EP1137763A1 - Glucoamylasen mit n-terminalen verlängerungen - Google Patents

Glucoamylasen mit n-terminalen verlängerungen

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Publication number
EP1137763A1
EP1137763A1 EP99959241A EP99959241A EP1137763A1 EP 1137763 A1 EP1137763 A1 EP 1137763A1 EP 99959241 A EP99959241 A EP 99959241A EP 99959241 A EP99959241 A EP 99959241A EP 1137763 A1 EP1137763 A1 EP 1137763A1
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EP
European Patent Office
Prior art keywords
glucoamylase
variant
pro
residue
ser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99959241A
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English (en)
French (fr)
Inventor
Bjarne Roenfeldt Nielsen
Allan Svendsen
Kirsten Novo Nordisk A/S BOJSEN
Jesper Novo Nordisk A/S VIND
Henrik Novo Nordisk A/S PEDERSEN
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Novozymes AS
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Novozymes AS
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Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP1137763A1 publication Critical patent/EP1137763A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Definitions

  • 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.
  • the present invention relates to a glucoamylase variant having improved thermostability.
  • Glucoamylase (1, 4- ⁇ -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 .
  • the glucoamylase enzyme is used to convert corn starch which is already partially hydrolyzed by an ⁇ -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 ⁇ -amylase or glucose isomerase and it is most active and stable at lower pH ' s than either ⁇ -amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH.
  • 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.
  • the invention relates to a variant of a parent glucoamylase, which has a peptide extension at the N-terminus.
  • 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.
  • 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
  • Tripeptidyl amino peptidase TPAP
  • TPAP Tripeptidyl amino peptidase
  • 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.
  • 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 residues to the amino acid sequence of a naturally-occurring glucoamylase, typically in the structural part of the glucoamylase .
  • 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.
  • 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.
  • 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.
  • ..can be found at the N-terminal .. means that the mature glucoamylase has a peptide extension at the N-terminal.
  • the peptide extension is native to the parent glucoamylase, i.e., before posttranslational processing to remove the pro- and/or pre- sequence .
  • the peptide extension may be the pre- and/or pro-sequence of the unprocessed parent glucoamylase, which is normally removed or cut off after expression and posttranslational processing.
  • the extension is a peptide at the N- 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.
  • the extension is linked to the N-terminal it may be done by means of any well-known protein engineering methods in the art .
  • thermostability means in the context of the present invention a glucoamylase variant, which has a higher T 1/2 (half-time) or residual enzymatic activity after a fix incubation period than the corresponding parent glucoamylase.
  • T 1/2 half-time
  • residual enzymatic activity after a fix incubation period than the corresponding parent glucoamylase.
  • T_ and residual activity The determination of thermostability, e.g., T_ and residual activity, is described below in the Materials and Method section.
  • T_ and residual activity is described below in the Materials and Method section.
  • the term u 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.
  • the capability of the peptide extension of providing the desired effect 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 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:
  • Length of peptide extension It has been found that peptide 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 1-50 amino acid residues, more preferably 1- 20 and even more preferably 1-10 amino acid residues.
  • fitahi 1 i ty 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 stability) or so as to not significantly reduce the structural stability of the glucoamylase variant.
  • an acceptable stability e.g., structural stability and/or expression stability
  • 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 .
  • 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
  • the residues should preferably come from residues with low preference for ⁇ -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 ⁇ -helix is prolonged N-terminally it would stick out of the structure with no contact to the N-terminal residues.
  • a peptide extension 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.
  • 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 ⁇ -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.
  • N-terminal residues mean 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. 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, Biopolymers 22 (1983) pp. 2577-2637.))
  • 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.
  • an improved structural stability may be provided by introduction of cysteine bridges in the glucoamylases of the invention.
  • 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 introducing a cysteine bridge is illustrated in Example 3.
  • 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.
  • 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- ⁇ -helix makers, even more preferably with short side chains .
  • n the number of X residues larger or equal to 5, preferably between 5 and 100, even more preferably between 5 and 10, even more preferably 5.
  • ACGPSTS (SEQ ID NO: 25)
  • ACPGTST (SEQ ID NO: 26)
  • ACGPSTSG (SEQ ID NO : 29)
  • ACPGTSTG (SEQ ID NO 30)
  • ACGTGTSS (SEQ ID NO 31)
  • the native pro-peptide of a glucoamylase e.g., the A . niger
  • Gl or G2 AMG is cleaved of by a kex2-like protease (dibasic protease) .
  • 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.
  • 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.
  • NVIPPR SEQ ID NO: 33
  • NPPIRP SEQ ID NO: 34
  • NVIPRP SEQ ID NO: 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.
  • 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.
  • 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- ⁇ -helix makers.
  • the glucoamylase variant comprises peptide extension, which is capable of forming a covalent binding to the mature part of the parent glucoamylase.
  • 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.
  • 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.
  • the peptide extension linked to the parent glucoamylase may advantageously be one of the following extensions :
  • ASPPSTS Ala-Ser-Pro-Pro-Ser-Thr-Ser
  • ACPPSTS Ala-Cys-Pro-Pro-Ser-Thr-Ser
  • a tripeptidyl aminopeptidase 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 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 aminopeptidase 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.
  • TPAP tripeptidyl aminopeptidase
  • the glucoamylase variant of the invention is prepared by i) modifying the nucleotide, preferably DNA, sequence encoding the parent glucoamylase so as to encode the desired peptide extension applied to the N-terminal end of the parent glucoamylase (e.g.
  • nucleic acid preferably DNA
  • peptide extension at the relevant location in the nucleic acid (preferably DNA) sequence encoding the parent glucoamylase)
  • resulting modified nucleic acid preferably DNA
  • the term "linked at” is intended to indicate that the extension is fused to the N-terminal end (e.g. last amino acid residue) of the mature glucoamylase.
  • 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.
  • 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) .
  • 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 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 sequence (s) will be described below.
  • PCR polymerase chain reaction
  • a glucoamylase variant according to the invention 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.
  • 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.
  • 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.
  • 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.
  • 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 glucoamylase, 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.
  • 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.
  • 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 during 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 cells such as cells of S. cereviciae, have been found to be suitable host cells.
  • 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.
  • Aspergillus niger glucoamylases Gl and G2 disclosed in Boel et al .
  • Glucoamylases Gl 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.
  • parent glucoamylases include AMG from Novo Nordisk, and also glucoamylase from the companies Genencor, Inc. USA, and Gist-Brocades, Delft, The Netherlands.
  • 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
  • the mature part of a polypeptide 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 least 97%, and most preferably at least 99% with the mature part of the amino acid sequence shown in SEQ ID NO 1.
  • the variant of the invention 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.
  • the parent homologous glucoamylase comprises a glucoamylase from a microorganism.
  • 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.
  • the parent glycoamylase is the Aspergillus niger Gl glucoamylase (Boel et al . (1984), EMBO J. 3 (5), p. 1097-1102.
  • the parent glycoamylase may be a truncated glucoamylase.
  • the DNA sequence encoding a parent glucoamylase may be isolated from any cell or microorganism producing the glucoamylase in question, using various methods well known in the art.
  • a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the glucoamylase to be studied.
  • labeled oligonucleotide probes may be synthesized and used to identify glucoamylase-encoding clones from a genomic library prepared from the organism in question.
  • a labelled oligonucleotide probe containing sequences homologous to another known 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.
  • 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.
  • oligonucleotides are synthesized, e . g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • 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 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.
  • 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.
  • suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus , Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans , Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis , Streptomyces lividans or Strep tomyces 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 .
  • a glucoamylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be intro- pokerd using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
  • a single-stranded gap of DNA, the glucoamylase-encoding sequence is created in a vector carrying the glucoamylase gene.
  • 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.
  • 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.
  • 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:
  • Step (b) of the above method of the invention is preferably performed using doped primers, as described in the working examples herein (vide infra) .
  • the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis.
  • 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.
  • a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) 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.
  • 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.
  • 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 ligase as deemed appropriate.
  • the doping is carried out using "constant random doping", in which the percentage of wild-type and mutation in each position is predefined.
  • 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.
  • the doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided.
  • DOPE program which, inter alia, ensures that introduction of stop codons is avoided.
  • PCR-generated mutagenesis 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 .1 , 1989, pp. 11-15).
  • a mutator strain of E. coli (Fowler et al . , Molec . Gen. Genet., 133, 1974, pp.
  • S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA 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.
  • 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 .
  • 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.
  • 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 during the mutagenesis treatment .
  • suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, 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.
  • the random mutagenesis may be advantageously localized to a part of the parent glucoamylase in question. This may, e . g. , be 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 been elucidated and related to the function of the enzyme.
  • the localized, or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art.
  • 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.
  • a DNA sequence encoding the variant produced by methods described above, or by any alternative 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.
  • 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 chromosome (s) into which it has been integrated. Examples of suitable expression vectors include pMT838.
  • the DNA sequence should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA 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 .
  • Suitable promoters for directing the transcription of the DNA sequence encoding a glucoamylase variant of the invention, especially in a bacterial host are the promoter of the lac operon of E. coli , the Streptomyces coelicolor agarase gene dagrA promoters, the promoters of the Bacillus licheniformis ⁇ -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.
  • 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 ⁇ -amylase, A . niger acid stable ⁇ -amylase, A . niger glucoamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A . oryzae triose phosphate isomerase or A . nidulans acetamidase.
  • TPI triose phosphate isomerase
  • the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the ⁇ -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.
  • 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 pUC19, pACYC177, pUBHO, 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 dal genes from B . subtilis or B . licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • 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.
  • 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 cell of the invention 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 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.
  • a microbial cell e.g. a bacterial or a fungal (including yeast) cell.
  • suitable bacteria are Gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, 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 lus 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 wi th Gibberella roseum and Gibberella roseum f. sp .
  • the host cell is a protease deficient of protease minus strain.
  • 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 known per s .
  • Aspergillus as a host micro-organism is described in EP 238 023 (Novo Nordisk A/S) , the contents of which are hereby incorporated by reference .
  • 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 conventional medium suitable for growing the host cell in question and obtaining expression of the glucoamylase variant of the invention. 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.
  • the present invention provides a method of using glucoamylase variants of the invention for producing glucose and the like from starch.
  • 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 ⁇ 6) glucosidic bonds.
  • the partial hydrolysis of the precursor starch utilizing ⁇ - amylase provides an initial breakdown of the starch molecules by hydrolyzing internal ⁇ - (1 ⁇ 4) -linkages .
  • the initial hydrolysis using ⁇ -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 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.
  • D.E. dextrose equivalent
  • the step of further hydrolyzing the release of D-glucose 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.
  • the temperature of the substrate liquid is dropped to between 55° and 60 °C.
  • the pH of the solution is dropped from 6 to 6.5 to a range between 3 and 5.5.
  • 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.
  • thermostable glucoamylase variant of the invention saccharification processes may be carried out at a higher temperature than traditional batch saccharification processes.
  • 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 processes (described above) and for continuous saccharification processes .
  • 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) 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.
  • the T 12 (half- time, as defined in the "Materials and Methods" section) is improved.
  • 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.
  • the risk of microbial contamination is also reduced when carrying the saccharification process at temperature above 63°C.
  • glucoamylase variants of the invention include the processes described in JP 3-224493; JP 1-191693 ;JP 62-272987; and EP 452,238.
  • the glucoamylase variant (s) of the invention may be used in the present inventive process in combination with an enzyme that hydrolyzes only ⁇ - (1 ⁇ 6) -glucosidic bonds in molecules with at least four glucosyl residues.
  • 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.
  • the invention relates to the use of a glucoamylase variant of the invention in a starch conversion process .
  • 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 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.
  • the invention also relates to a method for improving the thermostability of a parent glucoamylase by making an extension at the N-terminus.
  • the extension comprises a peptide extension.
  • Micro-organi sms Strain: Saccharomyces cerevisiae YNG318: MAT ⁇ leu2- ⁇ 2 ura3-52 his4-539 pep4- ⁇ l [cir+]
  • Plasmi s Plasmid encoding the truncated Aspergillus niger glucoamylase G2.pJSO026: (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
  • the expression plasmid pJS026, is derived from pYES
  • the DNA fragments and the opened vectors were mixed and transformed into the yeast Saccharomyces cerevisiae YNG318 by standard methods .
  • AGU One Novo Amyloglucosidase Unit
  • 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-HCl, pH 7.0. Centrifugation was performed for 15 min. at 1000 g and the protoplasts were collected from the top of the MgS0 4 cushion. 2 volumes of STC (1.2 M sorbitol,
  • Fed batch fermentation is performed in a medium comprising maltodextrin as a carbon source, urea as a nitrogen source and 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 5.0 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, ultrafiltration, clear filtration and germ filtration. Further purification may be done by anionexchange chromatographic methods known in the art .
  • the culture broth is filtrated and added ammoniumsulphate (AMS) to a concentration of 1.7 M AMS and pH is adjusted to pH 5.
  • AMS ammoniumsulphate
  • Precipitated material is removed by centrifugation on the solution containing glucoamylase activity is applied on a Toyo Pearl Butyl column previously equilibrated in 1.7 M AMS, 20 mM 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.
  • 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.
  • the T 1/2 is measured by incubating the glucoamylase (0.18- 0.36 AG/g DS) in question in 30% 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) .
  • T 1/2 was the period of time until which the % relative activity was decreased to 50%.
  • Residual enzyme activity pNPG method
  • 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.
  • 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 ml sample + 1 ml water + 3 ml pNPG reagent B: 0.5 ml sample + 1 ml AMG standard + 3 ml borate solution Place HS and H in a 50°C 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 pAMGY vector was constructed as follows: The lipase gene in pJSO026 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 pJSO026 plasmid was digested with Xbal and Smal at 37°C for 2 hours and the PCR amplicon was blunt ended using the Klenow fragment and then digested with Xbal .
  • the vector fragment and the PCR amplicon were ligated and transformed into E. coli by electrotransformation.
  • the resulting vector is designated pAMGY.
  • the expression plasmid pJS037 is described in WO 97/04079 and WO 97/07205. It 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, (1982), J. Mol. Appl Genet., 1, 419-434), and deleting a part of the URA3 promoter.
  • TPI triose phosphate isomerase
  • pLaC103 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 Gil form of AMG. The construction takes place in several steps, out lined below.
  • pT7-212 (EP37856/ US patent no. 5162498) is cleaved with Xbal, blunt-ended with Klenow DNA polymerase and dNTP.
  • the resulting vector fragment is purified from an agarose gel -electrophoresis and ligated with the 2.05 kb EcoRl-EcoRV fragment of pBoel53, thereby recreating the Xbal site in the EcoRV end of the AMG encoding fragment in the resulting plasmid pG2x.
  • the resulting plasmid pT7GII was submitted to a BamHI cleavage in presence of alkaline phosphatase followed by partial Sphl cleavage after inactivation of the phosphatase. From this reaction was the 2489 bp Sphl-BamHI fragment, encompassing the
  • the resulting plasmid is pLaC103.
  • thermostable glucoamylase variants Screening for thermostable glucoamylase variants
  • thermostable filter assay The libraries are screened in the thermostable filter assay described below.
  • 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, 1% agarose, 50 mM NaAc, pH 4.5.
  • the assay plates with Protran filters are marked the same way as the cellulose acetate 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 GmbH, 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.
  • Random mutagenesis may be carried out using the following steps :
  • Suitable dope algorithms for use in step 6 are well known in the art.
  • One such algorithm is described by Tomandl , D. et al . , 1997, Journal of Co puter-Aided Molecular Design 11:29-38.
  • 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 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) .
  • PCR reaction 9-15 The DNA from PCR reaction 1 together with either DNA from PCR reaction 2-8 were used as templates in the PCR reactions using 4244 as 5' primer and KB14 as 3' primer. These final PCR fragments were used in an in vivo recombination in yeast together with pJSO026 cut with the restriction enzymes Smal (or BamHI) and Xbal (to remove the coding region and at the same time create an overlap of about 75 bp in each end to make a recombination event possible) .
  • Smal or BamHI
  • Xbal to remove the coding region and at the same time create an overlap of about 75 bp 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)
  • AMI2 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)
  • AMI3 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 TGG TTG AGC-3' (SEQ ID NO : 5)
  • AMI5 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS NNS GCG ACC TTG GAT TCA TGG TTG AGC-3' (SEQ ID NO: 6)
  • AMI6 5'-GCA AAT GTG ATT TCC AAG CGC NNS NNS GCG ACC TTG GAT TCA TGG TTG AGC-3' (SEQ ID NO: 7)
  • AMI7 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)
  • 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.
  • yeast KR 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.1998, PNAS 95 (18): 10384-10389). Alternatively introduction of P in front of KR will reduce the % of cleaved molecules .
  • the pro-sequence 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 .
  • 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 e-di r cted mutagenesis For the construction of variants of the AMG G2 enzyme (SEQ ID NO: 11) the commercial kit, Chameleon double-stranded, site-directed mutagenesis kit was used according to the manufacturer's instructions .
  • Tjie gene encoding the AMG G2 enzyme in question is located on pENI1542 prepared by cutting the plasmid pIVI9 with BamHl/XhoI (Cleaving out the coprinus peroxidase gene) and cloning in a AMG G2 containing per fragment (cut Bglll/Sall) , made by the use of the pLaC103 (containing the G2 cDNA) as template and the two 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 Seal site of the Ampicillin gene of pNEI1542 was changed to a Mlul site by use of the following primer:
  • ACPPSTS 137767 (SEQ ID NO: 16) (5'P-GTGATTTCCAGCGGTGCCCGCCGTCCACGTCCGCGACCTTGGATTCATGG 3 ' )
  • D375C 137765 (SEQ ID NO: 18) (5'P-GTAGCATTGTATGTGCCGTGAAGAC 3 ' )
  • S431C 146826 (SEQ ID NO: 19) (5'P- CCGTCGTAACTGCGTCGTGCCTGC 3 ' )
  • E299C 146828 (SEQ ID NO: 20) (5'P-GTCTCAGTGACAGCTGCGCTGTTGCGGTG3 ' )
  • A479C 146829 (SEQ ID NO: 21) (5'P-CCACTACGACGTGCACCCCCACTGG 3 ' )
  • T480C 146830 (SEQ ID NO: 22) (5'P-CTACGACGGCTTGCCCCACTGGATCC 3 ' )
  • P481C 146831 (SEQ ID NO: 23) (5'P-CGACGGCTACCTGCACTGGATCCGGC 3 )
  • S8C 146827 (SEQ ID NO: 24) (5'P-TGGATTCATGGTTGTGTAACGAAGCGACC 3 ' )
  • 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.
  • the library may be screened in the thermostability filter assays described in the "Material and Methods" section above.
  • thermal stability activity was measured at pH 4,5, 70 °C as described in Methods section above. Thermal stability at 70°C, pH 4.5
  • 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.
  • 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 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.

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CA2352046A1 (en) 2000-06-15
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JP2002531121A (ja) 2002-09-24
CN1329665A (zh) 2002-01-02
WO2000034452A1 (en) 2000-06-15

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