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Size-exclusion chromatography, HR-ESI-MS and FT-IR of copra meal hydrolyzed by ManB-1601 showed the presence of oligosaccharides (CM-β-MOS) having a degree of polymerisation (DP) between 2 and 4. Thermal decomposition studies of the purified CM-β-MOS (DP 2, 3 and 4) showed mass loss at high temperatures (135.8 °C to 600 °C). DP2, DP3 and DP4 CM-β-MOS were adjudged as un-substituted Manβ-4Man, Manβ-4Manβ-4Man and Manβ-4Manβ-4Manβ-4Man, respectively, using NMR (

This study aimed to compare the effects of different hydrolysates (named GKOS and MKOS) on constipated rats, which were obtained by degradation from konjac glucomannan by β-glucanase and β-mannanase, respectively. GKOS and MKOS were characterized and administered by gavage at 100 mg kg

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.

The filamentous fungus Talaromyces cellulolyticus is a well-characterized cellulolytic and hemicellulolytic enzyme producer. In this study, the function of the tclB2 gene, which is a homolog of the manR/clrB/clr-2 gene in other filamentous fungi, in mannanolytic enzyme production by T. cellulolyticus was investigated. When a tclB2-disrupted strain (YDTclB) was grown in the presence of glucomannan, the production of β-mannanase, β-mannosidase, and α-galactosidase was decreased at the protein and transcriptional levels when compared to the control strain. In addition, a tclB2-overexpressing strain (YHTclB) showed higher β-mannanase and β-mannosidase production. When cellulose was used as a carbon source, the expression of genes encoding mannanolytic enzymes also decreased in YDTclB. These results suggested that TclB2 contributes to mannanolytic enzyme production in T. cellulolyticus. This work is the first study to identify a transcriptional regulator of mannanolytic enzyme genes in T. cellulolyticus.

Background: Fruit juice clarification is a challenging aspect of beverage industry which needs to be addressed for economical and hygienic production of fruit juices. Objective: Current study is focused on the complete purification, characterization and thermodynamic analysis of an efficient mannanase enzyme to analyze its applicability in biological clarification fruit juice. Methods: Mannanase production using Aspergillus awamori IIB037 in a 25 L stirred fermenter at pre optimized reaction conditions was carried out. Enzyme purification was carried out via series of steps. Characterization of enzyme along with kinetics and thermodynamic studies was conducted. Purified and characterized enzyme was assessed for its applicability in fruit juice clarification through clarification experiments on fresh apple juice. Results: Purification fold of 3.98 was obtained along with 86.80% purification yield of mannanase with specific activity of 158.16 U/mg. The molecular size of purified enzyme was determined as 66 kDa. The enzyme depicted 56% residual activity at 60°C after 8 hrs. Thermodynamic studies of an enzyme revealed enthalpy of activation (ΔH) and activation energy (Ea) as 30.53KJ/mol, 27.76KJ/mol, respectively. The enzyme activity increased in the presence of ß-mercaptoethanol surprisingly. On the other hand, methyl alcohol, ethanol, Hg2+ and Cu2+ inhibited enzyme activity. The enzyme showed Km and Vmax values of 11.07 mM and 19.08 μM min-1 for Locust Bean Gum (LBG) under optimal conditions. Juice treated with mannanase showed decrease in absorbance and increase in reducing sugar content. Conclusion: The current study demonstrated that mannanase from Aspergillus awamori in its purified form has significant characteristics to be employed industrially for juice clarification.

13 p.-4 fig.-1 tab., Deficiencies in Mannosidase β (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3′UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant β-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS., This work was supported by the projects PI16/01259 and PI20/01634, integrated into the Plan Nacional de I+D+I, AES 2013–2016 and 2017–2020; funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) “A way to make Europe”; and by the project PROYECTO/AEI/10.13039/50110001103 from the Agencia Española de Investigación. AGJ holds a Formación de Profesorado Universitario (FPU20/03387) fellowship from Ministerio de Ciencia e Innovación.

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.

In this study, first, β-mannanase gene man derived from Bacillus amyloliquefaciens CGMCC1.857 was cloned and expressed in Bacillus subtilis 168 to generate B. subtilis M1. However, the extracellular β-mannanase activity of B. subtilis M1 was not very high. To further increase extracellular β-mannanase extracytoplasmic molecular chaperone, PrsA lipoprotein was tandem expressed with man gene in B. subtilis 168 to yield B. subtilis M2. The secretion of β-mannanase of B. subtilis M2 was enhanced by 15.4%, compared with the control B. subtilis M1. Subsequently, process optimization strategies were also developed to enhance β-mannanase production by B. subtilis 168 M2. It was noted that the optimal temperature for β-mannanase production (25°C) was different from the optimal growth temperature (37°C) for B. subtilis. Based on these findings, a two-stage temperature control strategy was proposed where the bacterial culture was maintained at 37°C for the first 12 h to obtain a high rate of cell growth, followed by lowering the temperature to 25°C to enhance β-mannanase production. Using this strategy, the extracellular β-mannanase activity reached 5016 ± 167 U/ml at about 36 h, which was 19.1% greater than the best result obtained using a constant temperature (25°C). The result of this study showed that PrsA lipoprotein overexpression and two-stage temperature control strategy were more efficient for β-mannanase fermentation in B. subtilis.

Glycoside hydrolase family 5 subfamily 8 (GH5_8) mannanases belong to Firmicutes, Actinomycetia, and Proteobacteria. The presence or absence of carbohydrate-binding modules (CBMs) present a striking difference. While various GH5_8 mannanases need a CBM for binding galactomannans, removal of the CBM did not affect activity of some, whereas it in other cases reduced the catalytic efficiency due to increased KM. Here, monomodular GH5_8 mannanases from Eubacterium siraeum (EsGH5_8) and Xanthomonas citri pv. aurantifolii (XcGH5_8) were produced and characterized to clarify if GH5_8 mannanases from Firmicutes and Proteobacteria without CBM(s) possess distinct properties. EsGH5_8 showed a remarkably high temperature optimum of 55 °C, while XcGH5_8 had an optimum at 30 °C. Both enzymes were highly active on carob galactomannan and konjac glucomannan. Notably, EsGH5_8 was equally active on both substrates, whereas XcGH5_8 preferred galactomannan. The KM values were comparable with those of catalytic domains of truncated GH5_8s, while the turn-over numbers (kcat) were in the higher end. Notably, XcGH5_8 bound to but did not degrade insoluble ivory nut mannan. The findings support the hypothesis that GH5_8 mannanases with CBMs target insoluble mannans found in plant cell walls and seeds, while monomodular GH5_8 members have soluble mannans and mannooligosaccharides as primary substrates.

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Here, whole genome sequencing of Microbacterium sp. CIAB417 was conducted to determine its novelty at species level and identification of genes encoding for enzymes for mannan degradation. The draft genome was predicted to have 6.53 mbp length represented by 41 contigs and 6078 genes. However, only 82.35% genes were allocated for their functions. The whole genome phylogeny, ANI score (78.84%), GGDC (genome to genome distance calculations) show probability (DDH ≥ 70%) equal to 0% and difference in advanced biochemical properties among closely predicted species. The Microbacterium sp. CIAB417 was stipulated to be novel at species level. Isolate was named as Microbacterium camelliasinensis CIAB417 (accession no JAHZUT000000000) based on its isolation from a tea garden soil. Genome was predicted for three novel mannanase coding genes man1 (MZ702740), man2 (MZ702741), and man3 (MZ702737) that belong to the GH5 and GH113 family. Besides that, mannan side chain hydrolysing enzymes alpha-galactosidase (gla1; MZ702739) and beta-glucosidase (glu1; MZ702738) were also predicted.

Enzymatic hydrolysis using β-mannanase and α-galactosidase is necessary to produce low molecular weight galactomannan (LMW-GM) from galactomannans (GM) in the leguminous seeds. In this study, different ratios of avicel and melibiose were used as the inductors (carbon sources) for Trichoderma reesei to metabolize the enzyme cocktail containing β-mannanase and α-galactosidase using one-pot fermentation technology. The obtained enzyme cocktail was used to efficiently produce LMW-GM from GM in Sesbania cannabina seeds. Results showed that 15 g/L avicel and 10 g/L melibiose were the best carbon sources to prepare enzyme cocktail containing β-mannanase and α-galactosidase with activities of 3.69 ± 0.27 U/mL and 0.51 ± 0.02 U/mL, respectively. Specifically, melibiose could effectively induce the metabolite product of α-galactosidase by T. reesei, which showed good performance in degrading the galactose substituent from GM backbone. The degradation of galactose alleviated the spatial site-blocking effect for enzymatic hydrolysis by β-mannanase and improved the yield of LMW-GM. This research can lay the foundation for the industrial technology amplification of LMW-GM production for further application.

International audience; Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage β-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-β-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-β-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing β6–α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the −1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

β-Mannans are a heterogeneous group of polysaccharides with a common main chain of β-1,4-linked mannopyranoside residues. The cleavage of β-mannan chains is catalyzed by glycoside hydrolases called β-mannanases. In the CAZy database, β-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.

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

Palm kernel cake (PKC) is a by-product of palm kernel oil extraction with moderate nutritional value, containing 30-35% β-mannan, which is indigestible, slows growth, and reduces feed efficiency. PKC can be improved by mannanase hydrolysis, but the effectiveness of mannanase is dependent on the microbial source. Thus, the effect of steam pretreatment and bacterial mannanases on PKC quality was investigated. PKC was pretreated by steaming and hydrolyzed in the small intestine by various mannanases. The contents of reducing sugar, total sugar, and protein release were measured. Steamed PKC had a significant increase in protein (16.95 ± 0.14 to 20.98 ± 0.13%) and a substantial decrease in hemicellulose (29.52 ± 0.44 to 3.46 ± 0.88%) and lignin (8.94 ± 0.28 to 1.40 ± 0.22%). Mannanases from Escherichia coli-KMAN-3 and E. coli-Man6.7 recorded the highest activities, followed by commercial mannanase, Bacillus circulans NT6.7 and B. amyloliquefaciens NT6.3 mannanases, orderly. B. circulans NT6.7 and B. amyloliquefaciens NT6.3 had multi-activities that include glucanase (3.10 ± 0.04% and 2.47 ± 0.02%) and amylase (1.74 ± 0.03% and 1.38 ± 0.04%), respectively. B. amyloliquefaciens NT6.3 mannanase hydrolyzed steamed PKC to release more reducing sugar, total sugar, and protein than hydrolyzed raw PKC. In raw and steamed PKC, B. amyloliquefaciens NT6.3 mannanase produced the highest reducing sugar release. As a result, steam pretreatment and mannanase hydrolysis, particularly from B. amyloliquefaciens, can be used to increase the functioning of PKC and develop new feed ingredients for monogastric animals at a reasonable cost.

Palm kernel cake (PKC) is an abundant side stream that can only be added to non-ruminant feed in small concentrations due to its content of antinutritional factors, mainly galactomannan, which cannot be digested by non-ruminants. β-mannanases can be added to partially hydrolyze galactomannan to form mannose oligosaccharides, which are known to be prebiotic. We here investigate the action of a β-mannanase from B. subtilis on PKC by colorimetry, NMR and fluorescence microscopy. The amount of mannan oligosaccharides in solution was significantly increased by the β-mannanase and their degree of polymerization (DP) was significantly reduced. There was a dose-response behavior in that larger β-mannanase concentrations increased the amount of soluble mannose oligosaccharides while reducing their average DP. Using confocal immunofluorescence microscopy, solubilization of galactomannan in PKC was clearly visualized. Images show a clear disruption of the cellulose and galactomannan structures of the PKC cell walls. We thus show in this study that using commercial dosages of β-mannanase on PKC can lead to formation of prebiotic compounds. Thus, this study suggests that utilization of PKC in poultry feed formulation might be increased by addition of a β-mannanase and would improve the return on investment.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β-mannanase/β-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported β-N-acetylglucosaminide/β-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

β-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 β-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 β-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 β-mannanase producer B.subtilis US191 is a promising probiotic candidate for the feed industry.

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.

Exogenous enzymes have been used to improve nutrient utilization in several species of livestock, particularly swine and poultry. In addition, improved immunological and metabolic traits have been reported in nonruminants. The objective of this study was to determine the effects of β-mannanase supplementation on milk yield and composition, and immunological and metabolic responses in lactating Holstein dairy cows. Two weeks after calving, 20 Holstein cows (10 multiparous and 10 primiparous) were blocked by parity and assigned to 1 of 2 diets for 182 d. All cows were housed in the same environment and fed the same basal diet. The basal diet of the treatment group was supplemented with β-mannanase (CTCBio Inc., Seoul, South Korea) at 0.1% of concentrate dry matter. No differences were detected between the control and enzyme supplement groups in milk yield parameters or milk composition. Supplementation of β-mannanase enzyme reduced blood haptoglobin levels in supplemented multiparous cows compared with controls. Furthermore, nonesterified fatty acid concentration levels tended to be lower in cows fed β-mannanase, regardless of parity. Neither immunoglobulin G nor milk somatic cell count was affected by β-mannanase supplementation, regardless of parity. The number of insemination services tended to be lower in cows fed diets supplemented with β-mannanase. Results from this study suggest that supplementation of β-mannanase exogenous enzyme could help to reduce instances of systemic inflammation and decrease fat mobilization in lactating Holstein cows. Multiparous cows are considered susceptible to acute infections and inflammation; thus, the enzyme had a greater effect in multiparous cows.

Background Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production. Results Two strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS). Conclusion This work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.

β-mannan is a nonstarch polysaccharide found in hulled and dehulled soybeans that can survive drying-toasting phase of processing soybeans and have antinutritive effects in poultry. β-mannanase is an active enzyme (endohydrolase) that can hydrolyze β-mannan to reduce its antinutritional effects. Two experiments were conducted to evaluate the effects of β-mannanase supplementation in low energy/protein diets on egg production, egg quality, and apparent ileal digestibility of the dry matter (DM), crude protein (CP), and amino acids in 21-week-old Single Comb White Leghorn hens (Hy-Line W-36). A total of 192 hens (8 replicates of 6 hens per treatment) for a production study (Exp. 1) and a total of 64 hens (8 replicates of 2 hens per treatment) for a digestibility study (Exp. 2) were randomly allocated to 4 experimental treatments in a 2 × 2 factorial arrangement. Four dietary treatments were control (CS) based on corn and 44% CP soybean meal (ME: 2,850 kcal/kg CP: 18.5%) and CS-low energy/protein (CSL) (ME: 2,750 kcal/kg CP: 17.5%), with or without 0.05% β-mannanase enzyme. Hens were fed the experimental diets for 14 d for the digestibility study and 8 wk for the production study. Hen-day egg production (HDEP), weekly feed intake, FCR, and biweekly egg quality parameters were measured. Significant interaction on feed intake (P < 0.01) was observed between energy/protein and enzyme. At 3, 6 and 8 wk, the feed intake and FCR of CSL with enzyme were significantly lower than those of CSL without enzymes. The main effects indicated that birds fed diets without inclusion of β-mannanase had higher feed intake than those fed diets with enzymes at 4, 7, and 8 wk. The inclusion of β-mannanase significantly increased (P < 0.05) HDEP at 2, 3, 5, and 7 wk. However, there was no significant effect of nutrient density or enzyme supplementation on egg quality parameters. The digestibility study showed that the inclusion of β-mannanase significantly improved (P < 0.01) apparent ileal digestibility of lysine, histidine and tryptophan in the diet. The results of these experiments indicate that supplementation of β-mannanase could reduce the feed intake and FCR and improve HDEP and apparent ileal digestibility of key amino acids in corn/soy diets fed to laying hens.

We constructed a novel β-mannanase/GLP-1 fusion peptide, termed MGLP_1, and evaluated its ability to ameliorate obesity in a high-fat/high-sugar diet (HFSD)-induced mouse model. Eight-wk MGLP_1 treatment notably reduced obesity, as reflected by significant changes of body weight, serum triglyceride level, fatty liver and adipose tissue distribution. Amelioration of HFSD-induced gut dysbiosis by MGLP_1 was evidenced by reduced abundance ratio of bacterial phyla Firmicutes to Bacteroidetes, enhanced abundance of beneficial probiotic genera (Bifidobacterium, Lachnospiraceae, Ileibacterium), and reduced abundance of harmful genera (Clostridium, Romboutsia). Mechanisms of weight loss were investigated by comparing effects of treatment with MGLP_1 vs. prebiotics manno-oligosaccharides (MOS). MGLP_1 ameliorated gut microbiota imbalance by enhancing carbohydrate catabolism, whereas MOS promoted glycan synthesis and metabolism. Our findings, taken together, indicate that MGLP_1 fusion peptide has strong potential for amelioration of obesity by modifying relationships between gut microbiota and lipid and glucose metabolism.

The β-mannanase from Bacillus subtilis HM7 (Man26HM7) isolated from Dynastes hercules larvae excrement was cloned and expressed in Escherichia coli. Biochemical characterization shows that optimal pH and temperature for catalysis are 6.0 and 50 °C, respectively. Man26HM7 displayed excellent surfactant stability by retaining 70% of initial activity in 1%(w/v) SDS, and more than 90% of initial activity in 1%(w/v) Triton X-100 and Tween 80. Results from amino acid sequence alignment and molecular modeling suggest residue 238 of β-mannanase as a hotspot of SDS-tolerance. Mutagenesis at the equivalent residue of another homolog, β-mannanase from Bacillus subtilis CAe24 (Man26CAe24), significantly enhanced the SDS stability of this enzyme. Comparative computational analysis, including molecular docking and molecular dynamics simulation, were then performed to compute the binding free energy of SD to Man26HM7, Man26CAe24, and variant enzymes. The results suggest that residue 238 of Man26HM7 is involved in SDS binding to the hydrophobic surface of β-mannanase. This study provides not only the promising application of Man26HM7 in detergent and cleaning products but also valuable information for enhancing the surfactant stability of β-mannanase by enzyme engineering.

In this study, a GH26 endo-mannanase (Man26A) from an Aspergillus niger ATCC 10864 strain, with a molecular mass of 47.8 kDa, was cloned in a yBBH1 vector and expressed in Saccharomyces cerevisiae Y294 strain cells. Upon fractionation by ultra-filtration, the substrate specificity and substrate degradation pattern of the endo-mannanase (Man26A) were investigated using ivory nut linear mannan and two galactomannan substrates with varying amounts of galactosyl substitutions, guar gum and locust bean gum. Man26A exhibited substrate specificity in the order: locust bean gum ≥ ivory nut mannan > guar gum; however, the enzyme generated more manno-oligosaccharides (MOS) from the galactomannans than from linear mannan during extended periods of mannan hydrolysis. MOS with a DP of 2-4 were the major products from mannan substrate hydrolysis, while guar gum also generated higher DP length MOS. All the Man26A generated MOS significantly improved the growth (approximately 3-fold) of the probiotic bacterial strains Streptococcus thermophilus and Bacillus subtilis in M9 minimal medium. Ivory nut mannan and locust bean gum derived MOS did not influence the auto-aggregation ability of the bacteria, while the guar gum derived MOS led to a 50 % reduction in bacterial auto-aggregation. On the other hand, all the MOS significantly improved bacterial biofilm formation (approximately 3-fold). This study suggests that the prebiotic characteristics exhibited by MOS may be dependent on their primary structure, i.e. galactose substitution and DP. Furthermore, the data suggests that the enzyme-generated MOS may be useful as potent additives to dietary foods.

Fungal β-mannanases hydrolyze β-1, 4-glycosidic bonds of mannans and find application in the generation of mannose and prebiotic mannooligosaccharides (MOS). Previously, a MOS generating β-mannanase from Aspergillus oryzae MTCC 1846 (βManAo) was characterized and its structural and functional properties were unraveled through homology modeling and molecular dynamics in this study. The βManAo model was validated with 92.9% and 6.5% of the residues found to be distributed in the most favorable and allowed regions of the Ramachandran plot. Glu244 was found to play a key role in the interaction with mannotriose, indicating conserved amino acids for the catalytic reaction. A detailed metadynamic analysis of the principal components revealed the presence of an α8-helix in the C-terminus which was very flexible in nature and energy landscapes suggested high conformation sub-states and the complex dynamic behavior of the protein. The binding of the M3 substrate stabilized the β-mannanase and resulted in a reduction in the intermediate conformational sub-states evident from the free energy landscapes. The active site of the β-mannanase is mostly hydrophilic in nature which is accordance with our results, where the major contribution in the binding energy of the substrate with the active site is from electrostatic interactions. Define Secondary Structure of Proteins (DSSP) analysis revealed a major transition of the protein from helix to β-turn for binding with the mannotriose. The molecular dynamics of the βManAo–mannotriose model, and the role and interactions of catalytic residues with ligand were also described. The substrate binding pocket of βManAo was found to be highly dynamic and showed large, concerted movements. The outcomes of the present study can be exploited in further understanding the structural properties and functional dynamics of βManAo.

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.

In the current study, the purified β-mannanase (Man/Cel5B) from Thermotoga maritima was immobilized on glutaraldehyde cross-linked chitosan beads. The immobilization of Man/Cel5B on chitosan beads was confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. After immobilization, the protein loading efficiency and immobilization yield were found to be 73.3% and 71.8%, respectively. The optimum pH for both free and immobilized enzymes was found to be pH 5.5. However, the optimum temperature of immobilized Man/Cel5B increased by 10 °C, from 85 °C (free Man/Cel5B) to 95 °C (Immobilized). The half-life of free and immobilized enzymes was found to be 7 h and 9 h, respectively, at 85 °C owing to the higher thermostability of immobilized Man/Cel5B. The increase in thermostability was also demonstrated by an increase in the energy of deactivation (209 kJmol−1) for immobilized enzyme compared to its native form (92 kJmol−1), at 85 °C. Furthermore, the immobilized Man/Cel5B displayed good operational stability as it retained 54% of its original activity after 15 repeated catalytic reactions concerning its free form.

β-Mannanases hydrolyze lignocellulosic biomass with the release of mannan oligosaccharides, which are considered as renewable resource in higher plants. Here, we cloned, expressed and characterized a novel endo-β-mannanase (ManAC) from Aspergillus calidoustus. Homology alignment analysis indicated that ManAC belonged to glycosyl hydrolase (GH) 5 family members. The analysis of structural homologous model revealed that five residues, Arg116, Asn231, His305, Tyr307, and Trp370, constituted the active site of ManAC. Glu232 and Glu340, proton donor and nucleophile, formed the catalytic residues of ManAC. The recombinant ManAC exhibited maximal activity at pH 2.5 and 70 °C, and it was acid tolerant at a pH range of 2.0–6.0 and thermostable under 60 °C. Meanwhile, the activity of ManAC was not significantly affected by various metal ions, except for Mg2+ and Ag2+. The recombinant ManAC exhibited the highest β-mannanase activity towards locust bean gum (669.7 U/mg) with the Km and Vmax values of 3.4 mg/mL and 982.4 μmol/min/mg, respectively. These thermophilic and acidophilicc characteristics is better than most extreme β-mannanase. As the first reported mannanse from Aspergillus calidoustus (ManAC), these excellent properties of ManAC strongly promote the synthesis of mannooligosaccharides which have potential for food and feed industrial applications.

Made available in DSpace on 2021-06-25T10:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) After being isolated from a sugarcane pile, the bacterium Chitinophaga sp. CB10 demonstrated to be a rich source of carbohydrases, with 350 predicted CAZyme domains. CB10 was able to grow on carbohydrates of different structural complexities: glucose, carboxymethylcellulose, corn starch, galactomannan, Aloe vera gum and sugarcane bagasse. The sugarcane bagasse is a rich source of complex polymers, and the diversity of metabolites released by its enzymatic hydrolysis has an important role for green chemistry, including minority pathways such as the degradation of mannan conjugates. In this sense, CB10 demonstrated considerable levels of gene expression for mannanases, and was stable for a period of 96-144 hours in the presence of sugarcane bagasse as sole carbon source. The bacterium showed respectively 4.8x and 5.6x expression levels for two genes predicted for GH2 β-mannosidase: one located within a gene cluster identified as polysaccharide utilization loci(PUL), and another a classic β-mannosidase. These enzymes shared less than 45% of identity with enzymes characterized from the genus Chitinophaga belonging to the phylum Bacteroidetes. The degree of novelty - as demonstrated by the low identity with previously characterized enzymes; the remarkable capability to grow in different substrates; mannanase activity, evidenced by the release of residual oligosaccharides in the cultivation with galactomannan (HPLC-RID, 12.3 mMol); associated to the ability of mannanases expression in a low concentration of inductor conditions (sugarcane bagasse, 0.2%) indicate the high potential for the application of CB10 as a source of enzymes in the production of oligosaccharides from biomass. This capacity might prove to be very valuable for the biorefinery process of pre-biotic precursors and other functional oligosaccharides focused on the food and pharmaceutical industries. School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Department of Technology Laboratory of Biochemistry and Plant Microorganisms Graduate Program in Agricultural and Livestock Microbiology School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Graduate Program in Agricultural and Livestock Microbiology School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP)

Mannan is an important renewable resource whose backbone can be hydrolyzed by β-mannanases to generate manno-oligosaccharides of various sizes. Only a few glycoside hydrolase (GH) 113 family β-mannanases have been functionally and structurally characterize. Here, we report the function and structure of a novel GH113 β-mannanase from Bacillus sp. N16-5 (BaMan113A). BaMan113A exhibits a substrate preference toward manno-oligosaccharides and releases mannose and mannobiose as main hydrolytic products. The crystal structure of BaMan113A suggest that the enzyme shows a semi-enclosed substrate-binding cleft and the amino acids surrounding the +2 subsite form a steric barrier to terminate the substrate-binding tunnel. Based on these structural features, we conducted mutagenesis to engineer BaMan113A to remove the steric hindrance of the substrate-binding tunnel. We found that F101E and N236Y variants exhibit increased specific activity toward mannans comparing to the wild-type enzyme. Meanwhile, the product profiles of these two variants toward polysaccharides changed from mannose to a series of manno-oligosaccharides. The crystal structure of variant N236Y was also determined to illustrate the molecular basis underlying the mutation. In conclusion, we report the functional and structural features of a novel GH113 β-mannanase, and successfully improved its endo-acting activity by using structure-based engineering.

Mannanases catalyze the cleavage of β-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new β-mannanase from

More than 800 million people in the world suffer from chronic kidney disease (CKD). Genome-wide association studies (GWAS) have identified hundreds of loci where genetic variants are associated with kidney function; however, causal genes and pathways for CKD remain unknown. Here, we performed integration of kidney function GWAS and human kidney–specific expression quantitative trait analysis and identified that the expression of beta-mannosidase (MANBA) was lower in kidneys of subjects with CKD risk genotype. We also show an increased incidence of renal failure in subjects with rare heterozygous loss-of-function coding variants in MANBA using phenome-wide association analysis of 40,963 subjects with exome sequencing data. MANBA is a lysosomal gene highly expressed in kidney tubule cells. Deep phenotyping revealed structural and functional lysosomal alterations in human kidneys from subjects with CKD risk alleles and mice with genetic deletion of Manba. Manba heterozygous and knockout mice developed more severe kidney fibrosis when subjected to toxic injury induced by cisplatin or folic acid. Manba loss altered multiple pathways, including endocytosis and autophagy. In the absence of Manba, toxic acute tubule injury induced inflammasome activation and fibrosis. Together, these results illustrate the convergence of common noncoding and rare coding variants in MANBA in kidney disease development and demonstrate the role of the endolysosomal system in kidney disease development.

Mannanase and xylanase, the main hemicellulolytic enzymes, are widely used in food, feed, textile and papermaking industries, and usually they are used in combination. Mannanase Man5A from Talaromyces leycettanus JCM12802 consist of the carbohydrate binding module (CBM), linker region and catalytic domain. The CBM coding region of Man5A was removed and fused to C-terminal of the xylanase gene Tlxyn11B. The fusion gene Tlxyn11B-linker-man5A was successfully expressed in Pichia pastoris and the fusion protein Tlxyn11B-Man5A was purified and characterized. The theoretical molecular weights of Tlxyn11B, Man5A without CBM region, and Tlxyn11B-Man5A are 21.6 kDa, 41.0 kDa, and 62.6 kDa, respectively. The fusion protein had high xylanase and mannanase activities. The optimal temperature of the fused xylanase is 70 °C, which is 5 °C higher than Tlxyn11B-w (xylanase before fusion). The fused mannanase exhibited maximal activity at 90 °C, which is similar to Man5A-w (mannanase before fusion). More than 48% of xylanase activity of Tlxyn11B-Man5A was residual after the condition of 60 °C with 1 h, which is significantly higher than Tlxyn11B-w (only 20% of activity was left at 60 °C for 20 minutes). The optimal pHs of Tlxyn11B-Man5A for xylanase and mannanase activity are 4.0 and 5.0, respectively, which are 0.5 and 1.0 units higher than those of Tlxyn11B-w and Man5A-w. The pH range of fused enzymes got wider and the pH stability is improved. The specific activities of xylanase and mannanase of Tlxyn11B-Man5A are 1 784.3 U/mg and 1 639.6 U/mg, respectively, which is lower than those of Tlxyn11B and Man5A (8 300.0 U/mg and 1 979.0 U/mg). It may be due to of the high molecular weight of fusion enzyme. The Km and Vmax of the fused xylanase and mannanase are 1.2 mg/mL and 1.7 mg/mL, 2 000.0 μmol/(min·mg) and 2 831.6 μmol/(min·mg), respectively. Tlxyn11B and Man5A were successfully fusion expressed in P. pastoris, and the good properties of fusion of xylanase and mannanase make it has great application potential in animal feed, food and other industrial production, and it provided new ideas for the improvement of enzyme performance.甘露聚糖酶和木聚糖酶是主要的半纤维素降解酶，在食品、饲料、纺织、造纸等工业应用广泛且通常搭配使用。文中将蓝状菌Talaromyces leycettanus JCM12802 来源的性质优良的甘露聚糖酶编码基因man5A 的CBM(Carbohydrate-binding module) 编码区去除，留下连接区和催化区，并将木聚糖酶基因Tlxyn11B 成熟区编码序列与man5A 的连接区进行融合，形成Tlxyn11B-linker-man5A 融合基因，并在毕赤酵母中成功表达，获得了融合蛋白Tlxyn11B-Man5A。Tlxyn11B、不含CBM 区的Man5A 和Tlxyn11B-Man5A 的理论分子量分别为21.6 kDa、41.0 kDa、62.6 kDa。对纯化后的融合蛋白进行了性质分析，融合蛋白同时具有高的木聚糖酶和甘露聚糖酶活性。融合后的木聚糖酶的最适温度为70 ℃，较单独表达时提高了5 ℃。甘露聚糖酶的最适温度为90 ℃，与融合前一致。融合后的木聚糖酶热稳定性明显提高，60 ℃处理1 h 剩余48%的酶活力，单独表达的木聚糖酶60 ℃处理20 min 仅剩余20%的酶活力。融合后的木聚糖酶和甘露聚糖酶的最适pH 分别为4.0 和5.0，较单独表达时分别提高了0.5 和1.0 个单位，融合后酶的作用pH 范围有所拓宽。融合前后的蛋白均具有较好的pH 稳定性。融合后木聚糖酶和甘露聚糖酶的比活分别为1 784.3 U/mg 和1 639.6 U/mg，较单独表达时比活 (8 300 U/mg 和1 979 U/mg)降低，与融合酶分子量增大相关。融合后的木聚糖酶和甘露聚糖酶的Km 值分别为1.2 mg/mL 和1.7 mg/mL, Vmax分别为2 000.0 μmol/(min·mg)和2 831.6 μmol/(min·mg)。综合其性质特点，融合木聚糖酶和甘露聚糖酶在饲料、食品等工业生产中有较大应用潜力，并为酶的性能改良提供了新的思路。.