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PubMed1998

Cloning and expression of beta-glucosidase genes in Escherichia coli and Saccharomyces cerevisiae using shuttle vector pYES 2.0.

Genes for beta-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its SacI cloning site and transformed to E. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representative E. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of beta-glucosidase on E. coli. Expression of the bgl genes resulted in overproduction of beta-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carrying bgl genes from the representative recombinant E. coli were isolated with SacI, ligated in the shuttle vector pYES 2.0 in its SacI site and transformed to E. coli and S. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of beta-glucosidase on S. cerevisiae and enabled it to grow on cellobiose and salicin. The gall promoter of shuttle vector pYES 2.0 enabled the organisms to produce twice more beta-glucosidase than that supported by the lacZ-promoter of pUC18 plasmid in E. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains of S. cerevisiae. The enzyme produced by bgl+ yeast and E. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the beta-glucosidases from S. cerevisiae were substantially the same as those from C. biazotea.

Cloning, Molecular↗
PubMed2000

Simultaneous production of high activities of thermostable endoglucanase and beta-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus.

The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase and beta-glucosidase by Thermoascus aurantiacus using statistical factorial designs. The optimised medium containing 40.2 g l(-1) Solka Floc as the carbon source and 9 g l(-1) soymeal as the organic nitrogen source yielded 1130 nkat ml(-1) endoglucanase and 116 nkat ml(-1) beta-glucosidase activities after 264 h as shake cultures. In addition, good levels of beta-xylanase (3479 nkat ml(-1)) and low levels of filter-paper cellulase, beta-xylosidase, alpha-L-arabinofuranosidase, beta-mannanase, beta-mannosidase, alpha-galactosidase and beta-galactosidase were detected. Batch fermentation in a 5-1 laboratory fermentor using the optimised medium allowed the production of 940 nkat ml(-1) endoglucanase and 102 nkat ml(-1) beta-glucosidase in 192 h. Endoglucanase and beta-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they displayed optimum activity at 75 degrees C. Endoglucanase and beta-glucosidase showed good stability at pH values 4-8 and 4-7, respectively, after a prolonged incubation (48 h at 50 degrees C). Endoglucanase had half-lives of 98 h at 70 degrees C and 4.1 h at 75 degrees C, while beta-glucosidase had half-lives of 23.5 h at 70 degrees C and 1.7 h at 75 degrees C. Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell 50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T. aurantiacus in 50 h.

Ascomycota↗
PubMed2002

Isomaltose formed by alpha-glucosidases triggers amylase induction in Aspergillus nidulans.

Among various alpha-glucobioses examined, isomaltose was the most effective inducer for amylase synthesis in Aspergillus nidulans. Amylase induction by maltose was completely inhibited by addition of castanospermine or cycloheximide, while induction by isomaltose was not affected by the inhibitors, suggesting that amylase induction by maltose requires inducible alpha-glucosidases. Disruption of the alpha-glucosidase A gene ( agdA), the alpha-glucosidase B gene ( agdB), or both genes did not abolish maltose-dependent induction, although amylase production induced by maltose decreased about 2-fold in the agdA/ agdB double disruptant, compared with that in the agdB disruptant at all concentrations tested. Upon induction by isomaltose, amylase synthesis was enhanced considerably in the agdB and agdA/ agdB disruptants. Even at 3 nM, isomaltose induced amylase production in the double disruptant, supporting the suggestion that isomaltose is a physiological inducer for amylase. Therefore, maltose must be converted to isomaltose by alpha-glucosidases prior to triggering amylase synthesis, but no specific alpha-glucosidase is required for amylase induction by maltose. Probably any alpha-glucosidases having isomaltose-forming activity, including AgdA and AgdB, may participate in amylase induction by maltose.

Aspergillus nidulans↗
PubMed2000

Purification and characterization of a hydroxamic acid glucoside beta-glucosidase from wheat (Triticum aestivum L.) seedlings.

A beta-glucosidase (EC 3.2.1.21) with a high affinity for cyclic hydroxamic acid beta-D-glucosides was purified from 48-h-old wheat (Triticum aestivum L.) seedlings. The activity occurred transiently at a high level during the non-autotrophic stage of growth, and the nature of the transient occurrence was correlated with that of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). The glucosidase had maximum activity at an acidic pH (pH 5.5) and the purified enzyme showed a high affinity for DIMBOA-Glc, Vmax and Km being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta-glycosides, as well as flavone and isoflavone glucosides, but to a lesser extent. The results indicated that the primary natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme is involved in defense against pathogens and herbivores in non-autotrophic wheat. The glucosidase was found to be present as oligomeric forms with a molecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-terminal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro-(Ser?)-Lys-Pro-Ala-Glu-Pro-Ile-Gly-Pro), and showed no similarity to those of other plant glucosidases. Polyacrylamide gel electrophoresis under nondenaturing condition indicated the existence of at least eight isozymes. Three cultivars of Triticum aestivum had the same zone of glucosidase activity on zymograms, but the activity zones of the Triticum species, T. aestivum L., T. spelta L. and T. turgidum L., had different mobilities.

Amino Acid Sequence↗
PubMed1987

A simple differential immunoprecipitation assay of urinary acid and neutral alpha-glucosidases for glycogenosis II.

A specific assay for acid alpha-glucosidase in urine was developed to facilitate the diagnosis of glycogenosis II. This enzyme activity was calculated as a difference between the alpha-glucosidase activities before and after immunoprecipitation with antiserum to acid alpha-glucosidase. Acid alpha-glucosidase accounted for 86% of the total activity in control urine. All the cases of various clinical types of glycogenosis II showed either a marked decrease or a complete deficiency of this enzyme activity. A marked decrease of acid alpha-glucosidase was demonstrated by immunoblotting of the urine from patients with late-onset forms of this disease. These results indicate that assays of urinary acid alpha-glucosidase by this immunological method are useful for detection of the various types of glycogenosis II.

Adult↗
PubMed1990

Formation of a stable L-ascorbic acid alpha-glucoside by mammalian alpha-glucosidase-catalyzed transglucosylation.

Enzymatic transglucosylation from maltose to L-ascorbic acid (AA) with mammalian tissue homogenates was determined by a high-performance liquid chromatography method and compared with the reaction catalyzed by alpha-glucosidase from Aspergillus niger. The homogenates of small intestine and kidney had a high transglucosylase activity to form a new type of glucosylated AA, which was associated with alpha-glucosidase activity. The new compound was demonstrated to be an equimolar conjugate of AA and glucose by the spectral and quantitative analyses. In particular, it showed a high stability in a neutral solution and no reducing activity toward cytochrome c and a dye. These properties were very different from those of AA and L-ascorbic acid alpha-glucoside formed with alpha-glucosidase from A. niger, but they were consistent with those of L-ascorbic acid 2-O-phosphate and L-ascorbic acid 2-O-sulfate. Moreover, it exhibited a reducing power associated with AA after mild acid hydrolysis or treatment with rat intestinal alpha-glucosidase. These results indicate that it should be assigned the 2-O-alpha-glucoside structure. Consequently, it is concluded that mammalian alpha-glucosidase is able to form a very stable and nonreducing form of glucosylated AA through a specific transglucosylation reaction distinct from that of microbial alpha-glucosidase.

Animals↗
PubMed2004

Relationship between lipocalin-type prostaglandin D synthase and alpha-glucosidase in azoospermia seminal plasma.

BACKGROUND: [corrected] To determine the correlation of lipocalin-type prostaglandin D synthase (L-PGDS) and alpha-glucosidase in semen. METHODS: We analyzed 68 seminal plasmas for lipocalin-type prostaglandin D synthase (L-PGDS) and alpha-glucosidase, L-PGDS was analyzed by ELISA. The semen donors were categorized in 3 groups: normal, obstructive and non-obstructive azoospermia. We then evaluated their correlation. RESULTS: The difference of L-PGDS concentration (P<0.001) and alpha-glucosidase activity (P<0.001) among the 3 clinical groups was statistically significant. Correlation between L-PGDS concentration and alpha-glucosidase was also statistically significant. L-PGDS concentration correlated positively with alpha-glucosidase activity (r=0.882). CONCLUSIONS: L-PGDS in seminal plasma, like alpha-glucosidase, suggests an obstruction of the seminal ducts and may be a potential marker that may aid in the differential diagnosis of obstructive and non-obstructive azoospermia.

Adult↗
PubMed2005

Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth.

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

Adsorption↗
PubMed2006

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10.

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.

Bacillaceae↗
PubMed1997

Subsite structure of the beta-glucosidase from Aspergillus niger, evaluated by steady-state kinetics with cello-oligosaccharides as substrates.

The beta-glucosidase from a commercially available preparation from Aspergillus niger was highly purified. The Michaelis constant Km and the molar activity K0 for cello-oligosaccharide substrates Gn (n = 2-6) were obtained by steady-state kinetic analysis on the beta-glucosidase-catalyzed hydrolysis at 25 degrees C and pH 5.0. Stoichiometric production of Gn-1 by the beta-glucosidase reaction for Gn was confirmed by HPLC techniques. Based on Km and K0 for Gn, subsite affinities (Ai, i = 1-6) were estimated as follows (kcal/mol): A1 = 1.3, A2 = 5.2, A3 = 0.65, A4 = -0.10, A5 = -0.65, and A6 = -0.26, of which A1-A3 are much higher than those of the beta-glucosidase of Candida wickerhamii. The subsite structure is quite similar to that of the alpha-glucosidase of A. niger, whereas the dependence of k0 on n is highly characteristic for beta-glucosidase, and decreases with n, suggesting some interaction between the particular subsites.

Aspergillus niger↗
PubMed2002

A digestive beta-glucosidase from the salivary glands of the termite, Neotermes koshunensis (Shiraki): distribution, characterization and isolation of its precursor cDNA by 5'- and 3'-RACE amplifications with degenerate primers.

beta-Glucosidase activity [EC 3.2.1.21] was measured in the salivary glands and the gut of wood-eating termite, Neotermes koshunensis (Shiraki). 75% of the activity was detected in the salivary glands, whereas 15% of the activity was present in the hindgut, where numerous symbiotic flagellates reside. The salivary beta-glucosidase was partially purified by ion-exchange and gel filtration chromatography. The molecular weight of the salivary beta-glucosidase was 60 kDa, and the K(m) value on cellobiose was 2.5 mM. Its optimal pH was 5.6 and the activity was stable from 20 degrees C up to 45 degrees C. In addition to cellobiose, p-nitrophenyl-beta-D-fucopyranoside and laminaribiose were efficiently hydrolyzed by the salivary beta-glucosidase. Degenerate PCR using primers designed from N-terminal amino acid sequences of the salivary beta-glucosidase resulted in a cDNA fragment of 1730 bp, encoding 498 amino acids and with sequence similarity to glycosyl hydrolase family 1. Reverse-transcription (RT)-PCR showed that this beta-glucosidase is produced only in the salivary glands.

3' Untranslated Regions↗
PubMed1977

Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by alpha- and beta-glucosidase.

A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purifed Candida tropicalis alpha-glucosidase and sweet almond beta-glucosidase have been found to attack the same substrate, D-glucal, and to convert this unusual glycosyl substrate (which lacks alpha or beta anomeric configuration) to 2-deoxy-alpha-(or beta-) D-glucose, respectively. The stereospecificity of the hydration reaction catalyzed by each enzyme in D2O was revealed by the use of high-resolution (270 MHz) 1H magnetic resonance spectroscopy. The alpha-glucosidase caused a specific axial protonation (deuteration) of D-glucal at C-2, and formation of 2-deoxy-alpha-D-[2(a)-2H]glucose. The beta-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-beta-D-[2(e)-2H]glucose. These results are not accounted for by the generally accepted mechanisms of carbohydrase action derived from studies with glycosidically linked substrates alone. D-Glucal apparently binds to the enzymes with essentially the same overall orientation as the D-glucosyl moiety of glycosidically linked substrates (with the double bond of D-glucal lying essentially in the plane of the similarly bound D-glucosyl group). Thus, the alpha-glucosidase evidently protonates D-glucal from above the double bond and alpha-D-glucosidic substrates from below the glycosidic oxygen; beta-glucosidase apparently protonates D-glucal from below the double bond and beta-D-glucosides from above the glycosidic oxygen. A detailed mechanism is proposed for the hydration of D-glucal by each enzyme, involving an incipient glycosyl carbonium ion and assuming the presence at the active site of two carboxyl groups arranged to account for catalysis of glycosylations from glycosidically linked substrates. That D-glucal serves as a glycosyl substrate for these enzymes strongly supports the concept that glycosidases and glycosyltransferases are catalysts of glycosylation (i.e., glycosylases), since this concept does not make the usual assumption that carbohydrases are restricted to acting on substrates having a glycosidic bond and either alph- or beta-anomeric configuration.

Candida↗
PubMed2000

beta-glucosidases from five black Aspergillus species: study of their physico-chemical and biocatalytic properties.

Five black Aspergillus strains (A. aculeatus, A. foetidus, A. japonicus, A. niger, and A. tubingensis) were cultivated on crude wheat arabinoxylan as the carbon source under defined pH, temperature, and oxygen conditions. Protein and beta-glucosidase content differed remarkably within the obtained culture filtrates, of which eleven beta-glucosidases were isolated. Seven beta-glucosidases were purified to apparent electrophoretic homogeneity using anion-exchange and gel-permeation chromatography. They were found to be acidic proteins and most of them appeared to be glycoproteins with a molecular mass between 93 and 142 kDa. Classification of the beta-glucosidases into four groups (I-A, I-B, II, and III) is suggested according to their physicochemical and biocatalytic properties. The major beta-glucosidases were assigned to groups I-A and I-B, the minor beta-glucosidases to groups II and III, comprising acid-tolerant and glucose-tolerant enzymes, respectively.

Amino Acid Sequence↗
PubMed2000

Solid-state bioconversion of phenolics from cranberry pomace and role of Lentinus edodes beta-glucosidase.

Cranberry pomace contains large amounts of phenolic glycosides, which are important sources of free phenolics that have many food uses such as antioxidants, flavorings, and nutraceuticals. Our hypothesis was that these glycosides in cranberry pomace could be hydrolyzed by beta-glucosidase produced by Lentinus edodes during solid-state fermentation. On the basis of this hypothesis, our objective was to investigate the potential of using cranberry pomace as a substrate for the production of free phenolics and beta-glucosidase through solid-state fermentation by a food-grade fungus L. edodes. Our results suggested that L. edodes beta-glucosidase played a major role in release of phenolic aglycons from cranberry pomace during solid-state fermentation. After 50 days of cultivation, the yield of total free phenolics reached the maximum of 0.5 mg per g of pomace, while the beta-glucosidase activity was about 9 units per g of pomace. The enzyme exhibited optimal activity at 60 degrees C and at pH 3.5 and was stable at temperatures up to 50 degrees C and between pH 3 and 6.5. The major free phenolics produced from cranberry pomace were identified by HPLC as gallic acid, chlorogenic acid, p-hydroxybenzoic acid, and p-coumaric acid. These results suggest that cranberry pomace is a potential substrate for producing food-grade phenolics and fungal beta-glucosidase. The L. edodes beta-glucosidase showed good stability and tolerance to low pH and, therefore has potential applications in wine and juice processing for aroma and flavor enrichment through enzymatic hydrolysis of glucoside precursors.

Chromatography, High Pressure Liquid↗
PubMed2002

Substrate recognition by three family 13 yeast alpha-glucosidases.

Important hydrogen bonding interactions between substrate OH-groups in yeast alpha-glucosidases and oligo-1,6-glucosidase from glycoside hydrolase family 13 have been identified by measuring the rates of hydrolysis of methyl alpha-isomaltoside and its seven monodeoxygenated analogs. The transition-state stabilization energy, DeltaDeltaG, contributed by the individual OH-groups was calculated from the activities for the parent and the deoxy analogs, respectively, according to DeltaDeltaG = -RT ln[(Vmax/Km)analog/(Vmax/Km)parent]. This analysis of the energetics gave DeltaDeltaG values for all three enzymes ranging from 16.1 to 24.0 kJ.mol-1 for OH-2', -3', -4', and -6', i.e. the OH-groups of the nonreducing sugar ring. These OH-groups interact with enzyme via charged hydrogen bonds. In contrast, OH-2 and -3 of the reducing sugar contribute to transition-state stabilization, by 5.8 and 4.1 kJ.mol-1, respectively, suggesting that these groups participate in neutral hydrogen bonds. The OH-4 group is found to be unimportant in this respect and very little or no contribution is indicated for all OH-groups of the reducing-end ring of the two alpha-glucosidases, probably reflecting their exposure to bulk solvent. The stereochemical course of hydrolysis by these three members of the retaining family 13 was confirmed by directly monitoring isomaltose hydrolysis using 1H NMR spectroscopy. Kinetic analysis of the hydrolysis of methyl 6-S-ethyl-alpha-isomaltoside and its 6-R-diastereoisomer indicates that alpha-glucosidase has 200-fold higher specificity for the S-isomer. Substrate molecular recognition by these alpha-glucosidases are compared to earlier findings for the inverting, exo-acting glucoamylase from Aspergillus niger and a retaining alpha-glucosidase of glycoside hydrolase family 31, respectively.

Catalysis↗
PubMed2000

Molecular characterization of the Thermomonospora curvata aglA gene encoding a thermotolerant alpha-1,4-glucosidase.

The cloning, sequencing and structural characterization of a gene encoding a thermostable alpha-1,4-glucosidase from Thermomonospora curvata is described. DNA sequence analysis revealed four open reading frames designated aglA, aglR, aglE and aglF. The aglA gene encodes a thermostable alpha-1,4-glucosidase from T. curvata and is situated between two genes, aglR and aglE. Genes aglA, aglE and aglF are transcribed in the same direction, while aglR is transcribed in the opposite direction. By comparing the amino acid sequence of the alpha-1,4-glucosidase from T. curvata with other alpha-glucanases, it appears that the enzyme is a member of the alpha-amylase family. The proteins of this family have an (alpha/beta)8 barrel super secondary structure. The topology of the alpha-1,4-glucosidase was predicted by computer-assisted analysis. The topology of the secondary structures of the alpha-1,4-glucosidase resembles the structure of barley alpha-amylase, but the primary structure resembles most closely the oligo-1,6-glucosidase from Bacillus cereus. Putative catalytic residues (D221, E281 and D343) and calcium binding residues (N116, E179, D191, H224 or G225) are proposed.

Actinomycetales↗
PubMed1995

Construction of chimeric beta-glucosidases with improved enzymatic properties.

The amino acid sequences of beta-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show about 40% similarity. The pH/temperature optima and stabilities and substrate specificities of the two enzymes are quite different. C. gilvus beta-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35 degrees C, whereas the corresponding values for A. tumefaciens are 7.2-7.4 and 60 degrees C, respectively. The substrate specificity of A. tumefaciens enzyme toward different aryl glycosides is broader than C. gilvus enzyme. To analyze these properties further, three chimeric beta-glucosidases were constructed by substituting segments from the C-terminal homologous region of C. gilvus beta-glucosidase gene with that of A. tumefaciens. The chimeric enzymes were characterized with respect to pH/temperature activity and stability and substrate specificity. Chimeric enzymes exhibited chromatographic behavior similar to that of C. gilvus enzyme. However, enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45-50 degrees C and pH 6.6-7.0. Km values of chimeric enzymes for the various saccharides were admixtures of both parental enzymes. These results suggest that the two domains of C. gilvus and A. tumefaciens enzymes probably can fold independently. The homologous C-terminal region in beta-glucosidase appears to play an important role in determining enzyme characteristics. Changes in the properties on substitution of segments in this region might be related to the enzyme specificity, and beta-glucosidases with improved properties can be prepared by manipulating this region.

Agrobacterium tumefaciens↗
PubMed1997

Altering substrate specificity of Bacillus sp. SAM1606 alpha-glucosidase by comparative site-specific mutagenesis.

The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose. These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes. To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met76, Arg81, Ala116, Gly273, and Thr342. These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically. The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha, alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation. This loss in affinity for trehalose was critically governed by a Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-fold and quadruple mutants. These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.

Amino Acid Sequence↗