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WO1998003639A1 - Fabrication par genie genetique et a l'aide de proteines d'une glucoamylase permettant d'obtenir un ph optimal et d'accroitre la specificite d'un substrat ainsi que la stabilite thermique - Google Patents

Fabrication par genie genetique et a l'aide de proteines d'une glucoamylase permettant d'obtenir un ph optimal et d'accroitre la specificite d'un substrat ainsi que la stabilite thermique Download PDF

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Publication number
WO1998003639A1
WO1998003639A1 PCT/US1997/012983 US9712983W WO9803639A1 WO 1998003639 A1 WO1998003639 A1 WO 1998003639A1 US 9712983 W US9712983 W US 9712983W WO 9803639 A1 WO9803639 A1 WO 9803639A1
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glucoamylase
mutation
mutations
increased
mutant
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PCT/US1997/012983
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English (en)
Inventor
Martin Allen
Tsuei-Yun Fang
Yuxing Li
Hsuan-Liang Liu
Hsiu-Mei Chen
Pedro Coutinho
Richard Honzatko
Clark Ford
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Iowa State University Research Foundation, Inc.
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Priority to EP97936193A priority Critical patent/EP0970193A1/fr
Priority to JP10507223A priority patent/JP2000515377A/ja
Priority to AU38923/97A priority patent/AU3892397A/en
Publication of WO1998003639A1 publication Critical patent/WO1998003639A1/fr
Priority to US09/236,063 priority patent/US6537792B1/en

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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 field of the invention relates to mutations to produce a fungal glucoamylase enzyme that is more selective for the production of glucose rather than the o ⁇ - l , 6 linked disaccharide isomaltose, is more thermostable, and has increased pH optimum and produces increased amounts of glucose compared to wildtype enzymes .
  • Glucoamylase (EC 3.2.1.3) is a carbohydrase . Dis- covered in 1951, it is an exo-hydrolase that cleaves D- glucose from the nonreducing ends of maltooligosaccharides, attacking ⁇ -(l,4)-, and at a much slower rate, ⁇ - (1 , 6) -glucosidic bonds. It is one of more than one hundred carbohydrases (EC 3.2.1) that cleave O-glycosidic bonds of either a- or ⁇ - configuration.
  • Glucoamylase is primarily used in industry for the production of high- fructose corn sweeteners in a process that involves 1) cv-amylase to hydrolyze starch to maltooligosaccharides of moderate length (dextrin) ; 2) Glucoamylase to hydrolyze dextrin to glucose; and 3) glucose isomerase to convert glucose to fructose.
  • Corn sweeteners have captured over 50% of the U. S. sweetener market, and the three enzymes used to make them are among the enzymes made in highest volume .
  • glucose produced by glucoamylase can be crystallized or used in fermentation to produce organic products such as citric acid, ascorbic acid, lysine, glutamic acid or ethanol for beverages and fuel .
  • organic products such as citric acid, ascorbic acid, lysine, glutamic acid or ethanol for beverages and fuel .
  • glucoamylase has been successfully used for many years, it would be a more attractive product if it produced higher amounts of glucose instead of disaccharides, if it were more stable, and if it could be used in the same vessel with glucose isomerase.
  • Glucoamylase does not give 100% yield of glucose from dextrin because it makes various di- and trisaccharides, especially isomaltose and isomaltotriose, from glucose [Nikolov et al . , 1989] . These products, formed at high substrate concentrations, result from the ability of glucoamylase to form ⁇ - (1, 6) -glucosidic bonds. Glucoamylase is not as thermostable as either ⁇ -amylase or glucose isomerase. The optimum pH of GA (pH4-4.5) is lower than that of ⁇ amylase (pH5.5-6.5) and glucose isomerase (pH7-8) . Therefore glucoamylase hydrolysis must be done separately from the other enzymatic reactions in a different vessel and at lower temperatures, causing higher capital costs. Glucoamylase from the filamentous fungus
  • Aspergillus niger is the most widely used glucoamylase, and its biochemical properties have been extensively characterized.
  • This enzyme is found mainly in two forms, GAI (616 amino acids; referred to as AA hereinafter) and GAII (512 AA) , differing by the presence in GAI of a 104 -AA C-terminal domain required for adsorption to native starch granules [Svensson et al . , 1982; Svensson et al . , 1989] .
  • Both forms have a catalytic domain (AA1-440) followed by a Ser/Thr-rich, highly O-glycosylated region (AA441-512) [Gunnarsson et al .
  • the first thirty residues of this region are included in the three-dimensional structure of the enzyme [Aleshin et al . , 1994; 1996; Stoffer et al., 1995] ; they wrap around the catalytic domain like a belt .
  • these regions are AA35-59, AA104- 134, AA162-196, and AA300-320.
  • the second and third regions partially or completely overlap the three regions of homology to ⁇ -amylases [Svensson, 1988] .
  • the raw starch binding domain (AA512-616) has high homology to similar domains from several starch- degrading enzymes [Svensson et al . , 1989] .
  • Kinetic analysis showed that the substrate binding site is composed of up to seven subsites [Savel'ev et al . , 1982] with hydrolysis occurring between subsites 1 and 2.
  • the pK a 's of hydrolysis, 2.75 and 5.55 [Savel'ev and Firsov, 1982] , suggest that carboxylic acid residues at subsites 1 and 2 provide the catalytic acid and base for hydrolysis.
  • Glucoamylases from A. niger [Svensson et al . , 1983; Boel et al . , 1984] and Aspergillus awamori [Nunberg et al . , 1984] have been cloned and sequenced, and have identical primary structures. Innis et al . [1985] and more recently Cole et al . [1988] have developed vectors (pGAC9 and pPM18, respectively) for glucoamylase expression in yeast, allowing convenient manipulation and testing of glucoamylase mutants.
  • a fungal glucoamylase (1, 4- ⁇ r-D-glucan glucohydrolase; EC 3.2.1) with decreased thermal inactivation (increased thermostability) and reduced isomaltose formation provided by the mutation Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two is provided.
  • Cumulative thermostability is also provided for GA by including the mutation Asn20Cys coupled with Ala27Cys and at least one mutation from Table 13.
  • An engineered GA including Ser30Pro, Glyl37Ala, and Asn20Cys coupled with Ala27Cys provides even more thermostability.
  • Cumulative thermostability is also provided for GA by including the mutation Asn20Cys coupled with Ala27Cys and at least two mutations from Table 13.
  • the present invention also provides a fungal glucoamylase with reduced isomaltose formation including an Asn20Cys coupled with Ala27Cys mutation (S-S mutation) and at least one mutation selected from Table 14.
  • S-S mutation Asn20Cys coupled with Ala27Cys mutation and a 311-314Loop (also referred to as 300Loop) mutation are included in an engineered GA.
  • the engineered glucoamylase with reduced isomaltose formation includes Asn20Cys coupled with Ala27Cys mutations Ser30Pro and Glyl37Ala.
  • the present invention also provides engineered fungal glucoamylase including a 311-314Loop mutation whereby reduced isomaltose formation is provided by the mutation.
  • fungal glucoamylase including a 311-314Loop mutation and at least one mutation from Table 14 are prepared whereby cumulative reduced isomaltose formation is provided by the additional mutation.
  • the present invention provides a fungal glucoamylase including a mutation Ser411Ala whereby increased pH optimum and reduced isomaltose formation is provided by the mutation.
  • the Ser411Ala mutation is combined with at least one mutation from Table 15 whereby cumulative increased pH optimum is provided by the mutations.
  • an engineered fungal glucoamylase includes a mutation Ser411Ala and a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair whereby increased thermal stability, increased pH optimum and reduced isomaltose formation are provided by the mutations.
  • a fungal glucoamylase is engineered to include a Ser411Ala mutation and a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair and a 311-314Loop mutation whereby increased thermal stability, increased pH optimum and reduced isomaltose formation are provided by the mutations.
  • the present invention provides a method to obtain a fungal glucoamylase with reduced isomaltose formation by designing mutations to decrease the - ( 1 , 6 ) - glucosidic linkage affinity of GA.
  • the present invention also provides a method to obtain a fungal glucoamylase with decreased thermal inactivation by designing mutations to decrease the enzyme's conformational entropy of unfolding and/or increase stability of ot-helices, increase disulfide bonds, hydrogen bonding, electrostatic interactions, hydrophic interactions, Vanderwalls interactions and packing compactness .
  • the present invention also provides a fungal glucoamylase with increased pH optimum including changing the polarity, charge distribution and hydrogen bonding in the microenvironment of the catalytic base Glu400.
  • the present invention also provides a method of genetically engineering glucoamylase carrying at least two cumulatively additive mutations.
  • Individual mutations are generated by site-directed mutagenesis. These individual mutations are screened and those selected which show increased pH optimum and which show decreased irreversible thermal inactivation rates or reduced isomaltose formation.
  • Site directed mutagenesis is then performed to produce enzymes carrying at least two of the isolated selected mutations.
  • the engineered enzymes are screened for cumulatively additive effects of the mutations on thermal stabilizing or reduced isomaltose formation by the produced enzymes carrying at least two of the isolated selected mutations.
  • the engineered enzyme is screened for cumulatively additive effects of both of the mutations on pH optimum, thermostability and/or reduced isomaltose formation by the produced enzymes carrying at least two of the isolated selected mutations.
  • Vectors for each of the mutations and mutation combinations are also provided by the present invention as well as host cells transformed by the vectors.
  • FIGURE 1 is a graph showing the relationship between temperature and Jed for wild-type (•) and proline substituted mutant GA' s : S30P ( ⁇ ) , D345P (v), E408P (O) in Example 1.
  • FIGURE 2 is a graph showing effect of temperature on first-order ther oinactivation rate coefficients of wild-type (O) , A27C (•) , N20C (v), A27C/N20C ( ⁇ ) , A471C/T72C (D) , A27C/N20C/G137A ( ⁇ ) , A27C/N20C/S436P (O) AND G137A/S436P ( ⁇ ) glucoamylases measured in pH 4.5 buffer.
  • FIGURE 3 is a graph showing initial reaction rates of wild-type (O) , A27C/N20C (•) , A471C/T72C (v) and A29C/N20C/G137A ( ⁇ ) glucoamylases with 4% maltose in 0.05 M sodium acetate (pH 4.5) as substrate at temperatures from 60°C to 76°C.
  • FIGURE 4 is a graph showing the effect of temperature on the activity of wildtype and mutant GA. Error bars represent the standard deviation from three assays. Wildtype (•) , S30P/G137A (D) , S-S/S30P/G137A ( A ) .
  • FIGURE 5A-C are graphs showing the effect of temperature on irreversible thermal inactivation rate coefficients of wildtype and mutant GA.
  • Fig. 5A Wildtype (•) , S30P ( ⁇ ) , G137A ( ⁇ ), S30P/G137A (D) ;
  • Fig. 5B Wildtype (•) , S30P ( ⁇ ) , S-S (hexagon) , S-S/S30P (filed circle with empty center) ;
  • Fig. 5C Wildtype (•) , S30P/G137A (O) , S-S/S30P (filed circle with empty center) , S-S/S30P/G137A (A) .
  • FIGURE 6A-B are graphs showing saccharification of 28% (w/v) Maltrin M100 by wildtype (•) , S30P/G137A (D) and S-S/S30P/G137A (A) .
  • FIGURE 7 is a graph showing the 30% DE 10 maltodextrin saccharification of wildtype ( ⁇ ) and mutant glycoamylases : 300I_oop ( ⁇ ) , S30P/G137A (A) , S-S (•) , S30P/G137A/300Loop (x), S-S/300Loop ( ⁇ ) , at 55°C, enzyme concentration was 166.67 ⁇ g/mL in each reaction.
  • FIGURE 8 is a graph showing production of isomaltose by wildtype (•) and mutant glucoamylases: Y116W ( ⁇ ) , Y175F (A), R241K (T) , S411A ( ⁇ ) , S411G (hexagon) , during glucose condensation at 55°C with 30% (w/v) D-glucose in 0.05M sodium acetate buffer at pH4.4 with 0.02% sodium azide for 12 days.
  • FIGURE 9 is a graph showing the production of glucose by wildtype (•) and mutant glucoamylases: Y116W ( ⁇ ) , Y175F (A), R241K ( ⁇ ), S411A ( ⁇ ) , S411G (hexagon), during hydrolysis of DE 10 maltodextrin at 55°C with 28% (w/v) maltodextrin in 0.05M sodium acetate buffer at pH4.4 with 0.02% sodium azide for 12 days.
  • FIGURE 10 is a graph showing the initial rates of glucose production by wildtype (•) and S411A ( ⁇ ) glucoamylases during DE 10 maltodextrin hydrolysis at different pH values. Hydrolysis was performed at 36 °C with 28% (w/v) maltodextrin in 25mM citrate-phosphate buffer at indicated pHs with 0.02% sodium azide for 4 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • the present invention provides mutations for increased thermal stability, increased pH optimum and reduced isomaltose formation in the glucoamylase from fungal species which may provide increased glucose yields compared to wildtype glucoamylase.
  • Predicted structure and known sequences of glucoamylase are conserved among the fungal species [Coutino et al, 1994] .
  • Aspergillus awamori glucoamylase (1, 4- ⁇ -D-glucan glucohydrolase; EC 3.2.1.3; referred to as GA herein; SEQ ID No:l
  • any other fungal species including Aspergillus species glucoamylase can be used.
  • the numbering of the glucoamylase amino acids herein is based on the sequence of the exemplar Aspergillus awamori . Equivalent amino acid residue numbers are determined differently for different fungal species as is known in the art [Coutino et al . , 1994].
  • the present invention provides a fungal glucoamylase with decreased thermal inactivation (increased thermostability) and decreased isomaltose formation provided by engineering the inclusion of a mutation pair Asn20Cys coupled with Ala27Cys which forms a disulfide bond between them (this mutation is abbreviated as Asn20Cys/Ala27Cys or S-S) . Additional mutations providing decreased thermal inactivation are set forth in Summary Table 13.
  • Cumulative thermostability is also provided for GA by including at least two of the mutations in the enzyme as for example including mutations Ser30Pro and Glyl37Ala. Another example is to engineer S-S with Asn20Cys/Ala27Cys in the enzyme or to pair Glyl37Ala with S-S. Further, combinations of the individual mutations set forth in Table 13, particularly with S-S coupled with Ser30Pro also provide cumulative thermostability. In general two mutation combinations are made but triple mutations can also be constructed. As for example, an engineered GA including the three mutations: Ser30Pro, Glyl37Ala, and Asn20Cys/Ala27Cys provides even more thermostability.
  • Asn20Cys coupled with Ala27Cys is meant a pair of mutations which is abbreviated as "S-S” or Asn20Cys/Ala27Cys and between which is formed a disulfide bond as described herein in the Examples. In general, this is referred to as a single mutation since both are required to form the disulfide bond.
  • the present invention also provides a fungal glucoamylase with reduced isomaltose formation and increased glucose yield including the Asn20Cys/Ala27Cys mutation (S-S mutation) and at least one mutation selected from Table 14.
  • S-S mutation the Asn20Cys/Ala27Cys mutation and the 311-314Loop
  • the engineered glucoamylase with reduced isomaltose formation includes Asn20Cys/Ala27Cys and with mutations Ser30Pro and Glyl37Ala.
  • a glucoamylase with the 311-114 loop mutation is constructed to provide reduced isomaltose formation.
  • the 311-314Loop mutation is meant an insertional GA mutant with the sequence Tyr311-Tyr312-Asn313 -Gly314 ⁇ Tyr311-Asn-Gly-Asn-Gly-Asn- Ser-Gln-Gly314 (311-314 Loop; SEQ ID No:2) .
  • the present invention provides a fungal glucoamylase including a Ser411Ala mutation whereby increased pH optimum and reduced isomaltose formation is provided by the mutation.
  • Ser411Ala mutation is combined with at least one mutation from Table 15 whereby cumulative increased pH optimum is provided by the combined mutations.
  • Ser411Ala mutation is combined with at least one mutation from Table 14 whereby cumulative reduced isomaltose formation is provided by the mutations.
  • an engineered fungal glucoamylase includes a Ser411Ala mutation and the mutation pair Asn20Cys/Ala27Cys forming a disulfide bond between them whereby increased thermal stability, increased pH optimum and reduced isomaltose formation are provided by the mutations.
  • a fungal glucoamylase including a Ser411Ala mutation and a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair and a 311-314Loop mutation whereby increased thermal stability, increased pH optimum and reduced isomaltose formation are provided by the combination of mutations. Mutations are indicated by the amino acid being replaced followed by the residue number followed by the replacing amino acid. Amino acids are abbreviated either with the three letter code or single letter code. Mutations are generated using site directed mutagenesis as is known in the art. The sequence and residue number are from the Wildtype (WT) or nonmutant enzyme. Biochemical characterization is performed as described herein below and in the Examples. The Examples provide exemplars of the analysis for an individual mutation to determine it's characteristics and provide exemplars of analysis for combinations of mutations to determine if the combination provides a cumulative effects.
  • thermostability or decreased thermal inactivation
  • the present invention provides a method to obtain fungal glucoamylases with decreased thermal inactivation by designing mutations to decrease the rate of irreversible thermal inactivation at temperatures between 65°C and 77.5°C compared to wildtype. This is accomplished by designing glucoamylases with decreased thermal inactivation by designing mutations to decrease the enzyme's conformational entropy of unfolding and/or increase stability of ⁇ -helices, increase disulfide bonds, hydrogen bonding, electrostatic interactions, hydrophic interactions, Vanderwalls interactions and packing compactness .
  • the causes of irreversible inactivation at high temperatures include 1) aggregation, 2) the formation of incorrect structures, 3) the destruction of disulfide bonds, 4) deamidation (especially of Asn at Asn-Gly sequences) , and 5) cleavage of Asp-X peptide linkages. It is apparent that replacement of even one residue can make a large difference in protein thermostability [Matsumura and
  • thermostability due to the small increases in free energy (20-30 kJ/mol) usually required to stabilize protein tertiary structures [Nosoh and Sekiguchi, 1988] .
  • Genetic engineering to increase thermostability (or to decrease irreversible thermoinactivation) of enzymes has been successful in several cases [Perry and Wetzel, 1984; Imanaka et al . , 1986; Ahearn et al . , 1987].
  • the mechanisms that govern thermostability are not fully understood, so that amino acid (AA) replacements that promote thermostability are not accurately predicted [Leatherbarrow and Fersht, 1986; Nosoh and Sekiguchi, 1988; Pakula and Sauer, 1989].
  • the method of the present invention allows for more accurate prediction.
  • increased pH optimum is meant that the enzyme is functional at a higher pH, above that of wildtype.
  • the present invention also provides a method to design a fungal glucoamylase with increased pH optimum by changing the polarity, charge distribution and hydrogen bonding in the microenvironment of the catalytic base Glu400.
  • mutants S411G and S411A were designed to remove the hydrogen bond between Ser411 and Glu400 (see Example 8) .
  • increased selectivity is meant that there is decreased isomaltose formation due to decrease in the production of undesirable ex- (1 , 6) -linked byproducts (reversion products) at high glucose concentrations [Lee et al . , 1976] .
  • GA hydrolyzes and synthesizes both ⁇ -(l,4) and cv-(l,6) glucosidic bonds.
  • Increasing selectivity indicates that the enzyme synthesizes ⁇ l , 6 linked products at a lower rate than wildtype as shown by reduced levels of isomaltose formation in condensation reactions with 30% glucose as a substrate compared to wildtype GA. Additionally, improved selectivity may result in increased glucose yields in saccharification reactions using 28% DE 10 maltodextran as a substrate.
  • the present invention provides a method to obtain a fungal glucoamylase with reduced isomaltose formation by designing mutations to decrease the or- (1,6)- glucosidic linkage affinity of GA. That is mutations are designed in the active site to reduce isomaltose formation due to glucose condensation. The mutations are designed to have decreased ability to synthesize isomaltose while maintaining at least partial wildtype ability to digest ⁇ l-4 linked substrates resulting in a lower ratio of isomaltose formation to glucose formation than wildtype. These mutations are made at positions that are not completely conserved based on homology analysis.
  • the solved three-dimensional structure of the catalytic domain of glucoamylase from Aspergillus awamori var X100 which has about 95% homology with the corresponding regions of GAs from Aspergillus awamori and Aspergillus niger [Coutinho & Reilly, 1994] , contains thirteen alpha-helices, twelve of which are arranged in pairs forming an alpha/alpha barrel [Aleshin et al . , 1992, Aleshin et al . , 1994].
  • the active site is located in the cavity of the barrel center.
  • homology analysis of thirteen amino acid sequences of glucoamylases showed that five conserved regions define the active site [Coutinho & Reilly, 1994] .
  • the mechanism of GA catalysis involves two carboxyl groups [Hiromi et al . , 1966], Glul79 and Glu400 (in Aspergillus awamori or Aspergillus niger) [Frandsen et al., 1994, Harris et al . , 1993, Sierks et al., 1990].
  • Glul79 protonates the oxygen in the glycosidic linkage, acting as general acid acatalyst, and Glu400 activates water (Wat500) for nucleophilic attack at carbon C-1, acting as a general base catalyst [Frandsen et al., 1994].
  • the crystal structures of glucoamylase complexed with the pseudotetrasaccharides (acarbose and D-gluco-dihydroacarbose) showed that there are two different binding conformers, pH 4 -type and pH 6 -type, for pseudotetrasaccharides at pH 4 [Aleshin et al . , 1996, Stoffer et al . , 1995].
  • Binding of the first two sugar residues of the pseudotetrasaccharides is the same, but there is an extraordinary variation in binding of the third and fourth sugar residues of the pseudotetrasaccharides [Stoffer et al. , 1995] .
  • the substrate specificity of an enzyme is determined by its ability to form a stable complex with a ligand in both the ground state and the transition state.
  • the stability of the enzyme-ligand complex is affected by steric constraints, hydrogen bonding, van der Waal's and electrostatic forces, and hydrophobic contacts [see generally Fersht, 1985 Enzyme Structure and Mechanism, 2 nd edition, Freeman, San Francisco] .
  • Site-directed mutagenesis was used to construct mutations at residues 119 and 121 to alter the hydrogen bonding between enzyme and substrate .
  • Mutant S119E was designed to strengthen the hydrogen bond between the enzyme and the fourth sugar residue of the substrate to stabilize the pH 6-type conformer, and to bring a negative charge near subsite 4 in order to increase electrostatic interactions in active site.
  • Mutant S119G was designed to remove the same hydrogen bond in order to destabilize the pH 6-type conformer.
  • Mutant S119W was designed to remove the same hydrogen bond and to increase the hydrophobic interactions between the enzyme and the pH 6-type conformer.
  • Glyl21 is highly conserved in all glucoamylase sequences except in Clostridium sp . G005 GA, which has high ⁇ -1,6 activity and in which Gly is replaced by Thr.
  • G121A was designed to introduce a Beta- carbon at position 121 to displace the 6 -OH group of the third sugar residue from its hydrogen bonding position.
  • the double mutant G121A/S411G was designed to investigate additivity of the two substrate specificity mutations. S411G is shown herein to reduce the ratio of initial rates of isomaltose production (from glucose condensation reactions) to that of glucose production (from the hydrolysis of DE 10 maltodextrin) .
  • 300Loop mutation According to the amino acid sequence homology study [Countinho and Reilly, 1994] , it was found that GAs from Rhizopus and some other fungal families have a longer amino acid sequence and form a larger loop or cavity in the S4 conserved region compared to A . niger or A . awamori GAs. Since single mutation events alone are unlikely to bring about substantial increase in the specificity of bond hydrolysis or synthesis, an insertional mutant GA was designed, designated 300Loop or 311-314Loop (SEQ ID
  • Rhizopus GA was the first enzyme to which the subsite theory was successfully applied [Himori et al., 1973].
  • the 300Loop mutation was designed to decrease the ⁇ - (1 , 6) -glucosidic affinity by introducing a larger loop into the S4 conserved region.
  • Tyrl75Phe Tyrl75 is within the third conserved region. The nearest distance between Tyrl75 and the fourth residue of inhibitor D-grluco-dihydroacarbose is 4.06 A [Stoffer et al., 1995] . Tyrl75 is replaced by Phe or Gin in several other glucoamylases. Changing Tyrl75 to Phe was designed to increase the hydrophobic interaction between enzyme and substrate.
  • Glyl21Ala Glyl21 is highly conserved in all glucoamylase sequences except in Clostridium sp . G005 GA, which has high or-1,6 activity and in which Gly is replaced by Thr.
  • G121A was designed to introduce a ⁇ -carbon at position 121 to displace the 6- OH group of the third sugar residue from its hydrogen bonding position.
  • Gl ⁇ l21Ala with S411G (generally indicated as G121A/S411G) :
  • the double mutant was designed to investigate additivity (cumulative) of the two substrate specificity mutations.
  • S411G reduces the ratio of initial rates of isomaltose production (from glucose condensation reactions, see Examples) to that of glucose production (from the hydrolysis of maltodextrin 10) .
  • the present invention provides a method of engineering mutations for fungal glucoamylase and then preparing engineered enzymes carrying cumulatively additive mutations.
  • the initial step is to generate individual mutations by site directed mutagenesis and screen the individual mutations as described in the Examples. Those individual mutations which show decreased irreversible thermal inactivation rates or reduced isomaltose formation or increased pH optimum are then selected for combinational analysis. In general mutations are selected which have at least wildtype reaction rates.
  • Mutations are combined by site-directed mutagenesis to determine if their effects are additive as is discussed herein in the Examples.
  • Site directed mutagenesis to produce enzymes carrying at least two of the isolated selected mutations is performed as is known in the art .
  • These engineered enzymes are then screened for cumulatively additive effects on thermal stabilizing, pH optimum or reduced isomaltose formation.
  • the engineered enzymes carrying cumulative mutations are screened for cumulative effects on two or more of the parameters.
  • GA is purified from culture supernatants of 15-L batch fermentations by ultrafiltration, DEAE-Sephadex column chromatography, and column affinity chromatography using the potent inhibitor acarbose attached to a support [Sierks et al . , 1989] . Purities of the resulting preparations are tested by standard techniques such as SDS-polyacrylamide gel electrophoresis and isoelectric focusing with narrowband ampholytes . Protein are measured by absorbance at 280 nm or by Bradford's method [1976] . GA activity is measured by a glucose oxidase/o-dianisidine assay (Sigma) .
  • Selectivity is determined by any method known in the art but preferably by measuring the initial rate of isomaltose formation from 30% (w/v) glucose condensation reactions at pH 4.4 and 55°C in 0.05M sodium acetate buffer and then by measuring the initial rage of glucose formation in 30% (w/v) DE 10 altodextran hydrolysis reactions at pH 4.4 and 55°C 0.05M sodium acetate buffer. From the resulting initial rates, the ratio of isomaltose formation to glucose formation is calculated.
  • Thermostability is measured as is known in the art but preferably by incubating the enzyme at selected temperatures between 65°C and 77.5°C at 2.5°C intervals followed by activity analysis at 35°C using 4% maltose as substrate.
  • first-order decay is observed, as with WT GA, decay rate coefficients are determined.
  • Activation energies for decay are calculated from the rate coefficients at different temperatures.
  • pH optimum is measured as is known in the art but preferably at 45°C at 16 pH values, ranging for 2.2 to 7.0 using 0.025 M citrate-phosphate buffer with maltose or maltoheptaose as substrate.
  • Saccharification is measured as described in the Examples. Briefly, glucoamylase is incubated with DE 10 maltodextran as substrate in 0.05M sodium acetate buffer at pH 4.4 at 55°C. Samples are taken at various times from 0.5 to 288 hours and the production of glucose determined.
  • the present invention provides vectors comprising an expression control sequence operatively linked to the nucleic acid sequence of the various mutant sequences disclosed herein, combinations of mutations and portions thereof.
  • the present invention further provides host cells, selected from suitable eucaryotic and procaryotic cells, which are transformed with these vectors.
  • Vectors can be constructed containing the cDNA of the present invention by those skilled in the art and should contain all expression elements necessary to achieve the desired transcription of the sequences. Other beneficial characteristics can also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form. Examples are provided herein. Phagemids are a specific example of such beneficial vectors because they can be used either as plasmids or as bacteriophage vectors. Examples of other vectors include viruses such as bacteriophages, baculoviruses and retroviruses, DNA viruses, cosmids, plasmids, liposomes and other recombination vectors. The vectors can also contain elements for use in either procaryotic or eucaryotic host systems. One of ordinary skill in the art will know which host systems are compatible with a particular vector.
  • the vectors can be introduced into cells or tissues by any one of a variety of known methods within the art (calcium phosphate transfection; electroporation; lipofection; protoplast fusion; polybrene transfection; ballistic DNA delivery; lithium acetate or CaCl transformation) .
  • the host cell can be any eucaryotic and procaryotic cells, which can be transformed with the vector and which will support the production of the enzyme.
  • thermostable and selective mutants of fungal glucoamylase as well as methods of designing the mutations and screening for the cumulative effect of the mutations and vectors containing the mutations.
  • the methods used with and the utility of the present invention can be shown by the following non- limiting examples and accompanying figures.
  • S. cerevisiae C468 ( leu2-3 leu 2-112 his 3 -11 his 3 -15 mal ⁇ ) and the plasmid YEpPMl ⁇ were gifts from Cetus .
  • Acarbose was a gift from Miles Laboratories . All restriction enzymes were purchased from Promega as well as T4 DNA ligase and pGEM-7Z(+), an E. coli phagemid vector, were from Promega.
  • G 2 Maltose (G 2 ) , maltotriose (G 3 ) , maltotetraose (G 4 ) , alto- pentaose (G 5 ) , maltohexaose (G 6 ) , maltoheptaose (G 7 ) , glucose oxidase, peroxidase, and c.-naphthol were from Sigma.
  • Isomaltose (iG 2 ) was purchased from TCI America.
  • DE 10 Maltodextrin with the average degree of polymerizations (DP) of 10, 6, and 4, respectively, were from Grain Processing Corporation.
  • High- performance thin-layer chromatographic (HPTLC) plates (LHPK silica gel 60 A, 20 x 10 cm) were from Whatman.
  • Site-directed mutagenesis Site-directed mutagenesis was performed according to the Muta-Gene phagemid in vi tro mutagenesis kit from Bio-Rad which is based on the method of Kunkel et al [1985] .
  • a 1.7 kb XhoI ⁇ BamHI DNA fragment coding for the glucoamylase catalytic domain was cloned into a pBluescript II KS(+) vector from Stratagene.
  • Oligonucleotides used as mutagenic primers are provided with the specific Example. The presence of the individual mutations was confirmed by sequencing and each mutated GA gene fragment was subcloned into YepPMl ⁇ [Cole, et al . , 1988] and transformed into S. cerevisiae .
  • Wild-type (WT) and mutant enzymes are produced by growing yeast at 30°C in 5.3 L SD + His media for 72 hours at pH 4.5 in a 5.0 L fermentor. After 48 hours, lOOg of dextrose and 22g of (NH 4 ) 2 S0 4 in 300ml H 2 0 is added as a supplement. Following growth, the culture is centrifuged to remove yeast cells, the supernatant is concentrated by ultrafiltration, diafiltered against 0.5 M NaCl/0.1 M NaOAc , pH 4.5 and purified by acarbose-sepharose affinity chromatography .
  • GA is eluted with 1.7 M Tris-Cl, pH 7.6, dialyzed against H 2 0, further concentrated by ultrafiltration and diafiltered against 0.05 M NaOAc buffer, pH 4.5.
  • the protein concentration is determined according to the Pierce bicinchoninic acid protein assay [Smith et al . , 1985] using bovine serum albumin as a standard.
  • Enzyme activity assays Enzyme activities were determined at 50°C using 4% maltose in 0.05 M NaOAc buffer pH 4.5 as substrate. One international unit (IU) of enzyme activity was defined as the amount of enzyme required to produce 1 ⁇ mol/min glucose at assay conditions. Following mixing enzyme with substrate, six 100 ⁇ l samples were removed at seven minute intervals over 42 minutes, the reaction stopped with 40 ⁇ l of 4.0 M Tris-Cl, pH 7.0 and the glucose concentration was determined according to the Sigma peroxidase-glucose oxidase/o dianisidine glucose assay kit.
  • IU international unit
  • Irreversible thermal inactivation Duplicate aliquotes of 40 ⁇ g/ml of purified wild-type and mutant enzymes were subjected to inactivation at six or more temperatures between 65° and 80°C at intervals of 2.5°C. Samples were removed at six different time points, immediately placed on ice and stored at 4°C for 24 hours. The residual activity of the inactivated samples along with a corresponding sample which had not been subjected to thermal inactivation, was determined as described above but at 35°C.
  • pH dependence of glucoamylase activity was measured at 45°C at 16 different pH values, ranging from 2.2 to 7.0, using 0.025 M citrate-phosphate buffer [Mcllvane, 1921] with maltose or maltoheptaose as substrate.
  • the ionic strength of the citrate-phosphate buffer was maintained at 0.1 by adding potassium chloride.
  • the pK values of free enzyme and enzyme-substrate complex were measured at substrate concentrations (i) smaller than 0.2 K m , so that the initial rate ( v) was proportional to k cat /K m , and (ii) higher than 10 K m , so that the initial rate ( v) was proportional to k cat [Sierks & Svensson,
  • H is the concentration of hydrogen ion
  • K ⁇ and K 2 are dissociation constants of catalytic groups of enzyme.
  • Glucose condensations reactions were performed at 35°C and 55°C with 30% (w/v) D-glucose as substrate in 0.05 M acetate buffer at pH 4.4 for 12 days with the addition of 0.02% sodium azide, used to inhibit microbial growth in the reaction mixtures.
  • the enzyme concentration was 2.64 ⁇ M for both wild-type and mutant GAs. Samples were taken at various times and the reactions were stopped by adding samples to the same volume of 1 M Tris-HCl buffer at pH 7.0.
  • High Performance Thin Layer Chromatography (HPTLC) and Imaging Densito etry were used to determine the production of isomaltose by a method modified from that described by Robyt et al.
  • the following example is an exemplar of the methods and procedures that are used in the analysis of an individual mutation of a glucoamylase.
  • Aspergillus awamori glucoamylase thermal stability three proline substitution mutations were constructed. These mutations were predicted to increase GA stability by decreasing the enzyme's conformational entropy of unfolding .
  • Aspergillus awamori glucoamylase ( ⁇ -1, 4-D-glucan glucohydrolase, EC 3.2.1.3; GA) is an enzyme which catalyses the release of 3-glucose from the non- reducing ends of starch and related oligosaccharides .
  • GA is used in, and defines the rate limiting step of, the commercial conversion of starch to high glucose syrups which may be converted to fructose syrups by glucose isomerase, or used in fermentations to produce ethanol.
  • GA is used industrially at 55°-60°C; at higher temperatures the enzyme is rapidly and irreversible inactivated. Therefore, a GA variant with increased thermostability would be advantageous industrially to decrease reaction times and/or to increase solids concentrations.
  • oligonucleotides were used as mutagenic primers: CAGAGTCCGCGCCCGGCACCCAAGCACCGTC (Ser30 ⁇ Pro) (SEQ ID No: 3), AAGTCCAGCGACACAGGTGTGACCTCCAACGAC (Asp345 ⁇ Pro) (SEQ ID No: 4) and CGAGCGGAAAGCTGC GGGCCATCAGACTTGTC (Glu408 ⁇ Pro) (SEQ ID No: 5) .
  • activation energies for thermal inactivation were calculated using transition state theory and melting temperatures (T ) , the temperature at which the enzyme was 50% inactivated after 10 minutes were computed (Table 1) .
  • T transition state theory and melting temperatures
  • proline substitution mutations had different thermostabilities when measured by their resistance to irreversible thermal inactivation.
  • Glu408 ⁇ Pro decreased, Asp345 ⁇ Pro did not significantly alter and Ser30 ⁇ Pro increased GA stability ( Figure 1 and Table 1) .
  • the Asp345 ⁇ Pro mutant GA did not demonstrate stability significantly different from wild-type GA. This is particularly unexpected since position 345 lies at the N-terminus of an ⁇ -helix 2 ; a position previously shown to be particularly favorable for proline substitution [Watanabe et al, 1994] .
  • the new disulfide bond formed by A27C/N20C connects the C- terminus of helix 1 (Asn20) and a turn where residue Ala27 is located, while A471C/T72C bridges the N- terminus of helix 3 and the end of the 30 -residue highly O-glycosylated belt region together.
  • the disulfide bonds are formed spontaneously after fermentation and have different effects on GA thermostability and catalytic activity.
  • Site-Directed Mutagenesis Site-directed mutagenesis was performed as described herein above.
  • Oligonucleotide primers used are: 5'-CGT ACT GCC ATC CTG TGT AAC ATC GGG GCG GA-3' (N20C, AAT ⁇ TGT) (SEQ ID No: 6) , 5' -ATC GGG GCG GAC GGT TGT TGG GTG TCG GGC GCG- 3' (A27C, GCT ⁇ TGT) (SEQ ID No: 7), 5'-CGA AAT GGA GAT TGC AGT CTC-3' (T72C, ACC ⁇ TGC) (SEQ ID N ⁇ :8), 5'-G AGT ATC GTG TGT ACT GGC GGC ACC-3' (A471C, GCT ⁇ TGT) (SEQ ID No: 9), with the underlined letters indicating the nucleotide mutations.
  • SDS-PAGE and Thio-titration SDS-PAGE was carried out using 0.75 mm thick 10% polyacrylamide gels following the method of Garfin [1990] .
  • GA for thio-titration, GA at 2 mg/ml concentration was denatured by boiling in denaturing solution containing 2% SDS, 0.08 M sodium phosphate (pH 8.0) and 0.5 mg/ml EDTA [Habeeb, 1972] with or without 50 mM DTT [Pollitt and Zalkin, 1983] for 10 min.
  • the denatured GA (reduced or non-reduced) was concentrated using Centricon 30 concentrators (Amicon, MA, USA) and the reduced GA was applied to Bio-spin 30 chromatography columns (Bio-Rad, CA, USA) pre-equilibrated with denaturing solution to remove DTT. The resulting solution as well as the non-reduced denatured GA sample were divided into two portions.
  • One portion was used for a protein concentration assay and the other portion was assayed for thio reduction by mixing with 4 mg/ml DTNB in denaturing solution with a 30:1 volume ratio, followed by incubation at room temperature for 15 minutes, and absorbance measurement at 412 nm with a molar absorptive value of 13,600 M ⁇ c "1 [Habeeb, 1972] .
  • GA Activity Assay As described herein above, maltose was used as substrate in enzyme kinetics studies, with concentrations ranging from 0.2 K m to 4 K m at 35°C and pH 4.5 as described previously [Chen et al . , 1994b] . Kinetics parameters were analyzed by the program ENZFITTER. In residual enzyme activity assays, the conditions are the same as in the enzyme kinetics studies except that only one concentration of maltose (4%) is used as substrate. Specific activity assays were carried out with 4% maltose as substrate at 50°C and pH 4.5. One unit (IU) was defined as the amount of enzyme required to produce l ⁇ mol glucose per min under the conditions of the assay.
  • Residues Asn20, Ala27 and Thr72, Ala471 were chosen to be replaced with cysteine.
  • 132 pairs of residues were found that could potentially be sites for a disulfide bond. Pairs containing glycine were discarded on the assumption that glycine may be required for flexibility at that site. Also, the residues involved in hydrogen bonds and electrostatic interactions were eliminated.
  • Residues 20 paired with 27 as well as 72 paired with 471 were chosen as candidates for disulfide bond formation according to the geometrical analysis.
  • This disulfide bond also would make an additional linkage between the catalytic domain and the O-glycosylated linker.
  • This O-glycosylated linker has been proved to be important for GA thermostability by limiting the conformational space available to the GA unfolded peptide [Semimaru et al . , 1995 and Williamson et al . , 1992].
  • This disulfide bond could have a globe effect on the thermostability of GA because of this linkage.
  • the side chain -OH group of Thr72 in A . awamori var. X100 GA is hydrogen bonded to the main chain N atom of Asp73. In A .
  • the mutant A471C/T72C has faster mobility than wild-type during SDS-PAGE under non-reducing conditions, suggesting that an additional disulfide bond forms a new loop retarding the migration.
  • the possibility that a truncated enzyme was formed in this case was eliminated by DNA sequencing of the mutant cDNA and MALDI analysis.
  • the MALDI data showed that the mutant GA had the same molecular weight as wild- type GA.
  • Mutant A27C/N20C has the same migration as wild-type GA, which may be because the additional loop caused by the engineered disulfide bond is too small (seven residues) to affect migration.
  • Mutant A27C/N20C and A471C/T72C had specific activities at 50°C and kinetic parameters at 35°C very close to wild-type GA (Table 3) .
  • the single mutant A27C had slightly increased K m but the same k caet value as wild-type GA, and thus a reduced k cat /K m ratio of -30%.
  • Mutant N20C had the same K ⁇ but both a decreased k cat and k cae /K m ratio and a decreased specific activity at 50°C of more than 50%.
  • the irreversible thermoinactivation of wild-type and mutant GA was studied at 65°C, 67.5°C, 70°C, 72.5°C and 77.5°C with first-order irreversible theremoinactivation coefficients k d shown in Figure 2.
  • Mutants A27C, A27C/N20C and A471C/T72C had smaller k d values than did wild-type GA within the measured temperature range, which means the activity decayed more slowly than wild type, while mutant N20C had greater k d value than wild- type at all temperatures except 75°C, which means that N20C decayed faster than wild-type.
  • Table 4 shows the activation enthalpy ( ⁇ H,) , entropy ( ⁇ S,) and free energy of unfolding ( ⁇ G, ) at 65°C and 75°C of wild-type and mutant GAs, calculated according to transition-state theory.
  • the enthalpies of N20C and A27C/N20C decreased by 42 and 24 KJ/ ol respectively, while no significant change occurs for A27C and A471C/T72C.
  • Mutants N20C and A27C/N20C had decreased entropy of 115 kJ/mol and 75 kJ/mol respectively, while entropy of mutants A27C and A471C/T72C showed no significant change.
  • Mutant A27C and A471C/T72C had a slightly higher ⁇ G' than wild-type GA at 65°C and 75°C ( ⁇ 0.5 kJ/mol) , while the ⁇ G* of A27C/N20C was higher than that of wild-type by 1.5 and 2.2 kJ/mol at 65°C and 75°C respectively.
  • Mutant N20C had a decreased ⁇ G' by 3.0 and 1.8 kJ/mol at 65°C and 75°C, respectively, compared with wild-type GA.
  • the engineered disulfide bond mutant A27C/N20C significantly increased GA thermostability compared with wild-type GA while the single mutants produced either a slight increase (A27C) or a slight decrease (N20C) in thermostability.
  • the other disulfide bond mutant had the thermostability identical to wild-type GA.
  • thermostable mutants G137A [Chen et al . , 1996] and S436P (Li et al . , 1996) which have the potential to be combined and improve thermostability additively.
  • these mutations are combined with each other and with A27C/N20C (S-S; Example 2) to test their effects (cumulative/additive) on thermostability and GA activity.
  • Mutants A27C/N20C and A27C/N20C/G137A had higher activity than wild-type consistently from 70°C to 76°C with a peak at 72.5°C, while mutant A471C/T72C had activity lower than wild-type from 70°C to 71°C and 73°C to 74°C but higher at 72°C which is its optimal temperature.
  • mutant GAs A27C/N20C, A471C/T72C and the combined mutant A27C/N20C/G137A had increased temperature optima above wild-type GA by 1.5°C.
  • Thermoinactivation of GA The irreversible thermoinactivation of wild-type and mutant GA was studied at 65°C, 67.5°C, 70°C, 72.5°C and 77.5°C with first -order irreversible theremoinactivation coefficients k d shown in Figure 2.
  • Mutants A27C, A27C/N20C and A471C/T72C, A27C/N20C/G137A, A27C/N20C/S436P and G137A/S436P had smaller k d values than did wild-type GA within the measured temperature range, which means the activity decayed more slowly than wild type, while mutant N20C had greater k d value than wild-type at all temperatures except 75°C, which means that N20C decayed faster than wild-type.
  • Table 4 shows the activation enthalpy ( ⁇ H,) , entropy ( ⁇ S,) and free energy of unfolding ( ⁇ G,) at 65°C and 75°C of wild-type and mutant GAs, calculated according to transition-state theory.
  • the helix flexibility mutant G137A showed additive thermostability when combined with either S436P or A27C/N20C.
  • the combination S436P with A27C/N20C did not show additivity.
  • the S-S/S30P/G137A combined mutant was constructed using the S-S/S30P oligonucleotide listed above and a single stranded DNA template derived from a pBluescript II KS(+) vector with a 1.7 kb XhoI ⁇ BamHI DNA fragment coding for the GA catalytic domain which already contained mutations conferring the S30P and G137A amino acid substitutions.
  • the presence of the individual mutations was confirmed by sequencing and each mutated GA gene fragment was subcloned into YEpPMl ⁇ [Cole et al . , 1988] and transformed into S. cerevisiae.
  • Saccharification analysis Saccharifications were performed in duplicate using stirring heating blocks and tightly sealed vials to prevent evaporation. Eight ⁇ g/ml of wild-type and mutant GAs were assayed using
  • Table 5 shows the specific activities of the wild- type and mutant GAs at 50°C and pH 4.5 using maltose as substrate. None of the mutant GAs demonstrated reduced enzyme activity and the S30P/G137A and S-S/S30P/G137A mutants were somewhat more active than wild-type at 50°C. To further investigate this observation, the activities of these mutant enzymes were assayed at various temperatures between 35° and 68 °C ( Figure 4) . The S30P/G137A and S-S/S30P/G137A mutant GAs were more active than wild-type at all temperatures examined.
  • Wild-type and mutant GAs were subjected to thermal inactivation at pH 4.5 between 65° and 80 °C.
  • Semilogarithmic plotting of residual activity versus inactivation time yielded inactivation rate coefficients (Jd) .
  • Figure 5 shows the effect of temperature on Jd for wild-type and mutant GAs. As can be seen, the combined mutants are significantly more stable than the individual mutant enzymes. Additionally, the temperature at which the enzymes were 50% inactivated after 10 minutes (Tm) was calculated by extrapolation from the thermal inactivation plots and transition state theory was used to calculate activation energies for thermal inactivation ( ⁇ G') . Table 7 shows the changes in ⁇ G' ( ⁇ G') and Tm for the combined mutant GAs relative to wild-type GA. These data clearly demonstrate that combining the individual stabilizing mutations can cumulatively stabilize the enzyme .
  • Figure 6 shows the results of saccharification analysis at 55° and 65°C for wild-type, S30P/G137A and S-S/S30P/G137A GAs using the industrial DE 10 maltodextrin substrate Maltrin M100 (28% w/v) from Grain Processing Corporation. Complete conversion of 28% w/v DE 10 maltodextrin to glucose would result in a 1.71 M glucose syrup however, previous saccharification analyses in our laboratory have demonstrated that wild- type GA results in approximately 90% theoretical maximum glucose yield at 55°C (not shown) . At 55°C no significant difference in glucose production was observed between the wild-type and mutant enzymes. However, at 65°C the mutant GAs produced 8-10% more glucose than wild-type although none of the enzymes tested produced as much glucose as at 55°C probably due to thermal inactivation at the elevated reaction temperature.
  • Si tes of mutation As described in Example 2, the mutations Asn20 ⁇ Cys and Ala27 ⁇ Cys form a disulfide bond between the C- terminus of ⁇ -helix one and an extended loop between ⁇ - helices one and two.
  • S3OP and G137A were designed to stabilize the enzyme by reducing its conformational entropy of unfolding and are the most stabilizing in a series of proline substitution (Xaa ⁇ Pro) and Gly ⁇ Ala mutations respectively.
  • Ser30 is located at the second position of a type II /3-turn on an extended loop between c.-helices one and two and Glyl37 is located in the middle of the fourth ⁇ -helix.
  • the disulfide bond is formed between positions 20 and 27; relatively close to position 30.
  • the S30P/G137A mutant showed more than additive stabilization at low temperatures (65°-70°C) , but less than additive stabilization at high temperatures (77.5°-80°C) ( Figure 5A and Table 7).
  • the inactivation rate for the S30P/G137A combined mutant was nearly identical to the S3OP individual mutant protein. This indicates that both regions are very important for low temperature thermal inactivation, but at high temperatures inactivation became governed by other processes.
  • the S-S/S30P/G137A combined mutant was no more stable than S30P/G137A GA at low temperatures (65°- 70°C) , but was slightly more stable at higher temperatures (75°-80°C) ( Figure 5C and Table 7) .
  • the S30P/G137A double mutant cumulatively stabilized GA as demonstrated by decreased irreversible thermal inactivation rates relative to either individual mutant enzyme when analyzed between 65°C and 80°C. Similarly, the S-S/S30P combined mutant also demonstrated cumulative stabilization.
  • the S- S/S30P/G137A combined mutant was more stable than either of the "double" mutants, particularly at temperatures above 70°C.
  • the S-S/S30P combined mutant had the same activity as wild-type and the S30P/G137A and S-S/S30P/G137A mutants increased enzyme activity by 10-20% when assayed between 35° and 68°C.
  • the S30P/G137A and S-S/S30P/G137A mutant GAs decreased thermal inactivation rates approximately three fold relative to wild-type when inactivated in the presence of 1.71M glucose at 65°C. Additionally, at 55°C no difference in glucose yield was observed between these mutant GAs and wild-type for the saccharification of the industrial substrate Maltrin M100, whereas at 65°C the S30P/G137A and S-S/S30P/G137A GAs produced 8-10% more glucose than wild-type.
  • the kinetic parameters J_ cat and K M for the hydrolysis of G 2 to G 7 as well as iG 2 in 0.05 M acetate buffer, pH 4.4 , at 45°C are given in Table 8.
  • the 311-314 Loop mutant had k cat values 50-80% for all c.- (1,4) -linked substrates and only 30% for iG 2 , _C M values 50-75% for all substrates.
  • the k cat values for Glyl37 ⁇ Ala/Ser30 ⁇ Pro GA are 10-30% more, generally, than that of wild-type GA for all substrates.
  • the K M values of Glyl37 ⁇ Ala/Ser30 ⁇ Pro GA are about half to twofold for all the ⁇ - (1, 4) -linked substrates and essentially reached the wildtype level for iG 2 .
  • the k cat values for the GA engineered to carry the triple mutation, S-S/Glyl37 ⁇ Ala/Ser30 ⁇ Pro ranged from 80 to 120% generally for all substrates, and the K M values are 30-80% for all substrates compared to wild-type GA.
  • the J cat values for S-S GA are 85-110% for all substrates, and the S-S GA K M values are generally 90- 110% for all substrates. However, the S-S GA K M values are 140% for G 5 and 190% for G 6 .
  • k cat / ' K ⁇ are 75-105%, 60-110%, 60-110%, and 60-120% for the Tyr312 ⁇ Trp mutation, the combined Ser30 ⁇ Pro/Glyl37 ⁇ Ala double mutation, the combined S-S/Ser30 ⁇ Pro/Glyl37 ⁇ Ala triple mutation, and the S-S engineered GA, respectively.
  • the catalytic efficiencies for the 311- 314 Loop GA are 85-120% for all the ⁇ - (1, 4) -linked substrates, and only 50% for iG 2 , compared to wild-type GA.
  • Table 8 shows the ratios of the catalytic efficiencies for G 2 to iG 2 for wild-type and mutant GAs.
  • GAs engineered with the 311-314Loop mutation and LyslO ⁇ Arg mutation have the highest (240%) and the lowest (20%) catalytic efficiencies for ⁇ -(l,4)- over - (1, 6) -linked substrates, respectively.
  • the GAs engineered with the Tyr312 ⁇ Trp and S-S mutations show 50% and 20% increases for this ratio, respectively. All other mutants had lower ratios, indicating poorer ⁇ - (1, 4) -hydrolytic ability relative to c_-(l,6)-hy- drolytic ability than wild-type GA.
  • the 311-314Loop GA had the lowest initial rates for glucose production (64%, 61%, and 82% compared to wild-type GA at 35, 45, and 55°C, respectively) due to a specific activity only 60% that of wild-type GA (data not shown) .
  • Glucose concentrations decreased after reaching maximal values because of conversion to oligosaccharides .
  • Glucose condensation reactions IG 2 concentration profiles in 30% (w/v) glucose condensation reactions at 35, 45, and 55°C were analyzed.
  • the ratio of the initial rate of iG 2 production in a 30% (w/v) glucose condensation reaction to that of glucose formation in 30% DE 10 maltodextrin hydrolysis was calculated to estimate the selectivity for the synthesis of ⁇ - (1, 6) -linked products over the hydrolysis of x- (1, 6) -linked substrates.
  • These iG 2 /glucose ratios and their relative ratios for wild- type and mutant GAs are given in Table 9.
  • K108R and S- S mutants showed the highest and the lowest relative ratios among wildtype and all the mutant GAs at all reaction temperatures, respectively.
  • K108R had more specificity for ⁇ - (1, 6) -linkages than or- (1,4)- linkages and S-S GA had more affinity for a- ( 1 , 4. ) - linkages than - (1, 6) -linkages .
  • the 311-314Loop GA also showed very low relative ratios at these three temperatures .
  • Enzyme Kinetics The kinetic parameters are seen in (J_ cat and K m ) for the hydrolysis of ⁇ -1 , 6 -linked isomaltose and ⁇ -1, 4-linked maltooligodextrins (DP2-7) at 45°C and pH 4.4 are given in Table 10.
  • Mutant Y175F was active.
  • the k cat and K m values were 83-141% and 106- 171%, respectively, that of wildtype for the different substrates tested and catalytic efficiencies were 69- 102% that of wildtype.
  • Mutant R241K was also active.
  • Mutant S411G was highly active.
  • the k cat and K m values were 93-129% and 83-203%, respectively, that of wildtype for the different substrates tested and catalytic efficiencies were 55-122% that of wildtype.
  • Mutant S411A had a similar catalytic efficiency ratio as wildtype.
  • Mutants Y116W, R241K, and S411G had decreased catalytic efficiency ratios compared to that of wildtype GA.
  • R241K had a decreased initial rate of isomaltose production at 55°C compared to that of wildtype, and it also had a lower increase (about 5 times) in the initial rate of isomaltose production from 35°C to 55°C, compared to the wildtype increase (about 7 times) .
  • Y116W, Y175F, S411A and S411G had increased initial rates of isomaltose production or about 7, 6, and 5 times, respectively from 35°C to 55°C.
  • Mutants Y175F, S411A and S411G had a decreased ratio of the initial rate of isomaltose production to that of glucose production to that of glucose production by 12%, 35% and 56% at 35°C, respectively, and a decreased ratio by 24%, 60% and 62% at 55°C, respectively, compared to wildtype.
  • R241K had a very similar ratio to that of wildtype at both 35°C and 55°C.
  • k cat and K m The kinetic parameters, k cat and K m , for the hydrolysis of ⁇ -1, 4-linked maltose and maltoheptaose and ⁇ - 1 , 6-linked isomaltose at 45°C and pH 4.4 are given in Table 11.
  • Mutant S411G glucoamylase was highly active compared to wild-type, with an increased k cat and _C_ 13 - 30% and 11 - 59%, respectively, on the substrates tested.
  • the catalytic efficiencies (k cat /K were 71 - 116% that of wild-type.
  • Mutant S411A maintained 65 - 74% of wild-type catalytic efficiency with a slightly decreased k cat and a slightly increased K m .
  • Mutant S411C maintained 54 - 73% of wild-type catalytic efficiency with a decrease in both the k cat and K m values. Since mutant S411H and S411D had only about 6 - 12% of wild-type catalytic efficiency resulting from a seriously decreased k cat and an increased K m , the kinetic parameters for the hydrolysis of isomaltose were not determined. Only mutant S411H and S411D had large increases (5.5 to 7.5 kJ/mol) in the transition-state energy, ⁇ ( ⁇ G), for the hydrolysis of maltose and maltoheptaose.
  • mutants S411G and S411A had higher k cat values than that of wild-type at some pH values.
  • the uncomplexed and maltose-complexed S411H and S411D showed more narrow bell-shaped curves than that of wild-type.
  • the effects of pH on the hydrolysis of maltoheptaose by wild-type, S411G and S411A GAs were measured to further investigate the change of pK values and optimum pH of enzyme-substrate complexes using a long-length substrate.
  • the S411G mutation increased the pKl of both the maltose-complexed form and the maltoheptaose-complexed form by approximately 0.6 units, whereas S411G had no effect on the pK2 of either enzyme-substrate complexes and only had a minor effect on the pKl and pK2 of the free enzyme.
  • the combined effect of S411G on pKl and pK2 was an increased optimum pH of both the maltose- complexed form and the maltoheptaose-complexed form by approximately 0.3 units.
  • S411G mutation had no effect on the optimum pH of the free enzyme.
  • S411A and S411C had very similar effects on the pH dependence of maltose hydrolysis.
  • S411A and S411C increased the pK : of the free enzyme and the maltose-complexed forms by 0.3 - 0.5 and 1.21 units, respectively.
  • S411A and S411C also increased the pK 2 of the maltose- complexed form by approximately 0.5 units.
  • S411A increased the pK 2 and pK 2 of the maltoheptaose-complexed form by 1.31 and 0.4 units, respectively.
  • S411H increased the pK x of the free enzyme and maltose-complexed form by 0.33 and 1.47 units, respectively; however, it decreased the pK 2 of the free enzyme and the maltose-complexed form by 0.79 and 1.16 units, respectively.
  • S411D increased the pKl of the free enzyme and the maltose-complexed form by 0.36 and 1.23 units, respectively.
  • S411D also decreased the pK 2 of the maltose-complexed form by 0.32 units.
  • Maltodextrin 10 is a mixture of maltodextrin with an average (and major) degree of polymerization of 10.
  • the production of glucose by wild-type and S411A glucoamylases during the hydrolysis of maltodextrin 10 at 11 different pH values was determined, and used to calculate the initial rates of glucose production at different pH values ( Figure 10) .
  • the production of glucose increased following a hyperbolic curve. S411A had higher initial rates of glucose production than wild- type when the pH values were above 6.6 ( Figure 10) .
  • Tyr312Trp k C at (s "1 ) 17.2 ⁇ 0.3 36 8 + 0 9 50 7 + 0 9 50 7 + 0 8 56 0 + 0 8 63 3 ⁇ 0 6 M (mM) 0.940 ⁇ 0.059 0 343 ⁇ 0 028 0.193 + 0 010 0 100 + 0 006 0 108 ⁇ 0 005 0.103 ⁇ 0 003 ⁇
  • Ala lie Leu Asn Asn lie Gly Ala Asp Gly Ala Trp Val Ser Gly Ala 20 25 30
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO : 2 :
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid

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Abstract

Cette invention concerne une glucoamylase fongique qui comprend une paire de mutation Asn20Cys couplé à Ala27Cys, et dans laquelle une liaison disulfure est formée entre les deux éléments de la paire. Cette mutation permet d'accroître la stabilité thermique et de réduire la formation d'isomaltose en ce qui concerne l'enzyme. Cette invention concerne également une glucoamylase fongique contenant une mutation 311-314Loop, et dans laquelle la formation d'isomaltose est également réduite grâce à la mutation. Cette invention concerne en outre une glucoamylase fongique contenant un Ser411Ala de mutation, et dans laquelle il est possible d'obtenir un pH optimal et de réduire la formation d'isomaltose grâce à la mutation. Cette invention concerne enfin des combinaisons des mutations dans les glucoamylases obtenues par génie génétique, ainsi que des combinaisons avec d'autres mutations de glucoamylase qui permettent d'accroître la stabilité thermique, d'obtenir un pH optimal, et de réduire la formation d'isomaltose en vue d'améliorations cumulées dans les glucoamylases ainsi obtenues.
PCT/US1997/012983 1996-07-24 1997-07-24 Fabrication par genie genetique et a l'aide de proteines d'une glucoamylase permettant d'obtenir un ph optimal et d'accroitre la specificite d'un substrat ainsi que la stabilite thermique WO1998003639A1 (fr)

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EP97936193A EP0970193A1 (fr) 1996-07-24 1997-07-24 Fabrication par genie genetique et a l'aide de proteines d'une glucoamylase permettant d'obtenir un ph optimal et d'accroitre la specificite d'un substrat ainsi que la stabilite thermique
JP10507223A JP2000515377A (ja) 1996-07-24 1997-07-24 ｐＨ最適値、基質特異性及び熱安定性を増大するためのグルコアミラーゼのタンパク質操作
AU38923/97A AU3892397A (en) 1996-07-24 1997-07-24 Protein engineering of glucoamylase to increase ph optimum, substrate specificity and thermostability
US09/236,063 US6537792B1 (en) 1996-07-24 1999-01-22 Protein engineering of glucoamylase to increase pH optimum, substrate specificity and thermostability

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US2257896P 1996-07-24 1996-07-24
US60/022,578 1996-07-24
US2307796P 1996-08-02 1996-08-02
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WO1999027124A1 (fr) * 1997-11-26 1999-06-03 Novo Nordisk A/S Saccharification enzymatique d'amidon, comportant une phase de separation par membrane
WO2000004136A1 (fr) * 1998-07-15 2000-01-27 Novozymes A/S Variants de glucoamylase
WO2000043504A1 (fr) * 1999-01-22 2000-07-27 Iowa State University Research Foundation, Inc. Fabrication par genie proteique d'une glucoamylase permettant d'accroitre le ph optimum, la specificite du substrat et la thermostabilite
WO2000075296A1 (fr) * 1999-06-02 2000-12-14 Novozymes A/S Nouvelle glucoamylase
WO2001004273A3 (fr) * 1999-07-09 2001-09-07 Novozymes As Variante de glucoamylase
US6352851B1 (en) 1998-07-15 2002-03-05 Novozymes A/S Glucoamylase variants
WO2003029449A3 (fr) * 2001-10-01 2004-03-25 Novozymes As Variants de glucoamylase
EP1914306A3 (fr) * 1998-07-15 2008-09-10 Novozymes A/S Variantes de la glucoamylase
US10337041B2 (en) 2014-02-07 2019-07-02 Novozymes A/S Compositions for producing glucose syrups
CN114381448A (zh) * 2022-01-10 2022-04-22 鑫缘茧丝绸集团股份有限公司 一种葡聚糖酶突变体及其应用
US20220220454A1 (en) * 2019-05-31 2022-07-14 Nanjing Bestzyme Bio-Engineering Co., Ltd. Thermostable glucose oxidase
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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CN102994474B (zh) * 2012-12-31 2015-04-15 江南大学 一种热稳定性提高的淀粉酶突变体及其应用
CN103409392B (zh) * 2013-07-25 2015-06-03 江南大学 一种热稳定的淀粉酶突变体及其制备方法和应用

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DISSERTATION ABSTRACTS INTERNATIONAL, Abstract No. 95(03):B0023, 1994, Vol. 54, CHEN, "Site-Directed Mutagenesis to Enhance Thermostability of Aspergillus Awamori Glucoamylase Expressed in Saccharomyces Cerevisiae", page 5998. *
DISSERTATION ABSTRACTS INTERNATIONAL, Abstract No. 97:24090, 1996, Vol. 57, No. 11B, LI, "Genetic Construction and Biochemical Analysis of Thermostability Mutants of Glucoamylase from Aspergillus Awamori", page 6761. *
PROTEIN ENGINEERING, Abstract No. 93-04739, March 1993, Vol. 6, BAKIR et al., "Cassette Mutagenesis of the Active Site of Aspergillus Awamori Glucoamylase", page 41. *
PROTEIN ENGINEERING, December 1994, Vol. 7, No. 12, SIERCKS et al., "Protein Engineering of the Relative Specificity of Glucoamylase from Aspergillus Awamori Based on Sequence Similarities Between Starch-Degrading Enzymes", pages 1479-1484. *
PROTEIN ENGINEERING, January 1997, Vol. 10, No. 1, STOFFER et al., "Glucoamylase Mutants in the Conserved Active-Site Segment Trp170-Tyr175 Located at a Distance from the Site of Catalysis", pages 81-87. *
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US6136571A (en) * 1997-11-26 2000-10-24 Novo Nordisk A/S Method of producing saccharide preparations
WO1999027124A1 (fr) * 1997-11-26 1999-06-03 Novo Nordisk A/S Saccharification enzymatique d'amidon, comportant une phase de separation par membrane
US6303346B1 (en) 1997-11-26 2001-10-16 Novozymes A/S Method of producing saccharide preparations
US6129788A (en) * 1997-11-26 2000-10-10 Novo Nordisk A/S Method of producing saccharide preparations
US6352851B1 (en) 1998-07-15 2002-03-05 Novozymes A/S Glucoamylase variants
US8426183B2 (en) 1998-07-15 2013-04-23 Novozymes A/S Glucoamylase variants
WO2000004136A1 (fr) * 1998-07-15 2000-01-27 Novozymes A/S Variants de glucoamylase
JP2002520047A (ja) * 1998-07-15 2002-07-09 ノボザイムス アクティーゼルスカブ グルコアミラーゼ変異体
US8101392B2 (en) 1998-07-15 2012-01-24 Novozymes A/S Glucoamylase variants
US7122365B2 (en) * 1998-07-15 2006-10-17 Novozymes Als Glucoamylase variants
KR100764528B1 (ko) * 1998-07-15 2007-10-09 노보자임스 에이/에스 글루코아밀라제 변이체
US7833772B2 (en) 1998-07-15 2010-11-16 Novozymes A/S Glucoamylase variants
CN100402645C (zh) * 1998-07-15 2008-07-16 诺维信公司 葡糖淀粉酶变体
EP1914306A3 (fr) * 1998-07-15 2008-09-10 Novozymes A/S Variantes de la glucoamylase
WO2000043504A1 (fr) * 1999-01-22 2000-07-27 Iowa State University Research Foundation, Inc. Fabrication par genie proteique d'une glucoamylase permettant d'accroitre le ph optimum, la specificite du substrat et la thermostabilite
WO2000075296A1 (fr) * 1999-06-02 2000-12-14 Novozymes A/S Nouvelle glucoamylase
EP2009098A1 (fr) * 1999-07-09 2008-12-31 Novozymes A/S Variante de glucoamylase
US7927857B2 (en) 1999-07-09 2011-04-19 Novozymes A/S Glucoamylase variants
WO2001004273A3 (fr) * 1999-07-09 2001-09-07 Novozymes As Variante de glucoamylase
US8722369B2 (en) 1999-07-09 2014-05-13 Novozymes A/S Glucoamylase variants
US7371546B2 (en) 2001-10-01 2008-05-13 Novozymes A/S Glucoamylase variants
US7919281B2 (en) 2001-10-01 2011-04-05 Novozymes A/S Glucoamylase variants
WO2003029449A3 (fr) * 2001-10-01 2004-03-25 Novozymes As Variants de glucoamylase
US8222006B2 (en) 2001-10-01 2012-07-17 Novozymes A/S Glucoamylase variants
US11136607B2 (en) 2014-02-07 2021-10-05 Novozymes A/S Compositions for producing glucose syrups
US10337041B2 (en) 2014-02-07 2019-07-02 Novozymes A/S Compositions for producing glucose syrups
US11667938B2 (en) 2014-02-07 2023-06-06 Novozymes A/S Compositions for producing glucose syrups
US20220220454A1 (en) * 2019-05-31 2022-07-14 Nanjing Bestzyme Bio-Engineering Co., Ltd. Thermostable glucose oxidase
EP3978602A4 (fr) * 2019-05-31 2023-07-05 Nanjing Bestzyme Bio-engineering Co., Ltd. Glucose oxydase thermostable
US12203101B2 (en) * 2019-05-31 2025-01-21 Nanjing Bestzyme Bio-Engineering Co., Ltd. Thermostable glucose oxidase
CN114381448A (zh) * 2022-01-10 2022-04-22 鑫缘茧丝绸集团股份有限公司 一种葡聚糖酶突变体及其应用
CN114381448B (zh) * 2022-01-10 2024-02-20 鑫缘茧丝绸集团股份有限公司 一种葡聚糖酶突变体及其应用
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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