Mutant Glucoamylases Research Articles

Strain improvement by mutation for enhanced production of starch‐saccharifying glucoamylase from Bacillus licheniformis

AbstractA natural isolate, Bacillus licheniformis KIBGE‐IB3, was subjected to mutational analysis for the enhanced production of glucoamylase. Randomly generated mutants were initially screened for enzyme production and after secondary screening B. licheniformis KIBGE‐IB3M67 demonstrated maximum glucoamylase production with improved starch saccharifying capability. Enzyme activity from KIBGE‐IB3M67 reached up to 25 kU/mg by strain improvement, which was nearly twofold increase in enzyme production as compared to the parent strain. SEM revealed that raw potato starch is more profoundly hydrolyzed when treated with mutant glucoamylase. Successful mutation also reduced the fermentation time from 48 to 24 h for glucoamylase production, optimum fermentation pH and temperature slightly shifted from 7.0 (wild) to 7.5 (mutant), from 30 to 50°C (wild), and from 20 to 55°C (mutant), respectively. Reduction in fermentation time and enhanced starch saccharification ability makes this mutant (KIBGE‐IB3M67) an attractive candidate for the production of glucoamylase at commercial level.

Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation

Three mutations, Ser54→Pro, Thr314→Ala, and His415→Tyr, were identified in Aspergillus awamori glucoamylase gene expressed by Saccharomyces cerevisiae. The mutant glucoamylase (GA) was substantially more thermostable than a wild-type GA at 70°C, with a 3.0KJmol(-1) increase in the free energy of thermo-inactivation. The effect of starch from different botanical sources on the production of this GA was measured in liquid fermentation using commercial soluble starch, cassava, potato, and corn as the carbon source. The best substrate for GA production was the potato starch showing an enzymatic activity of 6.6U/mL. The commercial soluble starch was also a good substrate for the enzyme production with 6.3U/mL, followed by cassava starch and corn starch with 5.9 and 3.0U/mL, respectively. These results showed a significant difference on GA production related to the carbon source employed. The mutant GA was purified by acarbose-Sepharose affinity chromatography; the estimated molecular mass was 100kDa. The mutant GA exhibited optimum activity at pH 4.5 and an optimum temperature of 65°C.

Improvement of Aspergillus niger Glucoamylase Thermostability by Directed Evolution

AbstractDirected evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch‐plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply‐mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild‐type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site‐directed mutations D20C/A27C (forming a disulfide bond), S30P, and G137A to create a multiply‐mutated GA designated THS8. THS8 GA is substantially more thermostable than wild‐type GA at 80°C, with a 5.1 kJ/mol increase in the free energy of thermoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly‐mutated GAs have specific activities and catalytic efficiencies (kcat/Km) similar to those of wild‐type GA.

AFM Images of Complexes between Amylose andAspergillus niger Glucoamylase Mutants, Native and Mutant Starch Binding Domains: A Model for the Action of Glucoamylase

Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild-type A. niger starch binding domains (SBDs), and mutant SBDs (W563K and W590K) lacking either of the two starch binding sites. The images are interpreted in terms of a favourable binding between the amylose chains and the wild-type SBDs, leading to the formation of ring-like structures, in which parallel strands of the amylose molecule bind to both binding sites on the SBDs. The SBDs are seen to form a template for the assembly of an expanded amylosic double helix. This model for amylose-SBD binding has been used to propose a molecular mechanism for the role of the SBD in the hydrolytic action of glucoamylase on starch granules. The SBDs are considered to recognise the ends of amylosic double helices formed by the short amylosic chains present as branches on the amylopectin molecules, and displayed on the face of crystalline lamellae. This allows the binding and immobilisation of chain ends by the SBD, facilitating binding and cleavage by the exo-acting catalytic domain.

Thr/Ser-rich domain of Aspergillus glucoamylase is essential for secretion.

The recombinant Aspergillus awamori strain carrying the mutant glucoamylase-encoding gene in which the entire Thr/Ser-rich Gp-I domain was deleted abolished secretion of mutant glucoamylase. The transcription of the Bip-encoding bipA was low in the wild type (wt) strain, but elevated in the recombinant strain under the condition of glaA expression. The results indicate that the Gp-I domain is vital for glucoamylase secretion.

Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.

Mass spectrometric identification of a stable catalytic cysteinesulfinic acid residue in an enzymatically active chemically modified glucoamylase mutant.

Mass spectrometric identification of cysteinsulfinic acid resulting in restoration of activity of chemically modified Glu400 Cys catalytic-base glucoamylase (GA) mutants is described. This oxidation unexpectedly occurred during attempts to carboxyalkylate the Cys400 GA mutant using three different alkylation reagents. However, mass spectrometric peptide mapping did not show the presence of carboxyalkylation of the Cys400 residue but suggested an oxidation to cysteinsulfinic acid based on the observed mass increment. The presence of cysteinsulfinic acid was confirmed by employing matrix-assisted laser desorption/ionization mass spectrometry combined with post-source decay analysis. Furthermore, strong enhancement of metastable fragmentation was observed for peptides containing oxidized Cys in comparison with non-oxidized peptide.

Glucoamylase mutants with decreased Km-values for C-6 substituted isomaltosides

Glucoamylase can catalyze the hydrolysis of methyl 6 R- C-alkyl α-isomaltosides. A Trp120→Phe mutant altered in the +2 binding subsite had a K m of 0.089 mM for methyl 6 R- C-methyl-α-isomaltoside compared to a K m of 0.71 mM for the wild-type enzyme. This reflects an eight-fold lower K m for this substrate; however, the k cat for the mutant was decreased 200-fold compared with the wild-type glucoamylase. With increasing size of the substituent to 6 R- C-ethyl and -isopropyl, Trp120→Phe and wild-type glucoamylase have similar K m values, while k cat for the mutant increases 10- and 100-fold, respectively, approaching wild-type values. Perturbation of the structural integrity around the general acid catalyst Glu179, through elimination of the Trp120 NE1 hydrogen-bond to Glu179 OE2 in Trp120→Phe glucoamylase, seems to be counteracted by the larger C-6 substituents. The apparent complementarity between enzyme and substrate analogs emphasizes the favorable impact of hydrophobic forces in protein-carbohydrate interactions. Wild-type glucoamylase and Val181→Ala/Asn182→Ala/Gly183→Lys/Ser184→His, a quadruple mutant located beyond subsite +3, essentially maintain k cat in substrates with the three different 6 R- C substituents, while K m increases from 0.45 to 47.0 mM for methyl-6 R- C-isopropyl α-isomaltoside. Compared with the wild-type enzyme, the quadruple mutant has 1.5–3.7-fold improved specificity ( k cat/ K m) for the parent and 6 R-C-alkyl isomaltosides, but 10-fold lower activity for the α-(1→4)-linked maltose caused mainly by a low k cat. This mutation at distant subsites thus influences hydrolysis of disaccharides, corroborating the presence of long-range effects on the catalytic site.

Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

Mutations to alter Aspergillus awamori glucoamylase selectivity. I. Tyr48Phe49-->Trp, Tyr116-->Trp, Tyr175-->Phe, Arg241-->Lys, Ser411-->Ala and Ser411-->Gly.

Glucoamylase mutations to reduce isomaltose formation from glucose condensation and thus increase glucose yield from starch hydrolysis were designed to produce minor changes in the active site at positions not totally conserved. Tyr175-->Phe and Ser411-->Gly glucoamylases had catalytic efficiencies on DP 2-7 maltooligosaccharides like those of wild-type glucoamylase, while the catalytic efficiencies of Tyr116-->Trp, Arg241-->Lys and Ser411-->Ala glucoamylases were reduced by about half and Tyr48Phe49-->Trp glucoamylase had little remaining activity. Tyr175-->Phe, Ser411-->Ala and Ser411-->Gly glucoamylases had decreased ratios of the initial rate of isomaltose formation from glucose condensation to that of glucose formation from maltodextrin hydrolysis at both 35 and 55 degrees C compared with wild-type glucoamylase. Arg241-->Lys glucoamylase had a very similar ratio, while Tyr116-->Trp glucoamylase had a higher ratio. The highest glucose yields from maltodextrin hydrolysis were by the mutant glucoamylases having the lowest ratios of initial rates of isomaltose formation to glucose formation and this predicted high glucose yields better than the ratio of catalytic efficiency for maltose hydrolysis to that for isomaltose hydrolysis.

Some details of the reaction mechanism of glucoamylase from Aspergillus niger--kinetic and structural studies on Trp52-->Phe and Trp317-->Phe mutants.

Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obtained from structure energy minimization calculations based on known three-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme-substrate complex, in which the beta-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the beta-flank are established and the correct alignment on the alpha-flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.

Glucoamylase mutants in the conserved active-site segment Trp170-Tyr175 located at a distance from the site of catalysis.

To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.

Functional role of Aspergillus oryzae glucoamylase C-terminal domain investigated using its cDNA

The C-terminal domain of Aspergillus oryzae glucoamylase was analyzed by site-directed mutagenesis using glucoamylase cDNA. A mutant glucoamylase cDNA lacking the region corresponding to the C-terminal domain of the wild-type glucoamylase was constructed by inserting two stop codons in the gene for the wild-type glucoamylase. The wild-type and mutant glucoamylase cDNAs were expressed in Saccharomyces cerevisiae YPH 250, and then the produced wild-type and mutant glucoamylases were purified by acarbose affinity column chromatography. As compared to those of the wild-type glucoamylase, the K m values of the mutant enzyme determined using maltose, maltotriose or maltopentaose as a substrate were similar, but that determined using soluble starch as a substrate was twofold higher. The mutant glucoamylase showed a low rate of hydrolysis of raw cornstarch, although the wild-type glucoamylase showed a high rate of raw cornstarch hydrolysis. These results indicated that the C-terminal domain is important in the affinity of the enzyme to raw starch as reported for the glucoamylase of Aspergillus awamori.

Deletion analysis of the starch-binding domain of Aspergillus glucoamylase.

The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.

Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation.

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.

Increased thermostability of Asn182 → Ala mutant Aspergillus awamori glucoamylase

Asn182 --> Ala Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae had a first-order thermodeactivation coefficient 40% that of wild-type glucoamylase at pH 4.5 between 60 degrees and 65 degrees C, caused by the elimination of an Asn-Gly sequence subject to deamidation and eventual chain breakage. Above 70 degrees C, and at pHs 3.5 and 5.5, thermodeactivation coefficients of wild-type and mutant enzymes were roughly equal, because the fastest deactivation mechanism was no longer deamidation. The mutation had little effect on the enzyme's optimal pH for activity and subsite map, or on the glucose yield from starch dextrin hydrolysis. During enzyme production by yeast fermentation, highest cell densities and activities of wild-type and mutant glucoamylases were attained after a period of glucose starvation, followed by a second addition of glucose. (c) 1994 John Wiley & Sons, Inc.

Construction and characterization of mutant glucoamylases from the yeast Saccharomycopsis fibuligera.

To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHX linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements w...

Mutation in Neurospora crasa affecting some of the extracellular enzymes and several growth characteristics.

A mutant of Neurospora crassa, strain T9, which shows reduced activity of glucoamylase, was isolated. The mutant glucoamylase was not altered in pH optimum, or thermostability, but altered in elution profile of Bio-Gel filtration. The T9 mutation led to other distinct phenotypic effects: a poor growth response, resistance to the effect of sorbose, a high activity of extracellular acid phosphatase, and an increased sensitivity to high osmolarity of the growth medium. The discussion was based on the idea that the T9 mutation occurred at a gene governing the synthesis of cell wall precursors and resulted in an alteration in various characteristics related to extracellular enzymes and growth.