Transglycosylation Activity Research Articles

Expression and Characterization of a Novel Antifungal Exo-β-1,3-glucanase from Chaetomium cupreum

A novel β-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67%) with a glycoside hydrolase family 17 β-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8% recovery yield. The protein's molecular mass was approximately 65kDa, and its maximum activity appeared at pH 5.0 and temperature 45°C. Heavy metal ions Fe2+, Mn2+, Cu2+, Co2+, Ag+, and Hg2+ had inhibitory effects on Ccglu17A, but Ba2+ promoted the enzyme's activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing β-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84mg/mL and Vmax of 10.7μmolglucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-β-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.

Purification, characterization and gene analysis of a new α-glucosidase from shiraia sp. SUPER-H168

A new α-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified α-glucosidase were 4.5 and 60 °C, respectively, using p-nitrophenyl-α-glucopyranoside (α-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe2+, Cu2+, and Ag+ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K m, V max, and k cat/K m of the α-glucosidase were 0.52 mM, 3.76 U mg−1, and 1.3 × 104 L s−1 mol−1, respectively. K m with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The α-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The α-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 α-glucosidase is an ascomycetes α-glucosidase. This is the first report of α-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and α-pNPG, transglycosylation and conversion activity of maltose into trehalose.

Characteristics of Raw Starch-Digesting α-Amylase of Streptomyces badius DB-1 with Transglycosylation Activity and Its Applications.

Streptomyces badius DB-1 produces α-amylase extracellularly, and its production was enhanced 5.1-fold (from 9.47±0.51 to 48.23±1.45UmL-1) due to optimization by one-variable-at-a-time and statistical approaches. Soluble starch emerged as the most influential factor that strongly affected enzyme production. The purified enzyme is a monomer with a molecular mass of ~57kDa and optimally active at 50°C and pH 6.0. The enzyme hydrolyzes soluble as well as raw starches into simpler sugars with a high proportion (>40.0%) of maltotetraose. It is optimally active at moderate temperature and generates maltooligosaccharides from starch, thus, useful as an antistale in bread making. It also plays a role in increasing the formation of maltooligosaccharides due to transglycosylation activity, thus, finds application in functional foods. This is the first report on the production of raw starch-digesting α-amylase by S. badius with transglycosylation activity.

Purification and characterization of an extracellular β-glucosidase from Sporothrix schenckii.

An extracellular β‐glucosidase (E.C. 3.2.1.21), induced by cellulose in the mycelial form of human pathogen fungus Sporothrix schenckii, was purified to homogeneity using hydroxyapatite (HAp) adsorption chromatography in batch and Sephacryl S200‐HR size exclusion chromatography. The molecular mass of the purified enzyme was estimated to be 197 kDa by size exclusion chromatography with a subunit of 96.8 kDa determined by SDS/PAGE. The β‐glucosidase exhibited optimum catalytic activity at pH 5.5/45 °C and was relatively stable for up to 24 h at 45 °C. Isoelectric focusing displayed an enzyme with a pI value of 4.0. Its activity was inhibited by Fe2+ but not by any other ions or chelating agents. K m and V max values of the purified enzyme were 0.012 mm and 2.56 nmol·min−1·mg−1, respectively, using 4‐methylumbelliferyl β‐D‐glucopyranoside (4‐MUG) as the substrate and 44.14 mm and 22.49 nmol·min−1·mg−1 when p‐nitrophenyl β‐D‐glucopyranoside (p‐NPG) was used. The purified β‐glucosidase was active against cellobioside, laminarin, 4‐MUG, and p‐NPG and slightly active against 4‐methylumbelliferyl β‐D‐cellobioside and p‐nitrophenyl β‐D‐cellobioside but did not hydrolyze 4‐methylumbelliferyl β‐D‐xyloside, 4‐methylumbelliferyl β‐D‐galactopyranoside nor 4‐methylumbelliferyl α‐D‐glucopyranoside. In addition, the enzyme showed transglycosylation activity when it was incubated along with different oligosaccharides. Whether the transglycosylation and cellulase activities function in vivo as a mechanism involved in the degradation of cellulolytic biomass in the saprophytic stage of S. schenckii remains to be determined.

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 \u03b2-glucosidase

Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (rs/rh) from 0.33 to 1.45–2.71. Further increase in rs/rh was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5–10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.

A new family-3 glycoside hydrolase from Penicillium oxalicum BL 3005 catalyzing tyrosol glucosylation to form salidroside.

A glycoside hydrolase from Penicillium oxalicum BL 3005 was purified to apparent homogeneity. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The enzyme was identified to be a new member of family-3 by peptide sequence. High transglycosylation activity was found in the hydrolytic reaction of cellobiose. In the reaction, salidroside (4-hydroxyphenethyl O-β-d-glucopyranoside) was formed by adding tyrosol as the glycosyl acceptor. The optimum reaction pH and temperature were pH 6.5 and 55°C, respectively. The maximum yield of salidroside was almost 20 g/L. These results indicated that the β-glucosidase of P. oxalicum can be considered as a very promising catalyst for the synthesis of salidroside.

Module recombination and functional integration of oligosaccharide-producing multifunctional amylase

The oligosaccharide-producing multifunctional amylase (OPMA-N) has both hydrolytic and transglycosyl activities. Our previous reports demonstrated that the function and catalytic versatility of OPMA-N is closely related to its oligomerization, and its oligomeric state is affected by several conserved residues, such as Trp358, and by the cooperation of its small, noncatalytic N-terminal module and the catalytic module. We have demonstrated that the residue Trp358 exposed on the surface of OPMA-N molecule and has an obvious impact on OPMA-N oligomerization mainly by the charge effects. In this study, we investigated the effects of module recombination on the functional integration of OPMA-N. A series of module recombinants of the N-terminal and catalytic modules revealed that the intramolecular, semi-intramolecular and semi-intermolecular interactions of the N-terminal module with two or more catalytic modules enhanced the substrate affinity of the catalytic modules and facilitated the transglycosyl activity and functional integration of the enzyme by mediating positive cooperativity between the catalytic modules. Free N-terminal module alone did not contribute to these effects. Based on the results of this study, we speculated that the substrate affinity, but not the maximal catalytic activity, was the primary driving force in the natural evolution of enzymes, and that molecules of natural or modular enzymes may have occupied some evolutionary spaces that could be expanded or exhumed through module recombination to increase their overall catalytic efficacies or eventual catalytic efficiencies. All of the results in this study could be applied to integration of enzyme function and for creation of novel enzymes. Additionally, our results may provide important insights into the evolution of enzymes or organisms.

A new chitinase-D from a plant growth promoting Serratia marcescens GPS5 for enzymatic conversion of chitin

The current study describes heterologous expression and biochemical characterization of single-modular chitinase-D from Serratia marcescens (SmChiD) with unprecedented catalytic properties which include chitobiase and transglycosylation (TG) activities besides hydrolytic activity. Without accessory domains, SmChiD, hydrolyzed insoluble polymeric chitin substrates like colloidal, α- and β-chitin. Activity studies on CHOS with degree of polymerization (DP) 2–6 as substrate revealed that SmChiD hydrolyzed DP2 with a chitobiase activity and showed TG activity on CHOS with DP3–6, producing longer chain CHOS. But, the TG products were further hydrolyzed to shorter chain CHOS with DP1–2 products. SmChiD with its unique catalytic properties, could be a potential enzyme for the production of long chain CHOS and also for the preparation of efficient enzyme cocktails for chitin degradation.

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).

Acceptor Specificity of Amylosucrase from Deinococcus radiopugnans and Its Application for Synthesis of Rutin Derivatives.

The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β-N-Acetylhexosaminidase from Bifidobacterium bifidum.

β-N-Acetylhexosaminidases have attracted interest particularly for oligosaccharide synthesis, but their use remains limited by the rarity of enzyme sources , low efficiency, and relaxed regioselectivity of transglycosylation. In this work, genes of 13 β-N-acetylhexosaminidases, including 5 from Bacteroides fragilis ATCC 25285, 5 from Clostridium perfringens ATCC 13124, and 3 from Bifidobacterium bifidum JCM 1254, were cloned and heterogeneously expressed in Escherichia coli The resulting recombinant enzymes were purified and screened for transglycosylation activity. A β-N-acetylhexosaminidase named BbhI, which belongs to glycoside hydrolase family 20 and was obtained from B. bifidum JCM 1254, possesses the bifunctional property of efficiently transferring both GalNAc and GlcNAc residues through β1-3 linkage to the Gal residue of lactose. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation activities of BbhI were studied in detail. With the use of 10 mM pNP-β-GalNAc or 20 mM pNP-β-GlcNAc as the donor and 400 mM lactose as the acceptor in phosphate buffer (pH 5.8), BbhI synthesized GalNAcβ1-3Galβ1-4Glc and GlcNAcβ1-3Galβ1-4Glc at maximal yields of 55.4% at 45°C and 4 h and 44.9% at 55°C and 1.5 h, respectively. The model docking of BbhI with lactose showed the possible molecular basis of strict regioselectivity of β1-3 linkage in β-N-acetylhexosaminyl lactose synthesis. Oligosaccharides play a crucial role in many biological events and therefore are promising potential therapeutic agents. However, their use is limited because large-scale production of oligosaccharides is difficult. The chemical synthesis requires multiple protecting group manipulations to control the regio- and stereoselectivity of glycosidic bonds. In comparison, enzymatic synthesis can produce oligosaccharides in one step by using glycosyltransferases and glycosidases. Given the lower price of their glycosyl donor and their broader acceptor specificity, glycosidases are more advantageous than glycosyltransferases for large-scale synthesis. β-N-Acetylhexosaminidases have attracted interest particularly for β-N-acetylhexosaminyl oligosaccharide synthesis, but their application is affected by having few enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, we describe a microbial β-N-acetylhexosaminidase that exhibited strong transglycosylation activity and strict regioselectivity for β-N-acetylhexosaminyl lactose synthesis and thus provides a powerful synthetic tool to obtain biologically important GalNAcβ1-3Lac and GlcNAcβ1-3Lac.

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities

BackgroundThe filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently.ResultsThe thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3–69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose.ConclusionsWe report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3’-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

Crystal structure and identification of a key amino acid for glucose tolerance, substrate specificity, and transglycosylation activity of metagenomic β-glucosidase Td2F2.

β-Glucosidase Td2F2 isolated from a compost metagenome has high glucose tolerance and transglycosylation activity. In this study, we determined the high-resolution crystal structure of Td2F2. It has a unique structure at the -1 subsite that is important for substrate specificity but not for glucose tolerance. To elucidate the mechanism(s) of glucose tolerance, we isolated a glucose-sensitive Td2F2 mutant using random mutagenesis. In this mutant, Asn223 residue located between subsites +1 and +2 was mutated. The Asn223 mutation resulted in reduced glucose tolerance and transglycosylation activity, and drastically changed substrate specificity. These results indicate that the structure between subsites +1 and +2 is critical for the glucose tolerance and substrate specificity of Td2F2. Our findings shed light on the glucose tolerance and transglycosylation activity mechanisms of glycoside hydrolase family 1 β-glucosidases. The atomic coordinates and structure factors (codes 3WH5, 3WH6, 3WH8, 3WH7, 5AYB, and 5AYI) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Computational docking, molecular dynamics simulation and subsite structure analysis of a maltogenic amylase from Bacillus lehensis G1 provide insights into substrate and product specificity

Maltogenic amylase (MAG1) from Bacillus lehensis G1 displayed the highest hydrolysis activity on β-cyclodextrin (β-CD) to produce maltose as a main product and exhibited high transglycosylation activity on malto-oligosaccharides with polymerization degree of three and above. These substrate and product specificities of MAG1 were elucidated from structural point of view in this study. A three-dimensional structure of MAG1 was constructed using homology modeling. Docking of β-CD and malto-oligosaccharides was then performed in the MAG1 active site. An aromatic platform in the active site was identified which is responsible in substrate recognition especially in determining the enzyme’s preference toward β-CD. Molecular dynamics (MD) simulation showed MAG1 structure is most stable when docked with β-CD and least stable when docked with maltose. The docking analysis and MD simulation showed that the main subsites for substrate stabilization in the active site are −2, −1, +1 and +2. A bulky residue, Trp359 at the +2 subsite was identified to cause steric interference to the bound linear malto-oligosaccharides thus prevented it to occupy subsite +3, which can only be reached by a highly bent glucose molecule such as β-CD. The resulted modes of binding from docking simulation show a good correlation with the experimentally determined hydrolysis pattern. The subsite structure generated from this study led to a possible mode of action that revealed how maltose was mainly produced during hydrolysis. Furthermore, maltose only occupies subsite +1 and +2, therefore could not be hydrolyzed or transglycosylated by the enzyme. This important knowledge has paved the way for a novel structure-based molecular design for modulation of its catalytic activities.

Purification and characterization of a novel intracellular α-amylase with a wide variety of substrates hydrolysis and transglycosylation activity from Paenibacillus sp. SSG-1

Intracellular α-amylase was a special glycoside hydrolase in the cytoplasm. We cloned and expressed an intracellular α-amylase, Amy, from Paenibacillus sp. SSG-1. The recombinant enzyme was purified by metal-affinity chromatography, exhibited a molecular mass of 71.7 kDa. Amy exhibited unexpectedly sequence similarity and evolutionary relationships with alpha-glucanotransferase. The docked results of Amy with maltose showed it had similar catalytic residues with α-amylase and glucanotransferase. The substrate specificity experiment showed that Amy could hydrolyze typical substrates into glucose and maltose. It was noteworthy that Amy showed the catalytic capacity of cyclomaltodextrinase and pullulanase. Meanwhile, Amy could transfer sugar molecules and form maltotetraose upon the hydrolysis of substrates. These results indicated that Amy was a novel intracellular α-amylase with distinct catalytic ability characteristics of hydrolyzing glycogen/cyclodextrin/pullulan and transglycosylation. We deduced that Amy may play an important role in utilizing maltooligosaccharides that released from extracellular α-glucan or storage α-glucan (glycogen) in Paenibacillus sp. SSG-1.

Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity.

Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3'-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3'-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold.

Characterization of a Glycoside Hydrolase Family 27 α-Galactosidase from Pontibacter Reveals Its Novel Salt-Protease Tolerance and Transglycosylation Activity.

α-Galactosidases are of great interest in various applications. A glycoside hydrolase family 27 α-galactosidase was cloned from Pontibacter sp. harbored in a saline soil and expressed in Escherichia coli. The purified recombinant enzyme (rAgaAHJ8) was little or not affected by 3.5-30.0% (w/v) NaCl, 10.0-100.0 mM Pb(CH3COO)2, 10.0-60.0 mM ZnSO4, or 8.3-100.0 mg mL(-1) trypsin and by most metal ions and chemical reagents at 1.0 and 10.0 mM concentrations. The degree of synergy on enzymatic degradation of locust bean gum and guar gum by an endomannanase and rAgaAHJ8 was 1.22-1.54. In the presence of trypsin, the amount of reducing sugars released from soybean milk treated by rAgaAHJ8 was approximately 3.8-fold compared with that treated by a commercial α-galactosidase. rAgaAHJ8 showed transglycosylation activity when using sucrose, raffinose, and 3-methyl-1-butanol as the acceptors. Furthermore, potential factors for salt adaptation of the enzyme were presumed.

Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae

Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-D-xylopyranose-(1 → 6)-D-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes.