Note: Descriptions are shown in the official language in which they were submitted.
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ASPARAGINASE
Field of the invention
The present invention relates to a polypeptide having asparaginase activity
and to a
composition comprising such a polypeptide. The invention also relates to a
nucleic acid
encoding an asparaginase, to an expression vector comprising the nucleic acid
and to a
recombinant host cell comprising the nucleic acid or expression vector. The
invention further
relates to a method for the preparation of the polypeptide and to use of the
polypeptide or
composition in the production of a food product or to reduce the amount of
acrylamide formed in
a thermally processed food product based on an asparagine-containing raw
material. The
invention in addition relates to a process for the production of a food
product involving at least
one heating step and to a food product obtainable by such a process. Further,
the invention
relates to a dough comprising the polypeptide, to a method for the preparation
of a dough and to
a method for the preparation of a baked product. The invention also relates to
the polypeptide or
a composition for use in a method of treatment of the human or animal body by
therapy.
Background to the invention
The occurrence of acrylamide in a number of heated food products has been
recognized for some time (Tareke et al. Chem. Res. Toxicol. 13, 517-522
(2000)). Since
acrylamide is considered as probably carcinogenic for animals and humans, this
finding resulted
in world-wide concern. Further research revealed that considerable amounts of
acrylamide are
detectable in a variety of baked, fried and oven prepared common foods and it
was
demonstrated that the occurrence of acrylamide in food was the result of the
heating process.
A pathway for the formation of acrylamide from amino acids and reducing sugars
as a
result of the Mai!lard reaction has been proposed by Mottram et al. Nature
419:448 (2002).
According to this hypothesis, acrylamide may be formed during the Mai!lard
reaction. During
baking and roasting, the Mai!lard reaction is mainly responsible for the
color, smell and taste. A
reaction associated with the Mai!lard is the Strecker degradation of amino
acids and a pathway to
acrylamide was proposed. The formation of acrylamide became detectable when
the
temperature exceeded 120 C, and the highest formation rate was observed at
around 170 C.
When asparagine and glucose were present, the highest levels of acrylamide
could be observed,
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while glutamine and aspartic acid only resulted in trace quantities.
The official migration limit in the EU for acrylamide migrating into food from
food
contact plastics is set at 10 ppb (10 micrograms per kilogram). Although no
official limit is yet set
for acrylamide that forms during cooking, the fact that a lot of products
exceed this value,
especially cereals, bread products and potato or corn based products, causes
concern.
Several plant raw materials are known to contain substantial levels of
asparagine. In
potatoes asparagine is the dominant free amino acid (940 mg/kg, corresponding
with 40% of the
total amino-acid content) and in wheat flour asparagine is present as a level
of about 167 mg/kg,
corresponding with 14% of the total free amino acids pool (Belitz and Grosch
in Food Chemistry
- Springer New York, 1999). The fact that acrylamide is formed mainly from
asparagine
(combined with reducing sugars) may explain the high levels acrylamide in
fried, oven-cooked or
roasted plant products. Therefore, in the interest of public health, there is
an urgent need for food
products that have substantially lower levels of acrylamide or, preferably,
are devoid of it.
A variety of solutions to decrease the acrylamide content has been proposed,
either by
altering processing variables, e.g. temperature or duration of the heating
step, or by chemically
or enzymatically preventing the formation of acrylamide or by removing formed
acrylamide.
In several patent applications the use of asparaginase for decreasing the
level of
asparagine and thereby the amount of acrylamide formed has been disclosed.
Suitable
asparaginases for this purpose have been yielded from several fungal sources,
as for example
Aspergillus niger in W02004/030468 and Aspergillus oryzae in W004/032648.
Although all L-asparaginases catalyze the same chemical conversion, this does
not
mean that they are suitable for the same applications. Various applications
will place different
demands on the conditions under which the enzymes have to operate. Physical
and chemical
parameters that may influence the rate of an enzymatic conversion are the
temperature (which
has a positive effect on the chemical reaction rates, but may have a negative
effect on enzyme
stability), the moisture content, the pH, the salt concentration, the
structural integrity of the food
matrix, the presence of activators or inhibitors of the enzyme, the
concentration of the substrate
and products, etc.
Therefore there exists an ongoing need for improved asparaginases for several
applications having improved properties, for example in higher temperature
applications where
thermostability may be advantagenous.
Summary of the invention
The present invention is based on the identification of a polypeptide having
asparaginase
activity. The polypeptide may be derived from, for example, a microorganism of
the genus
Thermophilus such as from the species Thermophilus africanus.
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A polypeptide of the invention is preferably thermophilic, for example
thermostable (i.e.
capable of withstanding a thermal treatment in respect of its enzymatic
activity) and/or
thermoactive (i.e. only develops its full enzymatic activity at elevated
temperature). A
polypeptide of the invention may alternatively or additionally be one which is
active across a
broad pH range and/or at a relatively high or low pH.
Providing an asparaginase with improved thermophilic properties is an
important way of
broadening its application. Thermoactive and thermostable asparaginases have
substantial
advantages over other asparaginases. For instance, the conversion of the
asparagine into
aspartate can be conducted at comparatively high temperatures using
thermoactive or
thermostable asparaginases, and this results in a compatibility with processes
in which high
temperatures, in particular holding processes at high temperatures, still play
a role. Moreover,
the breakdown of asparagine at higher temperatures can be conducted at a
higher reaction rate.
Asparaginases active at a broad pH range are also advantageous since it may be
possible to use a polypeptide of the invention in different processes with
widely differing pH
ranges. It is also possible to use such a polypeptide in processes in which
the pH value is subject
to significant fluctuations in the process. Processes are also possible in
which pH values from 5
to 10 occur.
The polypeptide of the invention having asparaginase activity may be used, in
particular,
in the preparation of a foodstuff, preferably to reduce the content of
asparagine in the foodstuff.
The reduction of the asparagine content preferably also causes the acrylamide
content in the
foodstuff to be reduced when the foodstuff is subjected to a subsequent
thermal treatment.
Accordingly, the invention provides a polypeptide having asparaginase activity
selected
from the group consisting of:
i. a polypeptide having an amino acid sequence comprising the mature
polypeptide sequence of SEQ ID NO: 1;
ii. a polypeptide comprising an amino acid sequence that has at least 50%
sequence identity with the mature polypeptide sequence of SEQ ID NO: 1;
iii. a polypeptide encoded by a nucleic acid comprising a sequence that
hybridizes
under medium stringency conditions to the complementary strand of the mature
polypeptide encoding sequence of SEQ ID NO: 2; and
iv. a polypeptide comprising an amino acid sequence encoded by a nucleic acid
that has at least 50% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 2.
The invention also provides:
- a composition comprising a polypeptide of the invention;
- a nucleic acid encoding an asparaginase which comprises a
sequence that has at
least 50% sequence identity to the mature polypeptide encoding sequence of SEQ
ID NO: 2;
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- a nucleic acid that is an isolated, substantially pure, pure,
recombinant, synthetic or
variant nucleic acid of a nucleic acid of the invention;
- an expression vector comprising a nucleic acid of the invention operably
linked to
one or more control sequences that direct expression of the polypeptide in a
host
cell;
- a recombinant host cell comprising a nucleic acid or an expression vector
of the
invention;
- a method for the preparation of a polypeptide of the invention, which
method
comprises:
cultivating a host cell according to claim 9 in a suitable fermentation medium
under conditions that allow for production of the polypeptide; and,
optionally,
recovering the polypeptide;
- use of a polypeptide of the invention, a polypeptide obtainable by a
process of the
invention or a composition of the invention in the production of a food
product;
- use of a polypeptide of the invention, a polypeptide obtainable by a
process of the
invention or a composition of the invention to reduce the amount of acrylamide
formed in a thermally processed food product based on an asparagine-containing
raw material;
- a process for the production of a food product involving at least one
heating step,
which process comprises adding a polypeptide of the invention, a polypeptide
obtainable by a process of the invention or a composition of the invention to
an
intermediate form of said food product in said production process, wherein the
enzyme is added prior to or during said heating step in an amount that is
effective in
reducing the level of asparagine that is present in said intermediate form of
said food
product;
- a food product obtainable by the process of the invention or by the use
of the
invention;
- a dough comprising a polypeptide of the invention, a polypeptide
obtainable by a
process of the invention or a composition of the invention;
- a method for the preparation of a dough, which method comprises combining: a
polypeptide of the invention, a polypeptide obtainable by a process of the
invention
or a composition of the invention; and at least one dough ingredient.
- a method for the preparation of a baked product, which method comprises
the step
of baking or frying a dough of the invention or a dough obtainable by a
process of
the invention for the preparation of a dough; and
- a polypeptide of the invention, a polypeptide obtainable by a process of
the invention
or a composition of the invention for use in a method of treatment of the
human or
animal body by therapy.
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Brief description of the drawings
Figure 1 sets out the physical map of the asparaginase expression vector pAe7
5
containing the arabinose inducible promoter PBAD and regulator araC, the
kanamycin resistance
gene Km(R) and the origin from pBR322. The Ndel and Ascl sites are used to
introduce the
asparaginase gene.
Figure 2 sets out the relative activity of the asparaginase polypeptide from
Thermophilus
africanus at different temperatures.
Figure 3 sets out the relative activity of the asparaginase polypeptide from
Thermophilus
africanus at different pHs.
Description of the sequence listing
SEQ ID NO: 1 sets out the amino acid sequence of an asparaginase polypeptide
from
Thermophilus africanus (ACJ75594).
SEQ ID NO: 2 sets out the nucleotide sequence encoding the amino acid sequence
of an
asparaginase polypeptide from Thermophilus africanus, codon-pair optimized for
expression in
E. co/i. Thermophilus africanus. Start codon is at positions 4 to 6 and stop
codon is at positions
1009 to 1011.
Detailed description of the invention
Throughout the present specification and the accompanying claims, the words
"comprise",
"include" and "having" and variations such as "comprises", "comprising",
"includes" and "including" are
to be interpreted inclusively. That is, these words are intended to convey the
possible inclusion of other
elements or integers not specifically recited, where the context allows.
The articles "a" and "an" are used herein to refer to one or to more than one
(i.e. to one or at
least one) of the grammatical object of the article. By way of example, "an
element" may mean one
element or more than one element.
The term "complementary strand" can be used interchangeably with the term
"complement". The complementary strand of a nucleic acid can be the complement
of a coding
strand or the complement of a non-coding strand. When referring to double-
stranded nucleic
acids, the complement of a nucleic acid encoding a polypeptide refers to the
complementary
strand of the strand encoding the amino acid sequence or to any nucleic acid
molecule
containing the same. Typically, the reverse complementary strand is intended.
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The term "control sequence" can be used interchangeably with the term
"expression-
regulating nucleic acid sequence". The term as used herein refers to nucleic
acid sequences
necessary for and/or affecting the expression of an operably linked coding
sequence in a
particular host organism or in vitro. When two nucleic acid sequences are
operably linked, they
usually will be in the same orientation and also in the same reading frame.
They usually will be
essentially contiguous, although this may not be required. The expression-
regulating nucleic acid
sequences, such as inter alia appropriate transcription initiation,
termination, promoter, leader,
signal peptide, propeptide, prepropeptide, or enhancer sequences; Shine-
Dalgarno sequence,
repressor or activator sequences; efficient RNA processing signals such as
splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences
that enhance
translation efficiency (e.g., ribosome binding sites); sequences that enhance
protein stability; and
when desired, sequences that enhance protein secretion, can be any nucleic
acid sequence
showing activity in the host organism of choice and can be derived from genes
encoding
proteins, which are either endogenous or heterologous to a host cell. Each
control sequence may
be native or foreign to the nucleic acid sequence encoding the polypeptide.
When desired, the
control sequence may be provided with linkers for the purpose of introducing
specific restriction
sites facilitating ligation of the control sequences with the coding region of
the nucleic acid
sequence encoding a polypeptide. Control sequences may be optimized to their
specific purpose.
The term "derived from" also includes the terms "originated from," "obtained
from,"
"obtainable from," "isolated from," and "created from," and generally
indicates that one specified
material find its origin in another specified material or has features that
can be described with
reference to the another specified material. As used herein, a substance
(e.g., a nucleic acid
molecule or polypeptide) "derived from" a microorganism preferably means that
the substance is
native to that microorganism.
As used herein, the term "endogenous" refers to a nucleic acid or amino acid
sequence
naturally occurring in a host cell.
The term "expression" includes any step involved in the production of the
polypeptide
including, but not limited to, transcription, post transcriptional
modification, translation, post-
translational modification, and secretion.
An "expression vector" comprises a polynucleotide coding for a polypeptide,
operably
linked to the appropriate control sequences (such as a promoter, and
transcriptional and
translational stop signals) for expression and/or translation in vitro, or in
the host cell of the
polynucleotide.
The expression vector may be any vector (e.g., a plasmid or virus), which can
be
conveniently subjected to recombinant DNA procedures and can bring about the
expression of
the polynucleotide. The choice of the vector will typically depend on the
compatibility of the
vector with the cell into which the vector is to be introduced. The vectors
may be linear or closed
circular plasmids. The vector may be an autonomously replicating vector, i.e.
a vector, which
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exists as an extra-chromosomal entity, the replication of which is independent
of chromosomal
replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome,
or an artificial
chromosome. Alternatively, the vector may be one which, when introduced into
the host cell, is
integrated into the genome and replicated together with the chromosome(s) into
which it has
been integrated. The integrative cloning vector may integrate at random or at
a predetermined
target locus in the chromosomes of the host cell. The vector system may be a
single vector or
plasmid or two or more vectors or plasmids, which together contain the total
DNA to be
introduced into the genome of the host cell, or a transposon.
A "host cell" as defined herein is an organism suitable for genetic
manipulation and one
which may be cultured at cell densities useful for industrial production of a
target product, such
as a polypeptide according to the present invention. A host cell may be a host
cell found in
nature or a host cell derived from a parent host cell after genetic
manipulation or classical
mutagenesis. Advantageously, a host cell is a recombinant host cell.
A host cell may be a prokaryotic, archaebacterial or eukaryotic host cell. A
prokaryotic
host cell may be, but is not limited to, a bacterial host cell. A eukaryotic
host cell may be, but is
not limited to, a yeast, a fungus, an amoeba, an algae, a plant, an animal
cell, such as a
mammalian or an insect cell.
The term "heterologous" as used herein refers to nucleic acid or amino acid
sequences
not naturally occurring in a host cell. In other words, the nucleic acid or
amino acid sequence is
not identical to that naturally found in the host cell.
The term "hybridization" means the pairing of substantially complementary
strands of
oligomeric compounds, such as nucleic acid compounds.
Hybridization may be performed under low, medium or high stringency
conditions. Low
stringency hybridization conditions comprise hybridizing in 6X sodium
chloride/sodium citrate
(SSC) at about 45 C, followed by two washes in 0.2X SSC, 0.1% SDS at least at
50 C (the
temperature of the washes can be increased to 55 C for low stringency
conditions). Medium
stringency hybridization conditions comprise hybridizing in 6X SSC at about 45
C, followed by
one or more washes in 0.2X SSC, 0.1% SDS at 60 C, and high stringency
hybridization
conditions comprise hybridizing in 6X SSC at about 45 C, followed by one or
more washes in
0.2X SSC, 0.1% SDS at 65 C.
A nucleic acid or polynucleotide sequence is defined herein as a nucleotide
polymer comprising
at least 5 nucleotide or nucleic acid units. A nucleotide or nucleic acid
refers to RNA and DNA.
The terms "nucleic acid" and "polynucleotide sequence" are used
interchangeably herein.
A "peptide" refers to a short chain of amino acid residues linked by a peptide
(amide)
bonds. The shortest peptide, a dipeptide, consists of 2 amino acids joined by
single peptide
bond.
The term "polypeptide" refers to a molecule comprising amino acid residues
linked by
peptide bonds and containing more than five amino acid residues. The term
"protein" as used
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herein is synonymous with the term "polypeptide" and may also refer to two or
more
polypeptides. Thus, the terms "protein" and "polypeptide" can be used
interchangeably.
Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated,
acylated,
farnesylated, prenylated, sulfonated, and the like) to add functionality.
Polypeptides exhibiting
activity in the presence of a specific substrate under certain conditions may
be referred to as
enzymes. It will be understood that, as a result of the degeneracy of the
genetic code, a
multitude of nucleotide sequences encoding a given polypeptide may be
produced.
An "isolated nucleic acid fragment" is a nucleic acid fragment that is not
naturally
occurring as a fragment and would not be found in the natural state.
The term "isolated polypeptide" as used herein means a polypeptide that is
removed
from at least one component, e.g. other polypeptide material, with which it is
naturally
associated. The isolated polypeptide may be free of any other impurities. The
isolated
polypeptide may be at least 50% pure, e.g., at least 60% pure, at least 70%
pure, at least 75%
pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90%
pure, or at least 95%
pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or any other
analytical
method suitable for this purpose and known to the person skilled in the art.
An isolated
polypeptide may be produced by a recombinant host cell.
A "mature polypeptide" is defined herein as a polypeptide in its final form
and is obtained
after translation of a mRNA into polypeptide and post-translational
modifications of said
polypeptide. Post¨translational modification include N-terminal processing, C-
terminal truncation,
glycosylation, phosphorylation and removal of leader sequences such as signal
peptides,
propeptides and/or prepropeptides by cleavage.
A "mature polypeptide coding sequence" means a polynucleotide that encodes a
mature
polypeptide (with reference to its amino acid sequence).
The term "nucleic acid construct" is herein referred to as a nucleic acid
molecule, either
single-or double-stranded, which is isolated from a naturally occurring gene
or which has been
modified to contain segments of nucleic acid which are combined and juxtaposed
in a manner
which would not otherwise exist in nature. The term nucleic acid construct is
synonymous with
the term "expression cassette" when the nucleic acid construct contains all
the control sequences
required for expression of a coding sequence, wherein said control sequences
are operably
linked to said coding sequence.
The term "promoter" is defined herein as a DNA sequence that is bound by RNA
polymerase and directs the polymerase to the correct downstream
transcriptional start site of a
nucleic acid sequence to initiate transcription. A promoter may also comprise
binding sites for
regulators.
The term "recombinant" when used in reference to a cell, nucleic acid, protein
or vector,
indicates that the cell, nucleic acid, protein or vector, has been modified by
the introduction of a
heterologous nucleic acid or protein or the alteration of a native nucleic
acid or protein, or that
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the cell is derived from a cell so modified. Thus, for example, recombinant
cells express genes
that are not found within the native (non-recombinant) form of the cell or
express native genes
that are otherwise abnormally expressed, underexpressed or not expressed at
all. The term
"recombinant" is synonymous with "genetically modified" and "transgenic".
The terms "sequence identity" or "sequence homology" are used interchangeably
herein.
For the purpose of this invention, it is defined here that in order to
determine the percentage of
sequence homology or sequence identity of two amino acid sequences or of two
nucleic acid
sequences, the sequences are aligned for optimal comparison purposes. In order
to optimize the
alignment between the two sequences gaps may be introduced in any of the two
sequences that
are compared. Such alignment can be carried out over the full length of the
sequences being
compared. Alternatively, the alignment may be carried out over a shorter
length, for example
over about 20, about 50, about 100 or more nucleotides/bases or amino acids.
The sequence
identity is the percentage of identical matches between the two sequences over
the reported
aligned region.
A comparison of sequences and determination of percentage of sequence identity
between two sequences can be accomplished using a mathematical algorithm. The
skilled
person will be aware of the fact that several different computer programs are
available to align
two sequences and determine the identity between two sequences (Kruskal, J. B.
(1983) An
overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time
warps, string edits
and macromolecules: the theory and practice of sequence comparison, pp. 1-44
Addison
Wesley). The percent sequence identity between two amino acid sequences or
between two
nucleotide sequences may be determined using the Needleman and Wunsch
algorithm for the
alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol.
Biol. 48, 443-
453). Both amino acid sequences and nucleotide sequences can be aligned by the
algorithm.
The Needleman-Wunsch algorithm has been implemented in the computer program
NEEDLE.
For the purpose of this invention the NEEDLE program from the EMBOSS package
was used
(version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software
Suite (2000)
Rice,P. Longden,I. and Bleasby,A. Trends in Genetics 16, (6) pp276-277,
http://emboss.bioinformatics.n1/). For protein sequences EBLOSUM62 is used for
the substitution
matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters
used are a gap-
open penalty of 10 and a gap extension penalty of 0.5. The skilled person will
appreciate that all
these different parameters will yield slightly different results but that the
overall percentage
identity of two sequences is not significantly altered when using different
algorithms.
After alignment by the program NEEDLE as described above the percentage of
sequence identity between a query sequence and a sequence of the invention is
calculated as
follows: Number of corresponding positions in the alignment showing an
identical amino acid or
identical nucleotide in both sequences divided by the total length of the
alignment after
subtraction of the total number of gaps in the alignment. The identity defined
as herein can be
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obtained from NEEDLE by using the NOBRIEF option and is labeled in the output
of the program
as "longest-identity".
The nucleic acid and protein sequences of the present invention can further be
used as
a "query sequence" to perform a search against public databases to, for
example, identify other
5 family members or related sequences. Such searches can be performed using
the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-
10. BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
wordlength = 12
to obtain nucleotide sequences homologous to nucleic acid molecules of the
invention. BLAST
protein searches can be performed with the XBLAST program, score = 50,
wordlength = 3 to
10 obtain amino acid sequences homologous to protein molecules of the
invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be utilized as
described in
Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing
BLAST and Gapped
BLAST programs, the default parameters of the respective programs (e.g.,
XBLAST and
NBLAST) can be used. See the homepage of the National Center for Biotechnology
Information
at http://www.ncbi.nlm.nih.gov/.
The term "substantially pure" with regard to polypeptides refers to a
polypeptide
preparation which contains at the most 50% by weight of other polypeptide
material. The
polypeptides disclosed herein are preferably in a substantially pure form. In
particular, it is
preferred that the polypeptides disclosed herein are in "essentially pure
form", i.e. that the
polypeptide preparation is essentially free of other polypeptide material.
Optionally, the
polypeptide may also be essentially free of non-polypeptide material such as
nucleic acids, lipids,
media components, and the like. Herein, the term "substantially pure
polypeptide" is synonymous
with the terms "isolated polypeptide" and "polypeptide in isolated form". The
term "substantially
pure" with regard to polynucleotide refers to a polynucleotide preparation
which contains at the
most 50% by weight of other polynucleotide material. The polynucleotides
disclosed herein are
preferably in a substantially pure form. In particular, it is preferred that
the polynucleotide
disclosed herein are in "essentially pure form", i.e. that the polynucleotide
preparation is
essentially free of other polynucleotide material . Optionally, the
polynucleotide may also be
essentially free of non-polynucleotide material such as polypeptides, lipids,
media components,
and the like. Herein, the term "substantially pure polynucleotide" is
synonymous with the terms
"isolated polynucleotide" and "polynucleotide in isolated form".
A "synthetic molecule", such as a synthetic nucleic acid or a synthetic
polypeptide is
produced by in vitro chemical or enzymatic synthesis. It includes, but is not
limited to, variant
nucleic acids made with optimal codon usage for host organisms of choice.
A synthetic nucleic acid may be optimized for codon use, preferably according
to the
methods described in W02006/077258 and/or W02008000632, which are herein
incorporated
by reference. W02008/000632 addresses codon-pair optimization. Codon-pair
optimization is a
method wherein the nucleotide sequences encoding a polypeptide that have been
modified with
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respect to their codon-usage, in particular the codon-pairs that are used, are
optimized to obtain
improved expression of the nucleotide sequence encoding the polypeptide and/or
improved
production of the encoded polypeptide. Codon pairs are defined as a set of two
subsequent
triplets (codons) in a coding sequence. Those skilled in the art will know
that the codon usage
needs to be adapted depending on the host species, possibly resulting in
variants with significant
homology deviation from SEQ ID NO: 1, but still encoding the polypeptide
according to the
invention.
As used herein, the terms "variant", "derivative", "mutant" or "homologue" can
be used
interchangeably. They can refer to either polypeptides or nucleic acids.
Variants include
substitutions, insertions, deletions, truncations, transversions, and/or
inversions, at one or more
locations relative to a reference sequence. Variants can be made for example
by site-saturation
mutagenesis, scanning mutagenesis, insertional mutagenesis, random
mutagenesis, site-
directed mutagenesis, and directed-evolution, as well as various other
recombination approaches
known to a skilled person in the art. Variant genes of nucleic acids may be
synthesized artificially
by known techniques in the art.
The invention relates to a polypeptide having asparaginase activity.
Asparaginase (EC
3.5.1.1) is an enzyme that catalyzes the hydrolysis of aspargine to aspartic
acid and ammonia. A
polypeptide of the invention thus is capable of hydrolyzing asparagine to
aspartic acid and ammonia.
A polypeptide of the invention is one having asparaginase activity and which
is:
i. a polypeptide having an amino acid sequence comprising the mature
polypeptide sequence of SEQ ID NO: 1;
ii. a polypeptide comprising an amino acid sequence that has at least 50%
sequence identity with the mature polypeptide sequence of SEQ ID NO: 1;
iii. a polypeptide encoded by a nucleic acid comprising a sequence that
hybridizes
under medium stringency conditions to the complementary strand of the mature
polypeptide encoding sequence of SEQ ID NO: 2 (or the corresponding wild-type
sequence or a sequence codon optimized or codon pair optimized for expression
in a heterologous organism, such as a Bacillus, for example Bacillus
subtilis); or
iv. a polypeptide comprising an amino acid sequence encoded by a nucleic acid
that has at least 50% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 2 (or the corresponding wild-type sequence or a
sequence codon optimized or codon pair optimized for expression in a
heterologous organism, such as a Bacillus, for example Bacillus subtilis).
The invention also provides a polypeptide of the invention which is:
a polypeptide comprising an amino acid sequence that has at least 60%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence
identity
with the mature polypeptide sequence of SEQ ID NO: 1;
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a polypeptide encoded by a nucleic acid comprising a sequence that hybridizes
under high stringency conditions to the complementary strand of the mature
polypeptide
encoding sequence of SEQ ID NO: 2 (or the corresponding wild-type sequence or
a sequence
codon optimized or codon pair optimized for expression in a heterologous
organism, such as a
Bacillus, for example Bacillus subtilis); or
a polypeptide comprising an amino acid sequence encoded by a nucleic acid
that has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%
or 99% sequence identity to the mature polypeptide coding sequence of SEQ ID
NO: 2 (or the
corresponding wild-type sequence or a sequence codon optimized or codon pair
optimized for
expression in a heterologous organism, such as a Bacillus, for example
Bacillus subtilis).
The invention also relates to polypeptides which are isolated, substantially
pure, pure,
recombinant, synthetic or variant polypeptides of such polypeptides.
A polypeptide of the invention may be derivable from an organism of the genus
Thermophilus, such as from Thermophilus africanus. The wording "derived" or
"derivable" from
with respect to the origin of a polypeptide of the invention means that when
carrying out a
BLAST search with a polypeptide according to the present invention, the
polypeptide according
to the present invention may be derivable from a natural source, such as a
microbial cell, of
which an endogenous polypeptide shows the highest percentage homology or
identity with the
polypeptide as disclosed herein.
Preferably, a polypeptide of the invention may be a polypeptide that has least
50%, 60%,
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%
or 99% sequence identity to the mature polypeptide sequence of SEQ ID NO: 1.
When produced in a heterologous host, a polypeptide of the invention may be
produced
in a form which omits the methionine at position 1 in which case a polypeptide
of the invention
may be a polypeptide that has least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%,
88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the
polypeptide
sequence of amino acids 1 to 335 of SEQ ID NO: 1.
The mature polypeptide sequence typically has the amino acid sequence of amino
acids
1 to 335 of SEQ ID NO: 1.
A polypeptide according to the present invention may be encoded by any
suitable
polynucleotide sequence. Typically a polynucleotide sequence is codon
optimized, or a codon
pair optimized sequence for expression of a polypeptide as disclosed herein in
a particular host
cell. A polypeptide of the invention may be encoded by a polynucleotide
sequence that
comprises appropriate control sequences and/or signal sequences, for example
for secretion.
A polypeptide of the invention may be encoded by a polynucleotide that
hybridizes under
medium stringency, preferably under high stringency conditions to the
complementary strand of
the mature polypeptide coding sequence of SEQ ID NO: 2 (or the corresponding
wild-type
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13
sequence or a sequence codon optimized or codon pair optimized for expression
in a
heterologous organism, such as a Bacillus, for example Bacillus subtilis).
A polypeptide of the invention may also be encoded by a nucleic acid that has
at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to
a
mature polypeptide coding sequence of SEQ ID NO: 2 (or the corresponding wild-
type sequence
or a sequence codon optimized or codon pair optimized for expression in a
heterologous
organism, such as a Bacillus, for example Bacillus subtilis).
A polypeptide of the invention may also be a variant of a mature polypeptide
of SEQ ID
NO: 1, comprising a substitution, deletion and/or insertion at one or more
positions of the mature
polypeptide SEQ ID NO: 1. A variant of the mature polypeptide of SEQ ID NO: 1
may be an
amino acid sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12
amino acids from the
amino acids of the mature polypeptide of SEQ ID NO: 1.
In one embodiment the present invention features a biologically active
fragment of a
polypeptide as disclosed herein.
Biologically active fragments of a polypeptide of the invention include
polypeptides
comprising amino acid sequences sufficiently identical to or derived from the
amino acid
sequence of the asparaginase protein (e.g., the mature amino acid sequence of
SEQ ID NO: 1),
which include fewer amino acids than the full length protein but which
exhibits at least one
biological activity of the corresponding full-length protein. Typically,
biologically active fragments
comprise a domain or motif with at least one activity of the asparaginase
protein. A biologically
active fragment may for instance comprise a catalytic domain. A biologically
active fragment of
a protein of the invention can be a polypeptide which is, for example, 10, 25,
50, 100 or more
amino acids in length. Moreover, other biologically active portions, in which
other regions of the
protein are deleted, can be prepared by recombinant techniques and evaluated
for one or more
of the biological activities of the native form of a polypeptide of the
invention.
The invention also features nucleic acid fragments which encode the above
biologically
active fragments of the asparaginase protein.
A polypeptide according to the present invention may be a fusion protein.
Techniques for
producing fusion polypeptides are known in the art, and include ligating the
coding sequences
encoding the polypeptides so that they are in frame. Expression of the fused
polypeptide is under
control of the same promoter (s) and terminator. The hybrid polypeptides may
comprise a combination
of partial or complete polypeptide sequences obtained from at least two
different polypeptides wherein
one or more may be heterologous to a host cell. Such fusion polypeptides from
at least two different
polypeptides may comprise a binding domain from one polypeptide, operably
linked to a catalytic
domain from a second polypeptide . Examples of fusion polypeptides and signal
sequence fusions are
for example as described in W02010/121933, W02013/007820 and W02013/007821.
A polypeptide of the invention may be a naturally occurring polypeptide or a
genetically
modified or recombinant polypeptide.
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A polypeptide of the invention may be purified. Purification of proteins is
known to a
skilled person in the art.
A polypeptide of the invention may preferably be thermostable and/or
thermoactive.
Additionally or alternatively, a polypeptide of the invention may be active
across a broad pH
range and/or active at a relatively high or low pH.
A polypeptide of the invention may be thermostable. "Thermostable" herein
means that
a polypeptide of the invention may have a residual asparaginase activity of at
least 50% after an
incubation period of 5 min at 50 C. A polypeptide of the invention may have a
residual
asparaginase activity of at least 50% after an incubation period of 5 min at
55 C, 60 C, 65 C,
70 C or at a higher temperature.
"Residual activity" herein means any specific/volumetric enzymatic activity
that an
enzyme has after a specific incubation duration at a specific temperature
compared with the
original specific/volumetric activity in the range of its temperature optimum
under otherwise
identical reaction conditions (pH, substrate etc.). The specific/volumetric
activity of an enzyme
means a specific amount of a converted substrate (for example in pmol) per
unit time (for
example in minutes) per enzyme amount (for example in mg or ml). The residual
activity of an
enzyme results from the specific/volumetric activity of the enzyme after the
aforementioned
incubation duration divided by the original specific/volumetric activity
expressed as a percentage
(%). In this case, the specific activity of an enzyme may be indicated in U/mg
and the volumetric
activity of an enzyme may be indicated in U/ml. Alternatively, the
specific/volumetric activity of
an enzyme can also be indicated in katal/mg or katal/ml in the sense of the
description.
The term "enzymatic activity", sometimes also referred to as "catalytic
activity" or
"catalytic efficiency", is generally known to the person skilled in the art
and refers to the
conversion rate of an enzyme and is usually expressed by means of the ratio
kkat/Kv, wherein kkat
is the catalytic constant (also referred to as turnover number) and the Km
value corresponds to
the substrate concentration, at which the reaction rate lies at half its
maximum value.
Alternatively, the enzymatic activity of an enzyme can also be specified by
the specific activity
(pmol of converted substrate x mg' x min'; cf. above) or the volumetric
activity (pmol of
converted substrate x m1-1 x cf. above).
Reference can also be made to the general literature such as Structure and
Mechanism
in Protein Science: A guide to enzyme catalysis and protein folding, Alan
Fersht, W.H.Freeman,
1999; Fundamentals of Enzyme Kinetics, Athel Cornish-Bowden, Wiley-Blackwell
2012 and Voet
et al., "Biochemie" [Biochemistry], 1992, VCH-Verlag, Chapter 13, pages 331-
332 with respect to
enzymatic activity.
Thus, a thermostable polypeptide of the invention may have a residual activity
of at least
50%, at least 60%, at least 70%, at least 80%, at least 90% or higher under
the following
conditions as set out in Table 1. In Table 1 for example condition 4B means
that the
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asparaginase after 30 minutes at 60 C has a residual activity of at least 75%,
such as at least
80%, for example at least 90%.
Table 1. Conditions to determine residual activity.
Nr Time Temperature (degrees Celsius)
(mm)
A
1 5 50 60 70 80 90 100
2 10 50 60 70 80 90 100
3 20 50 60 70 80 90 100
4 30 50 60 70 80 90 100
5 40 50 60 70 80 90 100
6 50 50 60 70 80 90 100
7 60 50 60 70 80 90 100
8 70 50 60 70 80 90 100
5
Preferably, a polypeptide of the invention has a residual activity in the
range of from
75% to 100%, such as 75% to 90% under the conditions specified above.
A polypeptide of the invention having asparaginase activity is preferably
thermoactive.
"Thermoactive" herein means that the temperature optimum of such a polypeptide
is at least
10 about 50 C, at least about 55 C, at least about 60 C, at least about
65 C, at least about 70 C
or higher.
Thermoactivity may be determined as set out in Example 2.
The term "temperature optimum" is generally known to the skilled person and
relates to
the temperature range at which an enzyme exhibits its maximum enzymatic
activity. Reference
15 can be made in association with this to the relevant literature such as
Enzyme Assays: A
Practical Approach, Robert Eisenthal,Michael J. Danson, Oxford University
Press 2002; Voet et
al., "Biochemie", 1992, VCH-Verlag, Chapter 13, page 331; I. H. Segel, Enzyme
Kinetics:
Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems,
Wiley
Interscience, 1993; and A. G. Marangoni, Enzyme Kinetics: A Modern Approach,
Wiley
Interscience, 2002.
Herein, the temperature optimum is preferably understood to be the temperature
range,
in which a polypeptide of the invention has at least 80%, preferably at least
90% of the maximum
enzymatic activity under otherwise constant reaction conditions.
The temperature optimum of a polypeptide according to the invention preferably
lies in
the range of from 60 to 130 C., such as in the range of from 70 to 120 C,
for example in the
range of 75 to 110 C or in the range of from 80 to 100 C.
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Thus, a polypeptide of the invention is preferably thermostable (i.e. capable
of
withstanding a thermal treatment in respect of its enzymatic activity) and/or
thermoactive (i.e.
only develops its full enzymatic activity at elevated temperature).
At a temperature of from 60 to 120 C, such as from 65 to 110 C, such as
from 70 to
100 C, such as from 75 to 100 C or from 80 to 90 C, a polypeptide of the
invention may
have a specific activity of preferably at least 100, more preferred at least
200, further preferred
at least 300, further preferred at least 500, most preferred at least 800 and
in particular at least
100 units/mg, wherein 1 unit is defined as the amount of the polypeptide that
releases 1.0 pmol
of ammonia per minute from L-asparagine under the conditions set out in the
Examples
A polypeptide of the invention may be active at a relatively high pH.
Accordingly, a
polypeptide of the invention may have a pH optimum which is higher than the
wild-type
asparaginase from A. niger (as disclosed in W02004/030468) which has a pH
optimum of from
pH 4 to pH 5. A polypeptide of the invention may be more alkaliphilic than
such a wild-type
enzyme, i.e. may, for example, have a pH optimum of from pH 5 to pH 11, such
as from pH 6 to
pH 10. Optionally a variant protein of the invention may be more acidophilic
than the wild type
asparaginase from A. niger.
The term "pH optimum" is generally known to the skilled person and relates to
the pH
range, in which an enzyme has its maximum enzymatic activity. Reference can be
made in
association with this to the relevant literature such as Enzyme Assays: A
Practical Approach,
Robert Eisenthal,Michael J. Danson, Oxford University Press 2002 and Voet et
al., "Biochemie",
1992, VCH-Verlag, Chapter 13, page 331. Herein, the term pH optimum is
typically understood
to mean the pH range, in which the amidohydrolase used according to the
invention has at least
80%, preferably at least 90% of the maximum enzymatic activity under otherwise
constant
reaction conditions.
A polypeptide according to the invention may have a pH, which may be higher
than the
pH optimum and at which at least 50% of the asparaginase activity is still
present, (hereafter
indicated as alkaline pH), which is higher than that of the wild type
asparaginase from A. niger.
Thus, a polypeptide of the invention may have an alkaline pH at which at least
50% of the
activity (at the pH optimum) is observed which may at least pH 7Ø
A polypeptide of the invention may be active over a very broad pH range. In
the range
from pH 5 to pH 10, a polypeptide of the invention may preferably have an
activity of at least
10% of the maximum activity. As a result of this, it may possible to use a
polypeptide of the
invention in different processes with widely differing pH ranges. It is also
possible to use it in
processes in which the pH value is subject to significant fluctuations in the
process. Processes
are also possible in which pH values from 5 to 10 occur.
Over the entire pH range of from pH 5 to pH 10, a polypeptide of the invention
has an
activity of at least 10%, more preferred at least 15%, further preferred at
least 20%, most
preferred at least 25% and in particular at least 30% compared to the maximum
activity, i.e. to
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the maximum activity with the optimum pH value under otherwise identical
conditions, preferably
at optimum temperature and concentration.
The invention further provides a nucleic acid encoding an asparaginase which
comprises
a sequence that has at least 50% sequence identity to the mature polypeptide
encoding
sequence of SEQ ID NO: 2.
A nucleic acid of the invention may comprise a polynucleotide sequence
encoding a
polypeptide of the invention which has at least 60%, 70%, 75%, 80%, 85%, 90%,
95%, 96%,
97%, 98%, or 99% sequence identity to SEQ ID NO: 2, or to the mature
polypeptide coding
sequence of either thereof.
A polynucleotide sequence of the invention may comprise SEQ ID NO: 2 or may
comprise the mature polypeptide coding sequence of either thereof.
A nucleic acid of the invention may be an isolated, substantially pure, pure,
recombinant,
synthetic or variant nucleic acid of the nucleic acid of SEQ ID NO: 2. A
variant nucleic acid
sequence may for instance have at least 80% sequence identity to SEQ ID NO: 2.
The invention also provides a nucleic acid construct comprising a nucleic acid
of the
invention. An expression vector is also provide which comprises a nucleic acid
of the invention
or a nucleic acid of the invention operably linked to one or more control
sequences that direct
expression of the polypeptide in a host cell.
There are several ways of inserting a nucleic acid into a nucleic acid
construct or an
expression vector which are known to a skilled person in the art, see for
instance Sambrook &
Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold
Spring Harbor, NY,
2001. It may be desirable to manipulate a nucleic acid encoding a polypeptide
of the present
invention with control sequences, such as promoter and terminator sequences.
A promoter may be any appropriate promoter sequence suitable for a eukaryotic
or
prokaryotic host cell, which shows transcriptional activity, including mutant,
truncated, and hybrid
promoters, and may be obtained from polynucleotides encoding extracellular or
intracellular
polypeptides either endogenous (native) or heterologous (foreign) to the cell.
The promoter may
be a constitutive or inducible promoter. An inducible promoter may be, for
example, a starch
inducible promoter.
In the invention, bacteria may preferably be used as host cells for the
expression of a
polypeptide of the invention, in particular Bacilli. Suitable inducible
promoters useful in such host
cells include promoters that may be regulated primarily by an ancillary factor
such as a repressor
or an activator. The repressors are sequence-specific DNA binding proteins
that repress
promoter activity. The transcription can be initiated from this promoter in
the presence of an
inducer that prevents binding of the repressor to the operator of the
promoter. Examples of such
promoters from Gram-positive microorganisms include, but are not limited to,
gnt (gluconate
operon promoter); penP from Bacillus licheniformis; glnA (glutamine
synthetase); xylAB (xylose
operon); araABD (L-arabinose operon) and P
. spac promoter, a hybrid SP01//ac promoter that can
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18
be controlled by inducers such as isopropyl-R-D-thiogalactopyranoside [IPTG]
((Yansura D.G.,
Henner D.J. Proc Natl Aced Sci U S A. 1984 81(2):439-443). Activators are also
sequence-
specific DNA binding proteins that induce promoter activity. Examples of such
promoters from
Gram-positive microorganisms include, but are not limited to, two-component
systems (PhoP-
PhoR, DegU-DegS, Spo0A-Phosphorelay), LevR, Mry and GItC. Production of
secondary sigma
factors can be primarily responsible for the transcription from specific
promoters. Examples from
Gram-positive microorganisms include, but are not limited to, the promoters
activated by
sporulation specific sigma factors: GF, GE, GG and GK and general stress sigma
factor, GB. The 6B-
mediated response is induced by energy limitation and environmental stresses
(Hecker M, Volker
U. Mol Microbiol. 1998; 29(5):1129-1136.). Attenuation and antitermination
also regulates
transcription. Examples from Gram-positive microorganisms include, but are not
limited to, trp
operon and sacB gene. Other regulated promoters in expression vectors are
based the sacR
regulatory system conferring sucrose inducibility (Klier AF, Rapoport G. Annu
Rev Microbiol.
1988;42:65-95).
Strong constitutive promoters are well known and an appropriate one may be
selected
according to the specific sequence to be controlled in the host cell. Suitable
inducible promoters
useful in bacteria, such as Bacilli, include: promoters from Gram-positive
microorganisms such
as, but are not limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylase
promoter), and
amyE. Examples of promoters from Gram-negative microorganisms include, but are
not limited
to, tac, tet, trp-tet, Ipp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara,
5P6, 2-PR, and 2-PL.
Additional examples of promoters useful in bacterial cells, such as Bacilli,
include the
a-amylase and SPo2 promoters as well as promoters from extracellular protease
genes.
The promoter sequences may be obtained from a bacterial source. In a more
preferred
embodiment, the promoter sequences may be obtained from a gram positive
bacterium such as
a Bacillus strain, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus
circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus,
Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus
stearothermophilus,
Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain, e.g.,
Streptomyces lividans
or Streptomyces murinus; or from a gram negative bacterium, e.g., E. coli or
Pseudomonas sp.
An example of a suitable promoter for directing the transcription of a
polynucleotide
sequence of the present invention is the promoter obtained from the E. coli
lac operon. Another
example is the promoter of the Streptomyces coelicolor agarase gene (dagA).
Another example
is the promoter of the Bacillus lentus alkaline protease gene (aprH). Another
example is the
promoter of the Bacillus licheniformis alkaline protease gene (subtilisin
Carlsberg gene). Another
example is the promoter of the Bacillus subtilis levansucrase gene (sacB).
Another example is
the promoter of the Bacillus subtilis alphaamylase gene (amyF). Another
example is the
promoter of the Bacillus licheniformis alphaamylase gene (amyL). Another
example is the
promoter of the Geobacillus stearothermophilus glucan 1,4-0-maltohydrolase
gene (amyM).
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Another example is the promoter of the Bacillus amyloliquefaciens alpha-
amylase gene (amyQ).
Another example is a "consensus" promoter having the sequence TTGACA for the "-
35" region
and TATAAT for the "-10" region. Another example is the promoter of the
Bacillus licheniformis
penicillinase gene (penP). Another example are the promoters of the Bacillus
subtilis xylA and
xylB genes.
Preferably the promoter sequence is from a highly expressed gene. Examples of
preferred highly expressed genes from which promoters may be selected and/or
which are
comprised in preferred predetermined target loci for integration of expression
constructs, include
but are not limited to genes encoding glycolytic enzymes such as triose-
phosphate isomerases
(TPI),glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate
kinases (PGK),
pyruvate kinases (PYK or PKI), alcohol dehydrogenases (ADH), as well as genes
encoding
amylases, glucoamylases, proteases, xylanases, cellobiohydrolases, P-
galactosidases, alcohol
(methanol) oxidases, elongation factors and ribosomal proteins. Specific
examples of suitable
highly expressed genes include e. g. the LAC4 gene from Kluyveromyces sp., the
methanol
oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively, the
glucoamylase
(glaA) genes from A. niger and A. awamori, the A. oryzae TAKA-amylase gene,
the A. nidulans
gpdA gene and the T. reesei cellobiohydrolase genes.
Promoters which can be used in yeasts include e.g. promoters from glycolytic
genes,
such as the phosphofructokinase (PFK), triose phosphate isomerase (TPI),
glyceraldehyde-3 -
phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK),
phosphoglycerate
kinase (PGK) promoters from yeasts or filamentous fungi; more details about
such promoters
from yeast may be found in (WO 93/03159). Other useful promoters are ribosomal
protein
encoding gene promoters, the lactase gene promoter (LAC4), alcohol
dehydrogenase promoters
(ADHI, ADH4, and the like), and the enolase promoter (ENO). Other promoters,
both constitutive
and inducible, and enhancers or upstream activating sequences will be known to
those of skill in
the art. The promoters used in the host cells of the invention may be
modified, if desired, to
affect their control characteristics. Suitable promoters in this context
include both constitutive
and inducible natural promoters as well as engineered promoters, which are
well known to the
person skilled in the art. Suitable promoters in eukaryotic host cells may be
GAL7, GAL10, or
GAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC, TRP1, URA3, LEU2, EN01, TPI1,
and
A0X1. Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3.
Examples of
carbohydrate inducible promoters which can be used are GAL promoters, such as
GAL1 or
GAL10 promoters.
Promoters suitable in filamentous fungi are promoters which may be selected
from the
group, which includes but is not limited to promoters obtained from the
polynucleotides encoding
A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus
gpdA promoter, A.
niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or
A. awamori
glucoamylase (glaA), A. niger or A. awamori endoxylanase (xInA) or beta-
xylosidase (xInD), T.
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reesei cellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkaline
protease, A. oryzae triose
phosphate isomerase, A. nidulans acetamidase, Fusarium venenatum
amyloglucosidase (WO
00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn
(WO
00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma
reesei beta-
5
glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei
cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,
Trichoderma reesei
endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei
endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma
reesei beta-
xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from
the polynucleotides
10
encoding A. niger neutral alpha-amylase and A. oryzae triose phosphate
isomerase), and mutant,
truncated, and hybrid promoters thereof.
All of the above-mentioned promoters are readily available in the art.
Any terminator which is functional in a cell as disclosed herein may be used,
which are
known to a skilled person in the art.
15
Examples of suitable terminator sequences in filamentous fungi include
terminator
sequences of a filamentous fungal gene, such as from Aspergillus genes, for
instance from the
gene A. oryzae TAKA amylase, the genes encoding A. niger glucoamylase (glaA),
A. nidulans
anthranilate synthase, A. niger alpha-glucosidase, trpC and/or Fusarium
oxysporum trypsin-like
protease.
20 The
invention also relates to a vector which comprises a nucleic acid of the
invention,
said vector comprises at least an autonomous replication sequence and a
nucleic acid as
described herein.
The vector may be any vector (e.g. a plasmid or a virus), which can be
conveniently
subjected to recombinant DNA procedures. The choice of the vector will
typically depend on the
compatibility of the vector with the host cell into which the vector is to be
introduced. Preferably,
the vector is a plasmid. The vector may be a linear or a closed circular
plasmid. The vector may
further comprise a, preferably non-selective, marker that allows for easy
determination of the
vector in the host cell. Suitable markers include GFP and DsRed. The chance of
gene
conversion or integration of the vector into the host genome is preferably
minimized. The vector
according to the invention may be an extra-chromosomal vector. Such a vector
preferably lacks
significant regions of homology with the genome of the host to minimize the
chance of
integration into the host genome by homologous recombination. The person
skilled in the art
knows how to construct a vector with minimal chance of integration into the
genome. This may
be achieved by using control sequences, such as promoters and terminators,
which originate
from another species than the host species. Other ways of reducing homology
are by modifying
codon usage and introduction of silent mutations. The person skilled in the
art knows that the
type of host cell, the length of the regions of homology to the host cell
genome present in the
vector, and the percentage of homology between said regions of homology in the
vector and the
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host chromosome will determine whether and in which amount the vector will
integrate into the
host cell genome.
The autonomous replication sequence may be any suitable sequence available to
the
person skilled in the art that allows for plasmid replication that is
independent of chromosomal
replication.
The origin of replication may be any plasmid replicator mediating autonomous
replication
that functions in a cell. The term "origin of replication" or "plasmid
replicator" is defined herein as
a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
Examples of
bacterial origins of replication are the origins of replication of plasmids
pBR322, pUC19,
pACYC177, and pACYC184 permitting replication in E. coli, RSF1010 permitting
replication in
Pseudomonas is described, e.g., by F. Heffron et al., in Proc. Nat'l Acad.
Sci. USA 72(9):3623-27
(Sep 1975), and pUB110, pE194, pTA1060, and pAMR1 permitting replication in
Bacillus.
Preferably, the autonomous replication sequence used in filamentous fungi is
the AMA1
replicon (Gems et al., 1991 Gene. 98(1):61-7). Telomeric repeats may also
result in autonomous
replication (In vivo linearization and autonomous replication of plasmids
containing human
telomeric DNA in Aspergillus nidulans, Aleksenko et al. Molecular and General
Genetics MGG,
1998 - Volume 260, Numbers 2-3, 159-164, DOI: 10.1007/s004380050881). CEN/ARS
sequences and 3p vector sequences from yeast may also be suitable.
A vector or expression construct for a given host cell may thus comprise the
following
elements operably linked to each other in a consecutive order from the 5'-end
to 3'-end relative
to the coding strand of the sequence encoding the compound of interest or
encoding a
compound involved in the synthesis of the compound of interest: (1) a promoter
sequence
capable of directing transcription of a nucleic acid of the invention; (2)
optionally a sequence to
facilitate the translation of the transcribed RNA, for example a ribosome
binding site (also
indicated as Shine Delgarno sequence) in prokaryotes, or a Kozak sequence in
eukaryotes (3)
optionally, a signal sequence capable of directing secretion of the
asparaginase encoded by the
nucleic acid of the invention from the given host cell into a culture medium;
(4) a nucleic acid of
the invention, as described herein; and preferably also (5) a transcription
termination region
(terminator) capable of terminating transcription downstream of the nucleic
acid of the invention.
The vector may comprise these and/or other control sequences as described
herein.
Downstream of a nucleic acid of the invention there may be a 3'-untranslated
region
containing one or more transcription termination sites (e. g. a terminator,
herein also referred to
as a stop codon). The origin of the terminator is not critical. The terminator
can, for example, be
native to the DNA sequence encoding the polypeptide. However, preferably a
bacterial
terminator is used in bacterial host cells and a filamentous fungal terminator
is used in
filamentous fungal host cells. More preferably, the terminator is endogenous
to the host cell (in
which the nucleotide sequence encoding the polypeptide is to be expressed). In
the transcribed
region, a ribosome binding site for translation may be present. The coding
portion of the mature
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transcripts expressed by the constructs will include a start codon, usually
AUG (or ATG), but
there are also alternative start codons, such as for example GUG (or GTG) and
UUG (or TTG),
which are used in prokaryotes. Also a stop or translation termination codon is
appropriately
positioned at the end of the polypeptide to be translated.
Enhanced expression of an asparaginase of the invention may also be achieved
by the
selection of homologous and heterologous regulatory regions, e. g. promoter,
secretion leader
and/or terminator regions, which may serve to increase expression and, if
desired, secretion
levels of the protein of interest from the expression host and/or to provide
for the inducible
control of the expression of a compound of interest or a compound involved in
the synthesis of a
compound of interest.
The vector comprising at least an autonomous replication sequence and a
nucleic acid
of the invention, also referred to herein as "vector (or expression vector) of
the invention" can be
designed for expression of the nucleic acid in a prokaryotic or a eukaryotic
cell. For example, an
asparaginase of the invention can be produced in bacterial cells such as E.
coli or Bacilli, insect
cells (using baculovirus expression vectors), fungal cells, such as yeast
cells, or mammalian
cells. Suitable host cells are discussed herein and further in Goeddel, Gene
Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Alternatively,
the recombinant expression vector can be transcribed and translated in vitro,
for example using
T7 promoter regulatory sequences and T7 polymerase.
In order to identify and select cells which harbour a nucleic acid and/or
vector of the
invention, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is optionally
introduced into the vector and/or host cells along with the nucleic acid of
the invention. Preferred
selectable markers include, but are not limited to those which confer
resistance to drugs or which
complement a defect in the host cell.
Such markers include ATP synthetase, subunit 9 (o/iC), orotidine-5'-
phosphatedecarboxylase (pvrA), the bacterial G418 resistance gene (this may
also be used in
yeast, but not in fungi), the ampicillin resistance gene (E. coli), resistance
genes for neomycin,
kanamycin, tetracycline, spectinomycin, erythromycin, chloramphenicol,
phleomycin (Bacillus)
and the E. coli uidA gene, coding for 8-glucuronidase (GUS). Vectors may be
used in vitro, for
example for the production of RNA or used to transfect or transform a host
cell.
They also include e. g. versatile marker genes that can be used for
transformation of
most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the
amdS, niaD, facA
genes or cDNAs from A. nidulans, A. oryzae or A. niger), or genes providing
resistance to
antibiotics like G418, hygromycin, bleomycin, kanamycin, methotrexate,
phleomycin orbenomyl
resistance (benA). Alternatively, specific selection markers can be used such
as auxotrophic
markers which require corresponding mutant host strains: e. g. D-alanine
racemase (from
Bacillus), URA3 (from S. cerevisiae or analogous genes from other yeasts),
pyrG or pyrA (from
A. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC. In a
preferred embodiment
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the selection marker is deleted from the transformed host cell after
introduction of the expression
construct so as to obtain transformed host cells capable of producing the
compound of interest or
a compound involved in the synthesis of a compound of interest which are free
of selection
marker genes.
Expression of proteins in prokaryotes is often carried out in with vectors
containing
constitutive or inducible promoters directing the expression of either fusion
or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein encoded
therein, e.g. to the
amino terminus of the recombinant protein. Such fusion vectors typically serve
three purposes:
1) to increase expression of recombinant protein; 2) to increase the
solubility of the recombinant
protein; and 3) to aid in the purification of the recombinant protein by
acting as a ligand in affinity
purification. Often, in fusion expression vectors, a proteolytic cleavage site
is introduced at the
junction of the fusion moiety and the recombinant protein to enable separation
of the
recombinant protein from the fusion moiety subsequent to purification of the
fusion protein.
The present invention also provides a host cell comprising a nucleic acid or
an
expression vector as disclosed herein. A suitable host cell may be a
mammalian, insect, plant,
fungal, or algal cell, or a bacterial cell.
The host cell may be a prokaryotic cell. Preferably, the prokaryotic host cell
is bacterial
cell. The term "bacterial cell" includes both Gram-negative and Gram-positive
microorganisms.
Suitable bacteria may be selected from e.g. Escherichia, Anabaena,
Caulobactert,
Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium,
Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella,
Enterobacter,
Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces.
Preferably, the
bacterial cell is selected from the group consisting of B. subtilis, B.
amyloliquefaciens, B.
licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G.
oxydans, Caulobactert
crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides,
Pseudomonas
zeaxanthinifaciens, Pseudomonas fluorescence, Paracoccus denitrificans, E.
coli, C. glutamicum,
Staphylococcus camosus, Streptomyces lividans, Sinorhizobium melioti and
Rhizobium
radiobacter.
In one preferred embodiment of the invention the host cell deficient in the
essential gene
coding for the essential polypeptide is a prokaryotic cell, preferably a
bacterial cell, more
preferably a bacterial cell belonging to the genus Bacillus, Escherichia (such
as Escherichia
Pseudomonas, Lactobacillus.
In a preferred embodiment of the invention the bacterial host cell may
additionally
contain modifications, e.g. the bacterial host cell may be deficient in genes
which are detrimental
to the production, recovery and/or application of the compound of interest,
e.g. a compound of
interest being a polypeptide, e.g. an enzyme. In a preferred aspect the
bacterial host cell is a
protease deficient host cell, more preferably it is a Bacillus host cell
deficient in the gene aprE
coding for extracellular alkaline protease and deficient in the gene nprE
coding for extracellular
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neutral metalloprotease. In another preferred aspect the Bacillus host cell is
further deficient in
one or more proteases coded by the genes selected from the group consisting
of: nprB, vpr, epr,
wprA, mpr, bpr. In another preferred aspect the bacterial host cell does not
produce spores and
or is deficient in a sporulation related gene such as e.g. spo0A, spolISA,
sigE, sigF, spolISB,
spollE, sigG, spolVCB, spollIC, spolIGA, spollAA, spolVFB, spolIR, spollIJ. In
yet another
preferred aspect the Bacillus host cell is deficient in the gene amyE coding
for a-amylase. In yet
another preferred aspect the Bacillus host cell, more preferably a Bacillus
subtilis host cell, is
deficient in aprE, nprE, amyE and does not produce spores. In a more preferred
embodiment the
Bacillus host cell is BS154, CBS136327 or a derivative thereof.
According to an embodiment, the host cell according to the invention is a
eukaryotic host
cell. Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal,
or algal cell. Preferred
mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293
cells, PerC6
cells, and hybridomas. Preferred insect cells include e.g. Sf9 and Sf21 cells
and derivatives
thereof.
The eukaryotic cell may be a fungal cell, for example a yeast cell, such as a
cell of the
genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or
Yarrowia. More specifically, a yeast cell may be from Kluyveromyces lactis,
Saccharomyces
cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris,
Candida krusei.
Filamentous fungi include all filamentous forms of the subdivision Eumycota
and
Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's
Dictionary of The Fungi,
8th edition, 1995, CAB International, University Press, Cambridge, UK). The
filamentous fungi
are characterized by a mycelial wall composed of chitin, cellulose, glucan,
chitosan, mannan,
and other complex polysaccharides. Vegetative growth is by hyphal elongation
and carbon
catabolism is obligately aerobic. Filamentous fungal strains include, but are
not limited to, strains
of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus,
Cryptococcus,
Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix,
Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus,
Schizophyllum,
Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and
Trichoderma.
Preferred filamentous fungal cells belong to a species of an Acremonium,
Aspergillus,
Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia,
Thielavia, Fusarium or
Trichoderma genus, and most preferably a species of Aspergillus niger,
Acremonium
alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,
Aspergillus fumigatus,
Talaromyces emersonii, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium
lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma
reesei, Thielavia
terrestris or Penicillium chrysogenum. A more preferred filamentous fungal
host cell belongs to
the genus Aspergillus, more preferably the host cell belongs to the species
Aspergillus niger.
When the host cell according to the invention is an Aspergillus niger host
cell, the host cell
preferably is CBS 513.88, CB5124.903 or a derivative thereof.
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Several strains of filamentous fungi are readily accessible to the public in a
number of
culture collections, such as the American Type Culture Collection (ATCC),
Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor
Schimmelcultures
(CBS), Agricultural Research Service Patent Culture Collection, Northern
Regional Research
5 Center (NRRL), and All-Russian Collection of Microorganisms of Russian
Academy of Sciences,
(abbreviation in Russian - VKM, abbreviation in English - RCM), Moscow,
Russia. Useful strains
in the context of the present invention may be Aspergillus niger CBS 513.88,
CB5124.903,
Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011, CB5205.89, ATCC 9576,
ATCC14488-
14491, ATCC 11601, ATCC12892, P. chrysogenum CBS 455.95, P. chrysogenum
Wisconsin54-
10 1255(ATCC28089), Penicillium citrinum ATCC 38065, Penicillium
chrysogenum P2, Thielavia
terrestris NRRL8126, Talaromyces emersonii CBS 124.902, Acremonium chrysogenum
ATCC
36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC
26921,
Aspergillus sojae ATCC11906, Myceliophthora thermophila Cl, Garg 27K, VKM-F
3500 D,
Chrysosporium lucknowense Cl, Garg 27K, VKM-F 3500 D, ATCC44006 and
derivatives
15 thereof.
A host cell may be a recombinant or transgenic host cell. The host cell may be
genetically modified with a nucleic acid construct or expression vector as
disclosed herein with
standard techniques known in the art, such as electroporation, protoplast
transformation or
conjugation for instance as disclosed in Sambrook & Russell, Molecular
Cloning: A Laboratory
20 Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001.
The invention also relates to a process for the production of a polypeptide of
the
invention comprising cultivating a host cell in a suitable fermentation medium
under conditions
conducive to the production of the polypeptide and producing the polypeptide.
A skilled person in
the art understands how to perform a process for the production of a
polypeptide as disclosed
25 herein depending on a host cell used, such as pH, temperature and
composition of a
fermentation medium. Host cells can be cultivated in shake flasks, or in
fermenters having a
volume of 0.5 or 1 litre or larger to 10 to 100 or more cubic metres.
Cultivation may be
performed aerobically or anaerobically depending on the requirements of a host
cell.
Advantageously a polypeptide as disclosed herein is recovered or isolated from
the
fermentation medium.
The present invention further provides a composition comprising a polypeptide
according
to the invention. The composition may optionally comprise other ingredients
such as, for
example, a carrier, an excipient or a further enzyme, such as an auxiliary
enzyme. A
composition of the invention may a polypeptide of the invention and one or
more further
asparaginases. The one or more further asparaginases may be a second or
further polypeptide
of the invention.
A composition of the invention may comprise a polypeptide of the invention and
at least
one dough ingredient.
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Dough ingredients include, without limitation, (cereal) flour, egg, water,
salt, sugar, flavours,
fat (including butter, margarine, oil and shortening), baker's yeast, a
chemical leavening system such
as a combination of an acid (generating compound) and bicarbonate, milk
(including liquid milk and
milk powder), soy flour, oxidants (including ascorbic acid, bromate and
azodicarbonamide (ADA),
reducing agents (including L-cysteine), emulsifiers (including mono/di
glycerides, mono
glycerides such as glycerol monostearate (GMS), sodium stearoyl lactylate
(SSL), calcium
stearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) and
diacetyl tartaric acid esters
of mono- and diglycerides (DATEM) propylene glycol monostearate (PGMS),
lecithin), gums
(including guargum and xanthangum), flavours, acids (including citric acid,
propionic acid),
starch, modified starch, humectants (including glycerol) and preservatives
Cereals include maize, rice, wheat, barley, sorghum, millet, oats, rye,
triticale, buckwheat,
quinoa, spelt, einkorn, emmer, durum and kamut.
The preparation of a dough from dough ingredients is well known in the art and
includes
mixing of said ingredients and optionally one or more moulding and
fermentation steps.
Preparing a dough according to the invention may comprise the step of
combining a
polypeptide of the invention or a composition of the invention and at least
one dough ingredient.
Combining includes, without limitation, adding a polypeptide or a composition
of the
invention to the at least one component indicated herein, adding the at least
one component
indicated herein to a polypeptide or a composition of the invention, mixing a
polypeptide or a
composition of the invention and the at least one component indicated herein.
A composition may comprise a polypeptide of the invention and one or more
additional
enzymes
In such a composition of the invention, the additional enzyme may include
including an
alpha-amylase, such as a fungal alpha-amylase (which may be useful for
providing sugars
fermentable by yeast and retarding staling), beta-amylase, a cyclodextrin
glucanotransferase, a
protease, a peptidase, in particular, an exopeptidase (which may be useful in
flavour
enhancement), transglutaminase, triacyl glycerol lipase (which may be useful
for the modification
of lipids present in the dough or dough constituents so as to soften the
dough), galactolipase,
phospholipase, cellulase, hemicellulase, in particular a pentosanase such as
xylanase (which
may be useful for the partial hydrolysis of pentosans, more specifically
arabinoxylan, which
increases the extensibility of the dough), protease (which may be useful for
gluten weakening in
particular when using hard wheat flour), protein disulfide isomerase, e.g., a
protein disulfide
isomerase as disclosed in WO 95/00636, glycosyltransferase, peroxidase (which
may be useful
for improving the dough consistency), laccase, or oxidase, hexose oxidase,
e.g., a glucose
oxidase, aldose oxidase, pyranose oxidase, lipoxygenase or L-amino acid
oxidase (which may
be useful in improving dough consistency) or a protease.
In a composition of the invention, the additional enzyme may be a lipolytic
enzyme.
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In a composition of the invention, the additional enzyme may be an alpha
amylase.
In a composition of the invention, the additional enzyme may be a copper-
dependent
lytic polysaccharide monooxygenase (GH61).
A lipolytic enzyme, also referred to herein as a lipase, is an enzyme that
hydrolyses
triacylglycerol and/or galactolipid and/or phospholipids. The specificity of
the lipase can be
shown through in vitro assay making use of appropriate substrate, for example
triacylglycerol
lipid, phosphatidylcholine and digalactosyldiglyceride, or preferably through
analysis of the
reactions products that are generated in the dough during mixing and
fermentation.
The triacyl glycerol lipase may be a fungal lipase, preferably from Rhizopus,
Aspergillus,
Candida, Penicillum, Thermomyces, or Rhizomucor. In an embodiment the triacyl
glycerol lipase
is from Rhyzopus, in a further embodiment a triacyl glycerol lipase from
Rhyzopus oryzae is
used. Optionally a combination of two or more triacyl glycerol lipases may be
used
In a composition of the invention, the additional enzyme may be a
phospholipase.
In a composition of the invention, the additional enzyme may be a
galactolipase.
In a composition of the invention, the additional enzyme may be an enzyme
having both
phospholipase and galactolipase activity.
Typically, a composition of the invention comprises a compound with which a
polypeptide of the invention may be formulated. An excipient as used herein is
an inactive
substance formulated alongside with a polypeptide as disclosed herein, for
instance sucrose or
lactose, glycerol, sorbitol or sodium chloride. A composition comprising a
polypeptide as
disclosed herein may be a liquid composition or a solid composition. A liquid
composition usually
comprises water. When formulated as a liquid composition, the composition
usually comprises
components that lower the water activity, such as glycerol, sorbitol or sodium
chloride (NaCI). A
solid composition comprising a polypeptide as disclosed herein may comprise a
granulate
comprising the enzyme or the composition comprises an encapsulated polypeptide
in liquid
matrices like liposomes or gels like alginate or carrageenans. There are many
techniques known
in the art to encapsulate or granulate a polypeptide or enzyme (see for
instance G.M.H.
Meesters, "Encapsulation of Enzymes and Peptides", Chapter 9, in N.J. Zuidam
and V.A.
Nedovid (eds.) "Encapsulation Technologies for Active Food Ingredients and
food processing"
2010).
A composition of the invention may also comprise a carrier comprising a
polypeptide of
the invention. A polypeptide as disclosed herein may be bound or immobilized
to a carrier by
known technologies in the art.
A polypeptide or composition of the invention may be provided in a liquid
form, to allow
easy dispersion on the surface of the product, but dry powdered forms are also
possible.
Irrespective of the formulation of the polypeptide or composition, any
additives and stabilizers
known to be useful in the art to improve and/or maintain the enzyme's activity
can be applied.
When the polypeptide or composition is contained in a liquid form, it may be
applied to a food
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product or an intermediate form of a food product by any conceivable method,
for instance by
soaking or spraying.
The present invention also relates to a process for preparing a composition
comprising a
polypeptide of the invention, which may comprise spray-drying a fermentation
medium
comprising the polypeptide, or granulating, or encapsulating a polypeptide of
the invention, and
preparing the composition.
A polypeptide according to the invention or a composition of the invention
(comprising a
said polypeptide) may be used in the production of a food product. That is to
say, the invention
provides use of a polypeptide of the invention, a polypeptide obtainable by a
process of the
invention for the preparation of a polypeptide or a composition of the
invention in the production
of a food product.
The term "food product" is defined to include both food stuffs for human
consumption
and food stuffs for animal consumption. Hence the term "food product" should
be taken to mean
"food, pet food or feed" throughout this document. An example of a food
product is a baked
product.
Thus, a polypeptide according to the invention or a composition of the
invention may be
used to reduce the amount of acrylamide formed in a thermally processed food
product based on
an asparagine-containing raw material. That is to say, the invention provides
use of a
polypeptide of the invention, a polypeptide obtainable by a process of the
invention for the
preparation of a polypeptide or a composition of the invention to reduce the
amount of
acrylamide formed in a thermally processed food product based on an asparagine-
containing raw
material.
A polypeptide or composition of the invention may, for example, be used in a
process for
the production of a food product involving at least one heating step,
comprising adding one or
more asparaginase enzymes to an intermediate form of said food product in said
production
process whereby the enzyme is added prior to or during said heating step in an
amount that is
effective in reducing the level of asparaginase that is present in said
intermediate form of said
food product.
That is to say, the invention provides a process for the production of a food
product
involving at least one heating step, which process comprises adding a
polypeptide of the
invention, a polypeptide obtainable by a process of the invention for the
preparation of a
polypeptide or a composition of the invention to an intermediate form of said
food product in said
production process, wherein the enzyme is added prior to or during said
heating step in an
amount that is effective in reducing the level of asparagine that is present
in said intermediate
form of said food product.
An amount that is effective in reducing the level of asparagine that is
present in said
intermediate form of said food product, also referred to as effective amount,
includes an amount
of asparaginase (in the form of a polypeptide or composition of the invention)
of from about 0.1
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to about 100 ASPU/g dry ingredient, more preferably from about 0.1 to 50
ASPU/g dry ingredient
or from about 0.1 to 25 ASPU/g dry ingredient.
In an aspect, the asparginase is applied as an aqueous solution wherein the
asparaginase is present in an amount of from about 0.1 to about 100 ASPU/g dry
ingredient,
such as from about 0.1 to about 50 ASPU/g dry ingredient, for example from
about 0.1 to about
25 ASPU/g dry ingredient.
An amount that is effective in reducing the level of asparagine that is
present in said
intermediate form of said food product, also referred to as effective amount,
may be determined
in terms of the amount of asparaginase protein added (i.e. amount of protein/g
ingredient. The
amount to be added may depend on the specific activity of the asparaginase and
may readily be
determined by the person skilled in the art.
In the context of the present invention, 1 ASPU is defined as the amount of
asparaginase that liberates one micromole of ammonia per minute from L-
asparagine measured
under the conditions of the assay as specified in the Examples. Asparaginase
activity (in ASPU
units) may be determined by measuring the rate of hydrolysis of L-asparagine
to L-aspartic acid
and ammonia. The liberated ammonia subsequently reacts with phenol
nitroprusside and alkaline
hypochlorite resulting in a blue color (Berthelot reaction). The activity of
asparaginase may be
determined by measuring absorbance of the reaction mixture at 630 nm.
The heating step in the process of the invention is one in which acrylamide
may be
formed should the intermediate form of the food product comprise asparagine.
Such a heating
step may be a frying or a baking step, for example.
Typically, the temperature of such a heating step is such that an asparaginase
of the
invention will be added to an intermediate form of the food product prior to
the heating step.
However, it may be possible to add the asparaginase to the intermediate form
of the food during
the heating step.
Food production processes may though have additional heating steps which take
place
prior to the heating step of a process of the invention in which acrylamide
may be formed (in the
event that asparagine is present). For example, in the production of French
fries, blanching is a
common unit operation which is typically in hot water (65-80 C) for from 10 to
30 minutes. This
is disadvantageous for treatment with asparaginase which is not thermophilic
as the high
temperature may inactivate the enzyme. Accordingly, an additional unit
operation process at a
lower temperature would need to be used. However, availability of a
thermophilic asparaginase,
which has a high temperature optimum and is thermostable may allow blanching
to be combined
with enzyme treatment. Accordingly, an asparaginase of the present invention
may permit food
production processes with fewer unit operations.
A process of the invention is disclosed in W004/030468 which process and all
its
preferences are herein incorporated by reference. Also in W004/026043 suitable
processes are
described wherein the asparaginase according to the invention could be used.
The processes
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disclosed in W004/026043 and all preferences disclosed are herein incorporated
by reference.
An intermediate form of the food product is defined herein as any form that
occurs
during the production process prior to or during obtaining the final form of
the food product. The
intermediate form may comprise the individual raw materials used and/or
mixture thereof and/or
5 mixtures with additives and/or processing aids, or subsequently processed
form thereof. For
example, for the food product bread, the intermediate forms comprise for
example wheat, wheat
flour, the initial mixture thereof with other bread ingredients such as for
example water, salt,
yeast and bread improving compositions, the mixed dough, the kneaded dough,
the leavened
dough and the partially baked dough. For example for several potato-based
products, dehydrated
10 potato flakes or granules are intermediate products, and corn masa is an
intermediate product for
tortilla chips.
The food product may be made from at least one raw material that is of plant
origin,
for example potato, tobacco, coffee, cocoa, rice, cereal, for example wheat,
rye corn, maize,
barley, groats, buckwheat and oat. Wheat is here and hereafter intended to
encompass all known
15 species of the Triticum genus, for example aestivum, durum and/or
spelta. Also food products
made from more than one raw material or intermediate are included in the scope
of this
invention, for example food products comprising both wheat (flour and/or
starch) and potato.
Examples of food products in which the process according the invention can be
suitable for are any flour based products - for example bread, pastry, cake,
pretzels, bagels,
20 Dutch honey cake, cookies, gingerbread, gingercake and crispbread -, and
any potato-based
products - for example French fries, pommes frites, potato chips, croquettes.
The term food product includes without limitation a potato product, potato
flakes, potato
chips, potato crisps, French fries, hash browns, roast potatoes, breakfast
cereals, infant cereals,
crisp bread, muesli, biscuits, crackers, snack products, tortilla chips, corn
chips, roasted nuts,
25 rice crackers, Japanese "senbei", wafers, waffles, hot cakes, pancakes,
pretzels, salt or salty
sticks, potato pellets and extruded potato snacks.
The term food product also includes without limitation cereal based food
products, such
as breakfast cereals and cereal snacks.
Potato flakes may be manufactured by drying cooked potato mash with drum
drying. A
30 polypeptide or composition of the invention may be be added at the mix
and transfer stage.
Process steps and times are typically as follows: potatoes are cooked
(optionally in multiple
stages) for from 15 minutes to 1 hour; and mixed and transferred to a drum
dryer for from 5 to
30 minutes.
In typical industrial production of French fries, potatoes are initially
washed, sorted,
steam peeled and cut. Following cutting, the potato sticks may be blanched for
from 5 to 60
minutes, optionally in 2 to 3 sequential steps. Blanching may be carried out
to inactivate any
endogenous enzymes in the potato, to partially cook the potato and/or to leach
out reducing
sugars to prevent excessive browning of the final product. Following
blanching, the potato strips
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may quickly be dipped, e.g. for from 20 to 180 seconds, in a warm phosphate
salt solution, e.g.,
a warm solution of sodium acid pyrophosphate (SAPP), to prevent greying of the
final product.
The dip may optionally be combined with a dip in glucose for reaching the
desired colour. The
potatoes may be dried in a drier with hot circulating air at from 45 to 95 C
for from 5 to 20
minutes giving a weight loss of form 5 to 25%. Finally, the potato sticks may
be parfried before
being quick-frozen and packed. Final frying is then carried out at a food
service or by a
consumer.
Breakfast cereals are a diverse range of products that may be processed in a
number of
ways. A polypeptide or composition of the invention may be used in the
production of, for
example, pressure cooked (often also referred to as batch processed) breakfast
cereals,
extruded breakfast cereals or shredded breakfast cereals.
Typically, the ingredients are mixed and then cooked within a pressure cooker.
Depending on the scale of manufacture, the time taken to raise the temperature
of the
ingredients to above 80 C is typically in the range of 10 to 60 minutes.
According to the invention, there is provided a dough which comprises a
polypeptide of
the invention, a polypeptide obtainable by a process of the invention for the
preparation of a
polypeptide or a composition of the invention.
The invention also provides a method for the preparation of such a dough. Such
a
method may comprise the step of:
- combining:
(i) a polypeptide of the invention, a polypeptide obtainable by a process
of the invention for the preparation of a polypeptide or a composition of the
invention; and (ii) at least one dough ingredient.
A method for the preparation of a baked product may thus comprise baking or
frying a
dough of the invention.
Dough is usually made using basic dough ingredients including (cereal) flour,
such as wheat
flour or rice flour, water and optionally salt. For leavened products,
primarily baker's yeast is used next
to chemical leavening systems such as a combination of an acid (generating
compound) and
bicarbonate.
The term dough herein includes a batter. A batter is a semi-liquid mixture,
being thin
enough to drop or pour from a spoon, of one or more flours combined with
liquids such as water,
milk or eggs used to prepare various foods, including cake and wafers.
The dough may be made using a mix including a cake mix, a biscuit mix, a
brownie mix,
a bread mix, a pancake mix and a crepe mix.
The term dough includes frozen dough, which may also be referred to as
refrigerated
dough. There are different types of frozen dough; that which is frozen before
proofing and that
which is frozen after a partial or complete proofing stage. The frozen dough
is typically used for
manufacturing baked products including without limitation biscuits, breads,
bread sticks and
croissants.
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The term baked product includes, bread containing from 2 to 30 wt% sugar,
fruit
containing bread, breakfast cereals, cereal bars, eggless cake, soft rolls and
gluten-free bread.
Gluten free bread herein and herein after is bread than contains at most 20
ppm gluten. Several
grains and starch sources are considered acceptable for a gluten-free diet.
Frequently used
sources are potatoes, rice and tapioca (derived from cassava). Baked product
includes without
limitation tin bread, loaves of bread, twists, buns, such as hamburger buns or
steamed buns,
chapati, rusk, dried steam bun slice , bread crumb, matzos, focaccia, melba
toast, zwieback,
croutons, soft pretzels, soft and hard bread, bread sticks, yeast leavened and
chemically-
leavened bread, laminated dough products such as Danish pastry, croissants or
puff pastry
products, muffins, Danish bagels, confectionery coatings, crackers, wafers,
pizza crusts, tortillas,
pasta products, crepes, waffles and par-baked products. An example of a par-
baked product
includes, without limitation, partially baked bread that is completed at point
of sale or
consumption with a short second baking process.
The bread may be white or brown pan bread and may for example be manufactured
using a so called American style Sponge and Dough method or an American style
Direct method.
The bread may be a floor bread, i.e. a bread which is baked on an oven plate.
The term tortilla herein includes corn tortilla and wheat tortilla. A corn
tortilla is a type of
thin, flat bread, usually unleavened made from finely ground maize (usually
called "corn" in the
United States). A flour tortilla is a type of thin, flat bread, usually
unleavened, made from finely
ground wheat flour. The term tortilla further includes a similar bread from
South America called
arepa, though arepas are typically much thicker than tortillas. The term
tortilla further includes a
laobing, a pizza-shaped thick "pancake" from China and an Indian Roti, which
is made
essentially from wheat flour. A tortilla usually has a round or oval shape and
may vary in
diameter from about 6 to over 30 cm.
The baked product may be a crusty bread having a crispy crust and a soft core.
Examples of crusty bread include, but are not limited to, baguette, flute,
pistolet, ciabatta, batard,
Kaiser roll, hard roll, panini and maraguetta.
Raw materials as cited above are known to contain substantial amounts of
asparagine
which is involved in the formation of acrylamide during the heating step of
the production
process. Alternatively, the asparagine may originate from other sources than
the raw materials
e.g. from protein hydrolysates, such as yeast extracts, soy hydrolysate,
casein hydrolysate and
the like, which are used as an additive in the food production process. A
preferred production
process is the baking of bread and other baked products from wheat flour
and/or flours from
other cereal origin. Another preferred production process is the deep-frying
of potato chips from
potato slices.
Preferred heating steps are those at which at least a part of the intermediate
food
product, e.g. the surface of the food product, is exposed to temperatures at
which the formation
of acrylamide is promoted, e.g. 110 C or higher, 120 C or higher temperatures.
The heating step
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in the process according to the invention may be carried out in ovens, for
instance at a
temperature between 180-220 C, such as for the baking of bread and other
bakery products, or
in oil such as the frying of potato chips, for example at 160-190 C.
Following application of the enzyme to a product, a certain processing time is
required to
allow the enzyme to act before the food is heated, because a substantial
reduction of the amino
acids capable of generating acrylamide must be obtained, and because the
heating step will
generally inactivate the enzyme. Generally the processing time will take at
most 2 hours,
preferably at most 1.5 hour and most preferably at most 1 hour. In general
processing times of at
least 2 minutes can be reached. Preferably, the processing time is between 2
minutes and 2
hours, more preferably between 5 minutes and 1.5 hours, and most preferably
between 10
minutes and 1 hour.
It is to be understood that the more enzyme is added a shorter processing time
can
suffice for the enzyme to reach the desired effect and vice versa.
In another aspect, the invention provides food products obtainable by the
process of the
invention as described herein or by the use of a polypeptide of the invention
to produce food
products. These food products may be characterized by significantly reduced
acrylamide levels
in comparison with the food products obtainable by production processes that
do not comprise
adding a polypeptide of the invention in an amount that is effective in
reducing the level of amino
acids which are involved in the formation of acrylamide during a heating step.
The process
according to the invention may be used to obtain, for example, a decrease of
the acrylamide
content of the produced food product by preferably more than 50%, more
preferably more than
20%, even more preferably 10% and most preferably more than 5% as compared to
a food
product obtained using the same process in which a polypeptide of the
invention is not used.
An additional application for a polypeptide according to the invention is in
the therapy
of tumours. The metabolism of tumour cells requires L-asparagine, which can
quickly be
degraded by asparaginases. The asparaginase according to the invention can
also be used as an
adjunct in treatment of some human leukaemia. Administration of asparaginase
in experimental
animals and humans leads to regression of certain lymphomas and leukemia.
Therefore, the
invention provides a polypeptide of the invention, a polypeptide obtainable by
a process of the
invention for the preparation of a polypeptide or a composition of the
invention for use in a
method of treatment of the human or animal body by therapy, for example in the
treatment of
tumors, such as in the treatment of lymphomas or leukaemia in animals or
humans.
Standard genetic techniques, such as overexpression of enzymes in the host
cells,
genetic modification of host cells, or hybridisation techniques, are known
methods in the art,
such as described in Sambrook and Russel (2001) "Molecular Cloning: A
Laboratory Manual (3rd
edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
or F. Ausubel et
al, eds., "Current protocols in molecular biology", Green Publishing and Wiley
Interscience, New
York (1987). Methods for transformation, genetic modification etc of fungal
host cells are known
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from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671,
W090/14423, EP-
A-0481008, EP-A-0635 574 and US 6,265,186.
Embodiments of the invention:
1. A polypeptide having asparaginase activity selected from the group
consisting of:
a polypeptide having an amino acid sequence comprising the mature
polypeptide sequence of SEQ ID NO: 1;
a polypeptide comprising an amino acid sequence that has at least 50%
sequence identity with the mature polypeptide sequence of SEQ ID NO: 1;
a polypeptide encoded by a nucleic acid comprising a sequence that hybridizes
under medium stringency conditions to the complementary strand of the mature
polypeptide encoding sequence of SEQ ID NO: 2; and
iv. a polypeptide comprising an amino acid sequence encoded by a
nucleic acid
that has at least 50% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 2.
2. A polypeptide that is an isolated, substantially pure, pure,
recombinant, synthetic or
variant polypeptide of the polypeptide of embodiment 1.
3. A polypeptide according to embodiment 1 or 2 which is derivable from
Thermophilus
africanus.
4. A composition comprising a polypeptide according to any one of
embodiments 1 to 3.
5. A composition according to embodiment 4, comprising a carrier, an
excipient, or an
auxiliary enzyme and/or a dough ingredient.
6. A nucleic acid encoding an asparaginase which comprises a sequence that
has at least
50% sequence identity to the mature polypeptide encoding sequence of SEQ ID
NO: 2.
7. A nucleic acid that is an isolated, substantially pure, pure,
recombinant, synthetic or
variant nucleic acid of a nucleic acid of embodiment 6
8. An expression vector comprising a nucleic acid according to embodiment 6
or 7
operably linked to one or more control sequences that direct expression of the
polypeptide in a host cell.
9. A recombinant host cell comprising a nucleic acid according to
embodiment 6 or 7 or an
expression vector according to embodiment 8.
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10. A method for the preparation of a polypeptide according to embodiments
1 to 3, which
method comprises:
cultivating a host cell according to embodiment 9 in a suitable fermentation
5 medium under conditions that allow for expression of the polypeptide;
and, optionally,
recovering the polypeptide.
11. Use of a polypeptide according to any one of embodiments 1 to 3, a
polypeptide
obtainable by a process according to embodiment 10 or a composition according
to
10 embodiment 4 or 5 in the production of a food product.
12. Use of a polypeptide according to any one of embodiments 1 to 3, a
polypeptide
obtainable by a process according to embodiment 10 or a composition according
to
embodiment 4 or 5 to reduce the amount of acrylamide formed in a thermally
processed
15 food product based on an asparagine-containing raw material.
13. A process for the production of a food product involving at least one
heating step, which
process comprises adding a polypeptide according to any one of embodiments 1
to 3, a
polypeptide obtainable by a process according to embodiment 10 or a
composition
20 according to embodiment 4 or 5 to an intermediate form of said food
product in said
production process, wherein the enzyme is added prior to or during said
heating step in
an amount that is effective in reducing the level of asparagine that is
present in said
intermediate form of said food product.
25 14. A food product obtainable by the process according to embodiment
13 or by the use
according to embodiment 11 or 12.
15. A dough comprising a polypeptide according to any one of embodiments 1
to 3, a
polypeptide obtainable by a process according to embodiment 10 or a
composition
30 according to embodiment 4 or 5.
16. A method for the preparation of a dough, which method comprises
combining: a
polypeptide according to any one of embodiments 1 to 3, a polypeptide
obtainable by a
process according to embodiment 10 or a composition according to embodiment 4
or 5;
35 and at least one dough ingredient.
17. A method for the preparation of a baked product, which method comprises
the step of
baking or frying a dough according to claim 15 or a dough obtainable by a
method
according to claim 16.
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18. A polypeptide according to any one of embodiments 1 to 3, a
polypeptide obtainable by a
process according to embodiment 10 or a composition according to embodiment 4
or 5
for use in a method of treatment of the human or animal body by therapy.
A reference herein to a patent document or other matter which is given as
prior art is not
to be taken as an admission that that document or matter was known or that the
information it
contains was part of the common general knowledge as at the priority date of
any of the claims.
The disclosure of each reference set forth herein is incorporated herein by
reference in its
entirety.
The present invention is further illustrated by the following Examples:
EXAMPLES
Materials and Methods
General
Standard genetic techniques, such as overexpression of enzymes in the host
cells,
genetic modification of host cells, or hybridisation techniques, are known
methods in the art,
such as described in Sambrook and Russel (2001) "Molecular Cloning: A
Laboratory Manual (3rd
edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
or F. Ausubel et
al, eds., "Current protocols in molecular biology", Green Publishing and Wiley
Interscience, New
York (1987). Methods for transformation, genetic modification etc of fungal
host cells are known
from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671,
W090/14423, EP-
A-0481008, EP-A-0635 574 and US 6,265,186.
Water is Milli-Q water where nothing else is specified.
Asparaginase activity assay
Asparaginases catalyze the hydrolysis of L-asparagine to aspartic acid and
ammonia. To
determine asparaginase activity, first an enzymatic reaction is performed by
incubating the
enzyme with L-asparagine. After stopping the enzymatic reaction, the released
ammonia can be
detected in a second non-enzymatic reaction where the ammonium formed combines
with
phenol to obtain the metachromatic dye indophenol, which can be accurately
quantified by
spectrophotometric analysis.
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Enzyme and standard incubation
In a 96 well PCR microtiter plate (MTP), 20 pL appropriately diluted
asparaginase or
ammonia standard was added to 100 pL L-asparagine solution and after sealing
of the plate
incubated in a PCR machine at 50, 60, 70, 80, 90 or 100 C for 15 min.
Reactions were stopped
by transferring the MTP on ice. After centrifugation (5 min at 3000 rpm) to
remove condensation,
20 pL of the reaction mixtures was transferred to a new MTP containing 180 pL
water per well
and mixed by pipetting.
Ammonium sulfate was used as standard in the range of 0 to 3.9 g/I. L-
Asparagine
solution: L-Asparagine (10 g/L: >99% pure asparaginase) was dissolved in assay
buffer (100 mM
of MOPS at pH 7 or a mixed buffer system consisting of 50 mM citrate, 50 mM
KH2PO4 and 40
mM sodiumpyrophosphate at pH 5-10 (pH was adjusted with HCI or NaOH)).
The amount of sample used in Example 2 is such that the absorbance obtained
after the
indophenol method should not exceed that of the highest calibration point.
Indophenol method to detect ammonia
The formation of ammonia in enzyme reactions was quantified by adding 60 pL of
the
diluted reaction mixture to a new MTP containing per well 60 pL phenol
nitroprusside solution
(Sigma-Aldrich, product number: P6994). After addition of 60 pL alkaline
hypochlorite (Sigma-
Aldrich, product number: A1727) the plates were sealed and incubated for 15
min at 37 C with
shaking (750 rpm) on an Eppendorf thermomixer equipped with an MTP adapter.
The
absorbance was measured spectrophotometrically using a wavelength of 630 nm.
The
absorbance of the standards was plotted against the ammonia concentration in
the standards,
and the standard curve obtained was used to calculate the ammonia produced in
the enzyme
samples. The activity is given as micromole ammonia released per minute per ml
sample
(U/mL).
UPLC-MSMS method to measure asparagine
Potato dough samples (5 g dough in 100 ml 0.1 M HCI) and cereal (wheat) dough
samples (10 g dough in 50 ml 0.1 M HCI) are centrifuged during 10 min at 14000
rpm and 100 pl
of the supernatant is diluted with 100 pl of the internal labelled standard
working solution (2 pg of
L-asparagine-15N2) into an Eppendorf reaction vial and mixed. For the
calibration curve, 100 pl
internal labelled standard working solution is added to a set of calibration
solutions (100 pl; each
solution containing 0.7-7 pg of Asn) in an Eppendorf reaction vial and mixed.
For AccQtag
derivatization, 10 pl of the abovementioned solutions (both sample and
calibration solutions) are
taken to which 70 pl of AccO=Tag ultra reagent 1 (borate buffer) is added and
mixed. To this
mixture, 20p1 of AccO=Tag ultra reagent 2 is added and mixed immediately. This
solution is then
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transferred to an UPLC injection vial. UPLC-MS/MS analysis is performed
according to Ref.1:
Carolina Salazar, Jenny M. Armenta , Diego F. Cortes and Vladimir Shulaev,
Combination of an
AccQ-Tag-Ultra Performance Liquid Chromatographic Method with Tandem Mass
Spectrometry
for the Analysis of Amino Acids,
Methods In Molecular Biology (Clifton, N.J.), 828, 13-28 (2012)
Example 1: Expression of a putative new asparaginase from Thermophilus
africanus in E.
coil
In order to identify an L-asparaginase that function at high temperature, we
tested >35
asparaginase candidates from thermophiles and hyper thermophiles (both
bacteria and archaea).
We first performed a blast search (using two different L-asparaginase sequence
as query) to
retrieve all candidate asparaginase genes from the NCB! database. Our search
retrieved more
than 1000 candidates as "hits", suggesting that the search is comprehensive in
covering all
available L-asparaginase sequences.
Since it is not obvious by sequence alone which candidates would be functional
at the
desired high temperature, we further refined the hits using criteria:
Step 1. We rationally selected for sequences originating from thermophiles and
hyper
thermophiles (primarily bacteria and archaea), after which 130 hits remained.
Step 2. We then specifically selected for L-asparaginase that is not in the
class of
glutaminase-asparaginase (which usually contains an additional protein domain
and thus is larger
in size), by selecting for hits with sequence length between 300 to 400 amino
acids. Protein
alignment was then performed to confirm the identity of putative full length
protein.
A synthetic gene based on the protein sequence of a putative asparaginase from
Thermophilus africanus (SEQ ID NO:1; accession number: ACJ75594) was designed
by
optimizing the gene codon usage for E.coli according to the algorithm of
DNA2.0 (GeneGPS
technology).
For cloning purposes a DNA sequence containing a Ndel site CAT (was introduced
at the
5'- end and a DNA sequence containing a stop codon and a Ascl site
TAACCTGCAGGGGCGCGCC was introduced at the 3' end.
The synthetic DNA encoding the putative asparaginase (SEQ ID NO:2) was cloned
via
the 5'Ndel and 3'Ascl restriction sites into an arabinose inducible E. coli
expression vector,
containing the arabinose inducible promoter PBAD and regulator araC (Guzman J.
Bac. 177:4121
-4130, 1995), a kanamycin resistance gene Km(R) and the origin of replication
ori327 from
pBR322 (Watson, Gene. 70:399-403, 1988). Expression of the cloned gene may
thus be induced
by arabinose. The clones were sequence verified. The sequence of the final
plasmid pAe7 is
shown in Figure 1.
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The E.coli host RV308 (laclq-, su-, AlacX74, gal IS 11::0P308, strA,
http://www.ebi.ac.uk/ena/data/view/ERP005879) with additional deletions in
ampC and araB was
transformed using chemical competent cells (Z-Competent cells, prepared with
the Mix and
GolE.coli transformation kit, Zymo Research, Irvine CA, USA).
Correct transformants were pre cultured in 2xPY + neomycine (0/N). The
preculture
(1/100 vol) was used to inoculate the fermentation in MagicMediaTm E.coli
expression medium
(Thermo Fisher Scientific Inc), + neomycine (24 wells MTP, 3m1 volume,
breathable seal, 550
RPM 80%RH), after 4h growth at 30 C 0.02% arabinose (final concentration) was
used for
induction of the asparaginase gene and incubation was continued at 20 C for
48h. Cell-pellets
were frozen. Cell free extract (CFE) was prepared, incubation for 1 hour at 37
C in lysis buffer
((Tris-HCI 50 mM, DNasel 0,1 mg/ml, lysozyme 2 mg/ml, Mg504 25 pM). Cell
debris was
centrifuged and the CFE was transferred to a clean 96 well MTP covered by a
silicone mat and
stored at -20 C until further characterization.
The asparaginase gene sequence originates from Thermophilus africanus TCF52B
type
strain is strain 0b7 (= DSM 5309).The strain is commercially available via the
Deutsche
Sammlung von Mikroorganism und Zellkulturen GmbH. The type strain was
described by Huber
et al.,1989 (Thermophilus africanus gen. nov., represents a new genus of
thermophilic eubacteria
within the Thermotogales., Syst. Appl. Microbiol. 1232-37).
Example 2: Biochemical characterization of the putative asparaginase
2.1 Temperature dependent activity of Thermophilus africanus asparaginase
The temperature dependent activity of the Thermophilus africanus putative
asparaginase
was determined by performing catalytic activity measurements at selected
temperatures for 15
min at pH 7Ø Samples (CFE from Example 1) were diluted appropriately in 100
mM MOPS
buffer pH 7Ø A 96 well PCR plate containing 100 pL L-asparagine solution (10
g/L) in 100 mM
MOPS buffer pH 7.0 per well, was placed on ice and 20 pL of diluted sample was
added. After
sealing, the plate was incubated in a PCR cycler for 15 min at 50, 60, 70, 80,
90 or 100 C.
Following incubation, the plate was immediately placed on ice to stop the
enzymatic reaction.
After cooling down, the plate was centrifuged (5 min at 3000 rpm) to remove
condensation from
the seal.
Next, the concentration of ammonia in the reactions was determined as follows.
The
reaction mixtures were diluted 10-fold by adding 20 pL to a new MTP containing
180 pL water
per well and mixing by pipetting. 60 pL of the diluted mixture was then added
to a new MTP
containing 60 pL phenol nitroprusside solution per well. Finally, 60 pL
alkaline hypochlorite was
added to each well and the plate was sealed and incubated for 15 min at 37 C.
After cooling to
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ambient temperature, the MTP plate was centrifuged (5 min at 3000 rpm) and the
absorbance
measured at 630 nm.
The relative activity is shown in Figure 2. The Thermophilus africanus
asparaginase
shows maximal catalytic activity at 70 C.
5
2.2 pH dependent activity of Thermophilus africanus asparaginase
The pH dependent activity of Thermophilus africanus asparaginase was
determined by
performing catalytic activity measurements at selected pH values for 15 min at
70 C. Samples
10 (CFE from Example 1) were diluted in water. A 96 well PCR plate
containing 100 pL L-
asparagine solution (10 g/L) in buffer (50 mM citrate, 50mM Na2HPO4 and 40 mM
sodium
pyrophosphate, adjusted to pH 5, 6, 7, 8, 9 and 10) per well, was placed on
ice and 20 pL of
diluted sample was added. After sealing, the plate was incubated in a PCR
cycler for 15 min at
70 C. Following incubation, the plate was immediately placed on ice to stop
the enzymatic
15 reaction. After cooling down, the plate was centrifuged (5 min at 3000
rpm) to remove
condensation from the seal.
Next, the concentration of ammonia in the reactions was determined as follows.
The
reaction mixtures were diluted 10-fold by adding 20 pL to a new MTP containing
180 pL water
per well and mixing by pipetting. 60 pL of the diluted mixture was then added
to a new MTP
20 containing 60 pL phenol nitroprusside solution per well. Finally, 60 pL
alkaline hypochlorite was
added to each well and the plate was sealed and incubated for 15 min at 37 C
with shaking (750
rpm). After cooling to ambient temperature, the MTP plate was centrifuged (5
min at 3000 rpm)
and the absorbance measured at 630 nm.
The relative activity is shown in Figure 3. The Thermophilus africanus
asparaginase
25 shows maximal catalytic activity at pH 10.
Example 3: Use of the asparaginase to reduce asparaginase in a potato mash as
a model
for potato flake manufacture
30 Potato flakes are made by drying hot potato mash. In this Example,
potato flakes are
rehydrated and heated to bring them back to the earlier stage of their
manufacturing.
The asparaginase expressed from E. coli as described in Example 1 is used to
treat the
potato mash suitable for the production of potato flakes.
100g of water is added to 30g potato flakes. This mixture is heated for 45
minutes until a
35 consistent temperature of 90 C is reached. Enzyme is then added by
mixing for 1 minute and
the resulting mixture then held for 15 minutes at 90 C. The enzyme is
inactivated by exactly
weighing in approximately 5g of potato mash into 100m1 of 0.1M HCI.
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Different dosage levels of enzyme, ranging from 1000 Units to 15000 ASPU/ kg
dry
potato are tested. Asparagine levels are determined using UPLC-MSMS analysis
and compared
to the same food product made in the absence of enzyme.
The Thermophilus africanus asparaginase performed well in this test.
Example 4: Use of the asparaginase in a cereal mix suitable for cereal product
manufacturing
The asparaginase expressed from E. coli as described in Example 1 is used in
the
production of a food-product simulating the production of a breakfast cereal
using two different
protocols.
15g of water is added to 35g wholemeal flour and enzyme then added, followed
by
mixing for 4 minutes with a Siemens hand-blender. This mixture is then held
for 35 minutes in a
closed water bath at 90 C. The enzyme was inactivated by exactly weighing in
approximately
lOg of cereal mix into 50m1 of 0.1M HCI.
Different dosage levels of enzyme, ranging from 150 Units to 1500 ASPU / kg
wholemeal flour are tested.
Asparagine levels are determined using UPLC-MSMS analysis and compared to the
same food product made in the absence of enzyme.
The Thermophilus africanus asparaginase performed well in this test.
Example 5: Use of the asparaginase to reduce asparaginase in a potato mash as
a model
for potato flake manufacture
Potato flakes are made by drying hot potato mash. In this Example, potato
flakes are
rehyd rated and heated to bring them back to the earlier stage of their
manufacturing.
The asparaginase expressed from E. coli as described in Example 1 is used to
treat the
potato mash suitable for the production of potato flakes. PreventASe XRTM is a
non heat stable
asparaginase enzyme used as control.
100g of water was added to 30g potato flakes and mixed for 1 minute to reach a
temperature of 54 C +/- 3 C. The mix was further heated from 55 C to 90 C for
14 minutes and
then held for a further 15 minutes from 90 C to a final mix temperature of 94
¨ 95 C.
The enzyme was inactivated by weighing in of 5g potato mash into 100m1 of 0.1M
HCI
and shaken vigorously for two minutes.
Different dosage levels of enzyme, ranging from 1000 Units to 10000 Units per
kg dry
potato were tested. Asparagine levels were determined using UPLC-MSMS analysis
and
compared to the same food product made in the absence of enzyme. The results
are shown in
table 2 below.
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Dosage Amount of
asparagine
Enzyme Amount of
according to asparagine
example 1 % asparagine PreventASe XRTM
asparagine
(ASN g/kg) reduction (ASN g/kg) reduction
0 1,188 0 1,199 0
1000 1,056 11% 1,144 5%
1250 0,528 56% 1,177 2%
5000 0,451 62% 1,144 5%
7500 0,407 66% 1,122 6%
1000 0,275 77% 1,177 2%
Table 2
Example 6: Use of the asparaginase in a cereal mix suitable for cereal product
manufacturing
The asparaginase expressed from E coli in Example 1 was used in the production
of a
food-product simulating the production of a breakfast cereal.
37.5g of water is added to 30g wholemeal flour is mixed for 5 seconds, enzyme
then
added, and mixed for a further 55 seconds This mixture is then heated from
approximately from
51 C to 90 C over 12 minutes. It was then held for a further 15 minutes to
heat the mix from (or
where the temperature rises from?) 90 C to a final mix temperature of 94 ¨ 95
C.
The enzyme was inactivated by weighing 10g of cereal mix into 50m1 of 0.1M HCI
and
shaken vigorously for two minutes.
Different dosage levels of enzyme, ranging from 150 Units to 1500 ASPU / kg
wholemeal flour were tested.
Asparagine levels were determined using UPLC-MSMS analysis and compared to the
same food product made in the absence of enzyme. The results are shown in
table 3 below.
Dosage Amount of
asparagine
Enzyme Amount of
according to asparagine
example 1 %
asparagine PreventASe XRTM % asparagine
(ASN g/kg) reduction (ASN g/kg) reduction
0 0,2508 0 0,2508 0
150 0,0792 69% 0,2563 2%
300 0,2112 17% 0,2453 3%
600 0,1727 42% 0,2422 3%
900 0,1782 30% 0,1518 40%
1500 0,1144 55% 0,2321 8%
Table 3
