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1,2-β-Mannobiose phosphorylases (1,2-β-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-β-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-β-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-β-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Mannooligosaccharides (MOSs) are one of the most commonly used biomass-derived feed additives. The effectiveness of MOS varies with the length of oligosaccharides, medium length MOSs such as mannotetraose and mannopentaose being the most efficient. This study aims at improving specificity of β-mannanase from Aspergillus niger toward the desirable product size through rational-based enzyme engineering. Tyr 42 and Tyr 132 were mutated to Gly to extend the substrate binding site, allowing higher molecular weight MOS to non-catalytically bind to the enzyme. Hydrolysis product content was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. Instead of mannobiose, the enzyme variants yielded mannotriose and mannotetraose as the major products, followed by mannobiose and mannopentaose. Overall, 42% improvement in production yield of highly active mannotetraose and mannopentaose was achieved. This validates the use of engineered β-mannanase to selectively produce larger MOS, making them promising candidates for large-scale MOS enzymatic production process.

Metabolism of non-digestible dietary glycans directly influences the structure and composition of human gut microbiota and, in turn, the host health. β-Mannans form an integral component of the modern diet as naturally occurring dietary fibre or additives in processed foods. In the present study, in vitro fermentation and TLC studies were used to determine the ability of adult-associated Bifidobacterium adolescentis DSMZ 20083 to utilise β-manno-oligosaccharides from guar gum, locust bean gum, konjac root, and copra meal generated using GH26 endo-β-mannanase (ManB-1601). Further, to gain insights into the underlying molecular mechanism, a whole-genome microarray analysis, RT-qPCR, and molecular docking studies were employed to reconstruct the copra meal β-manno-oligosaccharides (CM-β-MOS) utilisation pathway in B. adolescentis DSMZ 20083. B. adolescentis DSMZ 20083 grew appreciably (O.D

Aspergillus quadrilineatus RSNK-1 produced a multi-enzymatic system containing (U/gds) β-mannanase (1021), endo-xylanase (1 9 1), α-galactosidase (3.42), β-xylosidase (0.07) and β-glucosidase (0.28) on low-cost copra meal (CM) in SSF. The enzyme preparation was covalently immobilized on aluminum oxide pellets (3 mm) under statistically optimized conditions leading to 73.17% immobilization yield. The immobilized enzyme (Man-AOP) displayed enhanced thermal and pH stability. Man-AOP was characterized by FTIR, SEM and PXRD revealing a covalent interaction. The bio-conjugate was successfully recycled for mannooligosaccharide (MOS) generation from locust bean gum (LBG) up to 10 cycles, yielding an average of 0.95 mg MOS/cycle. Man-AOP was also effective in clarification of apple, kiwi, orange and peach juices and enhanced their reducing sugar content. The bio-conjugate was useful in generation of MOS from mannan and enrichment of fruit juices.

Hitherto, how salt bridges contribute towards the structure-function of endo-mannanases has not been demonstrated. In the present study, we revealed that ManB-1601 (GH26 endo-mannanase from Bacillus sp.) has eight salt bridges which are highly conserved among GH26 endomannanases from Bacillus spp. Disruption of salt bridges do not alter overall structure, optimum pH and temperature of ManB-1601. Among the salt bridges, elimination of K95/E156 and E171/R221 pair decreased the substrate affinity and catalytic efficiency of ManB-1601. Differential scanning calorimetry and isothermal equilibrium denaturation studies suggested that salt bridges do not markedly contribute towards thermal (ΔTm 0 to -50C) and conformational stability (ΔΔG -0.37 to -1.25 Kcal mol-1) of ManB-1601. Interestingly, salt bridges were found to prominently contribute towards kinetic stability of ManB-1601 as salt bridge mutants exhibited drastic reduction in half-life of enzyme inactivation (T1/2) at 660C (1.1 to 6-fold) and 700C (4.09 to 22.5-fold). Molecular dynamic simulations studies showed that salt bridges contribute towards maintaining the biological activity against thermal denaturation by rigidifying the active site. Our study on ManB-1601 suggest that even in the case salt bridges do not confer thermal and conformational stabilities they may serve as crucial structural elements for enzyme functioning by contributing towards kinetic stability.

This study measured the metabolizable energy of soybean meal (SBM) and evaluated effects of soybean meal specific enzymes supplementation in corn-soybean diets on growth performance, intestinal digestion properties and energy values of 28-day-old broilers. A total of 336 one-day-old male AA broiler chickens were distributed to 7 groups in a completely random design. The birds were given 7 diets containing 6 diets with different combined soybean meals and a fasting treatment, 8 replicates per treatment and 6 birds per replicate (Trial 1). A total of 672 one-day-old male AA broiler chickens were randomly allocated to 7 dietary treatments including a control diet and 6 diets supplemented with 300 mg/kg α-galactosidase, 200 mg/kg β-mannanase, and 300 mg/kg protease individually or in combination (Trial 2). Apparent metabolizable energy (AME) of broilers was measured from d 25 to 27 in both trial 1 and trial 2. The results showed that AME values of combined soybean meals averaged 2,894 kcal/kg. Dietary β-mannanase and protease supplementation increased body weight gain (P0.05) during d 0 to 14, whereas did not affect the growth performance (P0.05) during d 14 to 28. Addition of β-mannanase in combination with other enzymes significantly increased lipase and trypsin content (P0.05) in ileum. In addition, dietary β-mannanase and protease supplementation individually or in combination enhanced trypsin enzyme content in jejunum (P0.05). The β-mannanase enzyme enhanced villus height and villus height to crypt depth ratio (P0.05) of ileum compared with control diet. Moreover, supplementation of enzyme except for protease enhanced raffinose and stachyose degradation ratio (P0.05). Dietary β-mannanase supplementation individually or in combination enhanced AME and AMEn values (P0.05). This study demonstrated that dietary enzyme supplementation especially β-mannanase improved intestinal digestion properties and contributed to high energy values.

Hitherto, the contribution of C-terminal amino acids in structure, stability and function of GH26 endo-mannanases has not been demonstrated. Semi-logarithmic plot of endo-mannanase activity showed a progressive decline with increase in the number of truncated amino acids [ManB-CΔ5 (129 U/mL), ManB-CΔ10 (47 U/mL), ManB-CΔ15 (0.05 U/mL) and ManB-CΔ20 (0.02 U/mL)]. ManB-CΔ5 and ManB-CΔ10 exhibited similar temperature and pH optima and product profile but biochemical properties (kinetic constants, mannan hydrolysis, response to metal ions and enzyme inhibitors) and stability (in presence of commercial detergents, anionic surfactants and organic solvents and half-life) were markedly affected. Interaction of truncated proteins with anionic surfactants was probed using intrinsic, Nile red, acrylamide quenching, resonance light scattering and synchronous fluorescence spectroscopy studies. Truncation of ten amino acids increased vulnerability to anionic surfactants as conformational changes, exposure of the hydrophobic core and susceptibility to unfolding process were observed. The microenvironment around Trp residues was affected more with surfactants as compared to Tyr residues in truncated proteins. Zn

A rational design method to improve β-mannanase (ManTJ102) thermostability was developed successfully in this study. The flexible area of residues 330–340 in ManTJ102 was firstly selected from analysis of molecular dynamics simulation and then the critical amino acid residue (Ala336Pro) with the lowest mutation energy was determined by virtual mutation, whose mutant was named as Mutant336. Afterward, the dynamics transition temperature (Tdtt) of ManTJ102 and Mutant336 was evaluated by simulated annealing and heating, and Mutant336 with higher Tdtt was implemented for experimental verification of the enzyme thermostability. As a result, the half-life of Mutant336 activity was 120 min at 60 °C, which was 24-fold of ManTJ102, and the irreversible thermal denaturation constant of Mutant336 was only about 2/5 of ManTJ102, indicating that Mutant336 has better thermostability than ManTJ102. Furthermore, Mutant336 has much higher β-mannanase activity and specific activity than ManTJ102. Therefore, Mutant336 was more suitable to further research for applications.

The classical microbial strategy for depolymerization of β-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-β-mannanases and β-mannosidases. In this work, we describe the first exo-β-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and β-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 β-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 β-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 β-mannosidases, Gly(439) and Gly(556), which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-β-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-β-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

The present study was conducted to determine whether the addition of β-mannanase in broiler feed changes hormonal profiles in the blood and broiler performance and nutrient availability. Five hundred and four Cobb male chickens were studied during d 7 to 21. Three corn-soybean meal (SBM) based diets 1) Low SBM (18% SBM); 2) High SBM (31% SBM); and 3) High SBM+GG (31% SBM + Guar Gum (GG) 0.5%) with 3 levels of β-mannanase (0, 200, and 400 ppm) were mixed to produce 9 diets. A factorial design 3 × 3 was performed with JMP pro 13 (SAS, 2017). Analysis of variance and contrast analysis were used to test significance level at P < 0.05. Glucose (190 and 188 mg/dL) was increased with 200 and 400 ppm of β-mannanase, respectively, compared to control (182 mg/dL) in the fasted state (P < 0.037). Glucose was higher in chicks fed with the High SBM and High SBM + GG diets but lower in the fasted re-fed state (P < 0.01). Insulin was higher with 200 and 400 ppm added β-mannanase in the fed state (P < 0.021). Insulin-like growth factor-1 was higher with 400 ppm added to High SBM+GG. β-mannanase improved feed conversion ratio (FCR) 9 points with 400 ppm in High SBM diet (P < 0.01) and 16 and 18 points with 200 and 400 ppm, respectively, added to the High SBM+GG diet (P < 0.01). Viscosity decreased from 19.2 to 7 cps with both enzyme doses in the High SBM + GG diet (P < 0.01). Digestible energy was +152 kcal/kg with 400 ppm β-mannanase in the High SBM diet and +200 kcal/kg with both levels of enzyme in High SBM+GG diet. Digestibility of amino acids was improved from 0.8 to 3.6% with β-mannanase in High SBM+GG diet (P < 0.05). In conclusion, chicks fed with High SBM and High SBM+GG diets with added β-mannanase significantly improved blood glucose and anabolic hormone homeostasis, FCR, digestible energy, and digestible amino acids compared to chicks fed with same diets without β-mannanase.

Locust bean gum (LBG) galactomannan has been claimed to have applications in the biopharmaceutical field. However, the effects of LBG galactomannan on immunomodulatory aspects are not yet clear. The purpose of this study was to over-express thermostable β-d-mannanase from the thermophilic actinomycete Thermobifida fusca BCRC 19214 using a Pichia pastoris expression system. The maximum intracellular β-d-mannanase activity obtained from the cell-free extract was approximately 40.0U/mL after 72h of cultivating a P. pastoris transformant (pPICZ-man) induced with methanol. Hydrolysis of native LBG galactomannan with 8U/mL β-d-mannanase for 24h significantly decreased the weight-average molecular weight of LBG galactomannan from 5,580,010 to 3188. Native and hydrolyzed LBG galactomannan in a range of 0-0.2% did not trigger significant cytotoxicity after 24h of treatment compared with the control. The native LBG galactomannan stimulated RAW 264.7 cells to produce cytokine TNF-α dose-dependently, but there was no significant IL-1β or nitric oxide production. The native LBG galactomannan also stimulated β-hexosaminidase secretion in RBL-2H3 cells. After the native LBG galactomannan was hydrolyzed with β-d-mannanase, all of the immunological properties disappeared. These results suggest the possible immunomodulatory effects of native LBG galactomannan.

Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. β-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-β-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 β-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.

Accurate determination of the distribution of substitutions in the primary molecular structure of heteropolysaccharides and their derivatives is a prerequisite for their increasing application in the pharmaceutical and biomedical fields, which is unfortunately hindered due to the lack of effective analytical techniques. Acetylated galactoglucomannan (GGM) is an abundant plant polysaccharide as the main hemicellulose in softwoods, and therefore constitutes an important renewable resource from lignocellulosic biomass for the development of bioactive and functional materials. Here we present a methodology for profiling the intramolecular structure of spruce GGM and its chemical derivatives (cationic, anionic, and benzoylated) by combining chemo-enzymatic hydrolysis, liquid chromatography, and mass spectrometry. Fast identification and qualitative mass profiling of GGM and its derivatives was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and electrospray ionization mass spectrometry (ESI-MS). Tandem mass fragmentation analysis and its hyphenation with hydrophilic interaction liquid chromatography (HILIC-ESI-MS/MS) provide further insights on the substitution placement of the GGM oligosaccharides and its derivatives. This method will be useful in understanding the structure-function relationships of native GGM and their derivatives, and therefore facilitate their potential application.

An engineered β-mannanase (mRmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris. Through high cell density fermentation, the expression level of mRmMan5A reached 79,680 U mL−1. The mRmMan5A showed maximum activity at pH 4.5 and 65 °C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200 °C for 7.5 min, and then hydrolyzed by mRmMan5A. As a result, the total manno-oligosaccharide yield reached 34.8 g/100 g dry PKC, indicating that 80.6% of total mannan in PKC was hydrolyzed. Moreover, the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production. A total of 261.3 g manno-oligosaccharides were produced from 1.0 kg of dry PKC. An effective β-mannanase for the bioconversion of mannan-rich biomasses and an efficient method for the production of manno-oligosaccharides from PKC are provided in this paper.

The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic material, is required for an efficient bio-based fuels and chemicals production. Two recombinant thermophilic enzymes, an endo-1,4-β-mannanase from Dictyoglomus turgidum (DturCelB) and an α-galactosidase from Thermus thermophilus (TtGalA), were assayed at 80 °C, to assess their heterosynergystic association on galactomannans degradation, particularly abundant in hemicellulose. The enzymes were tested under various combinations simultaneously and sequentially, in order to estimate the optimal conditions for the release of reducing sugars. The results showed that the most efficient degree of synergy was obtained in simultaneous assay with a protein ratio of 25% of DturCelB and 75% of TtGalA, using Locust bean gum as substrate. On the other hand, the mechanism of action was demonstrated through the sequential assays, i.e. when TtGalA acting as first to enhance the subsequent hydrolysis performed by DturCelB. The synergistic association between the thermophilic enzymes herein described has an high potential application to pre-hydrolyse the lignocellulosic biomasses right after the pretreatment, prior to the conventional saccharification step.

Spent coffee grounds (SCG) are inexpensive materials with a complex composition that makes them promising feedstocks for a biorefinery.Here, conversion of SCG into a wide range of high value-added products (coffee oil, bio-ethanol, D-mannose, manno-oligosaccharide (MOS), cafestol and kahweol) using a novel integrated system was evaluated. The process involves oil extraction, MOS production by mannanase obtained from Penicillium purpurogenum, NaOH (Na) and hydrogen peroxide (HP) pretreatment for the degradation of lignin and phenolic compounds, diterpenes extraction, enzymatic hydrolysis, and fermentation, which can be performed using environmentally friendly technologies. Approximately 97 mL of coffee oil, 164 g of D-mannose, 102 g of MOS, 99 g of bioethanol and a dash of cafestol/kahweol were produced from 1 kg of dry SCG. Producing high-value co-products from SCG using an integrated approach as demonstrated here may be an efficient strategy to reduce waste generation, while improving the economics of the biorefinery production process.

Bacillus nealsonii PN-11 produces thermo-alkalistable mannanase and protease active in wide temperature and pH range. Optimization of coproduction of protease and mannanase from this strain and application of cocktail of these enzymes as detergent additives were studied. On optimization mannanase yield of 834Ug-1 (11.12 fold increase) and protease yield of 70Ug-1 (4.7 fold increase) could be obtained in a single fermentation. Purification and characterization of mannanase have been done earlier and protease was done during this study and has a molecular mass of 48kDa. pH and temperature optima for protease were 10.0 and 65°C respectively. It was completely stable at 60°C for 3h and retained >80% of activity at pH 11.0 for 1h. Both the enzymes were compatible with detergents individually and in a combination. The wash performance of the detergent on different type of stains improved when protease or mannanase were used individually. However destaining was more efficient when a combination of mannanase and protease was used.

A GH 134 β-1,4-mannanase SsGH134 from Streptomyces sp. NRRL B-24484 possesses a carbohydrate binding module (CBM) 10 and a glycoside hydrolase 134 domain at the N- and C-terminal regions, respectively. Recombinant SsGH134 expressed in Escherichia coli. SsGH134 was maximally active within a pH range of 4.0–6.5 and retained >80% of this maximum after 90 min at 30°C within a pH range of 3.0–10.0. The β-1,4-mannanase activity of SsGH134 towards glucomannan was 30% of the maximal activity after an incubation at 100°C for 120 min, indicating that SsGH134 is pH-tolerant and thermostable β-1,4-mannanase. SsGH134, SsGH134-ΔCBM10 (CBM10-linker-truncated SsGH134) and SsGH134-G34W (substitution of Gly34 to Trp) bound to microcrystalline cellulose, β-mannan and chitin, regardless of the presence or absence of CBM10. These indicate that GH 134 domain strongly bind to the polysaccharides. Although deleting CBM10 increased the catalytic efficiency of the β-1,4-mannanase, its disruption decreased the pH, solvent and detergent stability of SsGH134. These findings indicate that CBM10 inhibits the β-1,4-mannanase activity of SsGH134, but it is involved in stabilizing its enzymatic activity within a neutral-to-alkaline pH range, and in the presence of various organic solvents and detergents. We believe that SsGH134 could be useful to a diverse range of industries.

Currently, the traditional fermentation using konjac powder for β-mannanase production presents operational difficulties and high energy consumption, because of the individual hot air sterilization for konjac powder. A simply enzymatic hydrolysis pretreatment for konjac powder was developed to solve the problems of the traditional process in a 7-L fermenter. In the new process, when hydrolysis yield of konjac powder was above 50%, the media became liquid state from gelatinous state and could be sterilized immediately, avoiding the hot air sterilization and solving the operational difficulties. Interestingly, the new process didn’t have negative influence on β-mannanase production. Additionally, it could save close to 23% of power consumption during the whole fermentation. For another example, it did work well using locust bean gum for β-mannanase production in a 7-L fermenter. Therefore, the new process might be scaled up for industrial production using mannan-based bioresource as substrate.

Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel β-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200UmL-1 after 168h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5×104Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, β-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

Supplementation of post-weaning diets with exogenous enzymes has been suggested to control post-weaning syndrome, by compensating for the under-developed endogenous enzyme secretory capacity and increasing nutrient digestibility in newly weaned pigs. The effect of in-feed enzymes in improving gut maturation, growth and/or health in weaned piglets is not always consistent. A systematic review and a meta-analysis were therefore conducted to determine which exogenous enzymes are most consistent in improving piglet growth and digestibility when supplemented to post-weaning diets. The mean difference effect of enzyme supplementation on growth and digestibility of dry matter (DM), gross energy (GE), crude protein (CP), and P digestibility was calculated for each study and this was used as the effect size estimate in the meta-analysis. The impact of feed enzyme supplementation on intestinal health and bone mineralization was also discussed where information was available in the literature. From a total of 90 studies included in the meta-analysis, gain to feed ratio (G:F) was improved in 55, remained un-changed in 28 and deteriorated in 7, in response to enzyme supplementation. Average daily gain, average daily feed intake and G:F was improved when phytase was supplemented in the diet. Phosphorous digestibility was increased with phytase supplementation while GE digestibility was reduced. Dietary phytase supplementation increased bone mineralization in pigs fed diets with a reduced P content. Supplementation with multi-enzyme complexes increased DM and CP digestibility. In conclusion, the most consistent improvements in piglet growth, P digestibility and bone mineralization were found due to exogenous phytase supplementation. Supplementation with xylanase alone or in combination with β-glucanase had inconsistent effects on piglet growth and nutrient digestibility. The most consistent improvements in growth and nutrient digestibility due to supplementation with multi-enzyme complexes were found when mannanase and/or protease were included in the complex.

The β-mannanase gene of Bacillus circulans NT 6.7 was successfully cloned in Lactobacillus plantarum WCFS1 using the pSIP403 expression vector and secreted to the supernatant rather than accumulated in the cells. The highest activity was achieved by controlling the pH at 6 during cultivation. Maximum mannanase activities detected in the supernatant and cell-free extract of 200 ml MRS broth were 8.2 and 0.86 U/ml, respectively. Enzyme activity in the supernatant increased to 27 U/ml by fermentation in a 5-L bioreactor with automatic pH control. The optimum temperature of recombinant β-mannanase was 50 °C and stable between 30 and 50 °C. The optimum pH was 6 with stability in the range 5–7. Enzyme activity slightly increased with Co 2+ but was strongly inhibited by EDTA. The enzyme exhibited high specificity to galactomannan substrates. The main products of copra meal and locust bean gum hydrolysis were manno-oligosaccharides. Therefore, recombinant β-mannanase produced from a food grade host, L. plantarum WCFS1, showed potential for use in manno-oligosaccharides production and other food-related applications.

Chronic kidney disease (CKD) is a complex gene-environmental disease affecting close to 10% of the US population. Genome-wide association studies (GWASs) have identified sequence variants, localized to non-coding genomic regions, associated with kidney function. Despite these robust observations, the mechanism by which variants lead to CKD remains a critical unanswered question. Expression quantitative trait loci (eQTL) analysis is a method to identify genetic variation associated with gene expression changes in specific tissue types. We hypothesized that an integrative analysis combining CKD GWAS and kidney eQTL results can identify candidate genes for CKD. We performed eQTL analysis by correlating genotype with RNA-seq-based gene expression levels in 96 human kidney samples. Applying stringent statistical criteria, we detected 1,886 genes whose expression differs with the sequence variants. Using direct overlap and Bayesian methods, we identified new potential target genes for CKD. With respect to one of the target genes, lysosomal beta A mannosidase (MANBA), we observed that genetic variants associated with MANBA expression in the kidney showed statistically significant colocalization with variants identified in CKD GWASs, indicating that MANBA is a potential target gene for CKD. The expression of MANBA was significantly lower in kidneys of subjects with risk alleles. Suppressing manba expression in zebrafish resulted in renal tubule defects and pericardial edema, phenotypes typically induced by kidney dysfunction. Our analysis shows that gene-expression changes driven by genetic variation in the kidney can highlight potential new target genes for CKD development.

Here, we report an efficient endoglucanase from Aureobasidium pullulans (termed ApCel5A) was expressed in Pichia pastoris. ApCel5A shows two different enzyme activities of endoglucanase (1270 U/mg) and mannanase (31.2 U/mg). Through engineering the signal peptide and fed-batch fermentation, the enzyme activity of endoglucanase was improved to 6.63-folds, totally. Its efficient synergism with Celluclast 1.5 L, excellent tolerance to low pH (2.5), cholate and protease suggests potential application in bioresources, food and feed industries. Site-directed mutagenesis experiments present that ApCel5A residues Glu245 and Glu358 are key catalytic sites, while Asp118, Asp122, Asp198 and Asp314 play an auxiliary role. More importantly, ApCel5A display high degradation efficiency of glucan and glucomannan substrates by using tetrasaccharide contained reducing end of glucose residue as an intermediate. This study elucidated the effective methods to improve an endoglucanase expression and detailed catalytic mechanism for degradation of various substrates, which provides a new insight for endoglucanase application.

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) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by β-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 β-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 β-mannanases, we propose a scheme of sequential action by the glycoside hydrolases encoded by the β-mannan PUL and involved in the β-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 β-mannanase BoMan26A.

The exogenous enzymes are less consistent in their effects as their beneficial effects depend upon the types and level non-starch polysaccharides (NSP) present in the diets. Therefore, exogenous enzymes should be selected on the basis of types and amount of the NSP in the pig diets. The objectives of the present experiments were to investigate the effects of dietary level of mannan and β-mannanase supplementation on growth performance, apparent total tract digestibility (ATTD) of energy and nutrients, and blood metabolites of growing pigs. In Exp. 1, 96 barrows were randomly allotted to four treatments on the basis of BW. There were four replicates in each treatment with six pigs per replicate. The dietary treatments were a corn–soybean meal (SBM)-based control diet and three other diets consisted of the control diet supplemented with 400, 800 or 1600 U of β-mannanase/kg diet. The final BW, average daily gain (ADG) and blood glucose increased (linear, P0.05) on the concentrations of blood total cholesterol, triacylglycerides and blood urea nitrogen. These results indicate that supplementation of β-mannanase to low- or high-mannan diets have potential to improve the performance of growing pigs. In addition, palm kernel meal may partially replace corn and SBM without reducing pig performance if β-mannanase is added to diet.

Gene encoding for β-mannanase (E.C 3.2.1.78) from Klebsiella oxytoca KUB-CW2-3 was cloned and expressed by an E. coli system resulting in 400 times higher mannanase activities than the wild type. A 3314bp DNA fragment obtained revealed an open reading frame of 1164bp, namely kman-2, which encoded for 387 amino acids with an estimated molecular weight of 43.2kDa. It belonged to the glycosyl hydrolase family 26 (GH26) exhibited low similarity of 50-71% to β-mannanase produced by other microbial sources. Interestingly, the enzyme had a broad range of substrate specificity of homopolymer of ivory nut mannan (6%), carboxymethyl cellulose (30.6%) and avicel (5%), and heteropolymer of konjac glucomannan (100%), locust bean gum (92.6%) and copra meal (non-defatted 5.3% and defatted 7%) which would be necessary for in vivo feed digestion. The optimum temperature and pH were 30-50°C and 4-6, respectively. The enzyme was still highly active over a low temperature range of 10-40°C and over a wide pH range of 4-10. The hydrolysates of konjac glucomannan (H-KGM), locust bean gum (H-LBG) and defatted copra meal (H-DCM) composed of compounds which were different in their molecular weight range from mannobiose to mannohexaose and unknown oligosaccharides indicating the endo action of mannanase. Both H-DCM and H-LBG enhanced the growth of lactic acid bacteria and some pathogens except Escherichia coli E010 with a specific growth rate of 0.36-0.83h(-1). H-LBG was more specific to 3 species of Weissella confusa JCM 1093, Lactobacillus reuteri KUB-AC5, Lb salivarius KL-D4 and E. coli E010 while both H-KGM and H-DCM were to Lb. reuteri KUB-AC5 and Lb. johnsonii KUNN19-2. Based on the nucleotide sequence of kman-2 containing two open reading frames of 1 and 2at 5' end of the +1 and +43, respectively, removal of the first open reading frame provided the recombinant clone E. coli KMAN-3 resulting in the mature protein of mannanase composing of 345 amino acid residues confirmed by 3D structure analysis and amino acid sequence at N-terminal namely KMAN (GenBank accession number KM100456). It exhibited 10 times higher extracellular and periplasmic total activities of 17,600 and 14,800 units than E. coli KMAN-2. With its low similarity to mannanases previously proposed, wide range of homo- and hetero-polysaccharide specificity, negative effect to E. coli and most importance of high production, it would be proposed as a novel mannanase source for application in the future.

Aspergillus quadrilineatus endo-β-mannanase effectively degraded konjac glucomannan (66.09% w/v), copra meal (38.99% w/v) and locust bean galactomannan (20.94% w/v). High performance liquid chromatography (HPLC) analysis of KG hydrolysate indicated its mannooligosaccharides (MOS) content (656.38 mg/g) with high amounts of DP 5 oligosaccharide. Multi-scale characterization of mannan hydrolysate was done using FTIR and 13C NMR which revealed α and β form of galactose or glucose in MOS, respectively. CM and LBG hydrolysates (1 mg/mL) have shown cytotoxic effect and reduced cell viability of Caco-2 cells by 45% and 62%, respectively. MOS DP (1–4) derived from LBG supported better Lactobacilli biofilm formation as compared to KG hydrolysate containing high DP MOS (5–7). Lactobacilli effectively fermented MOS to generate acetate and propionate as main short chain fatty acids. Lactobacilli produced leucine, isoleucine and valine as branched chain amino acids when grown on LBG hydrolysate.

To better understand the roles of genes involved in mannan degradation in filamentous fungi, in this study we searched, identified, and characterized one putative GH5 endo-β-mannanase (GH5-7) and two putative GH2 mannan-degrading enzymes (GH2-1 and GH2-4) in Neurospora crassa. Real-time RT-PCR analyses showed that the expression levels of these genes were significantly up-regulated when the cells were grown in mannan-containing media where the induction level of gh5-7 was the highest. All three proteins were heterologously expressed and purified. GH5-7 displayed a substrate preference toward galactomannan by showing 10-times higher catalytic efficiency than to linear β-mannan. In contrast, GH2-1 preferred short manno-oligosaccharides or β-mannan as substrates. Compared to the wild type strain, the growth of Δgh5-7 and Δgh5-7Δgh2-4 mutants, but not Δgh2-1, Δgh2-4, and Δgh2-1Δgh2-4 mutants, was poor in the cultures containing glucomannan or galactomannan as the sole carbon source, suggesting that GH5-7 plays a critical role in the utilization of heteromannans in vivo. On the other hand, all the mutants showed significantly slow growth when grown in the medium containing linear β-mannan. Collectively, these results indicate that N. crassa can utilize glucomannan and galactomannan without GH2-1 and GH2-4, but efficient degradation of β-mannan requires a concerted action of three enzymes, GH5-7, GH2-1, and GH2-4.

β-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg−1) and MEIR (9003.1 U mg−1) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg−1), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL−1). The prebiotic mannooligosaccharides (

A GH 26 endo-mannanase from Bacillus sp. CFR1601 was purified to homogeneity (Mw ∼39kDa, specific activity 10,461.5±100IU/mg). Endo-mannanase gene (manb-1601, 1083bp, accession No. KM404299) was expressed in Escherichia coli BL21 (DE3) and showed typical fingerprints of α/β proteins in the far-UV CD. A high degree of conservation among amino acid residues involved in metal chelation (His-1, 23 and Glu-336) and internal repeats (122-152 and 181-212) was observed in endo-mannanases reported from various Bacillus sp. Thermal inactivation kinetics suggested that metal ions are quintessential for stabilization of ManB-1601 structure as holoenzyme (Ea 87.4kcal/mol, ΔH 86.7kcal/mol, ΔS 186.6cal/k/mol) displayed better values of thermodynamic parameters compared to metal-depleted ManB-1601 (Ea 47kcal/mol, ΔH 45.7kcal/mol, ΔS 64.7cal/k/mol). EDTA treatment of ManB-1601 not only lead to transitions in both secondary and tertiary structure but also promulgated the population of conformational state that unfolds at lower temperature. ManB-1601 followed a three-state process for thermal inactivation wherein loss of tertiary structure preceded the concurrent loss of secondary structure and activity.

Genetic etiology of congenital/infantile nystagmus remains largely unknown. This study aimed to identify genomic mutations in patients with infantile nystagmus and an associated disease network. Patients with inherited and sporadic infantile nystagmus were recruited for whole-exome and Sanger sequencing. β-Mannosidase activities were measured. Gene expression, protein–protein interaction, and nystagmus-associated lysosomal storage disease (LSD) genes were analyzed. A novel heterozygous mutation (c.2013G>A; p.R638H) of MANBA, which encodes lysosomal β-mannosidase, was identified in patients with autosomal-dominant nystagmus. An additional mutation (c.2346T>A; p.L749H) in MANBA was found by screening patients with sporadic nystagmus. MANBA was expressed in the pretectal nucleus of the developing midbrain, known to be involved in oculomotor and optokinetic nystagmus. Functional validation of these mutations demonstrated a significant decrease of β-mannosidase activities in the patients as well as in mutant-transfected HEK293T cells. Further analysis revealed that nystagmus is present in at least 24 different LSDs involving the brain. This is the first identification of MANBA mutations in patients with autosomal-dominant nystagmus, suggesting a new clinical entity. Because β-mannosidase activities are required for development of the oculomotor nervous system, our findings shed new light on the role of LSD-associated genes in the pathogenesis of infantile nystagmus. Genet Med 17 12, 971–979.

Background: Filamentous fungi produce various mannanolytic enzymes including β-1,4-mannanases for β-mannan degradation. Results: Fungal β-1,4-mannanase, Man134A, has an unusual sequence and substrate specificity that differs from Man5C belonging to the GH5 family. Conclusion: Man134A is involved in β-mannan degradation in vivo. Significance: An Aspergillus nidulans β-1,4-mannanase reveals a novel glycoside hydrolase family, GH134., Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-β-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.

Many glycoside hydrolases involved in deconstruction of cellulose and xylan from the excellent plant cell wall polysaccharides-degrader Caldicellulosiruptor bescii have been cloned and analyzed. However, far less is known about the enzymatic breakdown of mannan, an important component of hemicellulose. We herein cloned, expressed and purified the first β-mannosidase CbMan2A from C. bescii. CbMan2A is thermophilic, with an optimal temperature of 80 °C. CbMan2A hydrolyzes mannooligosaccharides with degrees of polymerization from 2 to 6 mainly into mannose and shows strong synergy with CbMan5A, an endo-mannanase from the same bacterium, in releasing mannose from β-1,4-mannan. Thus CbMan2A forms the missing link in enzymatic conversion of mannan into the ready-to-use mannose by C. bescii. Based on these observations, a model illustrating how CbMan2A may assist C. bescii in mannan utilization is presented. In addition, CbMan2A appeared to bind to insoluble galactomannan in a pH-dependent fashion. Although the relation of this feature to mannan utilization remains elusive, CbMan2A represents an excellent model for investigation of the binding of GH2 β-mannosidases to galactomannan.

A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5-11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2-6.

Konjac oligo-glucomannan (KOG) was prepared by degradation of konjac glucomannan (KG) using β-mannanase. The hydrolysis process was monitored by the viscosity of the enzymatic hydrolysates. Factors affecting the enzymatic hydrolysis of KG were investigated, and the optimum hydrolysis conditions were as follows: time 2h; temperature 50°C; pH 6.0; and enzymatic concentration 150 U/g. Under these optimized conditions, minimum viscosity (31.9 mPa·s) of the hydrolasate was obtained. The average degree of polymerization (DP) of the resulting KOG was approximately equal to 5.2. The results of Fourier transform infrared (FTIR) spectra of KG and KOG indicated that KG was successfully degraded. In addition, their antioxidant activities were evaluated by determination of hydroxyl radical (•OH) and 1,1-diphenyl-2-picrylhrazyl radical (•DPPH) scavenging activity, and determination of reducing power. The results showed that KOG exhibited significant antioxidant activities. Taken together, this study suggested that KOG could potentially be used as a natural antioxidant.

It is becoming a common practice to use higher levels of wheat distillers dried grains with solubles (wDDGS) in poultry diets. The objective of this study was to determine the effects of level of inclusion of wDDGS with or without enzyme (E-, i.e., wDDGSE-) supplementation on performance and water consumption of turkey hens (0 to 72 d). Two diets (0 or 30% wDDGS) were formulated to meet the nutrient requirements of Hybrid Converter turkeys. Diets (0 or 30% wDDGS; starter, grower, and finisher) were then blended to obtain a different level of inclusion (15%) of wDDGS. The 30% wDDGS diet was divided into 3 fractions and 2 fractions supplemented with either protease (P+, i.e., wDDGSP+; 0.126 g/kg) or β-mannanase (M+, i.e., wDDGSM+; 0.05 g/kg). All 5 diets were fed ad libitum as mash. The 700 0-d turkey hens were randomly allocated into groups of 35 birds per replicate with 4 replicate floor pens per treatment, in a completely randomized design. Water consumption per pen was recorded beginning at 7 d. There was no effect of dietary treatment on feed intake. BW of turkey hens (52 d; grower) was significantly higher for 30% wDDGSP+ as compared to 0% wDDGSE- or 15% wDDGSE- diets; but was not different from 30% wDDGSE- or 30% wDDGSM+ diets. FCR (P0.01; 28 to 52 d), and total FCR (P0.05; 0 to 72 d) was significantly improved for birds fed 15 or 30% wDDGS regardless of enzyme treatment compared to 0% wDDGSE-. Water intake (WI, in mL per bird per day) tended to be higher (P = 0.08) between 7 and 28 d for 30% wDDGSP+ diets compared to other treatments. Similarly, WI of birds fed 30% wDDGSP+ was higher (P0.05; 28 to 52 and 52 to 72 d) and total WI (P = 0.07; 7 to 72 d) tended to be higher than other treatments. This study is the first to report the impact of wDDGS on WI. As high as 30% wDDGS can be substituted in turkey hen diets. No effect of P+ or M+ at the inclusion level tested was found on performance.

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75-25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.

An engineered thermophilic and acidophilic β-mannanase (ManAK) from Aspergillus kawachii IFO 4308 was highly expressed in Pichia pastoris. Through high cell density fermentation, the maximum yield reached 11,600 U/mL and 15.5 g/L, which is higher than most extreme β-mannanases. The recombinant ManAK was thermostable with a temperature optimum of 80 °C, and acid tolerant with a pH optimum of 2.0. ManAK could efficiently degrade locust bean gum, konjac gum, and guar gum into small molecular mannooligosaccharide (2000 Da), even at high initial substrate concentration (10%), and displayed different Mw distributions in their end products. Docking analysis demonstrated that the catalytic pocket of ManAK could only accommodate a galactopyranosyl residue in subsite -1, which might be responsible for the distinct hydrolysis product compositions from locust bean gum and guar gum. These superior properties of ManAK strongly facilitate mannooligosaccharide preparation and application in food and feed area.

Hydrogels were prepared from guar gum (GG) via esterification with 1,2,3,4-butanetetracarboxylic dianhydride (BTCA). Detailed spectroscopic analysis using FTIR and solid-state NMR revealed that an increase in the BTCA feed amount in the preparation mixture led to an increased degree of crosslinking, which affected the swelling behavior and rheological properties of the hydrogels. The hydrogels exhibited enzyme degradability, and after incubation with β-mannanase and α-galactosidase, 30-57% of the hydrogels were degraded. In addition, the hydrogels adsorbed bovine serum albumin and hen egg white lysozyme thorough electrostatic and hydrophobic interactions. The protein-adsorbed GG hydrogels exhibited a slow and steady release of the proteins over a 24h period in buffer solutions after a fast release of proteins in the first hour. As such, GG hydrogels are expected to be efficient drug delivery carriers for protein-based drugs.

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of β-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

Glucomanno-oligosaccharides (GMO), usually produced from hydrolysis of konjac tubers with a high content of glucomannan, have a positive effect on Bifidobacterium as well as a variety of other physiological activities. Response surface methodology (RSM) was employed to optimize the hydrolysis time, hydrolysis temperature, pH and enzyme to substrate ratio (E/S) to obtain a high GMO yield from konjac tubers. From the signal-factor experiments, it was concluded that the change in the direct reducing sugar (DRS) is consistent with total reducing sugar (TRS) but contrary to the degree of polymerization (DP). DRS was used as an indicator of the content of GMO in the RSM study. The optimum RSM operating conditions were: reaction time of 3.4 h, reaction temperature of 41.0 °C, pH of 7.1 and E/S of 0.49. The results suggested that the enzymatic hydrolysis was enhanced by temperature, pH and incubation time. Model validation showed good agreement between experimental results and the predicted responses.

Protease resistance of enzymes is required for the feed industry because of the presence of secretary proteases in the digestive tract. In this study, we report a rational method for protease-resistance improvement of enzymes based on molecular structure evaluation. The trypsin-resistance of β-mannanase MAN47 from Armillariella tabescens was investigated. Twelve tryptic sites within it were ordered by their positions in three-dimensional space from external to internal. Except of R144, R192 and R261, which were either conserved or highly related to the catalytic activity, the top external residue K280 and the most internal residue K371 were selected. With conducting computational design via H-bond analysis and molecular dynamics simulations, optimal mutants of K280N and K371Q were predicted. Meanwhile, a triple mutant K280N/K107H/R102N was also predicted. Half-lives of mutants K280N, K280N/K107H/R102N, K371Q and wild-type enzymes which were all pre-treated by trypsin at 40 °C were determined 185 min, 285 min, 102 min and 100 min, respectively. In addition, the temperature–activity and pH–activity profiles revealed that the mutations we designed had no obvious influence on the catalytic activity of the enzyme. Our results proved that trypsin-resistance of an enzyme could be improved by molecular rational evolution of homology modeling and molecular dynamics simulations.

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-β-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-β-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-β-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-β-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe.

An industrial medium, Corn Steep Liquor Powder Dextrose (CSD medium) was developed for constitutive expression of recombinant β-mananase by Pichia pastoris. The β-mananase activity (513 U/mL) with CSD medium was 1.64- and 2.5-fold higher than with YPD and BSM in shaken flasks. The β-mananase productivity with CSD medium was 61.0 U/mL h, which was 1.7- and 2.5-fold higher than with YPD and BSM in a 5-L fermenter based on a novel fed-batch strategy combining the real-time exponential feed mode with the DO-stat feed mode. The β-mananase activity, dry cell weight and the recombinant enzyme reached up to 5132 U/mL, 110.0 g/L and 4.50 g/L after 50 h cultivation in a 50-L fermenter. The high efficient expression of recombinant β-mananase by P. pastoris indicated that CSD medium and the novel fed-batch strategy have great potential for the production of recombinant β-mananase in industrial fermentation.

Sphingomonas sp. JB13, isolated from slag of a20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191bp) was cloned and encodes a 396-residue polypeptide (ManAJB13) showing the highest amino acid sequence identities of 56.2% with the putative glycosyl hydrolase (GH) family 26 endo-1,4-β-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified GH 26 endo-1,4-β-mannanase from Cellvibrio japonicus (2VX5_A). The recombinant ManAJB13 (rManAJB13) was expressed in Escherichia coli BL21 (DE3). Purified rManAJB13 displayed the typical characteristics of low-temperature-active enzymes: showing apparent optimal at 40°C, ~55% of the maximum activity at 20°C and ~20% at 10°C, and thermolability at 45°C (~15min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-β-mannanases might be ascribed to the more hydrophobic residues (AILFWV) and less polar residues (NCQSTY) compared with typical thermophilic and mesophilic counterparts. The purified rManAJB13 exhibited85% mannanase activity at the concentration of 0-4.0M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1M or 2M NaCl, or trypsin or proteinase K at 37°C and pH 6.5 for 1h. The K(m), V(max) and k(cat) values were 5.0mgml(-1), 277.8μmol min(-1)mg(-1), and 211.9s(-1), respectively, using locust bean gum as the substrate.

A β-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL. This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 °C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 °C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 °C with p-nitrophenyl β- d -mannopyranoside were 0.10 mM and 3.75 μmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic β-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137–148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20–43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) β-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod.

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035U mg(-1) towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining >60% of its maximum activity at pH 3.0-7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K(m) and V(max) values were 5.6 and 4.8mgmL(-1), and 2785 and 1608μmolmin(-1)mg(-1), respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96mgg(-1) reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.