologous to
a polypeptide such as
defined in a); c) a specific biologically active fragment of a polypeptide
such as defined in a) or b); and
d) a polypeptide modified by a polypeptide such as defined in a), b) or c).
In the present description, the terms polypeptide, peptide and protein are
interchangeable.
In some embodiments of the invention, the isolated and/or purified
polypeptides according to the
invention may be glycosylated, pegylated, and/or otherwise post-
translationally modified. In further
embodiments, glycosylation, pegylation, and/or other post-translational
modifications may occur in vivo
or in vitro and/or may be performed using chemical techniques. In additional
embodiments, any
glycosylation, pegylation and/or other post-translational modifications may be
N-linked or O-linked.
In some embodiments of the invention any one of the isolated and/or purified
polypeptides
according to the invention may be enzymatically or functionally active at
temperatures at or above about
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees
Celsius and/or may be
enzymatically or functionally active at a pH at, below, and/or above 8, 7, 6,
5, 4, 3, 2, 1, and/or 0. In
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further embodiments of the invention, glycosylation, pegylation, and/or other
post-translational
modification may be required for the isolated and/or purified polypeptides
according to the invention to
be enzymatically or functionally active at a pH at or below 8, 7, 6, 5, 4, 3,
2, 1, and/or 0 or at temperatures
at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
and/or 95 degrees Celsius.
Aspects of the invention relate to polypeptides that are isolated or obtained
by purification from
natural sources, or else obtained by genetic recombination, or alternatively
by chemical synthesis and that
they may thus contain unnatural amino acids, as will be described below.
A "polypeptide fragment" according to the embodiments of the invention is
understood as
designating a polypeptide containing at least five consecutive amino acids,
preferably ten consecutive
amino acids or 15 consecutive amino acids.
In the present invention, a "specific polypeptide fragment" is understood as
designating the
consecutive polypeptide fragment coded for by a specific fragment of a
nucleotide sequence according to
the invention.
"Homologous polypeptide" will be understood as designating the polypeptides
having, with
respect to the natural polypeptide, certain modifications such as, in
particular, a deletion, addition, or
substitution of at least one amino acid, a truncation, a prolongation, a
chimeric fusion, and/or a mutation.
Among the homologous polypeptides, those are preferred whose amino acid
sequence has at least 80% or
90%, homology with the sequences of amino acids of polypeptides according to
the invention.
"Specific homologous polypeptide" will be understood as designating the
homologous
polypeptides such as defined above and having a specific fragment of a
polypeptide according to the
invention.
In the case of a substitution, one or more consecutive or nonconsecutive amino
acids are replaced
by "equivalent" amino acids. The expression "equivalent" amino acid is
directed here at designating any
amino acid capable of being substituted by one of the amino acids of the base
structure without, however,
essentially modifying the biological activities of the corresponding peptides
and such that they will be
defined by the following. As will be apparent to one of ordinary skill in the
art, such substitutions are
easily created and identified using standard molecular biology techniques and
publicly available computer
programs such as BLAST. As such, each substitution referenced above should be
considered as set forth
herein and fully described. These equivalent amino acids may be determined
either by depending on their
structural homology with the amino acids which they substitute, or on results
of comparative tests of
biological activity between the different polypeptides, which are capable of
being carried out.
By way of nonlimiting example, the possibilities of substitutions capable of
being carried out
without resulting in an extensive modification of the biological activity of
the corresponding modified
polypeptides will be mentioned, the replacement, for example, of leucine by
valine or isoleucine, of
aspartic acid by glutamic acid, of glutamine by asparagine, of arginine by
lysine etc., the reverse
substitutions naturally being envisageable under the same conditions.
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In a further embodiment, substitutions are limited to substitutions in amino
acids not conserved
among other proteins which have similar identified enzymatic activity. For
example, one of ordinary skill
in the art may align proteins of the same function in similar organisms and
determine which amino acids
are generally conserved among proteins of that function. One example of a
program that may be used to
generate such alignments is available on the Internet at
worldwideweb.charite.de/bioinf/strap/ in
conjunction with the databases provided by the NCBI.
Thus, according to one embodiment of the invention, substitutions or mutations
may be made at
positions that are generally conserved among proteins of that function. In a
further embodiment, nucleic
acid sequences may be mutated or substituted such that the amino acid they
code for is unchanged
(degenerate substitutions and/mutations) and/or mutated or substituted such
that any resulting amino acid
substitutions or mutations are made at positions that are generally conserved
among proteins of that
function.
The specific homologous polypeptides likewise correspond to polypeptides coded
for by the
specific homologous nucleotide sequences such as defined above and, thus,
comprise in the present
definition the polypeptides that are mutated or correspond to variants which
can exist in Alicyclobacillus
acidocaldarius, and which especially correspond to truncations, substitutions,
deletions, and/or additions
of at least one amino acid residue.
"Specific biologically active fragment of a polypeptide" according to an
embodiment of the
invention will be understood in particular as designating a specific
polypeptide fragment, such as defined
above, having at least one of the characteristics of polypeptides according to
the invention. In certain
embodiments the peptide is capable of behaving as at least one of the types of
proteins outlined in
Table 1.
The polypeptide fragments according to embodiments of the invention, can
correspond to isolated
or purified fragments naturally present in Alicyclobacillus acidocaldarius or
correspond to fragments
which can be obtained by cleavage of said polypeptide by a proteolytic enzyme,
such as trypsin or
chymotiypsin or collagenase, or by a chemical reagent, such as cyanogen
bromide (CNBr). Such
polypeptide fragments can likewise just as easily be prepared by chemical
synthesis, from hosts
transformed by an expression vector, according to the invention, containing a
nucleic acid allowing the
expression of said fragments, placed under the control of appropriate
regulation and/or expression
elements.
"Modified polypeptide" of a polypeptide according to an embodiment of the
invention is
understood as designating a polypeptide obtained by genetic recombination or
by chemical synthesis as
will be described below, having at least one modification with respect to the
normal sequence. These
modifications may or may not be able to bear on amino acids at the origin of
specificity, and/or of
activity, or at the origin of the structural conformation, localization, and
of the capacity of membrane
insertion of the polypeptide according to the invention. It will thus be
possible to create polypeptides of
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equivalent, increased, or decreased activity, and of equivalent, narrower, or
wider specificity. Among the
modified polypeptides, it is necessary to mention the polypeptides in which up
to five or more amino
acids can be modified, truncated at the N- or C-terminal end, or even deleted
or added.
The methods allowing said modulations on eukaryotic or prokaryotic cells to be
demonstrated are
well known to the person of ordinary skill in the art. It is likewise well
understood that it will be possible
to use the nucleotide sequences coding for said modified polypeptides for said
modulations, for example,
through vectors according to the invention and described below.
The preceding modified polypeptides can be obtained by using combinatorial
chemistry, in which
it is possible to systematically vary parts of the polypeptide before testing
them on models, cell cultures
or microorganisms, for example, to select the compounds that are most active
or have the properties
sought.
Chemical synthesis likewise has the advantage of being able to use unnatural
amino acids, or
nonpeptide bonds.
Thus, in order to improve the duration of life of the polypeptides according
to the invention, it
may be of interest to use unnatural amino acids, for example in D form, or
else amino acid analogs,
especially sulfur-containing forms, for example.
Finally, it will be possible to integrate the structure of the polypeptides
according to the
invention, its specific or modified homologous forms, into chemical structures
of polypeptide type or
others. Thus, it may be of interest to provide at the N- and C-terminal ends
molecules not recognized by
proteases.
The nucleotide sequences coding for a polypeptide according to the invention
are likewise part of
the invention.
The invention likewise relates to nucleotide sequences utilizable as a primer
or probe,
characterized in that said sequences are selected from the nucleotide
sequences according to the
invention.
It is well understood that the present invention, in various embodiments,
likewise relates to
specific polypeptides of Alicyclobacillus acidocaldarius, coded for by
nucleotide sequences, capable of
being obtained by purification from natural polypeptides, by genetic
recombination or by chemical
synthesis by procedures well known to a person skilled in the art and such as
described in particular
below. In the same manner, the labeled or unlabeled mono- or polyclonal
antibodies directed against said
specific polypeptides coded for by said nucleotide sequences are also
encompassed by the invention.
Embodiments of the invention additionally relate to the use of a nucleotide
sequence according to
the invention as a primer or probe for the detection and/or the amplification
of nucleic acid sequences.
The nucleotide sequences according to embodiments of the invention can thus be
used to amplify
nucleotide sequences, especially by the PCR technique (Polymerase Chain
Reaction) (Erlich, 1989; Innis
et al., 1990; Rolfs et al., 1991; and White et al., 1997).
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These oligodeoxyribonucleotide or oligoribonucleotide primers advantageously
have a length of
at least eight nucleotides, preferably of at least twelve nucleotides, and
even more preferentially at least
20 nucleotides.
Other amplification techniques of the target nucleic acid can be
advantageously employed as
alternatives to PCR.
The nucleotide sequences of the invention, in particular the primers according
to the invention,
can likewise be employed in other procedures of amplification of a target
nucleic acid, such as: the TAS
technique (Transcription-based Amplification System), described by Kwoh et al.
in 1989; the 3SR
technique (Self-Sustained Sequence Replication), described by Guatelli et al.
in 1990; the NASBA
technique (Nucleic Acid Sequence Based Amplification), described by Kievitis
et al. in 1991; the SDA
technique (Strand Displacement Amplification) (Walker et al., 1992); and the
TMA technique
(Transcription Mediated Amplification).
The polynucleotides of the invention can also be employed in techniques of
amplification or of
modification of the nucleic acid serving as a probe, such as: the LCR
technique (Ligase Chain Reaction),
described by Landegren et al. in 1988 and improved by Barany et al. in 1991,
which employs a
thermostable ligase; the RCR technique (Repair Chain Reaction), described by
Segev in 1992; the CPR
technique (Cycling Probe Reaction), described by Duck et al. in 1990; the
amplification technique with
Q-beta replicase, described by Miele et al. in 1983 and especially improved by
Chu et al. in 1986, Lizardi
et al. in 1988, then by Burg et al., as well as by Stone et al. in 1996.
In the case where the target polynucleotide to be detected is possibly an RNA,
for example an
mRNA, it will be possible to use, prior to the employment of an amplification
reaction with the aid of at
least one primer according to the invention or to the employment of a
detection procedure with the aid of
at least one probe of the invention, an enzyme of reverse transcriptase type
in order to obtain a eDNA
from the RNA contained in the biological sample. The cDNA obtained will thus
serve as a target for the
primer(s) or the probe(s) employed in the amplification or detection procedure
according to the invention.
The detection probe will be chosen in such a manner that it hybridizes with
the target sequence or
the ainplicon generated from the target sequence. By way of sequence, such a
probe will advantageously
have a sequence of at least 12 nucleotides, in particular of at least 20
nucleotides, and preferably of at
least 100 nucleotides.
Embodiments of the invention also comprise the nucleotide sequences utilizable
as a probe or
primer according to the invention, characterized in that they are labeled with
a radioactive compound or
with a nonradioactive compound.
The unlabeled nucleotide sequences can be used directly as probes or primers,
although the
sequences are generally labeled with a radioactive isotope (32P, 35S, 3H,
1251) or with a nonradioactive
molecule (biotin, acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine,
fluorescein) to obtain probes
which are utilizable for numerous applications.
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Examples of nonradioactive labeling of nucleotide sequences are described, for
example, in
French Patent No. 7810975 or by Urdea et al. or by Sanchez-Pescador et al. in
1988.
In the latter case, it will also be possible to use one of the labeling
methods described in French
Patent Appl. Publication Nos. FR-2422956 and FR-2518755.
The hybridization technique can be carried out in various manners (Matthews et
al., 1988). The
most general method consists in immobilizing the nucleic acid extract of cells
on a support (such as
nitrocellulose, nylon, polystyrene) and in incubating, under well-defined
conditions, the immobilized
target nucleic acid with the probe. After hybridization, the excess of the
probe is eliminated and the
hybrid molecules formed are detected by the appropriate method (measurement of
the radioactivity, of the
fluorescence or of the enzymatic activity linked to the probe).
The invention, in various embodiments, likewise comprises the nucleotide
sequences according to
the invention, characterized in that they are immobilized on a support,
covalently or noncovalently.
According to another advantageous mode of employing nucleotide sequences
according to the
invention, the latter can be used immobilized on a support and can thus serve
to capture, by specific
hybridization, the target nucleic acid obtained from the biological sample to
be tested. If necessary, the
solid support is separated from the sample and the hybridization complex
formed between said capture
probe and the target nucleic acid is then detected with the aid of a second
probe, a so-called detection
probe, labeled with an easily detectable element.
Another aspect of the present invention is a vector for the cloning and/or
expression of a
sequence, characterized in that it contains a nucleotide sequence according to
the invention.
The vectors according to the invention, characterized in that they contain the
elements allowing
the integration, expression and/or the secretion of said nucleotide sequences
in a determined host cell, are
likewise part of the invention.
The vector may then contain a promoter, signals of initiation and termination
of translation, as
well as appropriate regions of regulation of transcription. The vector may be
able to be maintained stably
in the host cell and can optionally have particular signals specifying the
secretion of the translated protein.
These different elements may be chosen as a function of the host cell used. To
this end, the nucleotide
sequences according to the invention may be inserted into autonomous
replication vectors within the
chosen host, or integrated vectors of the chosen host.
Such vectors will be prepared according to the methods currently used by a
person skilled in the
art, and it will be possible to introduce the clones resulting therefrom into
an appropriate host by standard
methods, such as, for example, lipofection, electroporation, and thermal
shock.
The vectors according to the invention are, for example, vectors of plasmid or
viral origin. One
example of a vector for the expression of polypeptides of the invention is
baculovirus.
These vectors are useful for transforming host cells in order to clone or to
express the nucleotide
sequences of the invention.
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The invention likewise comprises the host cells transformed by a vector
according to the
invention.
These cells can be obtained by the introduction into host cells of a
nucleotide sequence inserted
into a vector such as defined above, then the culturing of said cells under
conditions allowing the
replication and/or expression of the transfected nucleotide sequence.
The host cell can be selected from prokaryotic or eukaryotic systems, such as,
for example,
bacterial cells (Olins and Lee, 1993), but likewise yeast cells (Buckholz,
1993), as well as plant cells,
such as Arabidopsis sp., and animal cells, in particular the cultures of
mammalian cells (Edwards and
Aruffo, 1993), for example, Chinese hamster ovary (CHO) cells, but likewise
the cells of insects in which
it is possible to use procedures employing baculoviruses, for example Sf9
insect cells (Luckow, 1993).
Embodiments of the invention likewise relate to organisms comprising one of
said transformed
cells according to the invention.
The obtainment of transgenic organisms according to the invention expressing
one or more of the
genes of Alicyclobacillus acidocaldarius, or part of the genes, may be carried
out in, for example, rats,
mice, or rabbits according to methods well known to a person skilled in the
art, such as by viral or
nonviral transfections. It will be possible to obtain the transgenic organisms
expressing one or more of
said genes by transfection of multiple copies of said genes under the control
of a strong promoter of
ubiquitous nature, or selective for one type of tissue. It will likewise be
possible to obtain the transgenic
organisms by homologous recombination in embryonic cell strains, transfer of
these cell strains to
embryos, selection of the affected chimeras at the level of the reproductive
lines, and growth of said
chimeras.
The transformed cells as well as the transgenic organisms according to the
invention are utilizable
in procedures for preparation of recombinant polypeptides.
It is today possible to produce recombinant polypeptides in relatively large
quantities by genetic
engineering using the cells transformed by expression vectors according to the
invention or using
transgenic organisms according to the invention.
The procedures for preparation of a polypeptide of the invention in
recombinant form,
characterized in that they employ a vector and/or a cell transformed by a
vector according to the invention
and/or a transgenic organism comprising one of said transformed cells
according to the invention are
themselves comprised in the present invention.
As used herein, "transformation" and "transformed" relate to the introduction
of nucleic acids
into a cell, whether prokaryotic or eukaryotic. Further, "transformation" and
"transformed," as used
herein, need not relate to growth control or growth deregulation.
Among said procedures for preparation of a polypeptide of the invention in
recombinant form, the
preparation procedures employing a vector, and/or a cell transformed by said
vector and/or a transgenic
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organism comprising one of said transformed cells, containing a nucleotide
sequence according to the
invention coding for a polypeptide of Alicyclobacillus acidocaldarius.
A variant according to an embodiment of the invention may consist of producing
a recombinant
polypeptide fused to a "carrier" protein (chimeric protein). The advantage of
this system is that it may
allow stabilization of and/or a decrease in the proteolysis of the recombinant
product, an increase in the
solubility in the course of renaturation in vitro and/or a simplification of
the purification when the fusion
partner has an affinity for a specific ligand.
More particularly, an embodiment of the invention relates to a procedure for
preparation of a
polypeptide of the invention comprising the following steps: a) culture of
transformed cells under
conditions allowing the expression of a recombinant polypeptide of nucleotide
sequence according to the
invention; b) if need be, recovery of said recombinant polypeptide.
When the procedure for preparation of a polypeptide of the invention employs a
transgenic
organism according to the invention, the recombinant polypeptide is then
extracted from said organism.
An embodiment of the invention also relates to a polypeptide which is capable
of being obtained
by a procedure of the invention such as described previously.
The invention, in another embodiment, also comprises a procedure for
preparation of a synthetic
polypeptide, characterized in that it uses a sequence of amino acids of
polypeptides according to the
invention.
A further embodiment of the invention likewise relates to a synthetic
polypeptide obtained by a
procedure according to the invention.
The polypeptides according to embodiments of the invention can likewise be
prepared by
techniques which are conventional in the field of the synthesis of peptides.
This synthesis can be carried
out in a homogeneous solution or in a solid phase.
For example, recourse can be made to the technique of synthesis in a
homogeneous solution
described by Houben-Weyl in 1974.
This method of synthesis comprises successively condensing, two by two, the
successive amino
acids in the order required, or in condensing amino acids and fragments formed
previously and already
containing several amino acids in the appropriate order, or alternatively
several fragments previously
prepared in this way, it being understood that it will be necessary to protect
beforehand all the reactive
functions carried by these amino acids or fragments, with the exception of
amine functions of one and
carboxyls of the other or vice-versa, which must normally be involved in the
formation of peptide bonds,
especially after activation of the carboxyl function, according to the methods
well known in the synthesis
of peptides.
Recourse may also be made to the technique described by Merrifield.
To make a peptide chain according to the Merrifield procedure, recourse is
made to a very porous
polymeric resin, on which is immobilized the first C-terminal amino acid of
the chain. This amino acid is
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immobilized on a resin through its carboxyl group and its amine function is
protected. The amino acids
that are going to form the peptide chain are thus immobilized, one after the
other, on the amino group,
which is deprotected beforehand each time, of the portion of the peptide chain
already formed, and which
is attached to the resin. When the whole of the desired peptide chain has been
formed, the protective
groups of the different amino acids forming the peptide chain are eliminated
and the peptide is detached
from the resin with the aid of an acid.
The invention additionally relates to hybrid polypeptides having at least one
polypeptide
according to the invention, and a sequence of a polypeptide capable of
inducing an immune response in
man or animals.
Advantageously, the antigenic determinant is such that it is capable of
inducing a humoral and/or
cellular response.
It will be possible for such a determinant to comprise a polypeptide according
to the invention in
glycosylated, pegylated, and/or otherwise post-translationally modified form
used with a view to
obtaining immunogenic compositions capable of inducing the synthesis of
antibodies directed against
multiple epitopes.
These hybrid molecules can be formed, in part, of a polypeptide carrier
molecule or of fragments
thereof according to the invention, associated with a possibly immunogenic
part, in particular an epitope
of the diphtheria toxin, the tetanus toxin, a surface antigen of the hepatitis
B virus (French Patent
7921811), the VP1 antigen of the poliomyelitis virus or any other viral or
bacterial toxin or antigen.
The procedures for synthesis of hybrid molecules encompass the methods used in
genetic
engineering for constructing hybrid nucleotide sequences coding for the
polypeptide sequences sought. It
will be possible, for example, to refer advantageously to the technique for
obtainment of genes coding for
fusion proteins described by Minton in 1984.
Said hybrid nucleotide sequences coding for a hybrid polypeptide, as well as
the hybrid
polypeptides according to the invention, are so characterized in that they are
recombinant polypeptides
obtained by the expression of said hybrid nucleotide sequences, which are
likewise part of the invention.
The invention likewise comprises the vectors characterized in that they
contain one of said hybrid
nucleotide sequences. The host cells transformed by said vectors, the
transgenic organisms comprising
one of said transformed cells as well as the procedures for preparation of
recombinant polypeptides using
said vectors, said transformed cells and/or said transgenic organisms are, of
course, likewise part of the
invention.
The polypeptides according to the invention, the antibodies according to the
invention described
below and the nucleotide sequences according to the invention can
advantageously be employed in
procedures for the detection and/or identification of Alicyclobacillus
acidocaldarius, in a sample capable
of containing them. These procedures, according to the specificity of the
polypeptides, the antibodies and
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the nucleotide sequences according to the invention which will be used, will
in particular be able to detect
and/or to identify Alicyclobacillus acidocaldarius.
The polypeptides according to the invention can advantageously be employed in
a procedure for
the detection and/or the identification of Alicyclobacillus acidocaldarius in
a sample capable of
containing them, characterized in that it comprises the following steps: a)
contacting of this sample with a
polypeptide or one of its fragments according to the invention (under
conditions allowing an
immunological reaction between said polypeptide and the antibodies possibly
present in the biological
sample); b) demonstration of the antigen-antibody complexes possibly formed.
Any conventional procedure can be employed for carrying out such a detection
of the
antigen-antibody complexes possibly formed.
By way of example, a method according to various embodiments brings into play
immunoenzymatic processes according to the ELISA technique, by
immunofluorescence, or
radioimmunological processes (RIA) or their equivalent.
Thus, the invention likewise relates to the polypeptides according to the
invention, labeled with
the aid of an adequate label, such as, of the enzymatic, fluorescent or
radioactive type.
Such methods comprise, for example, the following acts: deposition of
determined quantities of a
polypeptide composition according to the invention in the wells of a
microtiter plate, introduction into
said wells of increasing dilutions of serum, or of a biological sample other
than that defined previously,
having to be analyzed, incubation of the microtiter plate, introduction into
the wells of the microtiter plate
of labeled antibodies directed against pig immunoglobulins, the labeling of
these antibodies having been
carried out with the aid of an enzyme selected from those which are capable of
hydrolyzing a substrate by
modifying the absorption of the radiation of the latter, at least at a
determined wavelength, for example at
550 nm, detection, by comparison with a control test, of the quantity of
hydrolyzed substrate.
The polypeptides according to embodiments of the invention enable monoclonal
or polyclonal
antibodies to be prepared which are characterized in that they specifically
recognize the polypeptides
according to the invention. It will advantageously be possible to prepare the
monoclonal antibodies from
hybridomas according to the technique described by Kohler and Milstein in
1975. It will be possible to
prepare the polyclonal antibodies, for example, by immunization of an animal,
in particular a mouse, with
a polypeptide or a DNA, according to the invention, associated with an
adjuvant of the immune response,
and then purification of the specific antibodies contained in the serum of the
immunized animals on an
affinity column on which the polypeptide which has served as an antigen has
previously been
immobilized. The polyclonal antibodies according to the invention can also be
prepared by purification,
on an affinity column on which a polypeptide according to the invention has
previously been
immobilized, of the antibodies contained in the serum of an animal
immunologically challenged by
Alicyclobacillus acidocaldarius, or a polypeptide or fragment according to the
invention.
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The invention, in various embodiments, likewise relates to mono- or polyclonal
antibodies or
their fragments, or chimeric antibodies, characterized in that they are
capable of specifically recognizing a
polypeptide according to the invention.
It will likewise be possible for the antibodies of embodiments of the
invention to be labeled in the
same manner as described previously for the nucleic probes of the invention,
such as a labeling of
enzymatic, fluorescent or radioactive type.
An embodiment of the invention is additionally directed at a procedure for the
detection and/or
identification of Alicyclobacillus acidocaldarius in a sample, characterized
in that it comprises the
following steps: a) contacting of the sample with a mono- or polyclonal
antibody according to the
invention (under conditions allowing an immunological reaction between said
antibodies and the
polypeptides of Alicyclobacillus acidocaldarius possibly present in the
biological sample); b)
demonstration of the antigen-antibody complex possibly formed.
The present invention likewise relates to a procedure for the detection and/or
the identification of
Alicyclobacillus acidocaldarius in a sample, characterized in that it employs
a nucleotide sequence
according to the invention.
More particularly, the invention relates to a procedure for the detection
and/or the identification
of Alicyclobacillus acidocaldarius in a sample, characterized in that it
contains the following steps: a) if
need be, isolation of the DNA from the sample to be analyzed; b) specific
amplification of the DNA of
the sample with the aid of at least one primer, or a pair of primers,
according to the invention; c)
demonstration of the amplification products.
These can be detected, for example, by the technique of molecular
hybridization utilizing a
nucleic probe according to the invention. This probe will advantageously be
labeled with a nonradioactive
(cold probe) or radioactive isotope.
For the purposes of the present invention, "DNA of the biological sample" or
"DNA contained in
the biological sample" will be understood as meaning either the DNA present in
the biological sample
considered, or possibly the cDNA obtained after the action of an enzyme of
reverse transcriptase type on
the RNA present in said biological sample.
A further embodiment of the invention comprises a method, characterized in
that it comprises the
following steps: a) contacting of a nucleotide probe according to the
invention with a biological sample,
the DNA contained in the biological sample having, if need be, previously been
made accessible to
hybridization under conditions allowing the hybridization of the probe with
the DNA of the sample; b)
demonstration of the hybrid formed between the nucleotide probe and the DNA of
the biological sample.
The present invention also relates to a procedure according to embodiments of
the invention,
characterized in that it comprises the following steps: a) contacting of a
nucleotide probe immobilized on
a support according to the invention with a biological sample, the DNA of the
sample having, if need be,
previously been made accessible to hybridization, under conditions allowing
the hybridization of the
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probe with the DNA of the sample; b) contacting of the hybrid formed between
the nucleotide probe
immobilized on a support and the DNA contained in the biological sample, if
need be after elimination of
the DNA of the biological sample which has not hybridized with the probe, with
a nucleotide probe
labeled according to the invention; c) demonstration of the novel hybrid
formed in step b).
According to an advantageous embodiment of the procedure for detection and/or
identification
defined previously, this is characterized in that, prior to step a), the DNA
of the biological sample is first
amplified with the aid of at least one primer according to the invention.
Embodiments of methods include glycosylating or post-translationally modifying
a first
polypeptide using a second polypeptide selected from the group consisting of a
polypeptide having at
least 90% sequence identity to SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120,
137, 154, 171, 188, 205, 222,
239, 256, 273, 290, 307, 331, 333, 335, 337, and 338.
Further embodiments of methods include methods of modulating protein
stability, solubility,
degradation, activity profile, and/or externalization of a first polypeptide,
the methods comprising
glycosylating or post-translationally modifying the first polypeptide via a
second polypeptide selected
from the group consisting of a polypeptide having at least 90% sequence
identity to SEQ ID NOS:1, 18,
35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290,
307, 331, 333, 335, 337, and
338.
Further embodiments of methods include placing a cell producing or encoding a
recombinant,
purified, and/or isolated nucleotide sequence comprising a nucleotide sequence
selected from the group
consisting of a nucleotide sequences having at least 90% sequence identity to
at least one of the sequences
of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223,
240, 257, 274, 291, 332,
334, 336, 339, and 340 and/or a recombinant, purified, and/or isolated
polypeptide selected from the
group consisting of a polypeptide having at least 90% sequence identity to at
least one of the sequences of
SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239,
256, 273, 290, 307, 331,
333, 335, 337, and 338 in a environment comprising temperatures at or above
about 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/or a pH at,
below, and/or above 8, 7, 6, 5,
4, 3, 2, 1, and/or 0.
The present invention provides cells that have been genetically manipulated to
have an altered
capacity to produce expressed proteins. In particular, the present invention
relates to Gram-positive
microorganisms, such as Bacillus species having enhanced expression of a
protein of interest, wherein
one or more chromosomal genes have been inactivated, and/or wherein one or
more chromosomal genes
have been deleted from the Bacillus chromosome. In some further embodiments,
one or more indigenous
chromosomal regions have been deleted from a corresponding wild-type Bacillus
host chromosome. In
further embodiments, the Bacillus is an Alicyclobacillus sp. or
Alicyclobacillus acidocaldarius.
In additional embodiments, methods of glycosylating and/or post-
translationally modifying a
polypeptide at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, and/or
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95 degrees Celsius and/or at a pH at, below, and/or above 8, 7, 6, 5, 4, 3, 2,
1, and/or 0 via a recombinant,
purified, and/or isolated nucleotide sequence comprising a nucleotide sequence
selected from the group
consisting of a nucleotide sequences having at least 90% sequence identity to
at least one of the sequences
of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223,
240, 257, 274, 291, 332,
334, 336, 339, and 340 and/or a recombinant, purified, and/or isolated
polypeptide selected from the
group consisting of a polypeptide having at least 90% sequence identity to at
least one of the sequences of
SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239,
256, 273, 290, 307, 331,
333, 335, 337, and 338.
In some embodiments of the invention, any one of the isolated and/or purified
polypeptides
according to the invention may be enzymatically or functionally active at
temperatures at or above about
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees
Celsius and/or may be
enzymatically or functionally active at a pH at, below, and/or above 8, 7, 6,
5, 4, 3, 2, 1, and/or 0. In
further embodiments of the invention, glycosylation, pegylation, and/or other
post-translational
modification may be required for the isolated and/or purified polypeptides
according to the invention to
be enzymatically or functionally active at pH at or below 8, 7, 6, 5, 4, 3, 2,
1, and/or 0 or at a temperatures
at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
and/or 95 degrees Celsius.
The invention is described in additional detail in the following illustrative
examples. Although
the examples may represent only selected embodiments of the invention, it
should be understood that the
following examples are illustrative and not limiting.
EXAMPLES
Example 1: Glycosylation Using Nucleotide and Amino Acid Sequences from
Alicyclobacillus acidocaldarius
Provided in SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189,
206, 223, 240, 257,
274, 291, 332, 334, 336, 339, and 340 are a nucleotide sequences isolated from
Alicyclobacillus
acidocaldarius and coding for the polypeptides of SEQ ID NOS:1, 18, 35, 52,
69, 86, 103, 120, 137, 154,
171, 188, 205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338,
respectively. The nucleotide
sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189,
206, 223, 240, 257, 274,
291, 332, 334, 336, 339, and 340 are placed into expression vectors using
techniques standard in the art.
The vectors are then provided to cells such as bacteria cells or eukaryotic
cells such as Sf9 cells or CHO
cells. In conjunction with the normal machinery in present in the cells, the
vectors comprising SEQ ID
NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,
274, 291, 332, 334, 336, 339,
and 340 produce the polypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103,
120, 137, 154, 171, 188,
205, 222, 239, 256, 273, 290, 307, 331, 333, 335, 337, and 338. The
polypeptides of SEQ ID NOS:1, 18,
35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290,
307, 331, 333, 335, 337, and
338 are then isolated and/or purified. The isolated and/or purified
polypeptides of SEQ ID NOS:1, 18,
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35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290,
307, 331, 333, 335, 337, and
338 are then each demonstrated to have one or more of the activities provided
in Table 1 or some other
activity.
The isolated and/or purified polypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86,
103, 120, 137,
154, 171, 188, 205, 222, 239, 256, 273, 290 are demonstrated to have activity
in glycosylating other
proteins in conjunction with other proteins or cellular components.
Example 2: Modulating Protein Stability, Solubility, Degradation, Activity
Profile, and/or
Externalization of a First Polypeptide Using Nucleotide and Amino Acid
Sequences from
Alicyclobacillus acidocaldarius
The polypeptides and nucleotide sequence of Example 1 are used to post-
translationally modify
one or more other proteins through glycosylation or other post-translational
modification. The modified
proteins are demonstrated to have altered protein stability, solubility,
degradation, activity profile, and/or
externalization in comparison to non-modified proteins of the same or similar
amino acid sequence.
Example 3: Glycosylated Proteins of Alicyclobacillus acidocaldarius
Polypeptide RAAC02676 (SEQ ID NO:307) was obtained via the following protocol.
Alicyclobacillus acidocaldarius was cultured on wheat arabinoxylan and
harvested after three days. The
culture was centrifuged to remove cells and the resulting supernatant was
filtered with a 0.22 micron filter
to remove any remaining debris. The filtered supernatant was concentrated to
approximately 1 mL by
ultrafiltration through a 10,000 Da molecular weight cutoff membrane. The
resulting concentrated
filtered supernatant was additionally purified by trapping proteins on a
cation exchange column, eluting
them with a salt gradient, reloading them on a second cation exchange column
and eluting with a second
salt gradient. Samples were pooled and run on a 12% SDS-PAGE gel. Individual
bands were cut from
the gel and subjected to in gel tryptic digestion. The peptide fragments were
then eluted and separated on
a C-18 column an injected into an ion trap mass spectrometer via electrospray.
Mass spectra were run
through MASCOT which compares the observed spectra to theoretical spectra
generated from the known
protein sequence. MASCOT allows the user to specify modifications that might
exist on the protein and
looks for spectra consistent with these modifications. MASCOT identified a
number of peptides digested
from RAAC02676 that were potentially glycosylated as provided in Table 2
below.
As can be seen in Table 2, Queries 94, 96, 221, 332, 333, 337, and 400
returned expected
N-linked glycosylations on RAAC02676. All fragments in Table 2 are fragments
of SEQ ID NO:307
(RAAC02676). O-linked glycosylations are also expected.
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TABLE 2.
Query Observed Mr(expt) Mr(calc) Ppm Miss Score Expect Rank Peptide
17 398.1406 794.2667 794.4174 -189.65 0 (24) 73 1 K.YGDIVTK.N
18 398.1591 794.3037 794.4174 -143.08 0 (28) 38 2 K.YGDIVTK.N
19 398.1596 794.3047 794.4174 -141.82 0 55 0.073 1 K.YGDIVTK.N
20 398.1621 794.3097 794.4174 -135.53 0 (41) 1.7 1 K.YGDIVTK.N
77 569.1781 1136.3417 1136.5461 -179.86 0 (46) 0.41 1 R.EINAYAGSNAK.N
78 569.1796 1136.3447 1136.5461 -177.22 0 62 0.011 1 R.EINAYAGSNAK.N
94 579.6776 1157.3407 1157.5676 -196.02 0 34 6.9 1 R.QNGLSPSDLAR.T
Glyc-Asn (N)
96 579.7071 1157.3997 1157.5676 -145.05 0 (20) 2.5e+02 3 R.QNGLSPSDLAR.T
Glyc-Asn (N)
138 721.2386 1440.4627 1440.7394 -192.07 0 (62) 0.019 1 R.EPNGDIALMLVN
.S
139 721.2411 1440.4677 1440.7394 -188.60 0 82 0.00022 1 R.EPNGDIALMLVN
.S
207 987.8796 1973.7447 1974.0098 -134.27 0 (130) 6.2e-09 1 R.AVGLFYQSFLTEI
GQSSK.A
208 987.8796 1973.7447 1974.0098 -134.27 0 (123) 2.8e-08 1 R.AVGLFYQSFLTEI
GQSSK.A
209 987.8801 1973.7457 1974.0098 -133.76 0 (71) 0.0045 1 R.AVGLFYQSFLTEI
GQSSK.A
210 987.8986 1973.7827 1974.0098 -115.02 0 140 5.9e-10 1 R.AVGLFYQSFLTEI
GQSSK.A
216 661.1822 1980.5247 1980.8966 -187.71 0 (37) 4.1 1 R.WPGGSISDVYNW
TNTR.N
SISDVYNW
217 991.3196 1980.6247 1980.8966 -137.23 0 (59) 0.053 1 TNTR.N
NTR.N
218 991.3411 1980.6677 1980.8966 -115.52 0 (42) 3.6 1 R.WPGGSISDVYNW
TNTR.N
219 991.3461 1980.6777 1980.8966 -110.47 0 (31) 40 1 R.WPGGSISDVYNW
TNTR.N
R.WPGGSISDVYNW
221 991.8396 1981.6647 1981.8806 -108.91 0 85 0.00016 1 TNTR.N + Glyc-Asn
(N)
265 731.9392 2192.7957 2193.2117 -189.66 0 69 0.0086 1 R.GSNAAQILQTLQS
SPLLSPR.A
266 1097.4561 2192.8977 2193.2117 -143.15 0 (31) 56 1 R.GSNAAQILQTLQS
ISPLLSPR.A
286 1133.4606 2264.9067 2265.1892 -124.70 0 (31) 58 2 R.SPSTIYSADLNVL
GVGPYAITK.A
287 1133.4786 2264.9427 2265.1892 -108.81 0 (48) 0.95 1 R.SPSTIYSADLNVL
GVGPYAITK.A
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Query Observed Mr(expt) Mr(calc) Ppm Miss Score Expect Rank Peptide
288 1133.4811 2264.9477 2265.1892 -106.60 0 70 0.0073 1 R.SPSTIYSADLNVL
GVGPYAITK.A
289 756.1595 2265.4567 2265.1892 118 0 (21) 4.5e+02 9 R.SPSTIYSADLNVL
GVGPYAITK.A
309 1177.9986 2353.9827 2354.2481-112.72 0 68 0.012 1 K.ALVYGEGSSAVSP
LTLPTAHSVK.L
310 1178.0016 2353.9887 2354.2481-110.17 0 (51) 0.59 1 K.ALVYGEGSSAVSP
LTLPTAHSVK.L
311 1178.0086 2354.0027 2354.2481-104.22 0 (54) 0.26 1 K.ALVYGEGSSAVSP
LTLPTAHSVK.L
319 1182.9381 2363.8617 2364.1346 -115.41 0 (91) 5.le-05 1 R.TWSSFETQVDPQ
GAAQTALATR.I
320 1182.9386 2363.8627 2364.1346 -114.99 0 (109) 7.9e-07 1 R.TWSSFETQVDPQ
GAAQTALATR.1
321 1182.9416 2363.8687 2364.1346 -112.45 0 (115) 2.le-07 1 R.TWSSFETQVDPQ
GAAQTALATR.1
322 1182.9781 2363.9417 2364.1346 -81.57 0 (83) 0.00036 1 R.TWSSFETQVDPQ
GAAQTALATR.I
323 1182.9866 2363.9587 2364.1346 -74.38 0 126 1.6e-08 1 R.TWSSFETQVDPQ
GAAQTALATR.I
324 789.1825 2364.5257 2364.1346 165 0 (17) 1.le+03 9 R.TWSSFETQVDPQ
GAAQTALATR.1
307 1188.3281 2374.6417 2374.1851 192 0 88 7.6e-05 1 K.GNPGLSPQAYAQ
ALQFIQAMR.A
K.GNPGLSPQAYAQ
332 1188.4176 2374.8207 2375.1691-146.69 0 (72) 0.0042 1 ALQFIQAMR.A +
Glyc-Asn (N)
K.GNPGLSPQAYAQ
333 1188.4181 2374.8217 2375.1691-146.26 0 (76) 0.0019 1 ALQFIQAMR.A +
Glyc-Asn (N)
K.GNPGLSPQAYAQ
337 1188.9406 2375.8667 2376.1531 -120.54 0 (66) 0.017 1 ALQFIQAMR.A + 2
Glyc-Asn (N)
397 1288.5416 2575.0687 2575.3605 -113.30 0 (130) 8.2e-09 1 R.QASSSIVGNALAQ
ASLSPTISAYLR.Q
398 1288.5621 2575.1097 2575.3605 -97.38 0 135 2.6e-09 1 R.QASSSIVGNALAQ
ASLSPTISAYLR.Q
399 859.5002 2575.4787 2575.3605 45.9 0 (64) 0.034 1 R.QASSSIVGNALAQ
ASLSPTISAYLR.Q
R.QASSSIVGNALAQ
400 859.8579 2576.5517 2576.3445 80.4 0 (74) 0.0035 1 ASLSPTISAYLR.Q
Glyc-Asn (N)
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Example 4: Glycoprotein Staining of Proteins from Alicyclobacillus
acidocaldarius
Alicyclobacillus acidocaldarius was cultured on wheat arabinoxylan and
harvested after three
days. The culture was centrifuged to remove cells and the resulting
supernatant was filtered with a 0.22
micron filter to remove any remaining debris. The filtered supernatant was
concentrated to approximately
1 mL by ultrafiltration through a 10,000 Da molecular weight cutoff membrane.
Several lanes of this
concentrated material were run on a 12% SDS-PAGE gel along with a positive and
negative control that
are known glycosylated and non-glycosylated proteins using standard protocols.
The gel was cut in half
vertically and one half stained using SIMPLY BLUETM SAFE STAIN and the other
half using a
glycoprotein detection kit from Sigma. The positive and negative controls both
stained using the
SIMPLY BLUETM SAFE STAIN and only the positive control stained with the
glycoprotein stain
indicating that the staining protocol was working correctly. The
Alicyclobacillus acidocaldarius protein
lanes revealed a band at approximately 120 kDa on the SIMPLY BLUETM stained
gel which is the
expected weight of one extracelluar protein of Alicyclobacillus
acidocaldarius. The same position on the
glycoprotein stained gel showed pink bands indicating a positive result for a
glycosylated protein.
Example 5: Chemical Glycosylation of SEQ ID NO:307 Results in Greater Xylanase
activity.
SEQ ID NO:307 was expressed in E. coli and purified using a Co-resin system.
The nucleic acid
encoding SEQ ID NO:307 was altered for optimal codon usage in E. coli. The
purified SEQ ID NO:307
was chemically glycosylated using a mono(lactosylamido) mono(succinimidyl)
suberate. This chemically
reacts with amine groups on proteins to form an N-linked modification with a
terminal lactose on the
protein The glycosylation of the purified SEQ ID NO:307 was verified using a
glycosylation stain.
Purified glycosylated and un-glycosylated SEQ ID NO:307 was tested for
xylanase activity at pHs 2, 3.5,
and 5. The glycosylated SEQ ID NO:307 had higher levels of activity at pH 2
and 3.5 that the
ungylcosylated from of SEQ ID NO:307. Un-glycosylated SEQ ID NO:307 displayed
reduced solubility
at pH 2 and 3.5.
Example 6: Expression of SEQ ID NO:307 in Pichia pastoris.
Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastoris and
several clones had
significant xylanase and cellulose activities at pH 3.5, but not at pH 2. The
nucleic acid encoding SEQ ID
NO:307 was altered for optimal codon usage in Pichia pastoris.
Example 7: Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307
Expressed in
Alicyclobacillus acidocaldarius and E. coli.
Nucleic acid encoding SEQ ID NO:307 was inserted into E. coli with an N-
terminal His tag and
the resultant protein purified. The nucleic acid encoding SEQ ID NO:307 was
altered for optimal codon
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usage in E. coli. The resulting purified protein had no glycosylation. Protein
of SEQ ID NO:307 was also
purified from Alicyclobacillus acidocaldarius. The purified protein from
Alicyclobacillus acidocaldarius
was glycosylated as normal for protein produced from this organism. The
purified proteins were tested for
xylanase activity using wheat arabinoxylan (WAX). The results of the
comparison are presented in
FIG. 23. There was no data available for enzyme purified from Alicyclobacillus
acidocaldarius at pH
5.5. As can be seen in FIG. 23, the Alicyclobacillus acidocaldarius purified
enzyme (black bars) had
significantly more xylanase activity at pH 2 and 80 C than the E. coli
purified enzyme (white bars).
The purified proteins were also tested for cellulase activity using
carboxymethyl cellulose
(CMC). The results of the comparison are presented in FIG. 24. There was no
data available for enzyme
purified from Alicyclobacillus acidocaldarius at pH 5.5. As can be seen in
FIG. 24, the Alicyclobacillus
acidocaldarius purified enzyme (black bars) had significantly less cellulase
activity at pH 3.5 than the E.
coli purified enzyme (white bars).
FIG. 25 presents the ratio of cellulose/xylanase activity for the data
presented in FIGs. 23 and 24.
As can be seen therein, enzyme purified from Alicyclobacillus acidocaldarius
had predominantly
xylanase activity at pH 2 and 80 C, while having predominantly cellulose
activity at all other conditions
tested (black bars). The enzyme purified from E. coli had predominantly
cellulose activity at all
conditions tested (white bars). This data confirms that the glycosylation
state of SEQ ID NO:307 varies
the relative xylanase and cellulose activities of SEQ ID NO:307.
Example 8: Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307
Expressed in
E. coli and Pichia pastoris.
Nucleic acid encoding SEQ ID NO:307 was inserted into Pichiapastoris and the
resultant protein
purified. The nucleic acid encoding SEQ ID NO:307 was altered for optimal
codon usage in Pichia
pastoris. The purified protein from Pichia pastoris was glycosylated as normal
for protein produced from
this organism. Nucleic acid encoding SEQ ID NO:307 was inserted into E. coli
with an N-terminal His
tag and the resultant protein purified. The nucleic acid encoding SEQ ID
NO:307 was altered for optimal
codon usage in E. coli. The resulting purified protein had no glycosylation.
The purified proteins were
tested for xylanase activity using wheat arabinoxylan. The results of the
comparison are presented in
FIG. 26. As can be seen in FIG. 26, the Pichia pastoris purified enzyme (black
bars) had significantly
more xylanase activity at all conditions other than pH 3.5 and 80 C than the
E. coli purified enzyme
(white bars).
The purified proteins were also tested for cellulase activity using
carboxymethyl cellulose. The
results of the comparison are presented in FIG. 27. As can be seen in FIG. 27,
the Pichia pastoris
purified enzyme (black bars) had significantly greater cellulase activity at
60 C while the E. coli purified
enzyme (white bars) displayed significantly greater cellulase activity at 80
C.
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FIG. 28 presents the ratio of cellulose/xylanase activity for the data
presented in FIGs. 26 and 27.
As can be seen therein, enzyme purified from Pichia pastoris had predominantly
cellulase activity at all
conditions other than pH 2 and 80 C. The enzyme purified from E. coli had
predominantly cellulose
activity at all conditions tested. This data confirms that the glycosylation
state of SEQ ID NO:307 varies
the relative xylanase and cellulose activities of SEQ ID NO:307.
Example 9: Comparison of Xylanase and Cellulase Activity of SEQ ID NO:307
Expressed
in Alicyclobacillus acidocaldarius and Pichia pastoris.
Nucleic acid encoding SEQ ID NO:307 was inserted into Pichia pastoris and the
resultant protein
purified. The nucleic acid encoding SEQ ID NO:307 was altered for optimal
codon usage in Pichia
pastoris. The purified protein from Pichia pastoris was glycosylated as normal
for protein produced from
this organism. Protein of SEQ ID NO:307 was also purified from
Alicyclobacillus acidocaldarius. The
purified protein from Alicyclobacillus acidocaldarius was glycosylated as
normal for protein produced
from this organism. The purified proteins were tested for xylanase activity
using wheat arabinoxylan.
The results of the comparison are presented in FIG. 29. There was no data
available for enzyme purified
from Alicyclobacillus acidocaldarius at pH 5.5. As can be seen in FIG. 29, the
Alicyclobacillus
acidocaldarius purified enzyme (black bars) had significantly more xylanase
activity at A pH of 2 and a
pH of 3.5 and 80 C than the Pichia pastoris purified enzyme (white bars).
The purified proteins were also tested for cellulase activity using
carboxymethyl cellulose. The
results of the comparison are presented in FIG. 30. There was no data
available for enzyme purified from
Alicyclobacillus acidocaldarius at pH 5.5. As can be seen in FIG. 30, the
Alicyclobacillus acidocaldarius
purified enzyme (black bars) had significantly less cellulase activity at all
conditions other than pH 3.5
and 80 C than the Pichia pastoris purified enzyme (white bars).
FIG. 31 presents the ratio of cellulose/xylanase activity for the data
presented in FIGs. 29 and 30.
As can be seen therein, enzyme purified from Alicyclobacillus acidocaldarius
had predominantly
xylanase activity at pH 2 and 80 C, while having predominantly cellulose
activity at all other conditions
tested (black bars). The enzyme purified from Pichia pastoris also had
predominantly xylanase activity
at pH 2 and 80 C, while having predominantly cellulase activity at all other
conditions tested (white bars).
This data confirms that the glycosylation state of SEQ ID NO:307 varies the
relative xylanase and
cellulose activities of SEQ ID NO:307.
Example 10: Comparison of Xylanase and Cellulase Activity of Truncated SEQ ID
NO:307
Expressed in E. coli and Pichiapastoris.
Nucleic acid encoding a truncated SEQ ID NO:307 without the 203 C-terminal
amino acids was
inserted into Pichia pastoris with a C-terminal His tag and the resultant
protein purified. The nucleic acid
encoding SEQ ID NO:307 was altered for optimal codon usage in Pichia pastoris.
The purified protein
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from Pichia pastoris was glycosylated as normal for protein produced from this
organism. Nucleic acid
encoding a truncated SEQ ID NO:307 without the 203 C-terminal amino acids and
N-terminal 33 leader
sequence amino acids were inserted into E. coli with an and the resultant
protein purified. The nucleic
acid encoding SEQ ID NO:307 was altered for optimal codon usage in E. coli.
The resulting purified
protein had no glycosylation. The purified proteins were tested for xylanase
activity using wheat
arabinoxylan. The results of the comparison are presented in FIG. 32. As can
be seen in FIG. 32, the
Pichia pastoris purified enzyme (black bars) had significantly more xylanase
activity at all 60 C
conditions while the E. coli purified enzyme (white bars) had more xylanase
activity at all 80 C
conditions.
The purified proteins were also tested for cellulase activity using
carboxymethyl cellulose. The
results of the comparison are presented in FIG. 33. As can be seen in FIG. 33,
the Pichia pastoris
purified enzyme (black bars) had significantly greater cellulase activity 60 C
and pHs 3.5 and 5.5 while
the E. coli purified enzyme (white bars) displayed greater cellulase at all
other conditions.
FIG. 34 presents the ratio of cellulose/xylanase activity for the data
presented in FIGs. 32 and 33.
As can be seen therein, both purified enzymes had predominantly cellulase
activity at all conditions.
Example 11: Activity of SEQ ID NO:338 Expressed in E. coli and Pichiapastoris.
RAA000568 (SEQ ID NO:338) was expressed in both E. coli and Pichia pastoris.
Codon usage
in the encoding DNA was optimized for the particular organism. In E. coli, the
enzyme expressed
primarily as inclusion bodies. A small amount of enzyme was soluble and we
were able to purify it via
the His tag by immobilized metal affinity chromatography (IMAC). This purified
enzyme was tested for
both alpha glucosidase and alpha xylosidase activities at pH 6.0 and 60 C and
was found to have no
activity. Several attempts were also made to solubilize the inclusion bodies
and did not result in active
protein. In Pichia pastoris, a soluble enzyme was expressed and purified by
IMAC. This enzyme had
activity at pH 5.5 and 60 C for both alpha glucosidase and alpha xylosidase.
These results demonstrate
that the expression system and glycosylation state of RAA000568 may alter both
the solubility and
activity of the enzyme.
Example 12: Activity of SEQ ID NO:337 Expressed in E. coli and Pichiapastoris.
RAA000307 (SEQ ID NO:337) was expressed in both E. coli and Pichiapastoris.
Codon usage
in the encoding DNA was optimized for the particular organism. The E. coli
produced enzyme was
purified via a His tag by immobilized metal affinity chromatography (IMAC).
This purified enzyme
was tested for both alpha arabinofuranosidase (AFS) activity, as well as beta
xylosidase (BXYL) activity.
The results of this testing are presented in FIGs. 35-37. In Pichia pastoris,
a soluble enzyme was
expressed and purified by IMAC.
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The optimum conditions for the E. coli expressed AFS were pH 6.0 and 70 C,
while the optimum
conditions for BXYL were pH 5.0 and between 70 C and 80 C (FIGs. 35 and 36).
The enzyme did not
have activity at pH 2.0 for either AFS or BXYL activities (FIGs. 35 and 36).
The Pichia pastoris
expressed enzyme was screened at pH 2, 3.5, and 5.5 and at 60 C and 80 C. The
results are presented in
FIG. 37. The glycosylation modifications made by Pichia pastoris during
expression appeared to have
shifted the activity to a lower pH. The BXYL in the Pichia pastoris expressed
enzyme was 10.8 U/mg at
pH 3.0, 60 C, while it was only 1.1 U/mg for the E. coli expressed enzyme. The
Pichia pastoris
expressed enzyme also had some activity at pH 2.0 while there was no activity
for the E. coli expressed
enzyme. These results demonstrate that the expression system and glycosylation
state of RAAO00307
may alter its activity.
While this invention has been described in the context of certain embodiments,
the present
invention can be further modified within the spirit and scope of this
disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of the
invention using its general
principles. Further, this application is intended to cover such departures
from the present disclosure as
come within known or customary practice in the art to which this invention
pertains and which fall within
the limits of the appended claims and their legal equivalents.
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