Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.

Optimized production and characterization of endo-ß-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum.

Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Research and application progress of microbial ß-mannanases: a mini-review.

Mannan is a predominant constituent of cork hemicellulose and is widely distributed in various plant tissues. ß-Mannanase is the principal mannan-degrading enzyme, which breaks down the ß-1,4-linked mannosidic bonds in mannans in an endo-acting manner. Microorganisms are a valuable source of ß-mannanase, which exhibits catalytic activity in a wide range of pH and temperature, making it highly versatile and applicable in pharmaceuticals, feed, paper pulping, biorefinery, and other industries. Here, the origin, classification, enzymatic properties, molecular modification, immobilization, and practical applications of microbial ß-mannanases are reviewed, the future research directions for microbial ß-mannanases are also outlined.

Efficient production of High-Purity manno-oligosaccharides from guar gum by citric acid and enzymatic hydrolysis.

Currently, the production of manno-oligosaccharides (MOS) from guar gum faces challenges of low oligosaccharide enzymatic hydrolysis yield and complicated steps in separation and purification. In this work, a potential strategy to address these issues was explored. By combining citric acid pretreatment (300 mM, 130 °C, 1 h) with ß-mannanase hydrolysis, an impressive MOS yield of 61.8 % from guar gum (10 %, w/v) was achieved. The key success lay in the optimizing conditions that completely degraded other galactomannans into monosaccharides, which could be easily removable through Saccharomyces cerevisiae fermentation (without additional nutrients). Following ion exchange chromatography for desalination, and concluding with spray drying, 4.57 g of solid MOS with a purity of 90 % was obtained from 10 g of guar gum. This method offers a streamlined and effective pathway for obtaining high-yield and high-purity MOS from guar gum by combining citric acid pretreatment and enzymatic hydrolysis.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Analysis of the effects of ß-mannanase on immune function and intestinal flora in broilers fed the low energy diet based on 16S rRNA sequencing and metagenomic sequencing.

As an enzyme, ß-mannanase (BM) can be widely used as feed additive to improve the growth performance of animals. This experiment aimed to determine the effect of the addition of BM to low-energy diet on the immune function and intestinal microflora of broiler chickens. In this study, 384 one-day-old Arbor Acres broilers were randomly divided into 3 groups (8 replicates per group): positive control (PC, received a corn-soybean meal basal diet), negative control (NC, received a low-energy diet with Metabolizable Energy (ME) reduced by 50 kcal/kg) and NC + BM group (NC birds + 100 mg/kg BM). All birds were raised for 42 d. The results showed that BM mitigated the damage of immune function in peripheral blood of broilers caused by the decrease of dietary energy level by increasing the Concanavalin A (Con A) index of stimulation (SI) and macrophages phagocytic activity in the peripheral blood of broilers at 42 d (P < 0.05). The analysis of cecum flora showed that the low-energy diet significantly reduced the observed_species index (P < 0.01), Chao1 index and ACE index (P < 0.05), which reduced the abundance and evenness of species in the cecum of broilers at 21 d. It also significantly reduced the relative abundance of Candidatus_Arthromitus and significantly increased the relative abundance of Pseudomonas in the cecum of broilers at 21 d, while also significantly increasing the relative abundance of Monoglobus at 42 d. BM significantly increased the relative abundance of Lachnospiraceae_UCG-001 and Lachnospiraceae_bacterium_615 in the cecum of broilers at 21 d. In addition, BM inhibited microbial Fatty acid degradation by decreasing the activity of glutaryl-CoA dehydrogenase. Collectively, BM could improve intestinal health by enhancing the immune function of broilers, promoting the proliferation of beneficial bacteria and reducing the number of harmful bacteria, regulating intestinal flora, thereby alleviating the adverse effects of lower dietary energy levels.

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

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

Effects of ß-mannanase supplementation on intestinal health and growth of nursery pigs.

Two experiments were conducted using 120 pigs to test the hypothesis that supplementation of ß-mannanase could reduce digesta viscosity, enhance nutrient digestion, and improve intestinal health and growth of nursery pigs. In experiment 1, 48 crossbred barrows were randomly allotted to four treatments with increasing levels of ß-mannanase at 0, 200, 400, and 600 U/kg in feeds. All pigs were euthanized on day 12 to collect jejunal digesta to measure digesta viscosity and ileal digesta to measure apparent ileal digestibility (AID) of dry matter (DM), gross energy (GE), neutral detergent fiber (NDF), and acid detergent fiber (ADF). In experiment 2, 72 nursery pigs were randomly allotted to three treatments with increasing levels of ß-mannanase at 0, 400, and 600 U/kg in feeds. Plasma collected on day 9 was used to measure tumor necrosis factor-α (TNF-α), immunoglobulin G (IgG), malondialdehyde (MDA), and protein carbonyl (PC). All pigs were euthanized on day 10 to collect duodenal and jejunal tissues to evaluate the production of TNF-α, IL-6, and MDA, morphology, crypt cell proliferation, and expression of tight junction proteins in the jejunum. Data were analyzed using the MIXED procedure for polynomial contrasts and the NLMIXED procedure for broken-line analysis of SAS. In experiment 1, ß-mannanase supplementation tended to have quadratic effects on digesta viscosity (P = 0.085) and AID of GE (P = 0.093) in the pigs. In experiment 2, jejunal digesta viscosity of the pigs was reduced (P < 0.05) when ß-mannanase was supplemented at 360 U/kg of feed. ß-Mannanase supplementation linearly reduced (P < 0.05) TNF-α, IgG, MDA, and PC in the duodenum, and TNF-α, IgG, and MDA in the jejunum of the pigs. ß-Mannanase supplementation linearly increased (P < 0.05) villus height to crypt depth ratio and crypt cell proliferation in the jejunum. ß-Mannanase supplementation tended to linearly improve (P = 0.083) expression of zonula occludens-1 in the jejunum. In conclusion, supplementation of ß-mannanase at 360 U/kg reduced the digesta viscosity and up to 600 U/kg positively affected intestinal health and growth of pigs by reducing inflammation and oxidative stress whilst enhancing structure and barrier function in the jejunum.

Nursery pigs face challenges in digesting complex carbohydrates in their feeds, which can negatively affect their growth and intestinal health. In particular, ß-mannans can increase digesta viscosity and hinder nutrient digestion of nursery pigs. ß-Mannanase, an enzyme that breaks down ß-mannans, has been used in nursery feeds to alleviate negative impacts on nutrient utilization and intestinal health of nursery pigs. This study investigated the effects of increasing supplementation levels of ß-mannanase on intestinal health, nutrient utilization, and growth of nursery pigs. The results showed that supplementation of ß-mannanase at 360 U/kg in the feed reduced the digesta viscosity in the jejunum and up to 600 U/kg positively had beneficial effects on the intestinal health and growth of nursery pigs by reducing inflammation and oxidative stress through improving structure and barrier function in the jejunum.

A rumen-derived bifunctional glucanase/mannanase uncanonically releases oligosaccharides with a high degree of polymerization preferentially from branched substrates.

Glycoside hydrolases (GHs) are known to depolymerize polysaccharides into oligo-/mono-saccharides, they are extensively used as additives for both animals feed and our food. Here we reported the characterization of IDSGH5-14(CD), a weakly-acidic mesophilic bifunctional mannanase/glucanase of GH5, originally isolated from sheep rumen microbes. Biochemical characterization studies revealed that IDSGH5-14(CD) exhibited preferential hydrolysis of mannan-like and glucan-like substrates. Interestingly, the enzyme exhibited significantly robust catalytic activity towards branched-substrates compared to linear polysaccharides (P < 0.05). Substrate hydrolysis pattern indicated that IDSGH5-14(CD) predominantly liberated oligosaccharides with a degree of polymerization (DP) of 3-7 as the end products, dramatically distinct from canonical endo-acting enzymes. Comparative modeling revealed that IDSGH5-14(CD) was mainly comprised of a (ß/α)8-barrel-like structure with a spacious catalytic cleft on surface, facilitating the enzyme to target high-DP or branched oligosaccharides. Molecular dynamics (MD) simulations further suggested that the branched-ligand, 64-α-D-galactosyl-mannohexose, was steadily accommodated within the catalytic pocket via a two-sided clamp formed by the aromatic residues. This study first reports a bifunctional GH5 enzyme that predominantly generates high-DP oligosaccharides, preferentially from branched-substrates. This provides novel insights into the catalytic mechanism and molecular underpinnings of polysaccharide depolymerization, with potential implications for feed additive development and high-DP oligosaccharides preparation.

Growth performance, nutrient digestibility, fecal microbial diversity and volatile fatty acid, and blood biochemical indices of suckling donkeys fed diets supplemented with multienzymes.

BACKGROUND: As the foal grows, the amount of breast milk produced by the donkey decreases. In such cases, early supplemental feeding is particularly important to meet the growth needs of the foal. Foals have an incompletely developed gastrointestinal tract with a homogenous microbiota and produce insufficient amounts of digestive enzymes, which limit their ability to digest and utilize forage. Improving the utilization of early supplemental feeds, promoting gastrointestinal tract development, and enriching microbial diversity are the hotspots of rapid growth research in dairy foals. Plant-based feeds usually contain non-starch polysaccharides (NSPs), including cellulose, xylan, mannan, and glucan, which hinder nutrient digestion and absorption. In addition, proteins and starch (both biomolecules) form a composite system mainly through non-covalent interactions. The proteins wrap around the surface of starch granules and act as a physical obstacle, thereby inhibiting water absorption and expansion of starch and decreasing the enzyme's catalytic effect on starch. Glyanase, ß-mannanase, ß-glucanase, cellulase, protease, and amylase added to cereal diets can alleviate the adverse effects of NSPs. The current study determined the effects of adding multienzymes (glyanase, ß-mannanase, ß-glucanase, cellulase, protease, and amylase) to the diet of 2-month-old suckling donkeys on their growth performance, apparent nutrient digestibility, fecal volatile fatty acid (VFA) and pH, fecal bacterial composition, and blood biochemical indices. RESULTS: On day 120 of the trial, fecal samples were collected from the rectum of donkeys for determining bacterial diversity, VFA content, and pH. Moreover, fresh fecal samples were collected from each donkey on days 110 and 115 to determine apparent digestibility. The multienzymes supplementations did not affect growth performance and apparent nutrient digestibility in the donkeys; however, they tended to increase total height gain (P = 0.0544). At the end of the study, the multienzymes supplementations increased (P < 0.05) the Observed species, ACE, Chao1, and Shannon indices by 10.56%, 10.47%, 10.49%, and 5.01%, respectively. The multienzymes supplementations also increased (P < 0.05) the abundance of Firmicutes, Oscillospiraceae, Lachnospiraceae, Christensenellaceae, Christensenellaceae_R-7_group, and Streptococcus in feces, whereas decreased (P = 0.0086) the abundance of Proteobacteria. CONCLUSIONS: Multienzymes supplementations added to a basal diet for suckling donkeys can increase fecal microbial diversity and abundance.

Effects of ß-mannanase supplementation on productive performance, inflammation, energy metabolism, and cecum microbiota composition of laying hens fed with reduced-energy diets.

The objective of this study is to investigate the beneficial effects and underlying mechanism of dietary ß-mannanase supplementation on the productive performance of laying hens fed with metabolic energy (ME)-reduced diets. A total of 448 Hy-Line gray laying hens were randomly assigned to seven groups. Each group had 8 replicates with 8 hens. The groups included a control diet (CON) with a ME of 2750 kcal/Kg, diets reduced by 100 kcal/Kg or 200 kcal/Kg ME (ME_100 or ME_200), and diets with 0.15 g/Kg or 0.2 g/Kg ß-mannanase (ME_100+ß-M_0.15, ME_100+ß-M_0.2, ME_200+ß-M_0.15, and ME_200+ß-M_0.2). The productive performance, egg quality, intestinal morphology, inflammatory response, mRNA expression related to the Nuclear factor kappa B (NF-κB) and AMPK pathway, and cecum microbiome were evaluated in this study. ME-reduced diets negatively impacted the productive performance of laying hens. However, supplementation with ß-mannanase improved FCR, decreased ADFI, and restored average egg weight to the level of the CON group. ME-reduced diets increased the levels of interleukin-1ß (IL-1ß) and IL-6 while decreasing the levels of IL-4 and IL-10 in the jejunum of laying hens. However, dietary ß-mannanase supplementation improved jejunum morphology, reduced pro-inflammatory cytokine concentrations, and increased levels of anti-inflammatory factors in laying hens fed with ME-reduced diets. The mRNA levels of IL-6, IFN-γ, TLR4, MyD88, and NF-κB in the jejunum of ME-reduced diets were significantly higher than that in CON, dietary ß-mannanase supplementation decreased these genes expression in laying hens fed with ME-reduced diets. Moreover, dietary ß-mannanase supplementation also decreased the mRNA levels of AMPKα and AMPKγ, and increased the abundance of mTOR in the jejunum of laying hens fed with ME-reduced diets. Cecum microbiota analysis revealed that dietary ß-mannanase increased the abundance of various beneficial bacteria (e.g., g_Pseudoflavonifractor, g_Butyricicoccus, and f_Lactobacillaceae) in laying hens fed with ME-reduced diets. In conclusion, dietary ß-mannanase supplementation could improve the productive performance of laying hens fed with a ME-reduced diet by improving intestinal morphology, alleviating intestinal inflammation, changing energy metabolism-related signaling pathways, and increasing cecum-beneficial microbiota.

Production of mannooligosaccharides from orange peel waste with ß-mannanase expressed in Trichosporonoides oedocephalis.

A large quantity of orange peel waste (OPW) is generated per year, yet effective biorefinery methods are lacking. In this study, Trichosporonoides oedocephalis ATCC 16958 was employed for hydrolyzing OPW to produce soluble sugars. Glycosyl hydrolases from Paenibacillussp.LLZ1 which can hydrolyze cellulose and hemicellulose were mined and characterized, with the highest ß-mannanase activity of 39.1 U/mg at pH 6.0 and 50 ℃. The enzyme was overexpressed in T. oedocephalis and the sugar production was enhanced by 16 %. The accumulated sugar contains 57 % value-added mannooligosaccharides by the hydrolysis of mannans. The process was intensified by a pretreatment combining H2O2 submergence and steam explosion to remove potential inhibitors. The mannooligosaccharides yield of 6.5 g/L was achieved in flask conversion and increased to 9.7 g/L in a 5-L fermenter. This study improved the effectiveness of orange peel waste processing, and provided a hydrolysis-based methodology for the utilization of fruit wastes.

Effect of dietary ß-mannanase supplementation on broiler performance.

This study was conducted to investigate the effect of graded levels of ß-mannanase supplementation in broiler diets on growth performance, energy digestibility, and lesion scores in d-old birds fed mash, corn-soybean meal-based diets and raised to 42 d. Five dietary treatments were investigated: 1) positive control diet (PC) containing standard energy; 2) negative control (NC) with 100 kcal/kg diet reduction in AME compared to PC; 3) NC supplemented with 30 U/g ß-mannanase (NC + 30 U); 4) NC supplemented with 60 U/g ß-mannanase (NC + 60 U); and 5) NC supplemented with 90 U/g ß-mannanase (NC + 90 U). Each treatment had 6 replicate pens with 52 chicks per replicate. Data was analyzed using 1-way ANOVA, and means were separated by LSMEANS. Reduction of 100 kcal/kg feed (NC) resulted in an overall body weight gain reduction of 51 g (P < 0.05) and feed conversion loss of approximately 4 points (P < 0.05) compared to PC at 42 d of age. At the same time, supplementing ß-mannanase at 60 and 90 U/g improved growth performance parameters compared to NC, while 30 U/g did not result in significant improvements beyond NC; body weight gain was improved (P < 0.05) by 87, and 106 g when ß-mannanase was supplemented at 60 and 90 U/g, respectively, compared to NC. This corresponded to an improvement by 6 and 7 points in feed conversion for 60 and 90 U/g supplementation, respectively, compared to NC. Furthermore, AMEn was improved (P < 0.05) by 15, 97, and 116 kcal/kg at 42 d when ß-mannanase was added to NC at 30, 60, and 90 U/g, respectively. Digesta viscosity measured at 42 d was decreased (P < 0.05) by ß-mannanase supplementation of 60 and 90 U/g, compared to NC, while 42 d lesion scores were improved (P < 0.05) by ß-mannanase supplementation compared to NC. Data demonstrated that dietary supplementation of ß-mannanase improved growth performance, energy digestibility, and reduced viscosity and lesion scores when supplemented with diets with a reduced energy content of 100 kcal/kg compared to a standard energy diet.

Production of ß-mannanase, inulinase, and oligosaccharides from coffee wastes and extracts.

This study aimed to produce enzymes (beta (ß)-mannanase using a recombinant Aspergillus sojae AsT3 and inulinase using Aspergillus niger A42) and oligosaccharides (mannooligosaccharides (MOS), fructooligosaccharides (FOS)) using coffee waste, ground coffee, and coffee extract by solid-state fermentation (SSF). Plackett-Burman Design (PBD) was used to create a design for enzyme production with four different parameters (temperature, pH, solid-to-liquid ratio (SLR), and mix with coffee wastes and ground coffee). The highest ß-mannanase and inulinase activities were 71.17 and 564.07 U/mg of protein respectively. Statistical analysis showed that the temperature was statistically significant for the production of both enzymes (P < 0.05). The produced enzymes were utilized in French Pressed coffee extracts to produce oligosaccharides. As a result of the enzymatic hydrolyzation, the highest mannobiose, mannotriose, mannotetraose, and total MOS levels were 109.66, 101.11, 391.02, and 600.64 ppm, respectively. For the FOS production, the maximal 1,1,1-kestopentaose was 38.34 ppm. Consequently, this study demonstrates that a recombinant Aspergillus sojae AsT3 ß-mannanase and Aspergillus niger A42 inulinase produced from coffee wastes and ground coffee can be used in coffee extracts to increase the amount of oligosaccharides in coffee extracts.

Spatial activity mapping of ß-mannanase on soybean seeds.

For farm animals the supplementation of exogenous enzymes, like ß-mannanase, to soybean-based diets is beneficial to improve feed digestibility. In order to unravel the effect of ß-mannanase on soybean meal's cell structure, a novel imaging concept was developed which allows visualizing the spatial activity pattern of ß-mannanase with high sensitivity by fluorescence microscopy before any visible degradation of the cellular structure occurs. It is based on fluorescence labeling of newly formed reducing ends of ß-mannanase-hydrolyzed polysaccharides after the native reducing ends of all polysaccharides present were chemically reduced. It was revealed that ß-mannanase is not only active at the cell wall but also at previously unknown sites, like the middle lamella and, most prominently, at an intracellular matrix enclosing the protein storage vacuoles. Based on these findings it can be hypothesized that the evaluated ß-mannanase can degrade the enclosing matrix of encapsulated proteins and the cell wall structure and thereby improves efficiency of feed utilization.

Beta-mannosidosis in a domestic cat associated with a missense variant in MANBA.

A 6-month-old cat of unknown ancestry presented for a neurologic evaluation due to progressive motor impairment. Complete physical and neurologic examinations suggested the disorder was likely to be hereditary, although the signs were not consistent with any previously described inherited disorders in cats. Due to the progression of disease signs including severely impaired motor function and cognitive decline, the cat was euthanized at approximately 10.5 months of age. Whole genome sequence analysis identified a homozygous missense variant c.2506G > A in MANBA that predicts a p.Gly836Arg alteration in the encoded lysosomal enzyme ß -mannosidase. This variant was not present in the whole genome or whole exome sequences of any of the 424 cats represented in the 99 Lives Cat Genome dataset. ß -Mannosidase enzyme activity was undetectable in brain tissue homogenates from the affected cat, whereas α-mannosidase enzyme activities were elevated compared to an unaffected cat. Postmortem examination of brain and retinal tissues revealed massive accumulations of vacuolar inclusions in most cells, similar to those reported in animals of other species with hereditary ß -mannosidosis. Based on these findings, the cat likely suffered from ß -mannosidosis due to the abolition of ß -mannosidase activity associated with the p.Gly836Arg amino acid substitution. p.Gly836 is located in the C-terminal region of the protein and was not previously known to be involved in modulating enzyme activity. In addition to the vacuolar inclusions, some cells in the brain of the affected cat contained inclusions that exhibited lipofuscin-like autofluorescence. Electron microscopic examinations suggested these inclusions formed via an autophagy-like process.

Family 3 CBM improves the biochemical properties, substrate hydrolysis and coconut oil extraction by hemicellulolytic and holocellulolytic chimeras.

To understand the influence of family 3 Carbohydrate Binding Module (hereafter CBM3), single (GH5 cellulase; CelB, CelBΔCBM), bi-chimeric [GH26 endo-mannanase (ManB-1601) and GH11 endo-xylanase (XynB); ManB-XynB [1], ManB-XynB-CBM] and tri-chimeric [ManB-XynB-CelB [1], ManB-XynB-CelBΔCBM] enzyme variants (fused or deleted of CBM) were produced and purified to homogeneity. CBM3 did not alter the pH and temperature optima of bi- and tri-chimeric enzymes but improved the pH and temperature stability of ManB in CBM variants of bi-/tri-chimeric enzymes. Truncation of CBM in CelB shifted the pH optimum and increased the melting temperature (Tm 65 â„ƒ). CBM3 improved both substrate affinity (Km) and catalytic efficiency (kcat/Km) of fused enzymes in tri-chimera and CelB but only Km for bi-chimera. Far-UV CD of CelB and bi- and tri-chimeric enzymes suggested that CBM3 improved the α-helical content and compactness in the native state but did not prevent disintegration of secondary structural contents at acidic pH. Steady-state fluorescence studies suggested that under acidic conditions CBM3 prevented the exposure of hydrophobic patches in bi-chimeric protein but could not avert the opening up of chimeric enzyme structure. Aqueous enzyme assisted treatment of mature coconut kernel using single, bi- and tri-chimeric enzymes led to cracks, peeling and fracturing of the matrix and improved the oil yield by up to 22%.

Towards an understanding of the enzymatic degradation of complex plant mannan structures.

Plant cell walls are composed of a heterogeneous mixture of polysaccharides that require several different enzymes to degrade. These enzymes are important for a variety of biotechnological processes, from biofuel production to food processing. Several classical mannanolytic enzyme functions of glycoside hydrolases (GH), such as ß-mannanase, ß-mannosidase and α-galactosidase activities, are helpful for efficient mannan hydrolysis. In this light, we bring three enzymes into the model of mannan degradation that have received little or no attention. By linking their three-dimensional structures and substrate specificities, we have predicted the interactions and cooperativity of these novel enzymes with classical mannanolytic enzymes for efficient mannan hydrolysis. The novel exo-ß-1,4-mannobiohydrolases are indispensable for the production of mannobiose from the terminal ends of mannans, this product being the preferred product for short-chain mannooligosaccharides (MOS)-specific ß-mannosidases. Second, the side-chain cleaving enzymes, acetyl mannan esterases (AcME), remove acetyl decorations on mannan that would have hindered backbone cleaving enzymes, while the backbone cleaving enzymes liberate MOS, which are preferred substrates of the debranching and sidechain cleaving enzymes. The nonhydrolytic expansins and swollenins disrupt the crystalline regions of the biomass, improving their accessibility for AcME and GH activities. Finally, lytic polysaccharide monooxygenases have also been implicated in promoting the degradation of lignocellulosic biomass or mannan degradation by classical mannanolytic enzymes, possibly by disrupting adsorbed mannan residues. Modelling effective enzymatic mannan degradation has implications for improving the saccharification of biomass for the synthesis of value-added and upcycling of lignocellulosic wastes.

High NaCl concentrations induce the resistance to thermal denaturation of an extremely halotolerant (salt-activated) ß-mannanase from Bacillus velezensis H1.

ß-mannanase catalyzes the hydrolysis of mannans ß-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant ß-mannanases are already reported, existence of a high halotolerant ß-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant ß-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8 kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48 h incubation under optimal conditions of 50 °C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4 M NaCl, it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5 °C in the presence of 2.5 M NaCl. The Km and Vmax values were 5.63 mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13 mg/mL of reducing sugars from olive mill wastes (OMW) after 24 h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.