Galactomannan utilization by Cellvibrio japonicus relies on a single essential α-galactosidase encoded by the aga27A gene.

Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.

Thermophilic ß-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

ß-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable ß-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic ß-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

Engineered Alcaligenes sp. by chemical mutagen produces thermostable and acido-alkalophilic endo-1,4-ß-mannanases for improved industrial biocatalyst.

This study reported physicochemical properties of purified endo-1,4-ß-mannanase from the wild type, Alcaligenes sp. and its most promising chemical mutant. The crude enzymes from fermentation of wild and mutant bacteria were purified by ammonium sulfate precipitation, ion exchange and gel-filtration chromatography followed by an investigation of the physicochemical properties of purified wild and mutant enzymes. ß-mannanase from wild and mutant Alcaligenes sp. exhibited 1.75 and 1.6 purification-folds with percentage recoveries of 2.6 and 2.5% and molecular weights of 61.6 and 80 kDa respectively. The wild and mutant ß-mannanase were most active at 40 and 50 °C with optimum pH 6.0 for both and were thermostable with very high percentage activity but the wild-type ß-mannanase showed better stability over a broad pH activity. The ß-mannanase activity from the parent strain was stimulated in the presence of Mn2+, Co2+, Zn2+, Mg2+ and Na+. Vmax and Km for the wild type and its mutant were found to be 0.747 U//mL/min and 5.2 × 10-4 mg/mL, and 0.247 U/mL/min and 2.47 × 10-4 mg/mL, respectively. Changes that occurred in the nucleotide sequences of the most improved mutant may be attributed to its thermo-stability, thermo-tolerant and high substrate affinity- desired properties for improved bioprocesses.

Functional diversity of three tandem C-terminal carbohydrate-binding modules of a ß-mannanase.

Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating noncatalytic carbohydrate-binding modules (CBMs) to compensate for low-affinity binding typical of protein-carbohydrate interactions. The bacterium Saccharophagus degradans produces an endo-ß-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding are not clear. To learn more, we studied the full-length enzyme, three stepwise CBM family 10 (CBM10) truncations, and GFP fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose (dissociation constant, Kd = 1.48 µM). CBM10-3 and CBM10-2 bound galactomannan with similar affinity (Kd = 0.2-0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was twofold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was fourfold to ninefold reduced at low enzyme and substrate concentrations compared with the enzyme lacking CBM10-2 and CBM10-3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.

A novel neutral thermophilic ß-mannanase from Malbranchea cinnamomea for controllable production of partially hydrolyzed konjac powder.

Partially hydrolyzed konjac powder (PHKP) can be used to increase the daily intake of dietary fibers of consumers. To produce PHKP by enzymatic hydrolysis, a novel ß-mannanase gene (McMan5B) from Malbranchea cinnamomea was expressed in Pichia pastoris. It showed a low identity of less than 52% with other GH family 5 ß-mannanases. Through high cell density fermentation, the highest ß-mannanase activity of 42200 U mL-1 was obtained. McMan5B showed the maximal activity at pH 7.5 and 75 °C, respectively. It exhibited excellent pH stability and thermostability. Due to the different residues (Phe214, Pro253, and His328) in catalytic groove and the change of ß2-α2 loop, McMan5B showed unique hydrolysis property as compared to other ß-mannanases. The enzyme was employed to hydrolyze konjac powder for controllable production of PHKP with a weight-average molecular weight of 22000 Da (average degree of polymerization 136). Furthermore, the influence of PHKP (1.0%-4.0%) on the qualities of steamed bread was evaluated. The steamed bread adding 3.0% PHKP had the maximum specific volume and the minimum hardness, which showed 11.0% increment and 25.4% decrement as compared to the control, respectively. Thus, a suitable ß-mannanase for PHKP controllable production and a fiber supplement for steamed bread preparation were provided in this study. KEY POINTS: • A novel ß-mannanase gene (McMan5B) was cloned from Malbranchea cinnamomea and expressed in Pichia pastoris at high level. • McMan5B hydrolyzed konjac powder to yield partially hydrolyzed konjac powder (PHKP) instead of manno-oligosaccharides. • PHKP showed more positive effect on the quality of steamed bread than many other dietary fibers including konjac powder.

Structure and function of Bs164 ß-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164.

Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (Bs164) from the human gut symbiont Bacteroides salyersiae. Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only ß-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant Bs164, and multiangle light scattering we found that it is a trimeric retaining ß-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of Bs164, the first known structure of a GH164, at 1.91 Å resolution. Bs164 is composed of three domains: a (ß/α)8 barrel, a trimerization domain, and a ß-sandwich domain, representing a previously unobserved structural-fold for ß-mannosidases. Structures of Bs164 at 1.80-2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.

Structural investigation of a thermostable 1,2-ß-mannobiose phosphorylase from Thermoanaerobacter sp. X-514.

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Molecular advancements on over-expression, stability and catalytic aspects of endo-ß-mannanases.

The hydrolysis of mannans by endo-ß-mannanases continues to gather significance as exemplified by its commercial applications in food, feed, and a rekindled interest in biorefineries. The present review provides a comprehensive account of fundamental research and fascinating insights in the field of endo-ß-mannanase engineering in order to improve over-expression and to decipher molecular determinants governing activity-stability during harsh conditions, substrate recognition, polysaccharide specificity, endo/exo mode of action and multi-functional activities in the modular polypeptide. In-depth analysis of the available literature has also been made on rational and directed evolution approaches, which have translated native endo-ß-mannanases into superior biocatalysts for satisfying industrial requirements.

Molecular and biochemical characterizations of a new cold-active and mildly alkaline ß-Mannanase from Verrucomicrobiae DG1235.

Mannanases catalyze the cleavage of ß-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new ß-mannanase from Verrucomicrobiae DG1235 (ManDG1235) was biochemically characterized and its enzymatic properties were revealed. Amino acid alignment indicated that ManDG1235 belonged to glycoside hydrolase family 26 and shared a low amino acid sequence identity to reported ß-mannanases (up to 50% for CjMan26C from Cellvibrio japonicus). ManDG1235 was expressed in Escherichia coli. Purified ManDG1235 (rManDG1235) exhibited the typical properties of cold-active enzymes, including high activity at low temperature (optimal at 20 °C) and thermal instability. The maximum activity of rManDG1235 was achieved at pH 8, suggesting that it is a mildly alkaline ß-mannanase. rManDG1235 was able to hydrolyze a variety of mannan substrates and was active toward certain types of glucans. A structural model that was built by homology modeling suggested that ManDG1235 had four mannose-binding subsites which were symmetrically arranged in the active-site cleft. A long loop linking ß2 and α2 as in CjMan26C creates a steric border in the glycone region of active-site cleft which probably leads to the exo-acting feature of ManDG1235, for specifically cleaving mannobiose from the non-reducing end of the substrate.

A surface-exposed GH26 ß-mannanase from Bacteroides ovatus: Structure, role, and phylogenetic analysis of BoMan26B.

The galactomannan utilization locus (BoManPUL) of the human gut bacterium Bacteroides ovatus encodes BoMan26B, a cell-surface-exposed endomannanase whose functional and structural features have been unclear. Our study now places BoMan26B in context with related enzymes and reveals the structural basis for its specificity. BoMan26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase BoMan26A of the same locus. Using galactomannan, BoMan26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface-exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of BoMan26B and the periplasmic α-galactosidase BoGal36A increased the rate of galactose release by about 10-fold compared with the rate without BoMan26B. The results suggested that BoMan26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by BoGal36A. A crystal structure of BoMan26B with galactosyl-mannotetraose bound in subsites -5 to -2 revealed an open and long active-site cleft with Trp-112 in subsite -5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the -4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that BoMan26A and BoMan26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B.

Structural basis of exo-ß-mannanase activity in the GH2 family.

The classical microbial strategy for depolymerization of ß-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-ß-mannanases and ß-mannosidases. In this work, we describe the first exo-ß-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and ß-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 ß-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 ß-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 ß-mannosidases, Gly439 and Gly556, which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-ß-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-ß-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 ß-1,4-mannanase from Amphibacillus xylanus.

ß-1,4-Mannanase degrades ß-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) family 113 ß-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme-manno-oligosaccharides from this family has been reported. Here, crystal structures of a GH family 113 ß-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had higher affinity for -1 and +1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites (-4 to +2) exist in the groove, but mannose units preferentially occupied subsites -4 to -1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH family 113 ß-1,4-mannanase-manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism of GH family 113 members, and provide novel insight into protein engineering of GHs to improve their hydrolysis activity.

Selection of Bacillus subtilis US191 as a mannanase-producing probiotic candidate.

ß-Mannanases are crucial enzymes for the breakdown of mannans. As Mannans are being considered as antinutritional factors in poultry production, the search for mannanase-producing probiotic bacteria is now attracting considerable attention as a strategy to enhance nutrients bioavailability. Five soil born Bacilli (US134, US150, US176, US180, and US191) were selected for their ability to produce extracellular ß-mannanases that were biochemically characterized. The probiotic properties of these strains were determined to assess their potential as animal feed supplements. Bacillus subtilis US191 was shown to be sensitive to all antibiotics tested, to inhibit growth of the bacterial pathogens tested, and to produce a thermostable ß-mannanase. It exhibited a notable acid and bovine bile tolerance and high ability to form biofilm. These features may favor its adherence to the intestinal epithelial cells allowing its survival and persistence in the digestive tract. Furthermore, our study revealed that US191 was among the strains showing the highest ability to digest wheat dry matter in vitro when compared to the commercial feed additive Rovabio® Max. Altogether, our findings suggest that the ß-mannanase producer B.subtilis US191 is a promising probiotic candidate for the feed industry.

Regulation of hyperglycemia in diabetic mice by autolysates from ß-mannanase-treated brewer's yeast.

BACKGROUND: Diabetes mellitus is a serious chronic disease, characterized by hyperglycemia. This study administered either ß-mannanase-treated yeast cell autolysis supernatant (YCS) or yeast cell-wall residues after autolysis (YCR) to investigate their influence on the alleviation of diabetes in a diabetic mouse model. RESULTS: Application of either YCS or YCR led to body weight gain, blood glucose reduction, and an improvement in lipid composition in the diabetic mice. Administration of YCS was more effective in inhibiting oxidative stress than YCR. The expression of PPARα and CPT1α was enhanced, improving lipid biosynthesis, and Trx1 and HIF-1-α genes were downregulated due to the activation of thioredoxin following the interventions, indicating that the processes of lipid metabolism and oxidative stress were heavily involved in the reduction of diabetic characteristics following the interventions. The current study revealed that consumption of YCR also led to a reduction in hyperglycemia, this being associated with its richness in mineral elements, such as chromium and selenium. CONCLUSION: This study may highlight the potential of both YCS and YCR as functional ingredients in dietary formula for improving diabetic syndromes. © 2019 Society of Chemical Industry.

Galactomannan degradation by thermophilic enzymes: a hot topic for biotechnological applications.

Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a ß-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. ß-mannanase, ß-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.

Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus of Bacteroides ovatus: ENZYME SYNERGY AND CRYSTAL STRUCTURE OF A ß-MANNANASE.

A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) ß-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by ß-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two ß-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized ß-mannanases, we propose a scheme of sequential action by the glycoside hydrolases encoded by the ß-mannan PUL and involved in the ß-mannan utilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the ß-mannanase BoMan26A.

Structural and biochemical insights into the substrate-binding mechanism of a novel glycoside hydrolase family 134 ß-mannanase.

Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. ß-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-ß-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pH 5.0 and 50 °C, and exhibited high specific activity towards locust bean gum (2337 U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A-M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (-3 to +4) in the catalytic groove of RmMan134A. Mannose in the -1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impedes to bond with galactose branches. In addition, the various binding modes of RmMan134A-M5 indicated that subsites -2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A-M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and optimal conditions of RmMan134A were significantly improved. The purpose of this paper is to investigate the structure and function of fungal GH family 134 ß-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.

Trp residue at subsite - 5 plays a critical role in the substrate binding of two protistan GH26 ß-mannanases from a termite hindgut.

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two ß-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to ß-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite - 5. The Km values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.

Hac1p homologues from higher eukaryotes can improve the secretion of heterologous proteins in the yeast Pichia pastoris.

OBJECTIVES: To systematically explore the effects of overexpressing Hac1p homologues from different sources on protein secretion in Pichia pastoris system. RESULTS: Effects of Hac1p homologues encompassing P. pastoris (PpHac1p), S. cerevisiae (ScHac1p), Trichoderma reesei (TrHac1p) and Homo sapiens (HsXbp1), on secretion of three reporter proteins-ß-galactosidase, ß-mannanase and glucose oxidase were investigated. No individual Hac1p was optimal for all the enzymes. Rather, by testing a set of Hac1p, the secretory expression of each of the enzymes was improved. Notably, HsXbp1 overexpression improved ß-mannanase production from 73 to 108.5 U ß-mannanase mL-1 while PpHac1p had no impact in shake flask culture. Moreover, HsXbp1 led to 41 and 67% increases in ß-mannanase production in the single- and four-copy strain, respectively in 1-L laboratory fermenter. Transcription analysis of indicative chaperones suggested that HsXbp1 may cause a stronger and prolonged activation of the UPR target chaperone genes. CONCLUSION: Mammalian HsXbp1 worked better than yeast Hac1p in terms of improving ß-mannanase secretion in P. pastoris, and Hac1p screening may offer an effective strategy to engineer the secretion pathway of eukaryotic expression systems.

Effect of ß-mannanase domain from Trichoderma reesei on its biochemical characters and synergistic hydrolysis of sugarcane bagasse.

BACKGROUND: ß-mannanase is a key enzyme for hydrolyzing mannan, a major constituent of hemicellulose, which is the second most abundant polysaccharide in nature. Different structural domains greatly affect its biochemical characters and catalytic efficiency. However, the effects of linker and carbohydrate-binding module (CBM) on ß-mannanase from Trichoderma reesei (Man1) have not yet been fully described. The present study aimed to determine the influence of different domains on the expression efficiency, biochemical characteristics and hemicellulosic deconstruction of Man1. RESULTS: The expression efficiency was improved after truncating CBM. Activities of Man1 and Man1ΔCBM (CBM) in the culture supernatant after 168 h of induction were 34.5 and 42.9 IU mL-1 , although a value of only 0.36 IU mL-1 was detected for Man1ΔLCBM (lacking CBM and linker). Man1 showed higher thermostability than Man1ΔCBM at low temperature, whereas Man1ΔCBM had a higher specificity for galactomannan (Km = 2.5 mg mL-1 ) than Man1 (Km = 4.0 mg mL-1 ). Both Man1 and Man1ΔCBM could synergistically improve the hydrolysis of cellulose, galactomannan and pretreated sugarcane bagasse, with a 10-30% improvement of the reducing sugar yield. CONCLUSION: Linker and CBM domains were vital for mannanase activity and expression efficiency. CBM affected the thermostability and adsorption ability of Man1. The results obtained in the present study should help guide the rational design and directional modification of Man with respect to improving its catalytic efficiency. © 2017 Society of Chemical Industry.