Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases as Antibiofilm Agents in Acute Pulmonary Pseudomonas aeruginosa Infection.

The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl. In this study, recombinant glycoside hydrolases (GHs) that degrade Pel and Psl were evaluated alone and in combination with antibiotics in a mouse model of P. aeruginosa infection. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that, although GHs have short half-lives, administration of two GHs in combination resulted in increased GH persistence. Combining GH prophylaxis and treatment with the antibiotic ciprofloxacin resulted in greater reduction in pulmonary bacterial burden than that with either agent alone. This study lays the foundation for further exploration of GH therapy in bacterial infections.

Utilization Efficiency of Human Milk Oligosaccharides by Human-Associated Akkermansia Is Strain Dependent.

Akkermansia muciniphila is a mucin-degrading bacterium found in the human gut and is often associated with positive human health. However, despite being detected by as early as 1 month of age, little is known about the role of Akkermansia in the infant gut. Human milk oligosaccharides (HMOs) are abundant components of human milk and are structurally similar to the oligosaccharides that comprise mucin, the preferred growth substrate of human-associated Akkermansia. A limited subset of intestinal bacteria has been shown to grow well on HMOs and mucin. We therefore examined the ability of genomically diverse strains of Akkermansia to grow on HMOs. First, we screened 85 genomes representing the four known Akkermansia phylogroups to examine their metabolic potential to degrade HMOs. Furthermore, we examined the ability of representative isolates to grow on individual HMOs in a mucin background and analyzed the resulting metabolites. All Akkermansia genomes were equipped with an array of glycoside hydrolases associated with HMO deconstruction. Representative strains were all able to grow on HMOs with various efficiencies and growth yields. Strain CSUN-19, belonging to the AmIV phylogroup, grew to the highest level in the presence of fucosylated and sialylated HMOs. This activity may be partially related to the increased copy numbers and/or the enzyme activities of the α-fucosidases, α-sialidases, and ß-galactosidases. This study examines the utilization of individual purified HMOs by Akkermansia strains representing all known phylogroups. Further studies are required to examine how HMO ingestion influences gut microbial ecology in infants harboring different Akkermansia phylogroups. IMPORTANCE Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk and provide several benefits to developing infants, including the recruitment of beneficial bacteria to the human gut. Akkermansia strains are largely considered beneficial bacteria and have been detected in colostrum, breast milk, and young infants. A. muciniphila MucT, belonging to the AmI phylogroup, contributes to the HMO deconstruction capacity of the infant. Here, using phylogenomics, we examined the genomic capacities of four Akkermansia phylogroups to deconstruct HMOs. Indeed, each phylogroup contained differences in their genomic capacities to deconstruct HMOs, and representative strains of each phylogroup were able to grow using HMOs. These Akkermansia-HMO interactions potentially influence gut microbial ecology in early life, a critical time for the development of the gut microbiome and infant health.

A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans.

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(ß1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(ß1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-ß-N-acetylhexosaminidase activities, including exo-(ß1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

Structural and functional analyses of glycoside hydrolase 138 enzymes targeting chain A galacturonic acid in the complex pectin rhamnogalacturonan II.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the d-galacturonic acid-α-1,2-l-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second d-galacturonic acid side chain (linked ß-1,3 to l-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double displacement retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/ß)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu-361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting d-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes.

Diverse and common features of trehalases and their contributions to microbial trehalose metabolism.

Trehalose is a stable disaccharide that consists of two glucose units linked primarily by an α,α-(1 → 1)-linkage, and it has been found in a wide variety of organisms. In these organisms, trehalose functions not only as a source of carbon energy but also as a protector against various stress conditions. In addition, this disaccharide is attractive for use in a wide range of applications due to its bioactivities. In trehalose metabolism, direct trehalose-hydrolyzing enzymes are known as trehalases, which have been reported for bacteria, archaea, and eukaryotes, and are classified into glycoside hydrolase 37 (GH37), GH65, and GH15 families according to the Carbohydrate-Active enZyme (CAZy) database. The catalytic domains (CDs) of these enzymes commonly share (α/α)6-barrel structures and have two amino acid residues, Asp and/or Glu, that function as catalytic residues in an inverting mechanism. In this review, I focus on diverse and common features of trehalases within different GH families and their contributions to microbial trehalose metabolism.

A highly glucose-tolerant GH1 ß-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals.

In the feed industry, ß-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 ß-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC50 = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous ß-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 ß-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

The first crystal structure of a family 45 glycoside hydrolase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A.

Here we describe the first crystal structure of a beta-1,4-endoglucanase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A, which belongs to subfamily C of glycoside hydrolase family 45 (GH45). GtCel45A is ~ 18 kDa in size and the crystal structure contains 179 amino acids. The structure is refined at 1.30 Å resolution and Rfree 0.18. The enzyme consists of a single catalytic module folded into a six-stranded double-psi beta-barrel domain surrounded by long loops. GtCel45A is very similar in sequence (82% identity) and structure to PcCel45A from the white-rot fungus Phanerochaete chrysosporium. Surprisingly though, initial hydrolysis of barley beta-glucan was almost twice as fast in GtCel45A as compared to PcCel45A.

Natronosalvus hydrolyticus sp. nov., a beta-1,3-glucan utilizing natronoarchaeon from hypersaline soda lakes.

Use of curldlan, an insoluble ß-1,3-glucan, as an enrichment substrate under aerobic conditions resulted in the selection from hypersaline soda lakes of a single natronarchaeon, strain AArc-curdl1. This organism is an obligately aerobic saccharolytic, possessing a poorly explored (in Archaea) potential to utilize beta-1-3 glucans, being only a second example of a haloarchaeon with this ability known in pure culture. The main phenotypic property of the isolate is the ability to grow with insoluble ß-1,3-backboned glucans, i.e. curdlan and pachyman. Furthermore, the strain utilized starch family α-glucans, beta-fructan inulin and a limited spectrum of sugars. The major ether-bound membrane polar phospholipids included PGP-Me and PG. The glyco- and sulfolipids were absent. The major respiratory menaquinone is MK-8:8. According to phylogenomic analysis, AArc-curdl1 represents a separate species in the recently described genus Natronosalvus within the family Natrialbaceae. The closest related species is Natronosalvus amylolyticus (ANI, AAI and DDH values of 90.2, 91.6 and 44 %, respectively). On the basis of its unique physiological properties and phylogenomic distance, strain AArc-curdl1T is classified as a novel species Natronosalvus hydrolyticus sp. nov. (=JCM 34865 = UQM 41566).

Genetic-Phenotype Analysis of Bifidobacterium bifidum and Its Glycoside Hydrolase Gene Distribution at Different Age Groups.

Human gut microbiota interfere with host development and aging. Bifidobacterium is a microbial genus found in the human digestive tract that has probiotic activities such as improving constipation and enhancing immunity. The species and numbers present change with age, but there has been limited research on probiotic gut microbiota at specific ages. This study analyzed the distribution of 610 bifidobacteria in subjects in several age groups (0-17, 18-65, and 66-108 y) using 486 fecal samples and determined the distribution of glycoside hydrolases based on genetic analysis of strains representing 85% of the Bifidobacterium species abundance in each age group. 6'-Sialyllactose is a major component of acidic breast milk oligosaccharides, which can promote human neurogenesis and bifidobacteria growth. Using genotypic and phenotypic association analysis, we investigated the utilization of 6'-sialyllactose by six B. bifidum strains isolated from subjects 0-17 and 18-65 y. A comparative genomic analysis of the six B. bifidum strains revealed differences in genomic features across age groups. Finally, the safety of these strains was evaluated by antibiotic gene and drug resistance phenotype analysis. Our results reveal that the distribution of glycoside hydrolase genes in B. bifidum varies with age, thus affecting the phenotypic results. This provides important insights for the design and application of probiotic products for different ages.

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes that enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization to precise Michaelis-Menten analysis.

Cloning, Expression, and Characterization of Endo-ß-1,6-galactanase PoGal30 from Penicillium oxalicum.

Because ß-1,6-galactans are significant components in arabinogalactans from plant cell walls, identifying selective endo-ß-1,6-galactanases is crucial to degrading these polysaccharides and to analyzing and modifying their structures. Here, we cloned and expressed in E. coli a novel endo-ß-1,6-galactanase in the glycosidic hydrolase family 30 (GH30) from Penicillium oxalicum. Our recombinant PoGal30 hydrolase (1464 bp gene) that contains an N-terminal His-tag for purification by nickel affinity chromatography has a specific activity of 3.8 U/mg on the substrate de-arabinosylated gum Arabic (dGA) polysaccharide. The enzyme has 487 residues with a molecular mass of 60 kDa, an isoelectric point of 6, and functional pH and temperature optima of pH 2.5 to pH 5.0 and 40 °C, respectively. While the activity of PoGal30 is activated by Mg2+ (5 or 50 mmol/L), it is completely inhibited by Cu2+ and Fe3+ (50 mmol/L) and partially inhibited by Hg2+, EDTA, and SDS (50 mmol/L). The enzyme demonstrates high specificity towards ß-1,6-galactosidic linkages in dGA, but is inactive against aryl-glycosides and galactobioses with different linkages. Using PoGal30 is, therefore, an effective approach to analyzing the fine structure of polysaccharides and preparing bioactive oligosaccharides.

A Case Study of the Glycoside Hydrolase Enzyme Mechanism Using an Automated QM-Cluster Model Building Toolkit.

Glycoside hydrolase enzymes are important for hydrolyzing the ß-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol-1 for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol-1 for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme.

Glycoside hydrolase subfamily GH5_57 features a highly redesigned catalytic interface to process complex hetero-ß-mannans.

Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage ß-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-ß-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-ß-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing ß6-α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the -1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

Gene Cloning, Functional Expression, and Characterization of a Novel GH46 Chitosanase from Streptomyces avermitilis (SaCsn46A).

A n ovel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acid signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then, the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 °C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 °C. The Km and Vmax values of this enzyme were 1.32 mg/mL, 526.32 U/mg/min, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3-mM concentration. The enzyme can hydrolyze a variety of polysaccharides which are linked by ß-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin-layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases in the Prevention of Experimental Invasive Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.

Genome-Wide Identification and Functional Characterization of ß-Agarases in Vibrio astriarenae Strain HN897.

The genus Vibrio is a genetically and metabolically versatile group of heterotrophic bacteria that are important contributors to carbon cycling within marine and estuarine ecosystems. HN897, a Vibrio strain isolated from the coastal seawater of South China, was shown to be agarolytic and capable of catabolizing D-galactose. Herein, we used Illumina and PacBio sequencing to assemble the whole genome sequence for the strain HN897, which was comprised of two circular chromosomes (Vas1 and Vas2). Genome-wide phylogenetic analysis with 140 other Vibrio sequences firmly placed the strain HN897 into the Marisflavi clade, with Vibrio astriarenae strain C7 being the closest relative. Of all types of carbohydrate-active enzyme classes, glycoside hydrolases (GH) were the most common in the HN897 genome. These included eight GHs identified as putative ß-agarases belonging to GH16 and GH50 families in equal proportions. Synteny analysis showed that GH16 and GH50 genes were tandemly arrayed on two different chromosomes consistent with gene duplication. Gene knockout and complementation studies and phenotypic assays confirmed that Vas1_1339, a GH16_16 subfamily gene, exhibits an agarolytic phenotype of the strain. Collectively, these findings explained the agar-decomposing of strain HN897, but also provided valuable resources to gain more detailed insights into the evolution and physiological capability of the strain HN897, which was a presumptive member of the species V. astriarenae.

Cell-surface display of designer cellulosomes by Lactobacillus plantarum.

Cell-surface display of designer cellulosomes complexes has attracted increased interest in recent years. These engineered microorganisms can efficiently degrade lignocellulosic biomass that represents an abundant resource for conversion into fermentable sugars, suitable for production of biofuels. The designer cellulosome is an artificial enzymatic complex that mimics the architecture of the natural cellulosome and allows the control of the positions, type, and copy number of the cellulosomal enzymes within the complex. Lactobacillus plantarum is an attractive candidate for metabolic engineering of lignocellulosic biomass to biofuels, as its natural characteristics include high ethanol and acid tolerance and the ability to metabolize hexose sugars. In recent years, successful expression of a variety of designer cellulosomes on the cell surface of this bacterium has been demonstrated using the cell-consortium approach. This strategy minimized genomic interference on each strain upon genetic engineering, thereby maximizing the ability of each strain to grow, express, and secrete each enzyme. In addition, this strategy allows stoichiometric control of the cellulosome elements and facile exchange of the secreted proteins. A detailed procedure for display of designer cellulosomes on the cell surface of L. plantarum is described in this chapter.

Gene cloning, expression, and X-ray crystallographic analysis of a ß-mannanase from Eisenia fetida.

The endo-1,4-ß-mannanases (Ef-Man) gene from Eisenia fetida was determined to consist of 1131 bp and encode a 377 amino acid protein. The amino acid sequence showed similarity with the endo-1,4-ß-mannanases of Daphnia pulex (62%), Cryptopygus antarcticus (64%), Crassostrea gigas (61%), Mytilus edulis (60%), and Aplysia kurodai (58%). The gene encoding mature Ef-Man was expressed in Pichia pastoris (GS115 strain). Based on SDS-PAGE analysis, the molecular mass of the purified recombinant Ef-Man (rEf-Man) was estimated to be 39 kDa. All catalytically important residues of endo-1,4-ß-mannanases in the glycoside hydrolase (GH) family 5 were conserved in Ef-Man. The optimal temperature for rEf-Man was identified as 60 °C. HPLC and HPAEC analyses suggest that Ef-Man requires at least six subsites for efficient hydrolysis and is capable of performing transglycosylation reactions. The overall structure of rEf-Man is similar to those of GH5 family proteins, and tertiary structures around the active site are conserved among endo-1,4-ß-mannanase families. X-ray crystallographic analysis supports the hydrolysis and transglycosylation reaction mechanism determined by HPLC and HPAEC analyses.

Functional Analysis of a Highly Active ß-Glucanase from Bispora sp. MEY-1 Using Its C-terminally Truncated Mutant.

A ß-1,3-1,4-glucanase-encoding gene, Bisglu16B, was identified in Bispora sp. MEY-1. The deduced BisGlu16B consists of an N-terminal signal peptide, a catalytic module of glycoside hydrolase family 16 (GH16), and a C-terminal serine/proline-rich module. After expression in Pichia pastoris GS115, the purified recombinant BisGlu16B showed maximal activity at pH 4.0 and 55 °C and had broad substrate specificity (ß-1,3-/ß-1,4-mixed, ß-1,3-, ß-1,4-, and ß-1,6-linked glucan, and ß-1,4-mannan). The enzyme possessed high specific activities toward barley ß-glucan (34 700 U·mg-1), lichenan (23 900 U·mg-1), and laminarin (9 000 U·mg-1). After removing the C-terminal module, the truncated mutant, BisGlu16B-ΔC, retained similar enzymatic properties to the wild type but displayed significantly enhanced activities (up to 2.5-fold). Functional and structural analyses indicated that the C-terminal module plays a key role in the substrate binding of BisGlu16B. This study provided an excellent candidate glucanase for industrial purposes and revealed the functions of a C-terminal serine/proline-rich region.

Cloning and expression of a chitinase gene from Eisenia fetida.

Chitin is the second most abundant biopolymer in nature and is an important resource. In this study, we identified a chitinase gene, named Eisenia fetida-Chitinase (EF-Chi) gene, of 1494 base pairs (bp) that encodes a protein of 498 amino acids as indicated by the corresponding mRNA sequence. The amino acid sequence of EF-Chi was similar to those of chitinases from Eisenia andrei (99%), Branchiostoma floridae (50%) and Oryzias latipes (49%), and a gene encoding mature EF-Chi was expressed in the GS115 strain of Pichia pastoris. The molecular mass of the purified recombinant EF-Chi (rEF-Chi) was estimated to be 60kDa and catalytically important residues of chitinases of the glycoside hydrolase (GH) family 18 were conserved in EF-Chi. The optimal catalytic temperature of rEF-Chi was identified as 60°C, and the hydrolytic product from colloidal chitin was N-acetyl-chitobiose, suggesting that EF-Chi is an exo-type enzyme.