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Glucoamylases with N-terminal extensions
FIEDD OF THE INVENTION
The present invention relates to a glucoamylase variant of a
parent glucoamylase, a DNA sequence encoding the variant
glucoamylase and a process using such variant enzyme for
hydrolyzing starch.
More specifically, the present invention relates to a
glucoamylase variant having improved thermostability.
BACKGROL3'ND OF THE INVENTTON
Glucoamylase (1,4-a-D-glucan glucohydrolase, EC 3.2.1.3) is
an enzyme which catalyzes the release of D-glucose from the non-
reducing ends of starch or related oligo- and polysaccharide
molecules. Glucoamylases are produced by several filamentous
fungi and yeasts, with those from Aspergillus being commercially
most important.
Commercially, the glucoamylase enzyme is used to convert corn
starch which is already partially hydrolyzed by an a-amylase to
glucose. The glucose is further converted by glucose isomerase to
a mixture composed almost equally of glucose and fructose. This
mixture, or the mixture further enriched with fructose, is the
commonly used high fructose corn syrup commercialized throughout
the world. This syrup is the world's largest tonnage product
produced by an enzymatic process. The three enzymes involved in
the conversion of starch to fructose are among the most important
industrial enzymes produced.
One of the main problems exist with regard to the commercial
use of glucoamylase in the production of high fructose corn syrup
is the relatively low thermal stability of glucoamylase.
Glucoamylase is not as thermally stable as a-amylase or glucose
isomerase and it is most active and stable at lower pH's than
either a-amylase or glucose isomerase. Accordingly, it must be
used in a separate vessel at a lower temperature and pH.
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SIJI~~RY OF T13E TIV~IEI~iTIOP~
Thus, the object of the present invention is to improve
properties of enzymes with glucoamylase activity, in particular
to improve the thermal stability of such enzymes.
It has surprisingly been found that it is possible to
significantly enhance the thermal stability of an enzyme with
glucoamylase activity by linking a peptide extension to the N
i0 terminal of the enzyme. '
Consequently, in a first aspect the invention relates to a
variant of a parent glucoamylase, which has a peptide extension
at the N-terminus.
In the present context the term "peptide extension°' is
intended to indicate that a stretch of one or more consecutive
amino acid residues has been added to the N-terminal end of the
parent (mature) glucoamylase.
The term "mature glucoamylase " is used in its conventional
meaning, i.e., to indicate the active form of the glucoamylase
resulting after posttranslational and postsecretional processing
(to trim glycosylation and remove N and/or C-terminal sequences,
such as pre- and pro-peptide sequences) by the producer organism
in question. More specifically this means that amino acid
sequences such as the pre- and pro-peptide sequences, if present,
have been removed from the initially translated glucoamylase,
i.e., the unprocessed glucoamylase. A mature glucoamylase
encompassed by the present definition is a glucoamylase cut
(processed) by Tripeptidyl amino peptidase (TPAP) , which cuts
the A. niger glucoamylase (see SEQ ID NO: 1) at position 3, i.e.,
between Leu and Asp.
The term "parent glucoamylase°° is intended to indicate the
glucoamylase to be modified according to the invention. The
parent glucoamylase may be a naturally-occurring (or wild type)
glucoamylase or may be a variant thereof prepared by any suitable
means. For instance, the parent glucoamylase may be a variant of
a naturally-occurring glucoamylase which has been modified by
substitution, deletion or truncation of one or more amino acid
residues or by addition or insertion of one or more amino acid
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residues to the amino acid sequence of a naturally-occurring
glucoamylase, typically in the structural part of the
glucoamylase.
In other aspects the invention relates to a DNA sequence
encoding a glucoamylase variant as defined above, a DNA
construct, a recombinant expression comprising a DNA sequence of
the invention, and a host cell harbouring a DNA sequence of the
invention or a vector of the invention.
The glucoamylase variant of the invention may conveniently be
used in a process for converting starch and accordingly, in yet
other aspects the invention relates to a process for converting
starch or partially hydrolyzed starch into syrup containing
dextrose, said process including the step saccharifying starch
hydrolyzate in the presence of a glucoamylase variant of the
invention.
In final aspects, the invention provides a method for
improving the thermostability of parent glucoamylase by making an
extension at the N-terminus.
The inventors of the present invention have provided a number
of improved variants of a parent glucoamylase with improved
thermostability. The improved thermal stability is obtained by
linking a peptide extension to a parent glucoamylase. This will
be described in details below.
DET~~LEO DESCRaP~iorr of ~x~ i:rr~rrrzo~
Pep~~ae eX~en~~o~
As stated above it has surprisingly been found that a
glucoamylase variant, in particular with improved
thermostability, may be achieved when an appropriate peptide
extension can be found at the N-terminus of the parent
glucoamylase. The present invention is based on this finding.
In the context of the present invention " . . can be found at
the N-terminal.." means that the mature glucoamylase has a
peptide extension at the N-terminal. In one embodiment the
peptide extension is native to the parent glucoamylase, i.e.,
before posttranslational processing to remove the pro- and/or
pre-sequence. Thus, the peptide extension may be the pre- and/or
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pro-sequence of the unprocessed parent glucoamylase, which is
normally removed or cut off after expression and
posttranslational processing.
In another embodiment the extension is a peptide at the «T
terminal identical to the peptide sequence normally being cut of
by the donor cell during processing, e.g., the pre- and/or pro
sequence. In most cases the peptide extension is different from
the pre- and/or pro-sequence. This will be described further
below.
In the case of the extension is linked to the LV-terminal it
may be done by means of any well-known protein engineering
methods in the art.
The term °'a glucoamylase variant with improved
thermostability " means in the context of the present invention a
glucoamylase variant, which has a higher T°" (half-time) or
residual enzymatic activity after a fix incubation period than
the corresponding parent glucoamylase. The determination of
thermostability, e.g.g T3~ and residual activity, is described
below in the Materials and Method section.
The term " an appropriate peptide extension " is used to
indicate that the peptide extension to be used is one, which is
capable of effecting an improved thermostability as defined
above. The '°appropriateness " of the peptide extension may be
checked by a comparative analysis of the thermostability of a
modified glucoamylase variant to which the peptide extension has
been linked and of the corresponding parent glucoamylase,
respectively. The thermostability may, e.g., be determined by any
suitable technique such as the thermostability assay described in
the present application.
It is presently believed that the capability of the peptide
extension of providing the desired effect such as improved
thermostability depends on, e.g., the identity of the parent
glucoamylase to be modified, the structure (including length) of
the peptide extension, the impact of the peptide extension on the
structure of the entire glucoamylase variant enzyme, the nature
or functionality of amino acid residues of the peptide extension,
etc. A prerequisite for the peptide extension being capable of
providing the desired effect is, of course, that the glucoamylase
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variant containing the peptide extension is expressible in a
suitable host organism. The following general considerations may
be of relevance for the design of a suitable peptide extension:
,Pnc~-1, ~f pride extension: It has been found that peptide
5 extensions containing varying numbers of amino acid residues are
capable of providing the desired effect and thus, it is not
possible to specify an exact number of amino acid residues to be
present in the peptide extension to be used in accordance with
the present invention. It is contemplated that the upper limit of
the number of amino acid residues is determined, inter alia, on
the impact of the peptide extension on the expression, the
structure and/or the activity of the resulting modified
glucoamylase variant.
The peptide extension may thus comprise 1-100 amino acid
residues, preferably 1-SO amino acid residues, more preferably 1
and even more preferably 1-10 amino acid residues.
ah;l;r.=: The peptide extension should preferably be chosen
so as to provide a glucoamylase variant with an acceptable
stability (e. g., structural stability and/or expression
20 stability) or so as to not significantly reduce the structural
stability of the glucoamylase variant. Although many peptide
extensions are not believed to confer any substantial structural
instability to the resulting glucoamylase variant, it may in
certain instances and with certain parent glucoamylases be
relevant to choose a peptide extension, which in itself can
confer a structural stability to the modified glucoamylase
enzyme. For instance, the peptide extension can increase the
number of interactions and/or be covalently bound by adding
cysteine bridges to from the N-terminal extension to the N-
terminal residues as discussed below.
Nat-yr~ of amino a .; d r .~; d ~ . of t-h y t;_de extens; on
To obtain an improved interaction between the N-terminal residues
and the N-terminal extension, the residues should preferably come
from residues with low preference for a-helix making, and thus be
used in the context of the present invention. This can be
rationalised by the fact that if the N-terminal a-helix is
prolonged N-terminally it would stick out of the structure with
no contact to the N-terminal residues. A peptide extension
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according to the invention comprises an improved stability by
improving the contact of the N-terminal residues to the N-
terminal extension. Within a giving N-terminal extension the main
part of residues must come from a group of non-helix makers. It
is contemplated by using residues having ~,-helix propensities in
N-terminal of the helix, and/or in the middle of the helix,
and/or C-terminal part of the helix, lower or equal to one, the
improvement of contact between N-terminal residues and the N-
terminal extension will be optimal. Residues having propensities
lower than one in the N-terminal part of the alpha-helix are of
special interest as the extension are placed in the N-terminal
part of the natural a-helix in the glucoamylase. Residues
comprising M (Methionine), K (Lysine), H (Histidine), v (Valine),
I (Isoleucine), Y (Tyrosine), C (Cysteine), F (Phenylalanine), T
(Threonine), G (Glycine), N (Asparagine), P (Proline), S (Serine)
and D (Aspartic acid) can be used in the present invention as
non-«-helix makers.
In the context of the invention '°N-terminal residues " mean
the residues around the N-terminal residue, ~.e., in a sphere of
18, 12 and/or B ~ from the central of the N-terminal residue, and
which is not a part of the N-terminal extension. More preferred,
the extensions are within 10 A but on the surface of the enzyme
defined as the residues having a positive number in accessibility
using the Connelly water accessible surface program ((version
oct. 1988), reference W. Kabsch and C. Sander, Hiopolymers 22
(1983) pp. 257-7-2637.))
" Non-helix makers" are here defined by the data obtained
from table 6.5 in ((Proteins: Creighton T.E. (1993)) where
different propensities are described for the different amino acid
residues.
Alternatively, an improved structural stability may be
provided by introduction of cysteine bridges in the glucoamylases
of the invention. F'or instance, a cysteine bridge between the
peptide extension and the mature part of the glucoamylase may be
established if at least one of the amino acid residues of the
peptide extension is a cysteine residue which is located so as to
be able to form a covalent binding to a cysteine residue in the
mature part of the glucoamyalse variant. The positive effect of
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introducing a cysteine bridge is illustrated in Example 3. if no
suitable cysteine is present in the mature glucoamylase, a
cysteine may be inserted at a suitable location of said parent
glucoamylase, conveniently by replacing an amino acid of the
parent glucoamylase, which is considered unimportant for the
activity.
Generally, the amino acid sequence of a peptide extension
comprising a cysteine residue in the present invention can be
referred to as:
X-C- (x) n,
wherein x independently represents one amino acid, preferably of
the above mentioned non-a-helix makers, even more preferably with
short side chains.
Between the carboxy-terminal side of the Cys and processed,
naturally occurring N-terminal, there can be any number (n) of X
residues larger or equal to 5, preferably between 5 and 100, even
more preferably between 5 and 10, even more preferably 5.
Examples are:
ACGPSTS (SEQ ID NO: 25)
ACPGTST (SEQ ID N0: 26)
ACGTGTS (SEQ ID N0: 27)
ACTGSTG (SEQ ID N0: 28)
ACGPSTSG (SEQ ID N0: 29)
ACPGTSTG (SEQ ID NO: 30)
ACGTGTSS (SEQ ID NO: 31)
ACTGSTGT {SEQ ID NO: 32)
The native pro-peptide of a glucoamylase (e. g., the .~. niger
G1 or G2 AMG) is cleaved of by a kex2-like protease (dibasic
protease). Thus, kex2 proteases are proteases capable of
cleaving kex2 or kex2-like sites. Kex2 sites (see, e.g., Methods
in Enzymology Vol 185, ed. D. Goeddel, Academic Press Inc.
(1990), San Diego, CA, "Gene Expression Technology") and kex2
like sites are di-basic recognition sites (i.e., cleavage sites)
found between the pro-peptide encoding region and the mature
region of some proteins.
Mutating this cleavage site may leave the N-terminal pro-
peptide intact.
Examples:
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NVIPPR (SEQ ID NO: 33)
NPPIRP (SEQ ID N0: 34)
NVIPRP (SEQ ID N0: 35)
Another possibility is to delete or inactivate the kex2-like
proteases encoding gene in the host chosen to express the
glucoamylase gene. This may also leave the N-terminal peptide
extension intact.
Other genes encoding proteases involved in N-terminal
processing such as a tripeptidyl aminopeptidase encoding gene
might also be deleted or inactivated in the host of interest for
expression.
N-terminal residues for cysteine variants are defined as the
residues around the N-terminal residue, i.e., in a sphere of 18,
12 and /or 8 A from the central of the N-terminal residue, and
which is not a part of the N-terminal extension. Generally, the
amino acid sequence for an extension comprising a cysteine
residue in the present invention can be referred to as:
x-C-x-x-x-x-x, wherein x independently represents one of the
above mentioned non-a-helix makers.
In a specific embodiment, the glucoamylase variant comprises
peptide extension, which is capable of forming a covalent binding
to the mature part of the parent glucoamylase. In another
specific embodiment, the glucoamylase variant comprises one or
more cysteine residue in the peptide extension and a cysteine
residue in the mature part of the parent glucoamylase in such a
manner that said cysteine residues together form a cysteine
bridge. In yet another specific embodiment, the cysteine residue
in the mature part of the parent glucoamylase has been inserted
or has substituted an amino acid residue of the parent
glucoamylase. _In an most preferred specific embodiment, the
aspartic acid residue at a position corresponding to position 375
or the glutamic acid at a position corresponding to position 299
or the serine residue at a position corresponding to position 431
or the alanine residue at a position corresponding to position
471 or the alanine residue at a position corresponding to
position 479 or the threonine residue at a position corresponding
to position 480 or the proline residue at a position
corresponding to position 481 or the serine residue at a position
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corresponding to position 8, has been substituted with a cysteine
residue in the amino acid sequence of Aspergillus a~.iger Gl
glucoamylase.
Specifically, the peptide extension linked to the parent
glucoamylase may advantageously be one of the following
extensions:
Asn-Val-Ile-Ser-Arg-Arg(NVISRR), or
Asn-Val-Ile-Pro-Lys-Arg(NVIPKR), or
Ala-Ser-Pro-Pro-Ser-Thr-Ser(ASPPSTS), or
Ala-Cys-Pro-Pro-Ser-Thr-Ser(ACPPSTS), or
Pro-Cys-Ser-Ala-Gly-Glu(PCSAGE), or
Pro-Leu-Ala-Leu-Ser-Asp(PLALSD), or
Leu-Gly-Val-Thr-Gly-Glu(LGVTGE), or
Ala-Gly-Pro-Leu-Pro-Ser-Glu(AGPLPSE), or
Leu-Gly-Pro-Asp(LGPD), or
Ile-Phe-Glu-Leu-Thr-Pro-Arg(IFELTPR), or
Ile-Ser-Asn (IShT) , or
Met-Asn (M1V) .
In the present context, a tripeptidyl aminopeptidase (TPAP) is
intended to indicate an aminopeptidase which cleaves tripeptides
from the N-terminus of a peptide or protein sequence, such as an
extended amino acid sequence found in a prohormone or proenzyme.
The tripeptidyl aminopeptidase (TPAP) has in some cases been
found to lead to a reduced stability when cleaving tripeptide
fragments from unsubstituted N-termini of peptides,
oligonucleutides, or proteins. More specific, the tripeptidyl
aminopeptida-se cleavage of N-termini reduces the stability of
glucoamylase enzymes. Accordingly, the invention also relates to
a variant of a parent glucoamylase, wherein the peptide extension
is capable of preventing a tripeptidyl aminopeptidase (TPAP)
cleavage of the glucoamylase enzyme.
Methods of linking a peptide extension to a parent glucoaanylase
Although a variant of the invention may be obtained by adding
(fusing or inserting) a synthetically produced peptide extension
into the parent glucoamylase enzyme in question, it is presently
preferred that the glucoamylase variant of the invention is
prepared by i) modifying the nucleotide, preferably DNA, sequence
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encoding the parent glucoamylase so as to encode the desired
peptide extension applied to the N-terminal end of the parent
glucoamylase (e. g. by inserting a nucleic acid (preferably DNA)
sequence encoding the peptide extension at the relevant location
5 in the nucleic acid (preferably DNA) sequence encoding the parent
glucoamylase), ii) expressing the resulting modified nucleic acid
(preferably DNA) sequence in a suitable expression system, and
iii) recovering the resulting glucoamylase variant.
In the present context, the term ' ~ linked at °' is intended to
10 indicate that the extension is fused to the N-terminal end (e.c~.
last amino acid residue) of the mature glucoamylase.
Many glucoamyalses are expressed as "prepro-
glucoamylases°°,
i.e., as glucoamyalses consisting of the mature glucoamylase, a
secretory signal peptide (i.e., prepeptide) and a pro-peptide.
The prepro-glycoamylase is processed intracellularly to be
secreted into the fermentation medium, from which the mature
glucoamylase can be isolated and/or purified. Adding the peptide
extension to the parent glucoamylase can be carried out by
linking nucleic acid sequences encoding the desired peptide
extensions upstream (for N-terminal peptide extensions) to the
DNA sequence encoding the parent glucoamylase.
The insertion should be performed in such a way that the
desired glucoamylase variant (i.e.~ having the desired peptide
extensions(s)) is expressed and secreted by the host cell after
transcription, translation, and processing of the glucoamylase
variant . The term °'processing'° means in this context removal
of
pre- and pro-peptides (except, of course, when the pro-peptide is
identical to the desired peptide extension. This will be dealt
with further below).
In most cases it is possible to extend the parent glucoamylase
by inserting a DNA sequence encoding the peptide extension
between the DNA sequence encoding the pro-peptide or the
prepeptide (if no prosequence is present) and the DNA sequence
encoding the mature glucoamylase.
The insertion/addition of a DNA sequence encoding the peptide
extension can be carried out by any standard techniques known by
any skilled person in the field of molecular biology, cf., e.g.
Sambrook et al., 1989). This include, e.g., the polymerase chain
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reaction. (PCR) using specific primers, for instance described in
US patent 4,683,202 or R.K. Saiki et al., (1988), Science, 239,
487-491. How to provide for the expression and secretion of
adjacent DNA sequences) will be described below.
While care must be exerted to select a proper expression
system for producing a glucoamylase variant of the invention (in
particular when a modified DNA sequence is used for the
production), it has been found that a glucoamylase variant
according to the invention (having an improved thermal stability)
may be obtained by expressing a DNA sequence encoding the parent
glucoamylase enzyme in question in an expression system which is
incapable of processing the translated polypeptide in the normal
manner, and thereby results in the production of an glucoamylase
which comprises a part of or the entire propeptide or a similar
peptide sequence associated with the mature protein prior to its
processing. In this case, the propeptide or similar peptide
sequence constitutes the peptide extension. The pro-peptide or
similar peptide sequence may be heterologous or homologous to the
parent glucoamylase and can be present in the N-terminal of the
parent glucoamylase. The production of a glucoamylase variant
according to the invention using this latter technique is
described further below.
Accordingly, if a suitable stretch of amino acids is already
encoded in the prepro form of the parent glucoamylase and this
stretch of amino acids is cut off in the processing of the
glucoamylase by a given expression system, the peptide extension
can be applied by changing the expression host system to a system
in which said processing of said stretch of amino acids does not
occur. In such a case the secretory signal pre-peptide will be
cut off during or after the secretion, resulting in a modified
glucoamylase-consisting of the parent glucoamylase comprising the
pro-peptide or part thereof or a similar peptide sequence encoded
by the corresponding DNA sequence, i.e. a glucoamylase being
extended at the N-terminus.
Yeast cells have been found of particular use for applying
peptide extensions (in the form of the propeptide or a part
thereof) to parent fungal glucoamyalses enzymes, in particular
the Aspergillus niger glucoamylase enzyme.
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In an highly preferred embodiment the peptide extension is
designed and applied by means of random mutagenesis according to
the following principle:
a) subjecting a DNA sequence encoding the parent gluciamylase
enzyme with a peptide extension to localized random mutagenesis
in the peptide extension or in a of the N-terminal end of the
parent glucaamylase,
b) expressing the mutated DNA sequence obtained in step a) in a
host cell, and
c) screening for host cells expressing a mutated glucoamyalse
enzyme which has an improved performance as compared to the
parent glucoamyalse enzyme.
By this approach a number of highly advantageous peptide
additions have been created.
The localized random mutagenesis may be performed essentially as
described in WO 95/22615. More specifically, the mutagenesis is
performed under conditions in which only one or more of the above
areas are subjected to mutagenesis. Especially for mutagenizing
large peptide extensions, it may be relevant to use PCR generated
mutagenesis (e. g, as described by Deshler 1992 or Leung et al.,
1989), in which one or more suitable oligonucleotide probes are
used which flanks the area to be mutagenized. For mutagenesis of
shorter peptide extensions, it is more preferably perform the
localized random mutagenesis by use of doped or spiked
oligonucleotides. The doping or spiking is used, e.g., to avoid
codons for unwanted amino acid residues or to increase the
likelihood that a particular type of amino acid residue, such as
a positively charged or hydrophobic amino acid residue, is
introduced at a desired position.
Subsequent to the mutagenesis the mutated DNA is expressed by
culturing a suitable host cell carrying the DNA sequence under
conditions allowing expression to take place. The host cell used
for this purpose may be one which has been transformed with the
mutated DNA sequence, optionally present on a vector, or one
which carried the DNA sequence encoding the parent enzyme durir_g
the mutagenesis treatment. Examples of suitable host cells are
given below, and is preferably a host cell which is capable of
secreting the mutated enzyme (enabling an easy screening). Yeast
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cells, such as cells of S. cereviciae, have been found to be
suitable host cells.
Parent glucoamylases
Parent glucoamylase contemplated according to the present
invention include fungal glucoamylases, in particular fungal
glucoamylases obtainable from an Aspergillus strain, such as an
Aspergillus niger or Aspergillus awamori glucoamylases and
variants or mutants thereof, homologous glucoamylases, and
further glucoamylases being structurally and/or functionally
similar to SEQ ID NO: 1. Specifically contemplated are the
Aspergillus niger glucoamylases G1 and G2 disclosed in Boel et
al. (1984), " Glucoamylases G1 and G2 from Aspergillus niger are
synthesized from two different but closely related mRNAs ", EMBO
J. 3 (5), p. 1097-1102,. The G2 glucoamylase is disclosed in SEQ
ID NO: 1.
Co~ercial parent glucoamylases
Commercially available parent glucoamylases include AMG from
Novo Nordisk, and also glucoamylase from the companies Genencor,
Inc. USA, and Gist-Brocades, Delft, The Netherlands.
Parent homologous glucoamylases
The homology of the parent glucoamylase is determined as
the degree of similarity between two protein sequences
indicating a derivation of the first sequence from the second.
The homology may suitably be determined by means of computer
programs known in the art such as GAP provided in the GCG
program package (Program Manual for the Wisconsin Package,
Version 8, August 1994, Genetics Computer Group, 575 Science
Drive, Madison,- Wisconsin, USA 53711) (Needleman, S.B. and
Wunsch, C.D., (1970), Journal of Molecular Biology, 48, p. 443-
453). Using GAP with the following settings for polypeptide
sequence comparison: GAP creation penalty of 3.0 and GAP
extension penalty of 0.1, the mature part of a polypept.ide
encoded by an analogous DNA sequence of the invention exhibits a
degree of identity preferably of at least 80%, at least 90%,
more preferably at least 95%, more preferably at 2east 97%, and
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most preferably at least 99% with the mature part of the amino
acid sequence shown in SEQ ID NO 1.
In a preferred embodiment the variant of the vnverstion has
improved thermal stability within the temperature interval from
about 60-80°C, preferably 63-75°C, at a pH of 4-5, in particular
4.2-4.7, using e.g. maltodextrin as the substrate.
In another preferred embodiment, the parent homologous
glucoamylase comprises a glucoamylase from a microorganism. In a
more preferred embodiment, the microorganism comprises
Eubacteria, Archaebacteria, fungi, algae and protozoa, and in an
yet more preferred embodiment, the parent homologous glucoamylase
is derived from a filamentous fungi.
In an highly preferred embodiment the parent glycoamylase is
the Aspergillus niger G1 glucoamylase (Boel et al. (1984), EMBG
J. 3 (S), p. 1097-1102. The parent glycoamylase may be a
truncated glucoamylase.
Methods for prepargng glucoamylas~ variants
Several methods for introducing mutations into genes are
known in the art. After a brief discussion of the cloning of
glucoamylase-encoding DNA sequences, methods for generating
mutations at specific sites within the glucoamylase-encoding
sequence will be discussed.
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Cloning a DNA sequence encoding a glucoamylaseCloning a DNA
sequence encoding an a-amylaseCloning a DNA sequence encoding an
a-amylaseCloning a DNA sequence encoding an a-amylase
The DNA sequence encoding a parent glucoamylase may be
5 isolated from any cell or microorganism producing the
glucoamylase in question, using various methods well known in the
art. First, a genomic DNA and/or cDNA library should be
constructed using chromosomal DNA or messencrer RNA from the
organism that produces the glucoamylase to be studied. Then, if
t0 the amino acid sequence of the glucoamylase is known, labeled
oligonucleotide probes may be synthesized and used to identify
glucoamylase-encoding clones from a genomic library prepared from
the organism in question. Alternatively, a labelled oligonu-
cleotide probe containing sequences homologous to another known
15 glucoamylase gene could be used as a probe to identify
glucoamylase-encoding clones, using hybridization and washing
conditions of lower stringency.
Yet another method for identifying glucoamylase-encoding
clones would involve inserting fragments of genomic DNA into an
expression vector, such as a plasmid, transforming glucoamylase
negative bacteria with the resulting genomic DNA library, and
then plating the transformed bacteria onto agar containing a
substrate for glucoamylase (i.e. maltose), thereby allowing
clones expressing the glucoamylase to be identified.
Alternatively, the DNA sequence encoding the glucoamylase may
be prepared synthetically by established standard methods, e.g.
the phosphoroamidite method described S.L. Beaucage and M.H.
Caruthers, (1981), Tetrahedron Letters 22, p. 1859-1869, or the
method described by Matthes et al., (1984), EMBO J. 3, p. 801-
805. In the phosphoroamidite method, oligonucleotides are syn-
thesized, e.g.- in an automatic DNA synthesizer, purified,
annealed, 1-igated and cloned in appropriate vectors. Finally, the
DNA sequence may be of mixed genomic and synthetic origin, mixed
synthetic and cDNA origin or mixed genomic and cDNA origin,
prepared by ligating fragments of synthetic, genomic or cDNA
origin (as appropriate, the fragments corresponding to various
parts of the entire DNA sequence), in accordance with standard
techniques. The DNA sequence may also be prepared by polymerase
CA 02352046 2001-05-28
~'~ 00/34452 ~'~'d'l~tC99/006~6
16
chain reaction (PCR) using specific primers, for instance as
described in US 4,683,202 or R.K. Saiki et al., (1988), Science
239, 1988, pp. 487-491.
Subsequent to the incubation with or exposure to the
mutagenising agent, the mutated DNA is expressed by culturing a
suitable host cell carrying the DNA sequence under conditions
allowing expression to take place. The host cell used for this
purpose may be one which has been transformed with the mutated
DNA sequence, optionally present on a vector, or one which was
carried the DNA sequence encoding the parent glucoamyalse during
the mutagenesis treatment. Examples of suitable host cells are
the following: gram positive bacteria such as Bacillus subtilis,
Bacillus licheniformis, Bacillus lentos, Bacillus brevis,
Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus coagulans, Bacillus circulans,
Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis,
Streptomyces lividans or Streptomyces murinus~ and gram-negative
bacteria such as E. coli. The mutated DNA sequence may further
comprise a DNA sequence encoding functions permitting expression
of the mutated DNA sequence.
Site-directed mutagenesis
Once a glucoamylase-encoding DNA sequence has been isolated, and
desirable sites for mutation identified, mutations may be intro
duced using synthetic oligonucleotides. These oligonucleotides
contain nucleotide sequences flanking the desired mutation sites.
In a specific method, a single-stranded gap of DNA, the
glucoamylase-encoding sequence, is created in a vector carrying
the glucoamylase gene. Then the synthetic nucleotide, bearing the
desired mutation, is annealed to a homologous portion of the
single-stranded DNA. The remaining gap is then filled in with DNA
polymerase I (Klenow fragment) and the construct is ligated using
T4 ligase. A specific example of this method is described in
Morinaga et al., (1984), Biotechnology 2, p. 646-639. t1S
4,760,025 discloses the introduction of oligonucleotides encoding
multiple mutations by performing minor alterations of the
cassette. However, an even greater variety of mutations can be
introduced at any one time by the Morinaga method, because a
CA 02352046 2001-05-28
WO 00134452 PC"1'/I)K99100686
17
multitude of oligonucleotides, of various lengths, can be
introduced.
Another method for introducing mutations into glucoamylase-
encoding DNA sequences is described in Nelson and Long, {1989),
Analytical Biochemistry 180, p. 147-151. It involves the 3-step
generation of a PCR fragment containing the desired mutation
introduced by using a chemically synthesized DNA strand as one of
the primers in the PCR reactions. From the PCR-generated
fragment, a DNA fragment carrying the mutation may be isolated by
IO cleavage with restriction endonucleases and reinserted into an
expression plasmid.
Further, Sierks. et al., (1989) " Site-directed mutagenesis
at the active site Trp120 of Aspergillus awamori glucoamylase.
Protein Eng., 2, 621-625; Sierks et al., (1990), '°Determination
of Aspergillus awamori glucoamylase catalytic mechanism by site-
directed mutagenesis at active site Asp176, G1u179, and G1u180" .
Protein Eng. vol. 3, 193-198; also describes site-directed
mutagenesis in an Aspergillus glucoamylase.
Random l~3utagenesis_
Random mutagenesis is suitably performed either as localised
or region-specific random mutagenesis in at least three parts of
the gene translating to the amino acid sequence shown in
question, or within the whole gene. The random mutagenesis of a
DNA sequence encoding a parent glucoamylase may be conveniently
performed by use of any method known in the art. In relation to
the above, a further aspect of the present invention relates to a
method for generating a variant of a parent glucoamylase, wherein
the variant exhibits increased thermal stability relative to the
parent, the method comprising:
(a) subjecting a DNA sequence encoding the parent glucoamylase to
random mutagenesis,
(b) expressing the mutated DNA sequence obtained in step (a) in a
host cell, and
(c) screening for host cells expressing a glucoamylase variant
which has an altered property (i.e. thermal stability) relative
to the parent glucoamylase.
Step (a) of the above method of the invention is preferably
CA 02352046 2001-05-28
9'd~ 00!34452 f~'d°/I~FC991006~6
18
performed using doped primers, as described in the ~rorking
examples herein (vide infra). For instance, the random
mutagenesis may be performed by use of a suitable physical er
chemical mutagenizing agent, by use of a suitable
oligonucleotide, or by subjecting the DNA sequence to PCR
generated mutagenesis. Furthermore, the random mutagenesis may be
performed by use of any combination of these mutagenizing agents.
The mutagenizing agent may, e.g., be one which induces
transitions, transversions, inversions, scrambling, deletions,
and/or insertions. Examples of a physical or chemical
mutagenizing agent suitable for the present purpose include
ultraviolet (W) ir-radiation, hydroxylamine, N-methyl-N'-nitro-
N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid,
ethyl methane sulphonate (EMS), sodium bisulphite, formic acid,
and nucleotide analogues. When such agents are used, the
mutagenesis is typically performed by incubating the DNA sequence
encoding the parent enzyme to be mutagenized in the presence of
the mutagenizing agent of choice under suitable conditions for
the mutagenesis to take place, and selecting for mutated DNA
having the desired properties. When the mutagenesis is performed
by the use of an oligonucleotide, the oligonucleotide may be
doped or spiked with the three non-parent nucleotides during the
synthesis of the oligonucleotide at the positions which are to be
changed. The doping or spiking may be done so that codons for
unwanted amino acids are avoided. The doped or spiked
oligonucleotide can be incorporated into the DNA encoding the
glucoamylase enzyme by any published technique, using e.g. PCR,
LCR or any DNA polymerase and lipase as deemed appropriate.
Preferably, the doping is carried out using "constant random
doping", in which the percentage of wild-type and mutation in
each position is predefined. Furthermore, the doping may be
directed toward a preference for the introduction of certain
nucleotides, and thereby a preference for the introduction of one
or more specific amino acid residues. The doping may be made,
e.g., so as to allow for the introduction of 90% wild type and
10% mutations in each position. An additional consideration in
the choice of a doping scheme is based on genetic as well as
protein-structural constraints. The doping scheme may be made by
CA 02352046 2001-05-28
bV~ 00/34Ll52 PCT/I~HC99I00686
19
using the DOPE program which, inter alia, ensures that
introduction of stop codons is avoided. When PCR-generated
mutagenesis is used, either a chemically treated or non-treated
gene encoding a parent glucoamylase is subjected to PCR under
conditions that increase the mis-incorporation of nucleotides
(Deshler 1992; Leung et al., Technique, Vol.l, 1989, pp. 11-15).
A mutator strain of E. col.i (Fowler et al. , Molec. Gen. Genet. ,
133, 1974, pp. 179-191), S. cereviseae or any other microbial
organism may be used for the random mutagenesis of the DNA
0 encoding the glucoamylase by, e.g., transforming a plasmid
containing the parent glycosylase into the mutator strain,
growing the mutator strain with the plasmid and isolating the
mutated plasmid from the mutator strain. The mutated plasmid may
be subsequently transformed into the expression organism. The DNA
sequence to be mutagenized may be conveniently present in a
genomic or cDNA library prepared from an organism expressing the
parent glucoamylase. Alternatively, the DNA sequence may be
present on a suitable vector such as a plasmid or a
bacteriophage, which as such may be incubated with or other-wise
exposed to the mutagenising agent. The DNA to be mutagenized may
also be present in a host cell either by being integrated in the
genome of said cell or by being present on a vector harboured in
the cell. Finally, the DNA to be mutagenized may be in isolated
form. It will be understood that the DNA sequence to be subjected
to random mutagenesis is preferably a cDNA or a genomic DNA
sequence. In some cases it may be convenient to amplify the
mutated DNA-sequence prior to performing the expression step b)
or the screening step c). Such amplification may be performed in
accordance with methods known in the art, the presently preferred
method being PCR-generated amplification using oligonucleotide
primers prepared on the basis of the DNA or amino acid sequence
of the parent enzyme. Subsequent to the incubation with or
exposure to the mutagenising agent, the mutated DNA is expressed
by culturing a suitable host cell carrying the DNA sequence under
conditions allowing expression to take place. The host cell used
for this purpose may be one which has been transformed with the
mutated DNA sequence, optionally present on a vector, or one
which was carried the DNA sequence encoding the parent enzyme
CA 02352046 2001-05-28
dV0 00/34452 ~'C1"/I)~C99/006~6
during the mutagenesis treatment. Examples of suitable host
cells are the following: gram positive bacteria such as Bacillus
subtilis, Bacillus Zicheniformis, Bacillus Zentus, Bacillus
brevis, Bacillus stearothermophilus, Bacillus alkalophilus,
5 Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus
circulans, Bacillus Zautus, Bacillus megaterium, Bacillus
thuringiensis, Streptomyces Ziviaans or Streptomyces znurinus; and
gram-negative bacteria such as ,E. coli. The mutated DNA sequence
may further comprise a DNA sequence e~:coding functions permitting
10 expression of the mutated DNA sequence.
hocalized random mutagenesis
The random mutagenesis may be advantageously localized to a
part of the parent glucoamylase in question. This may, e.g., be
IS advantageous when certain regions of the enzyme have been
identified to be of particular importance for a given property of
the enzyme, and when modified are expected to result in a variant
having improved properties. Such regions may normally be
identified when the tertiary structure of the parent enzyme has
20 been elucidated and related to the function of the enzyme.
The localized, or region-specific, random mutagenesis is
conveniently performed by use of PCF2 generated mutagenesis
techniques as described above or any other suitable technique
known in the art. Alternatively, the DNA sequence encoding the
part of the DNA sequence to be modified may be isolated, e.g., by
insertion into a suitable vector, and said part may be
subsequently subjected to mutagenesis by use of any of the
mutagenesis methods discussed above.
Expression of glucoamylase variants
According to the invention, a DNA sequence encoding the
variant produced by methods described above, or by any alterna-
tive methods known in the art, can be expressed, in enzyme form,
using an expression vector which typically includes control
sequences encoding a promoter, operator, ribosome binding site,
translation initiation signal, and, optionally, a repressor gene
or various activator genes.
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21
Expression vector
The recombinant expression vector carrying the DNA sequence
encoding a glucoamylase variant of the invention may be any
vector, which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the
host cell into which it is to be introduced. The vector may be
one which, when introduced into a host cell, is integrated into
the host cell genome and replicated together with the
chromosomes) into which it has been integrated. Examples of
suitable expression vectors include pMT838.
Promoter
In the vector, the DNA sequence should be,operably connected
to a suitable promoter sequence. The promoter may be any DNA
IS sequence which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcrip
tion of the DNA sequence encoding a glucoamylase variant of the
invention, especially in a bacterial host, are the promoter of
the 1ac operon of E.coli, the Streptomyces coelicolor agarase
gene dagA promoters, the promoters of the Bacillus licheniformis
oc-amylase gene (amyL), the promoters of the Bacillus
stearothermophilus maltogenic amylase gene (amyM), the promoters
of the Bacillus amyloliquefaciens ~,-amylase (amyQ), the promoters
of the Bacillus subtilis xylA and xylB genes etc. For
transcription in a fungal host, examples of useful promoters are
those derived from the gene encoding A. oryzae TAKA amylase, the
TPI (triose phosphate isomerase) promoter from S. cerevisiae
(Alber et al. (1982) , J. Mol. Appl. Genet 1, p. 419-434, Rhizo-
mucor miehei aspartic proteinase, A. niger neutral ~c,-amylase, A.
niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor
miehei lipase, A. oryzae alkaline protease, A. oryzae triose
phosphate isomerase or A. nidulans acetamidase.
Expression vector
The expression vector of the invention may also comprise a
CA 02352046 2001-05-28
b~'O 00134452 fCTII)~C99/006~6
22
suitable transcription terminator and, in eukaryotes, poly
adenylation sequences operably connected to the DNA sequence
encoding the a-amylase variant of the invention. Termination and
polyadenylation sequences may suitably be derived from the same
sources as the promoter.
The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell in question. Examples of
such sequences are the origins of replication of plasmids pUCl9,
pACYC177, pUB110, pE194, pAMBl and pIJ702.
The vector may also comprise a selectable marker, e.g. a gene
the product of which complements a defect in the host cell, such
as the daI genes from B. subtilis or B. licheniformis, or one
which confers antibiotic resistance such as arnpicillin,
kanamycin, chloramphenicol or tetracyclin resistance. Fur-
thermore, the vector may comprise Aspergillus selection markers
such as amdS, argB, niaD and sC, a marker giving rise to
hygromycin resistance, or the selection may be accomplished by
co-transformation, e.g. as described in WO 91/17243.
The procedures used to ligate the DNA construct of the inven
tion encoding a glucoamylase variant, the promoter, terminator
and other elements, respectively, and to insert them into
suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf.,
for instance, Sambrook et al., Mo1 ralar ~on,ny~ A T,abora ory
Mdniaal., 2nd Ed. , Cold Spring Harbor, 1989) .
Host Cells
The cell of the invention, either comprising a DNA construct
or an expression vector of the invention as defined above, is
advantageously_used as a host cell in the recombinant production
of a glucoamylase variant of the invention. The cell may be
transformed with the DNA construct of the invention encoding the
variant, conveniently by integrating the DNA construct (in one or
more copies) in the host chromosome. This integration is
generally considered to be an advantage as the DNA sequence is
more likely to be stably maintained in the cell. Integration of
the DNA constructs into the host chromosome may be performed
according to conventional methods, e.g. by homologous or
CA 02352046 2001-05-28
wo ooiaaasa Pcmnx99ioosa6
23
heterologous recombination. Alternatively, the cell may be
transformed with an expression vector as described above in
connection with the different types of host cells.
The cell of the invention may be a cell of a higher organism
such as a mammal or an insect, but is preferably a microbial
cell, e.g. a bacterial or a fungal (including yeast) cell.
Examples of suitable bacteria are Gram positive bacteria such
as Bacillus subtilis, Bacillus Iicheniformis, Bacillus Ientus,
Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalo-
philus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus
circulans, Bacillus lautus, Bacillus megaterium, Bacillus
thuringiensis, or Streptomyces lividans or Streptomyces murinus,
or gramnegative bacteria such as E.coli. The transformation of
the bacteria may, for instance, be effected by protoplast trans-
formation or by using competent cells in a manner known per se.
The yeast organism may favorably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces
cerevisiae.
The host cell may also be a filamentous fungus e.g, a strain
belonging to a species of Aspergillus, most preferably Aspergil
1us oryzae or Aspergillus niger, or a strain of Fusarium, such as
a strain of Fusarium oxysporium, Fusarium graminearum (in the
perfect state named Gribberella zeae, previously Sphaeria zeae,
synonym with Gibberella roseum and Gibberella roseum f. sp.
cerealis), or Fusarium sulphureum (in the prefect state named
Gibberella puricaris, synonym with Fusarium trichothecioides,
Fusarium bactridioides, Fusarium sambucium, Fusarium roseum, and
Fusarium roseum var. graminearum), Fusarium cerealis (synonym
with Fusarium crokkwellnse), or Fusarium venenatum.
In a preferred embodiment of the invention the host cell is a
protease deficient of protease minus strain.
This may for instance be the protease deficient strain
Aspergillus oryzae JaL 125 having the alkaline protease gene
named '°alp " deleted. This strain is described in WO 97/35956
(Novo Nordisk).
Filamentous fungi cells may be transformed by a process
involving protoplast formation and transformation of the
protoplasts followed by regeneration of the cell wall in a manner
CA 02352046 2001-05-28
W~ 00134452 PCT/DIC991006~6
24
known z~. The use of Aspergillus as a host micro-organism is
described in EP 238 023 (Novo Nardisk A/S), the contents of which
are hereby incorporated by reference.
Method of producing the glucaaxnylase variant of the inventi~n
In a yet further aspect, the present invention relates to a
method of producing a glucoamylase variant of the invention,
which method comprises cultivating a host cell under conditions
conducive to the production of the variant and recovering the
variant from the cells and/or culture medium.
The medium used to cultivate the cells may be any convention-
al medium suitable for growing the host cell in question and
obtaining expression of the glucoamylase variant of the invent-
ion. Suitable media are available from commercial suppliers or
may be prepared according to published recipes (e. g. as described
in catalogues of the American Type Culture Collection).
The glucoamylase variant secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures such
as ion exchange chromatography, affinity chromatography, or the
like.
Starch conversion
The present invention provides a method of using glucoamylase
variants of the invention for producing glucose and the like from
starch. Generally, the method includes the steps of partially
hydrolyzing precursor starch in the presence of ~,-amylase and
then further hydrolyzing the release of D-glucose from the non-
reducing ends of the starch or related oligo- and polysaccharide
molecules in the presence of glucoamylase by cleaving ~,-(1"'4)
and ~-(1-'67 glucosidic bonds.
The partial hydrolysis of the precursor starch utilizing ~,-
amylase provides an initial breakdown of the starch molecules by
hydrolyzing internal a-(1'-'4)-linkages. In commercial
CA 02352046 2001-05-28
W~ 0013dd52 PC'I"/DIC99/OOb~b
applications, the initial hydrolysis using u,-amylase is run at a
temperature of approximately 105°C. A very high starch
concentration is processed, usually 30~ to 40~ solids. The
initial hydrolysis is usually carried out for five minutes at
5 this elevated temperature. The partially hydrolyzed starch can
then be transferred to a second tank and incubated for
approximately one hour at a temperature of 85° to 90°C to derive
a dextrose equivalent (D.E.) of 10 to 15.
The step of further hydrolyzing the release of D-glucose
10 from the non-reducing ends of the starch or related oligo- and
polysaccharides molecules in the presence of glucoamylase is
normally carried out in a separate tank at a reduced temperature
between 30° and 60°C. Preferably the temperature of the
substrate
liquid is dropped to between 55° and 60°C. The pH of the
solution
15 is dropped from 6 to 6.5 to a range between 3 and 5.5.
Preferably, the pH of the solution is 4 to 4.5. The glucoamylase
is added to the solution and the reaction is carried out for 24-
72 hours, preferably 36-48 hours.
By using a thermostable glucoamylase variant of the
20 invention saccharification processes may be carried out at a
higher temperature than traditional batch saccharification
processes. According to the invention saccharification may be
carried out at temperatures in the range from above 60-80°C,
preferably 63-75°C. This apply both for traditional batch
25 processes (described above) and for continuous saccharification
processes.
Actually, continuous saccharification processes including
one or more membrane separation steps, i.e. filtration steps,
must be carried out at temperatures of above 60°C to be able to
maintain a -reasonably high flux over the membrane or to minimize
microbial contamination. Therefore, the thermostable variants of
the invention provides the possibility of carrying out large
scale continuous saccharification processes at a fair price
and/or at a lower enzyme protein dosage within and period of time
acceptable for industrial saccharification processes. According
to the invention the saccharification time may even be shortened.
The activity of the glucoamylase variant (e. g. AMG variant)
CA 02352046 2001-05-28
Vd~ 00/34452 PC'&'/~IC99/006~b
26
of the invention is generally substantially higher at
temperatures between 60°C-80°C than at the traditionally used
temperature between 30-60°C. Therefore, by increasing the
temperature at which the glucoamylase operates the
saccharification process may be carried out within a shorter
period of time.
Further, by improving the thermal stability the T1" (half-
time, as defined in the " Materials and Methods " section) is
improved. As the thermal stability of the glucoamylase variants
of the invention is improved a minor amount of glucoamylase need
to be added to replace the glucoamylase being inactivated during
the saccharification process. More glucoamylase is maintained
active during saccharification process according to the present
invention. Furthermore, the risk of microbial contamination is
also reduced when carrying the saccharification process at
temperature above 63°C.
An example of saccharification process wherein the
glucoamylase variants of the invention may be used include the
processes described in JP 3-224493; JP 1-192693 ;JP 62-272987;
and EP 452,238.
The glucoamylase variants) of the invention may be used in
the present inventive process in combination with an enzyme that
hydrolyzes only a-(1~6)-glucosidic bonds in molecules with at
least four glucosyl residues. Preferentially, the glucoamylase
variant of the invention can be used in combination with
pullulanase or isoamylase. The use of isoamylase and pullulanase
for debranching, the molecular properties of the enzymes, and the
potential use of the enzymes with glucoamylase is set forth in
G.M.A. van Beynum et al., Starch Conversion Technology, Marcel
Dekker, New York,-1985, 101-142.
In a further aspect the invention relates to the use of a
glucoamylase variant of the invention in a starch conversion
process.
Further, the glucoamylase variant of the invention may be
used in a continuous starch conversion process including a
continuous saccharification step.
The glucoamylase variants of the invention may also be
CA 02352046 2001-05-28
W~ 00/34452 PCT/I)1C99/00686
27
used in immobilised form. This is suitable and often used for
producing speciality syrups, such as maltose syrups, and further
for the raffinate stream of oligosaccharides in connection with
the production of fructose syrups.
The glucoamylase of the invention may also be used in a
process for producing ethanol for fuel or beverage or may be used
in a fermentation process for producing organic compounds, such
as citric acid, ascorbic acid, lysine, glutamic acid.
Finally, the invention also relates to a method for improving
the thermostability of a parent glucoamylase by making an
extension at the N-terminus. In an important embodiment, the
extension comprises a peptide extension.
MATERIhLS AND METHODS
Material:
Fn~~rmPS~nmG G1: Aspergillus niger glucoamylase G1 disclosed in
Boel et al. (1984), EMBO J. 3 (5), 1097-1102, available from
Novo Nordisk.AMG G2: Truncated Aspergillus niger glucoamylase G1
shown in SEQ ID No. 1, available from Novo Nordisk)
A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available from
Institute for Fermention, Osaka; 17-25 Juso Hammachi 2-Chome
Yodogawa-ku, Osaka, Japan, having the alkaline protease gene
named " alp " (described by Murakami K et al., (1991), Agric.
Biol. Chem: 55, p. 2807-2811) deleted by a one step gene
replacement method (described by G. May in °'Applied Molecular
Genetics of Filamentous Fungi " (1992), p. 1-25. Eds. J. R.
Kinghorn and G. Turner; Blackie Academic and Professional), using
the A. oryzae pyre gene as marker. Strain JaL 125 is further
disclosed in WO 97/35956 (Novo Nordisk).
Strain: Saccharomyces cerevisiae YNG318: MATpr,leu2-~2 ura3-52
his4-539 pep4-Q1[cir+]
CA 02352046 2001-05-28
W~ 00134452 ' P~'&'/D1G99100686
28
pLaC103: Plasmid encoding the truncated ~lspergillus ra.iger
glucoamylase G2.pJS0026: (S. cerevisiae expression
plasmid){J.S.Okkels, (1996)"A URA3-promoter deletion in a pYES
vector increases the expression level of a fungal lipase in
Saccharomyces cerevisiae. Recombinant DNA Biotechnology III: The
Integration of Biological and Engineering Sciences, vol. 782 of
the Annals of the New York Academy of Sciences) More
specifically, the expression plasmid pJS026, is derived from pYES
2.0 by replacing the inducible GAL1-promoter of AYES 2.0 with the
constitutively expressed TPI (triose phosphate isomerase)-
promoter from Saccharomyces cerevisiae (Albert and Karwasaki,
(1982), J. Mol. Appl Genet., 1, 419-434), and deleting a part of
the URA3 promoter.
Me tFZOds a
Transforma.t-ion o saocharnm~~r.~a r visiaP ynTC"~'I8
The DNA fragments and the opened vectors were mixed and
transformed into the yeast Saccharomyces cerevisiae YNG318 by
standard methods.
Determi nab i_om. of ACt1 ar_~ ~'~v
One Novo Amyloglucosidase Unit (AGU) was defined as the
amount of enzyme which hydrolyzes 1 micromole maltose per minute
under the following standard conditions:
Substrate. . . . . . maltose
Temperature. . . . . 25°C
pH. . . . . . . . . .4.3 (acetate buffer)
Reaction time. . . . 30 minutes
A detailed description of the analytical method (AF22) is
available on request.
100 ml of YPD (Sherman et al., (1981), Methods in Yeast
Genetics, Cold Spring Harbor Laboratory) were inoculated with
spores of A. oryzae and incubated with shaking for about 24
hours. The mycelium was harvested by filtration through
miracloth and washed with 200 ml of 0.6 M MgS04. The mycelium
was suspended in 15 ml of 1.2 M MgS04, 10 mM NaH2P04, pH 5.8.
CA 02352046 2001-05-28
WO 00!34452 PC'H"/DIG99/00686
29
The suspension was cooled on ice and 1 ml of buffer containing
120 mg of NovozymT"' 234 was added. After 5 min., 1 ml of 12
mg/ml BSA (Sigma type H25) was added and incubation with gentle
agitation continued for 1.5-2.5 hours at 37C until a large
number of protoplasts are visible in a sample inspected under
the microscope.
The suspension was filtered through miracloth, the filtrate
transferred to a sterile tube and overlayed with 5 ml of 0.6 M
sorbitol, 100 mM Tris-HC1, pH 7Ø Centrifugation was performed
for 15 min. at 1000 g and the protoplasts were collected from
the top of the MgS04 cushion. 2 volumes of STC (1.2 M sorbitol,
10 mM Tris-HCl, pH 7.5, 10 mM CaCl2) were added to the
protoplast suspension and the mixture is centrifugated for 5
min. at 1000 g. The protoplast pellet was resuspended in 3 ml of
STC and repelleted. This was repeated. Finally, the protoplasts
were resuspended in 0.2-1 ml of STC.
100 ul of protoplast suspension were mixed with 5-25 ~g of
p3SR2 (an A. nidulans amdS gene carrying plasmid described in
Hynes et al., Mol. and Cel. Biol., Vol. 3. No. 8, 1430-1439,
Aug. 1983) in 10 ~.1 of STC. The mixture was left at room
temperature for 25 min. 0.2 ml of 60% PEG 4000 (BDH 29576), 10
mM CaCl2 and 10 mM Tris-HC1, pH 7.5 was added and carefully
mixed (twice) and finally 0.85 ml of the same solution were
added and carefully mixed. The mixture was left at room
temperature for 25 min., spun at 2.500 g for 15 min. and the
pellet was resuspended in 2 ml of 1.2M sorbitol. After one more
sedimentation the protoplasts were spread on minimal plates
(Cove, (1966), Hiochem. Biophys. Acta 113, 51-56) containing 1.0
M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM
CsCl to inhibit background growth. After incubation for 4-7 days
at 37C spores were picked, suspended in sterile water and spread
for single colonies. This procedure is repeated and spores of a
single colony after the second re-isolation were stored as a
defined transformant.
Fed bat-c°h fe,rm nt-a i on
Fed batch fermentation is performed in a medium comprising
maltodextrin as a carbon source, urea as a nitrogen source and
CA 02352046 2001-05-28
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yeast extract. The fed batch fermentation is performed by
inoculating a shake flask culture of A. oryzae host cells in
question into a medium comprising 3.5% of the carbon source and
0.5% of the nitrogen source. After 24 hours of cultivation at pH
5 5.0 and 34°C the continuous supply of additional carbon and
nitrogen sources are initiated. The carbon source is kept as the
limiting factor and it is secured that oxygen is present in
excess. The fed batch cultivation is continued for 4 days, after
which the enzymes can be recovered by centrifugation,
10 ultrafiltration, clear filtration and germ filtration. Further
purification may be done by anionexchange chromatographic methods
known in the art.
Pnri ~,~~t-iori
15 The culture broth is filtrated and added ammoniumsulphate
(AMS) to a concentration of 1.7 M AMS and pH is adjusted to pH
5. Precipitated material is removed by centrifugation on the
solution containing glucoamylase activity is applied on a Toyo
Pearl Hutyl column previously equilibrated in 1.7 M AMS, 20 mM
20 sodium acetate, pH 5. Unbound material is washed out with the
equilibration buffer. Bound proteins are eluted with 10 mM
sodium acetate, pH 4.5 using a linear gradient from 1.7 - 0 M
AMS over 10 column volumes. Glucoamylase containing fractions
are collected and dialysed against 20 mM sodium acetate, pH 4.5.
Thermal Srahi7it~r d t- rmina ion of varian of Yh inyPn_ion
The thermal stability of variants of the invention is tested
using the following method: 950 microliter 50 mM sodium acetate
buffer (pH 4.3) (NaOAc) is incubated for 5 minutes at 70°C. 50
microliter enzyme in buffer {4 AGU/ml) is added. 2 x 40
microliter samples are taken at 0, 5, 20 and/or 40 minutes,
respectively, and chilled on ice. The activity (AGU/ml) measured
before incubation (0 minutes) is used as reference (100%) . The
decline in percent is calculated as a function of the incubation
time.
fhal -1 ifP) of the g]acoamylase
The Tl,z is measured by incubating the glucoamylase (0.18-
CA 02352046 2001-05-28
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31
0.36 AG/g DS) in question in 300 10 DE maltodextrin at pH 4.5 at
the temperature in question (e. g. 70°C). Samples were withdrawn
at set time intervals and further incubated at 50°C for 24 hours
to ensure that all substrate was hydrolysed, since maltodextrin
might affect the activity assay. Incubation at 50°C for 24 hours
will not reduce the enzyme activity significantly. After
incubation the samples were cooled and residual enzyme activity
measured by the pNPG method (as described below).
The % residual glucoamylase activity was determined at
different times. Tl,a was the period of time until which the
relative activity was decreased to 50%.
RP~i dial Pn~yme ~~t ivi t-~r (~NPC' mPS-h~c~l
pNPG reagent:
0.2 g pNPG (p-nitrophenylglucopyranoside) was dissolved in
0.1 M acetate buffer (pH 4.3) and made up to 100 ml.
Borate solution:
3.8g NaaB40, ~10 H20 was dissolved in Milli-Q water and made
up to 100 ml.
AMG standard:
An aqueous enzyme solution containing a known amount of
enzyme equivalent to 0.04 AGU/ml.
Samples might be diluted prior to analysis (1:1-1:2
with
water). The following solutions were prepared:
HS: 0.5 ml sample + 1 ml AMG standard + 3 ml pNPG reagent
H: 0.5 rnl sample + 1 ml water + 3 ml pNPG reagent
B: 0.5 mi sample + 1 ml AMG standard + 3 ml borate solution
Place HS and H in a 50C water bath. After 2 hours, 3 ml
borate solution was added to each vial. B was placed at
room
temperature and 3 ml pNPG reagent added after 2 hours. The
optical density of all three solutions were measured at nm,
400
and the activity calculated:
Activity = 2 * AGUBt * (H-B) / (HS-H)
where HS, H, and B are the OD of the solutions analysed, and
AGU$t is the activity of the AMG standard used.
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32
Construction of pAMGY
The pAMGY vector was constructed as follows: The lipase gene in
pJS0026 was replaced by the AMG gene, which was PCR amplified
with the forward primer; FG2: 5'-CAT CCC CAG GAT CCT TAC TCA GCA
ATG-3' and the reverse primer: RG2: 5'-CTC AAA CGA CTC ACC AGC
CTC TAG AGT-3' using the template plasmid pLAC103 containing the
AMG gene. The pJS0026 plasmid was digested with XbaI and SmaI at
37°C for 2 hours and the PCR amplicon was blunt ended using the
Klenow fragment and then digested with XbaI. The vector fragment
and the PCR amplicon were ligated and transformed into E.colz by
electrotransformation. The resulting vector is designated pAMGY.
The expression plasmid pJS037 is described in Vd0 97/04079
and WO 97/07205. I~: is derived from pYES 2.0 by replacing the
inducible GALL-promoter of pYES 2.0 with the constitutively
expressed TPI (triose phosphate isomerase)-promoter from
Saccharomyces cerevisiae (Albert and Karwasaki, 11982), J. Mol.
Appl Genet., 1, 419-434), and deleting a part of the URA3
promoter.
Constructiora of plaaC303
The A. niger AMGII cDNA clone (Boel et al., (1984),
supra) is used as source for the construction of pLaC103 aimed at
S. cerevisiae expression of the GII form of AMG.
The construction takes place in several stepsP out lined
below.
pT7-212 (EP37856/ US patent no. 5162498) is cleaved with
XbaI, blunt-ended with Klenow DNA polymerise and dNTP. After
cleavage with EcoRI the resulting vector fragment is purified
from an agaros_e gel-electrophoresis and ligated with the 2.05 kb
EcoRl-EcoRV fragment of pBoe153, thereby recreating the XbaI
site in the EcoRV end of the AMG encoding fragment in the
resulting plasmid pG2x.
In order to remove DNA upstream of the AMG cds, and furnish
the AMG encoding DNA with an appropriate restriction endonuclease
recognition site, the following construct was made: The 930 by
EcoRI-PstI fragment of p53 was isolated and subjected to AluI
cleavage, the resulting 771 by Alu-PstI fragment was ligated into
CA 02352046 2001-05-28
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33
pBR322 with blunt-ended EcoRI site (see above) and cleaved with
Pstl In the resulting plasmid pBR-AMG°, the EcoRI site was
recreated just 34 by from the initiation colon of the AMG cds.
From pBR-AMG' the 775 by EcoRI - PstI fragment was isolated
and joined with the 1151 by PstI - XbaI fragment from pG2x in a
ligation reaction including the Xbal - EcoRI vector fragment of
pT7-212.
The resulting plasmid pT7GII was submitted to a BamHT
cleavage in presence of alkaline phosphatase followed by partial
SphI cleavage after inactivation of the phosphatase. From this
reaction was the 2489 by Sphl-BamHI fragment, encompassing the
S.c. TPI promoter linked to the AMGII cds.
The above fragment together with the 1052 by BamHI fragment
of pT7GII was ligated with the alkaline phosphatase treated
vector fragment of pMT743 (EP37856jUS 5162498), resulting from
SphI-Bam~-TI digestion. The resulting plasmid is pLaC103.
Screening for therm~stable glucoamylase variants
The libraries are screened in the thermostable filter
assay described below.
Filter assay for tlaermostability
Yeast libraries are plated on cellulose acetate filter(OE
67, Schleicher & Schuell, Dassel, Germany) on SC ura-agar plates
with 100 ~,g/ml ampicillin at 30°C for at least 72 hrs. The
colonies are replica plated to nitrocellulose filters (Protran-
Ba 85, Schleicher & Schuell, Dassel, Germany) and incubated at
room temperature for 1 hours. Colonies are washed from Protran
filters with tap water. Each filter is specifically marked with
a needle before incubation in order to be able to localise
positive variants on the filters after the screening. The
Protran filters with bound variants are transferred to a
container with 0.1 M NaAc, pH 4.5 and incubated at 55-75°C for
15 minutes. The cellulose acetate filters on SC ura-agar plates
are stored at room temperature until use. After incubation, the
residual activities are detected on plates containing 5%
maltose, to agarose, 50 mM NaAc, pH 4.5. The assay plates with
Protran filters are marked the same way as the cellulose acetate
CA 02352046 2001-05-28
Wf7 00134452 ~C'~'/I31C991006~6
34
filters and incubated for 2 hours at 50°C. After removal of the
Protran filters, the assay plates are stained with Glucose GOD
perid (Boehringer Mannheim GrnbH, Germany). Variants with
residual activity are detected on assay plates as dark green
spots on white background. The improved variants are located on
the storage plates. Improved variants are rescreened twice under
the same conditions as the first screen.
CA 02352046 2001-05-28
WO OOI34452 PCT/D1499/UOb86
Random mutagenesis may be carried out using the following
steps:
1. Select regions of interest for modification in the
5 parent enzyme,
2. Decide on mutation sites and non-mutated sites in the
selected region,
3. Decide on which kind of mutations should be carried
out, e.g., with respect to the desired stability and/or
10 performance of the variant to be constructed,
4. Select structurally reasonable mutations,
5. Adjust the residues selected by step 3 with regard to
step 4.
6. Analyze by use of a suitable dope algorithm the
15 nucleotide distribution.
7. If necessary, adjust the wanted residues to genetic
code realism, e.g. taking into account constraints resulting from
the genetic code, e.g. in order to avoid introduction of stop
codonsr the skilled person will be aware that some codon
20 combinations cannot be used in practice and will need to be
adapted
8. Make primers
9. Perform random mutagenesis by use of the primers
10. Select resulting glucoamylase variants by screening for
25 the desired improved properties.
Dcy a~ r i hm_
Suitable dope algorithms for use in step 6 are well known in
the art. Cne such algorithm is described by Tomandl, D. et al.,
30 1997, Journal of Computer-Aided Molecular Design 11:29-38.
Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A,
and Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).
35 EXAMPLES
EXAMPLE 1.
Construction of glucoamylase variants with N-terminal extensions
CA 02352046 2001-05-28
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36
33:3nd~m mmtag~
The oligonucleotides Atoll-18(with possibilities for inserting 1-7
extra amino acids after the KexII site and in front of the mature
protein) and primer AM18 together with 2 primers corresponding to
sequences about 75 by outside the coding region in both the 5°
(4244: 5'-TCA AGA ATA GTT CAA ACA AGA AGA-3°) and 3° end (KB14:
5'-CTT TTC GGT TAG AGC GGA TG-3') are used to generate PCR
library-fragments by the overlap extension method (Norton et al.,
Gene, 77 (1989), pp. 61-68).
The following PCR reactions were performed:
PCR reaction 1: 4244 as 5' primer and AM18 as 3' primer.
PCR reaction 2: AM11 as 5' primer and KB14 as 3° primer (7
extra aa) .
PCR reaction 3: AMI2 as 5° primer and KB14 as 3° primer (6
extra aa).
PCR reaction 4: AM13 as 5' primer and KB14 as 3' primer (5
extra aa).
PCR reaction 5: Alvil4 as 5' primer and KB14 as 3' primer (4
extra aa) .
PCR reaction 6: AM15 as 5' primer and KB14 as 3° primer (3
extra aa).
PCR reaction 7: AM16 as 5' primer and KB14 as 3' primer (2
extra aa).
PCR reaction 8: AM17 as 5' primer and KB14 as 3' primer (1
extra aa).
Template in the first reaction: pAMGY, template in reaction 2-8:
either pAMGY or an improved variant cloned in the same vector.
PCR reaction 9-15: The DNA from PCR reaction 1 together with
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6V0 00/34452 PCT'IDIC99/00686
37
either DNA from PCR reaction 2-8 were used as templates in the
PCR reactions using 4244 as 5' primer and FCB14 as 3' primer.
These final PCR fragments were used in an in vivo recombination
in yeast together with pJS0026 cut with the restriction enzymes
SmaI(or BarnHI) and XbaI (to remove the coding region and at the
same time create an overlap of about 75 by in each end to make a
recombination event possible).
AM11: 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS NNS NNS NNS NNS NNS
GCG ACC TTG GAT TCA TGG TTG AGC-3' (SEQ ID NO: 2)
AM12: 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS NNS NNS NNS NNS GCG
ACC TTG GAT TCA TGG TTG AGC-3' (SEQ ID NO: 3)
AM13: 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS NNS NNS NNS GCG ACC
TTG GAT TCA TGG TTG AGC-3° (SEQ ID NO: 4)
AM14: 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS NNS NNS GCG ACC TTG
GAT TCA '.CGG TTG AGC-3' (SEQ ID NO: 5)
AM15: 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS NNS GCG ACC TTG GAT
TCA TGG TTG AGC-3' (SEQ ID NO: 6)
AM16: 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS GCG ACC TTG GAT TCA
TGG TTG AGC-3' (SEQ ID NO: 7)
AM17: 5'-GCA AAT GTG ATT TCC AAG CGC NNS GCG ACC TTG GAT TCA TGG
TTG AGC-3° (SEQ ID NO: 8)
AM18: 5'-GCG CTT GGA AAT CAC ATT TGC-3'(SEQ ID NO: 9)
4244: 5'-TCA AGA ATA GTT CAA ACA AGA AGA-3' (SEQ ID NO: 10)
KB14: 5'- CTT TTC GGT TAG AGC GGA TG-3' (SEQ ID NO: 11)
EXAMPLE 2
Introduction of N-terminal extension by changing the KexTT
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38
recognition site
A N-terminal extension can be introduced by removing or
changing the KexII recognition site "KR" in front of the mature
protein. An extension of 6 amino acids can then be introduced as
the pro-sequence consist of 6 amino acids. In yeast K.R is the
optimal recognition site for Kex II. A change to RR will reduce
the % of cleaved molecules (Bevan, A, Brenner,C and
Fuller,R.s.199B, PNAS 95 (18): 10384-10389). Alternatively
introduction of P in front of KR will reduce the % of cleaved
molecules.
The pro-sea_uence was changed from NVISKR to either NVISRR or
NVIPKR and gave approximately 50% AMG molecules with the
extension NVISRR or NVIPKR and 50% normally processed mature AMG
molecules.
EXAMPLE 3
Construction of glucoamylase (AMG 2) variants containing a
cysteine bridge
The glucoamylase variant of the invention contains the following
mutations : A479C or T480C or P481C or A471C or S431C or S8C or
E299C or D375C and the peptide extension ACPPSTS and ASPPSTS.
The parent glucoamylase (AMG 2) contains the following mutations:
A479C or T480C or P481C or A471C or S431C or S8C or E299C or
D375C.
The cysteine bridge was constructed as follows:
Si -di r Pd mw ac~ienPSi s
For the construction of variants of the AMG G2 enzyme (SEQ ID N0:
11) the commercial kit, Chameleon double-stranded, site-directed
mutagenesis kit was used according to the manufacturer's
instructions.
Tie gene encoding the AMG G2 enzyme in question is located on
pENI1542 prepared by cutting the plasmid pIVI9 with BamHI/XhoI
(Cleaving out the coprinus peroxidase gene) and cloning in a AMG
G2 containing pcr fragment (cut BglII/SalI), made by the use of
the pLaC103 (containing the G2 cDNA) as template and the two
CA 02352046 2001-05-28
WO OOI34452 PCT/IpI699l00686
39
primers 139123 (CGCACGAGATCTGCAATGTCGTTCCGATCTCTA) (SEQ ID NO:
12) and 139124 (CAGCCGGTCGACTCACAGTGACATACCAGAGCG) (SEQ ID NO:
13). This was confirmed by DNA sequencing, as was the variants.
In accordance with the manufacturer's instructions the ScaI site
of the Ampicillin gene of pNEI1542 was changed to a Mlul site by
use of the following primer:
7258: 5'p gaa tga ctt ggt tga cgc gtc acc agt cac 3° (SEQ ID NO.
14). (Thus changing the ScaI site found in the ampicillin
resistance gene and used for cutting to a MluI site). The
pENIl542 vector comprising the AMG gene in question was then used
as a template for DNA polymerase and oligo 7258 (SEQ ID N0. 14)
and 21401 (SEQ ID NO. 15) . Primer no. 21401 (SEQ ID N0. 15) was
used as the selection primer. 21401: 5'p gg gga tca tga tag gac
tag cca tat taa tga agg gca tat acc acg cct tgg acc tgc gtt ata
gcc 3 ' (SEQ ID NO: 15)
The introduction of a cysteine residue is introduced into the AMG
gene in question by addition of an appropriate oligos comprising
the desired mutation as follows:
Mutagenesis oligo:
ACPPSTS 137767 (SEQ ID NO: 16)
(5~P-GTGATTTCCAGCGGTGCCCGCCGTCCACGTCCGCGACCTTGGATTCATGG 3°)
ASPPSTS 137766 (SEQ ID NO: 17)
(5~P-GTGATTTCCAGCGGTCCCCGCCGTCCACGTCCGCGACCTTGGATTCATGG 3~)
D375C 137765(SEQ ID NO: 18)
(5'P-GTAGCATTGTATGTGCCGTGAAGAC 3~)
S431C 146826 (SEQ ID N0: 19)
(5'P-ACCGTCGTAACTGCGTCGTGCCTGC 3°)
E299C 146828 (SEQ ID N0: 20)
(5~P-GTCTCAGTGACAGCTGCGCTGTTGCGGTG3')
A479C 146829 (SEQ ID N0: 21)
(5"P-CCACTACGACGTGCACCCCCACTGG 3°)
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!W~ 00/34452 ~CT/DK99/00686
T480C 146830 (SEQ ID NO: 22)
(5~P-CTACGACGGCTTGCCCCACTGGATCC 3~)
5 P481C 146831 (SEQ ID N0: 23)
(5'"P-CGACGGCTACCTGCACTGGATCCGGC 3~)
S8C 14&827 (SEQ ID NO: 24)
(5~P-TGGATTCATGGTTGTGTAACGAAGCGACC 3~)
Mutants being made
ACPPSTS,D375C
ACPPSTS,S431C
ACPPSTS,E299C
ACPPSTS,A479C
ACPPSTS,T480C
ACPPSTS,P481C
ASPPSTS
S8C+A479C
S8C+T480C
S8C+P481C
The mutations are verified by sequencing the whole gene. The
plasmid was transformed into A. oryzae using the method described
above in the "Materials and Methods" section. The variant was
fermented and purified as described above in the ''Materials and
Methods" section.
Scre~enina
The library may be screened in the thermostability filter
assays described in the °°Material and Methods " section above.
Example 4
G7 ucoamyl a~ vari an a wi t-h i n .r _a_sP~ thermal ~ abi 1 i ty
The thermal stability activity was measured at pH 4,5, 70°C as
described in Methods section above.
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i'1'O 00/34452 PC'TIDIC99I006~6
41
Thermalstability 70C, 4.5
at pH
Enzyme Residualactivity (%)
5 min 20 min 40 min
AMG, (wt) 71 21 2
G2
NVIPKR 85 31 8
PLALSD 73 26 8
The result shows that it is possible to increase the thermal
stability by linking an extension at the N-terminal of a
glucoamylase enzyme according to the invention.
Example 5
Glm~am~rl a~ . vari an s wi 1, i nrrPa~~~3 h anal sta ' l i t-y
The thermal stability activity of improved variants expressed in
yeast was measured on crude samples at pH 4.5, 68°C, as
described in Methods section above.
Thermal stability at 68°C, pH 4.5
Enzyme Residualactivity (%)
5 min 10 min 20 min
AMG, G2 (wt) 57 29 16
ISN 65 39 28
MN 65 39 28
MPGRLP 56 34 23
IFELTPR 55 38 22
LGPD 62 28 23
LGVTGE 55 32 22
AGPLTPR 50 33 22
PCSAGE 57 26 21
PLASD 67 47 36
NVIPKR 57 35 23
Example 6
The thermal stability activity of improved variants expressed in
A. niger was measured on crude samples at pH 4,5, 70°C as
described in Methods section above.
Variant Residual activity
(%)
40 min
G2 4
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dY~ 00/34452 ~T~'E'/D1C99/006~6
42
ACPPSTS+E299C 19
ACPPSTS+A479C 11
ACPPSTS+T480C 24
N-terminal analysis of the variar!t ACPPSTS+E299C showed that no
free Cysteine or oxidized Cysteine were present in the N-
terminal of this variant indicating that an -SS- bond had been
formed between the Cysteine in the N-terminal and Cysteine in
position 299.
CA 02352046 2001-05-28
iV0 00134452 PCT/DK99I00686
1
SEQUENCE LISTING
(1)
GENERAL
INFORMATION:
(i) APPLICANT: -
{A)NAME: NOVO NORDISK A/S
{B)STREET: Novo All
(C)CITY: DK-2880 Bagsvaerd
(E)COUNTRY: Denmark
(F)POSTAL CODE (ZIP): DK-2880
(G)TELEPHONE: +45 4444
8888
(H)TELEFAX: +45 4449 3256
(1.1 )
TITLE
OF
INVENTION:
Glucoamylase
variants
(iii)
NUMBER
OF
SEQUENCES:
35
IS (iv) COMPUTER
READABLE
FORM:
{A)MEDIUM TYPE: Floppy
disk
(B)COMPUTER: IBM PC compatible
{C)OPERATING SYSTEM: PC-DOS/MS-DOS
(D)SOFTWARE: PatentIn Release (EPO)
#1.0, Version #1.25
(2) INFORMATION
FOR
SEQ
ID
NO:
1:
(i)SEQUENCE CHARACTERISTICS;
(A) LENGTH: 534 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: protein
(iii) Organism: Aspergillus
niger
(xi)SEQUENCE DESCRIPTION:
SEQ ID NO:~1:
Met SerPhe Arg Ser Leu Leu GlyLeuVal ThrGly
Ala Leu Ser Cys
-24 -20 -15 -10
Leu AlaAsn Val Ile Ser Lys LeuAspSer LeuSer
Arg Ala Thr Trp
-5 1 5
Asn GluAla Thr Val Ala Arg LeuAsnAsn GlyAla
Thr Ala Ile Ile
10 15 20
Asp GlyAla Trp Val Ser Gly GlyIleVal AlaSer
Ala Asp Ser Va1
25 30 35 40
Pro SerThr Asp Asn Pro Asp ThrTrpThr AspSer
Tyr Phe Tyr Arg
45 50 55
Gly LeuVal Leu Lys Thr Leu PheArgAsn AspThr
Val Asp Leu Gly
60 65 70
Ser LeuLeu Ser Thr Ile Glu SerAlaGln IleVal
Asn Tyr Ile Ala
75 80 85
Gln GlyIle Ser Asn Pro Ser SerSerGly GlyLeu
Gly Asp Leu Ala
90 95 100
Gly GluPro Lys Phe Asn Val AlaTyrThr SerTrp -
Asp Glu Thr Gly
105 110 115 120
Gly ArgPro Gln Arg Asp Gly ArgAlaThr MetIle
Pro Ala Leu Ala
125 130 135
Gly PheGly Gln Trp Leu Leu TyrThrSer AlaThr
Asp Asn Gly Thr
140 145 150
Asp IleVal Trn Pro Leu Val LeuSerTyr AlaGln
Arg Asn Asp Val
155 160 165
Tyr TrpAsn Gln Thr Gly Tyr GluGluVal GlySer
Asp Leu Trp Asn
170175 180
<IMG>
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WCe 00/34452 F'CT/D~G99/00686
2
Ser PhePheThrIleAlaValGlnHisArgAlaLeuValGluGlySer
185 190 195 200
Ala PheAlaThrAlaValGlySer5erCysSerTrpCysAspSerGln
205 210 215
Ala ProGluIleLeuCysTyrLeuGlnSerPheTrpThrGlySerPhe
220 225 230
Ile LeuAlaAsnPheAspSerSerArgSerGlyLysAspAlaAsnThr
235 240 245
Leu LeuGlySerIleHisThrPheAspProGluAlaAlaCysAspAsp
250 255 260
Ser ThrPheGlnProCysSerProArgAlaLeuAlaAsnHisLysGlu
265 270 275 280
Val ValAspSerPheArgSerIleTyrThrLeuAsnAspGlyLeuSer
285 290 295
Asp SerGluA1aValAlaValGlyArgTyrProGluAspThrTyrTyr
300 305 310
Asn GlyAsnProTrpPheLeuCysThrLeuAlaAlaAlaGluGlnLeu
315 320 325
Tyr AspAlaLeuTyrGlnTrpAspLysGlnGlySerLeuGluValThr
330 335 340
Asp ValSerLeuAspPhePheLysAlaLeuTyrSerAspAlaAlaThr
345 350 355 360
Gly ThrTyrSerSerSerSerSerThrTyrSerSerIleValAspAla
365 370 375
Val LysThrPheAlaAspGlyPheValSerIleValGluThrHisAla
380 385 390
Ala SerAsnGlySerMetSerGluGlnTyrAspLysSerAspGlyGlu
395 400 405
Gln LeuSerAlaArgAspLeuThrTrpSerTyrAlaAlaLeuLeuThr
410 415 420
Ala AsnAsnArgArgAsnSerValValProAlaSerTrpGlyGluThr
425 430 435 440
Ser AlaSerSerValProGlyThrCysAlaAlaThrSerAlaIleGly
445 450 455
Thr TyrSerSerValThrValThrSerTrpProSerIleValAlaThr
460 465 470
Gly GlyThrThrThrThrAlaThrProThrGlySerGlySerValThr
475 480 485
Ser ThrSerLysThrThrAlaThrAlaSerLysThrSerThrThrThr
490 495 500
Arg SerGlyMetSerLeu
505 510
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02352046 2001-05-28
CVO OOI34452 &'C'TlD~C99/00686
3
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "AAll"
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 22-42
(D): OTHER INFORMATION: /Note= N= A,C,G or T
S= G or C
(xi) SEQUENCE DESCRIPTION: 5EQ ID NO: 2:
GCAAATGTGA TTTCCAAGCG CNNSNNSNNS NNSNNSNNSN NSGCGACCTT :~'3ATTCATGG TTGAGC 66
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(H) OTHER INFORMATION: /desc = '°AA12°'
( i x ) FEATURE
(A) NAME/KEY: misc-feature
(B) LOCATION: 22-39
(D): OTHER INFORMATION: /Note= N= A,C,G or T
S= G or C
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCAAATGTGA TTTCCAAGCG CNNSNNSNNS NNSNNSNNSG CGACCTTGGA TTCATGG TTG AGC 63
(2)INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: artificial sequence
(ix)FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "AA13"
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B)LOCATION: 22-36
(D):OTHER INFORMATION: /Note= N= A,C,G
or T
S= G or C
(xi)SEQUENCE DESCRIPTION: SEQ ID NO:
4:
GCAAATGTGA TTTCCAAGCG CNNSNNSNNS NNSNNSGCGA CCTTGGATTC ATGGTTGAGC 60
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
iii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "AA14"
(ix) FEATURE:
CA 02352046 2001-05-28
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4
(A) NAME/KEY: misc-feature
(B) LOCATION: 22-33
(D): OTHER INFORMATION: /Note= N= A,C,G or T
S= G or C
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GCAAATGTGA TTTCCAAGCG CNNSNNSNNS NNSGCGACCT TGGATTCATGG TTGAGC 66
(2)INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: artificial sequence
(ix)FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "AA15'
(ix) FEATURE:
(A) NAME/KBY: misc-feature
(B)LOCATION: 22-30
(D):OTHER INFORMATION: /Note= N= A,C,G
Or T
S= G or C
(xi)SEQUENCE DESCRIPTION: SEQ ID NO:
6:
GCAAATGTGA TTTCCAAGCG CNNSNNSNNS GCGACCTTGG ATTCATGGTT GAGC 54
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix)FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc
= "AA16"
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 22-27
(D): OTHER INFORMATION: /Note= N=
A,C,G or T
S= G or C
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO: 7:
GCAAATGTGA TTTCCAAGCG CNNSNNSGCG ACCTTGGATT CATGGTTGAG C 51
(2) INFORMATION FOR SEQ ID N0: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C)-STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = °'AA17"
(ix) FEATURE:
(A) NAh9E/KEY: misc-feature
(B) LOCATION: 22-24
(D): OTHER INFORMATION: /Note= N= A,C,G or T
S= G or C
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GCAAATGTGA TTTCCAAGCG CNNSGCGACC TTGGATTCAT GGTTGAGC 48
CA 02352046 2001-05-28
bV~ 00/34452 PC'f/1)%C991006~6
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
5 tB) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "AA18"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GCGCTTGGAA ATCACATTTG C 21
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTTCS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = °'4244"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
TCAAGAATAG TTCAAACAAG RAGA 24
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "KB14"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CTTTTCGGTT AGAGCGGATG 20
(2) INFORMATION FOR SEQ ID N0: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "Primer 139123'°
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
CGCACGAGAT CTGCAATGTC GTTCCGATCT CTA 33
(2) INFORMATION FOR SEQ ID N0: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02352046 2001-05-28
WO 00134452 ~CT/DiC99/00686
6
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "Primer 139124"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CAGCCGGTCG ACTCACAGTG ACATACCAGA GCG 23
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(Ay NAME/ KEY: mist-feature:
(By OTHER INFORMATION: /desc = "7258"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
gaatgacttggtt gacgcgtcac cagtcac 27
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iiy MOLECULE TYPE: artificial sequence
(ixy FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "21441"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ggggatcatg ataggactag ccatattaat gaagggcata
taccacgcct tggacctgcg ttatagcc 68
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "ACPPSTS 137767"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 16:
GTGATTTCCA GCGGTGCCCG CCGTCCACGT CCGCGACCTT GGATTCATGG 50
(2) INFORMATION FOR SEQ ID N0: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "ASPPSTS 137766"
CA 02352046 2001-05-28
W~ 00134452 ~C'T/I~iG99/00686
7
(xi) SEQUErdCE DESCRT_PTION: SEQ ID N0: 17:
GTGATTTCCA GCGGTCCCCG CCGTCCACGT CCGCGACCTT GGATTCATGG 50
(2) INFORMATION FOR SEQ ID N0: 18:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "D375C 137765"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 18:
GTAGCATTGT ATGTGCCGTG AAGAC 25
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(H) OTHER INFORMATION: /desc = "S431C 146826"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
ACCGTCGTAA CTGCGTCGTG CCTGC 25
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "E299C 146828°'
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:
GTCTCAGTGA CAGCTGCGCT GTTGCGGTG 39
(2) INFORMATION FOR SEQ ID N0: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A} NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = "A479C 146829"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CCACTACGAC GTGCACCCCC ACTGG 25
CA 02352046 2001-05-28
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(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLDGY: linear
(ii) MOLECULE TYPE: artificial sequence
( v~x) FEATURE
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "T480C 146830"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CTACGACGGC TTGCCCCACT GGATCC 26
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
ZO (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: misc-feature:
(B) OTHER INFORMATION: /desc = "P481C 146831"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CGACGGCTAC CTGCACTGGA TCCGGC 26
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: artificial sequence
(ix) FEATURE:
(A) NAME/ KEY: mist-feature:
(B) OTHER INFORMATION: /desc = '°S8C 146827"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
TGGATTCATG GTTGTGTAAC GAAGCGACC 29
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide extension
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2S:
$d
ACGPSTS 7
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(H) TYPE: amino acid
SD) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide extension
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
ACPGTST
CA 02352046 2001-05-28
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9
(2) INFORMATION FOR SEQ ID N0: 27:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: peptide extension
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
lO ACGTGTS 7
(2) INFORMATION
FOR
SEQ
ID
NO:
28:
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 7 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii)MOLECULETYPE: peptide
extension
(xi)SEQUENCEDESCRIPTION: SEQ 28:
ID NO:
ACTGSTG 7
(2) INFORMATION
FOR
SEQ
ID
NO:
29:
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 8 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii)MOLECULETYPE: peptide
extension
(xi)SEQUENCEDESCRIPTION: SEQ 29:
ID N0:
ACGPSTSG 8
(2) INFORMATION
FOR
SEQ
ID
NO:
30:
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 8 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii)MOLECULETYPE: peptide
extension
(xi)SEQUENCEDESCRIPTION: SEQ 30:
ID NO:
ACPGTSTG 8
(2) INFORMATION
FOR
SEQ
ID
N0:
31:
(i)
SEQUENCE
CHARACTERISTICS:
(Aj LENGTH: 8 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(iijMOLECULETYPE: peptide
extension
(xi)SEQUENCEDESCRIPTION: SEQ 31:
ID NO:
ACGTGTSS 8
(2) INFORMATION
FOR
SEQ
ID
NO:
32:
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 8 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii)MOLECULETYPE: peptide
extension
(xi)SEQUENCEDESCRIPTION: SEQ 32:
ID NO:
ACTGSTGT 8
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(2) INFORMATION FOR SEQ ID N0: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amina acids
5 (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide extension
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
10 NVIPPR 6
(2) INFORMATION FOR SEQ ID N0: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acid$
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide extension
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
NPPIRP 6
(2) INFORMATION FOR SEQ ID N0: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide extension
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
NVIPRP 6
