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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.

β-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannan-based polysaccharides at β-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 β-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49-72% amino-acid sequence similarity with β-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Å revealed an open cleft-shaped active site. The enzyme structure is based on a (β/α) 8 -barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to β-strand 4 and at the end of β-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency (k cat /K m ) towards DP6 (2571.26 min -1  mM -1 ). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 Verrucomicrobiae DG1235 (ManDG1235) was biochemically characterized and its enzymatic properties were revealed. Amino acid alignment indicated that ManDG1235 belonged to glycoside hydrolase family 26 and shared a low amino acid sequence identity to reported β-mannanases (up to 50% for Cj Man26C from Cellvibrio japonicus ). ManDG1235 was expressed in Escherichia coli . Purified ManDG1235 (rManDG1235) exhibited the typical properties of cold-active enzymes, including high activity at low temperature (optimal at 20 °C) and thermal instability. The maximum activity of rManDG1235 was achieved at pH 8, suggesting that it is a mildly alkaline β-mannanase. rManDG1235 was able to hydrolyze a variety of mannan substrates and was active toward certain types of glucans. A structural model that was built by homology modeling suggested that ManDG1235 had four mannose-binding subsites which were symmetrically arranged in the active-site cleft. A long loop linking β2 and α2 as in Cj Man26C creates a steric border in the glycone region of active-site cleft which probably leads to the exo-acting feature of ManDG1235, for specifically cleaving mannobiose from the non-reducing end of the substrate.

A 36.7 kDa extracellular mannanase was originally purified from a generally regarded as safe (GRAS) lactic acid bacteria (LAB) strain Lactobacillus casei HDS-01. The LC-GC/GC determined that HDS-01 mannanase contained 338 amino acid residues. This protein belonged to glycoside hydrolase (GH) 26 Family and shared high similarity (94%) but low coverage (54%) with mannanase from Bacillus licheniformis (GQ859489.1). Circular dichroism (CD) illustrated that the proportion of α + β structural elements closely related to enzyme activity and were both negatively influenced by temperature. Fourier-transform infrared (FT-IR) showed the existence of typical protein bonds such as CO, CC, NH, CCH and CC. The CD and FT-IR collectively suggested HDS-01 mannanase a β-conformation rich (60-70%) protein. The optimal reaction condition of HDS-01 mannanase was 40 °C and pH 5.0. More than 50% activity was maintained after 2 h incubation under the condition of pH value 5.0-7.0 and 30-60 °C. This enzyme was strongly activated by Mn 2+ and exhibited a high affinity for locust bean gum (K m  = 2.68 ± 0.02 mg mL -1 , V max  = 400.03 ± 1.22 μmol mL -1 ·min -1 ). The HDS-01 mannanase clarified fruit juices more efficiently than commercial mannanase. These results promised HDS-01 mannanase a bio-safe addictive used in various especially food grade industrial fields., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metal binding sites are present in one-third of proteins and are crucial for biological functions and structural maintenance. GH26 endo-mannanase (ManB-1601) from Bacillus sp. harbors a Zn 2+ binding site which connects N (H1, H23) and C (E336)-terminal residues. Present study reveals how native circularization of ManB-1601 through Zn 2+ coordination regulates the structure-function. We generated individual Zn 2+ coordinating mutants and characterized them using biochemical and biophysical approaches. Contribution of individual Zn 2+ coordination towards maintaining ManB-1601 stability and rigidity was in the following order H23>H1 > E336. Elimination of E336 and H23-Zn 2+ coordination affected substrate hydrolysis to a greater degree than H1-Zn 2+ coordination. Metal quantification of mutant proteins indicated that H23A did not contain Zn 2+ . Molecular dynamic simulation studies revealed disruption of H23-Zn 2+ coordination leads to increased flexibility of N and C-terminal, active site loops and consequent drifting of substrate away from the active site region. Finally, mechanistic understanding on the functioning of Zn 2+ site in ManB-1601 is developed wherein 1) H23 by anchoring Zn 2+ ion majorly regulates the structure-function properties, 2) H1 provides thermo-stability, 3) E336 contributes towards maintaining substrate hydrolysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

β-Mannanase was expressed in Thermoanaerobacterium aotearoense SCUT27 induced by locust bean gum (LBG). The open reading frame encoding a GH26 β-mannanase was identified and encoded a preprotein of 515 amino acids with a putative signal peptide. The enzyme without a signal sequence (Man25) was overexpressed in Escherichia coli with a specific activity of 1286.2 U/mg. Moreover, a facile method for β-mannanase activity screening was established based on agar plates. The optimum temperature for the purified Man25 using LBG as a substrate was 55 °C. The catalytic activity and thermostability of Man25 displayed a strong dependence on calcium ions. Through saturation mutagenesis at the putative Ca 2+ binding sites in Man25, the best mutant ManM3-3 (D143A) presented improvements in thermostability with 3.6-fold extended half-life at 55 °C compared with that of the wild-type. The results suggest that mutagenesis at metal binding sites could be an efficient approach to increase enzyme thermostability.

Mannooligosaccharides are released by mannan-degrading endo-β-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

The galactomannan utilization locus ( Bo ManPUL) of the human gut bacterium Bacteroides ovatus encodes Bo Man26B, a cell-surface-exposed endomannanase whose functional and structural features have been unclear. Our study now places Bo Man26B in context with related enzymes and reveals the structural basis for its specificity. Bo Man26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase Bo Man26A of the same locus. Using galactomannan, Bo Man26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface-exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of Bo Man26B and the periplasmic α-galactosidase Bo Gal36A increased the rate of galactose release by about 10-fold compared with the rate without Bo Man26B. The results suggested that Bo Man26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by Bo Gal36A. A crystal structure of Bo Man26B with galactosyl-mannotetraose bound in subsites -5 to -2 revealed an open and long active-site cleft with Trp-112 in subsite -5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the -4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that Bo Man26A and Bo Man26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B., (© 2019 Bågenholm et al.)

Two glycoside hydrolases encoded by the mannan utilization gene cluster of alkaliphilic Bacillus sp. N16-5 were studied. The recombinant Gal27A (rGal27A) hydrolyzed both galactomannans and oligo-galactomannans to release galactose, while the recombinant Man113A (rMan113A) showed poor activity toward galactomannans, but it hydrolyzed manno-oligosaccharides to release mannose and mannobiose. rGal27A showed synergistic interactions with rMan113A and recombinant β-mannanase ManA (rManA), which is also from Bacillus sp. N16-5, in galactomannan degradation. The synergy degree of rGal27A and rManA on hydrolysis of locust bean gum and guar gum was 1.13 and 2.21, respectively, and that of rGal27A and rMan113A reached 2.00 and 2.68. The main products of galactomannan hydrolyzed by rGal27A and rManA simultaneously were galactose, mannose, mannobiose, and mannotriose, while those of galactomannan hydrolyzed by rGal27A and rMan113A were galactose and mannose. The yields of mannose, mannobiose, and mannotriose dramatically increased compared with the hydrolysis in the presence of rManA or rMan113A alone.

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., (© 2018 Domingues et al.)

Heterologous expression is an efficient strategy for target protein production. Dlt operon plays the important role in the D-alanylation of lipoteichoic acid, which might affect the net negative charge of cell wall for protein secretion. In this study, dlt operon was deleted to improve the target protein production, and nattokinase, α-amylase and β-mannanase with different isoelectric points (PIs) were served as the target proteins. Firstly, our results implied that deletions of dltA, dltB, dltC and dltD improved the net negative charge of cell wall for extracellular protein secretion respectively, and among which, the dltB deficient strain DW2ΔdltB showed the best performance, its nattokinase (PI: 8.60) activity was increased by 27.50% compared with that of DW2/pP43SacCNK. Then, the dltABCD mutant strain was constructed, and the net negative charge and nattokinase activity were increased by 55.57% and 37.13% respectively, due to the deficiency of dltABCD. Moreover, it was confirmed that the activities of α-amylase (PI: 6.26) and β-mannanase (PI: 5.75) were enhanced by 44.53% and 53.06% in the dltABCD deficient strains, respectively. Collectively, this study provided a strategy that deletion of dlt operon improves the protein secretion in B. licheniformis, and which strategy was more conducive to the target protein with lower PI.

Background: Mannanases are important enzymes which are widely used as a tool in agriculture and food industries. To improve the performance of mannanase Man23, a mutant library was created with rational design, and mutations were introduced on loops around the catalytic region. The Brevibacillus brevis B16 cell-free system which was created in this experiment provided the ability to express the mutant library efficiently. The activities of mutants were measured with a multi-volume spectrophotometer., Results: The mutant Man1606 gained from this system is a sextet which has mutations of N146G, S147H, S156P, T157Y, Q206S and T249H simultaneously on loops 6, 8 and 10. Man1606 showed higher activity and stability than Man23. The optimal temperature of Man1606 rose by 5 °C (from 55 to 60 °C) and the optimal pH increased slightly but its range became broader., Conclusion: This experiment demonstrated the B. brevis cell-free system shortens the expression time and is an efficient tool for mannanase engineering. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Two thermophilic β-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

β-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant β-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation. The mutant enzymes were expressed in Pichia pastoris SMD1168, and their thermal stability, pH stability, trypsin resistance and pepsin resistance were determined. Two mutant MAN47 enzymes, g-123 and g-347, were glycosylated as expected when expressed in yeast, and their thermal stability, pH stability, and protease resistance were significantly improved compared to the wild-type enzyme. An enzyme with multiple stability characterizations has broad prospects in practical applications, and the rational design N-glycosylation strategy may have applications in simultaneously improving several properties of other biotechnological targets., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70°C. The enzyme showed ˃55% activity at 5.0-15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37°C for 60min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85kJmol(-1) with a Kcat of 142.58×10(4)s(-1). MnCSB39 had Km and Vmax values of 0.082mg/mL and 1099±1.0Umg(-1), respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q10, ΔGE-S, and ΔGE-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44U mannanase from solid state fermentation of 1g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An alkaline-thermostable mannanase from Streptomyces sp. CS428 was produced, purified, and biochemically characterized. The extracellular mannanase (Mn428) was purified to homogeneity with 12.4 fold, specific activity of 2406.7 U/mg, and final recovery of 37.6 %. The purified β-mannanase was found to be a monomeric protein with a molecular mass of approximately 35 kDa as analyzed by SDS-PAGE and zymography. The first N-terminal amino acid sequences of mannanase enzyme were HIRNGNHQLPTG. The optimal temperature and pH for enzyme were 60 °C and 12.5, respectively. The mannanase activities were significantly affected by the presence of metal ions, modulators, and detergents. Km and Vmax values of Mn428 were 1.01 ± 3.4 mg/mL and 5029 ± 85 µmol/min mg, respectively when different concentrations (0.6-10 mg/mL) of locust bean gum galactomannan were used as substrate. The substrate specificity of enzyme showed its highest specificity towards galactomannan which was further hydrolyzed to produce mannose, mannobiose, mannotriose, and a series of mannooligosaccharides. Mannooligosaccharides can be further converted to ethanol production, thus the purified β-mannanase isolated from Streptomyces sp. CS428 was found to be attractive for biotechnological applications.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Lactobacillus casei is widely used in food production and feed industry. The aim of this study was to construct the recombinant expression mannanase Lb. casei., Methods: The mature peptide gene of β-1,4-mannanase from Bacillus pumilus was cloned into expression vectors pELX1 and pELSH, then electroporated into Lb. casei, establishing an intracellular and a secretion expression mannanase Lb. casei respectively., Results: After incubation, the specific activity of β-1,4-mannanase was 23 U/mg whole cell protein for intracellular expression and 8.8 U/mL for secretion expression in supernatant., Conclusion: Mannanase gene expression in Lb. casei provides application prospect and deserves further study.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications., Methods: We obtained a mannanase gene (named man(B)) from Bacillus pumilus Nsic2 and expressed the gene man(B) in Escherichia coli and Bacillus subtilis. Furthermore, we characterized the enzyme., Results: The gene man(B) had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid beta-mannanase (Man(B)). The protein sequence showed the highest identity with the beta-mannanase from B. pumilus CCAM080065. We expressed the gene man(B) in E. coli BL21 (DE3) with the enzyme activity of 11021.3 U/mL. Compared with other mannanases, Man(B) showed higher stability under alkaline conditions and was stable at pH6.0 -9.0. The specific activity of purified Man(B) was 4191 ± 107 U/mg. The K(m) and V(max) values of purified Man(B) were 35.7 mg/mL and 14.9 μmol/(mL x min), respectively. Meanwhile, we achieved recombinant protein secretion expression in B. subtilis WB800N., Conclusion: We achieved heterologous expression of the gene man(B) and characterized its enzyme. The alkaline-adapted Man(B) showed potential value in industrial applications due to its pH stability.

A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment's performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35-40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Paenibacillus polymyxa A-8, which secretes β-mannosidase, was isolated from the soil sample under a pine tree located in the "Laoban" mountain region of Sichuan, China. The β-mannosidase gene (MANB) was isolated from P. polymyxa A-8, using primers according to the complete genome. The MANB (2,550 bp) encoding 849 amino acid residues was expressed in Escherichia coli. The specific activities of β-mannosidase produced by P. polymyxa A-8 and E. coli pET30a-MANB were 12 nkat/mg and 635 nkat/mg respectively. SDS-PAGE analysis indicated that the molecular mass of the recombinant MANB was approximately 96 kDa. The recombinant MANB was active between pH 7.0-8.5 with the maximum activity at pH 7.0. It had good pH stability and adaptability. The MANB had the optimal temperature of 35°C and was relatively stable at 35-40°C. In addition, the MANB activity was enhanced by K+, Ca2+, Mn2+, and Mg2+ and inhibited by Zn2+, Cu2+, and Hg2+.

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants Ka ~ 1.2·10(5) and 3.0·10(5) M(-1) with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with Ka of 5.0·10(-5) M(-1). Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of Ka.

Functional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length CtManf and truncated CtManT were cloned in the pET28a(+) vector and expressed in E. coli BL21(DE3) cells, exhibiting 53 kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, CtManf and CtManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca(2+) and Mg(2+) salts. Plausible mannanase activity of CtManf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of manno-oligosaccharides. Notably highest protein concentrations of CtManf and CtManT were achieved in tryptone yeast extract (TY) medium, as compared with other defined media. Both CtManf and CtManT displayed stability at 60 and 50 °C, respectively, and Ca(2+) ions imparted higher thermostability, resisting their melting up to 100 °C.

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K(a) of 3.02 × 10(4) M(-1) and 1.52 × 10(4) M(-1), respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K(m), and k(cat) values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg(-1), 0.23 mM, and 305 s(-1), respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To identify the catalytic residues of mannanase AaManA from Alicyclobacillus acidocaldarius., Methods: Based on the sequence alignment by ClustalX and ESPript and the structure information of GH -53 family, the possible catalytic residues were selected and mutated by overlap extension PCR. The protein of wild type and mutant were expressed in E. coli BL21 (DE3) and ordinal purified by Ni - NTA affinity chromatography, gel - filtrate chromatography and ion - exchange chromatography. The purified protein was analyzed by thin layer chromatography (TLC) and the dinitrosalicylic acid (DNS) methods for enzyme assay., Results: Seven mutants, E151A, E159A, E231A, C150A, E151Q, E231Q and double mutation E151Q&E231Q were successful constructed. Mutant E159A showed similar activities with wild type, and C150A mutation resulted in only a 3 -fold reduction in the activities, but mutations E151A, E231A, E151Q, E231Q and E151Q&E231Q resulted in sharp decreases or loss in the activities, indicating that Glu151 and Glu231 play critical roles in AaManA activity. Furthermore, the presence of Glu151 at the C terminus of beta4 and Glu231 at the C terminus of beta7 was entirely consistent with the positions of the acid/base catalyst and the nucleophile catalyst of a GH - A enzyme, respectively., Conclusion: By combining the results of TLC and enzyme assay of those mutants and the structural comparisons, it was confirmed that Glu151 and Glu231 fulfilled the roles of an acid/base catalyst and nucleophile catalyst in AaManA, respectively.

In this work, we engineered the α/β fold of mannanase Man23 based on its molecular structure analysis to obtain more stable variants. By introducing 31 single-site mutations in the α/β fold and shuffling them, the incorporation of four mutations (K178R, K207R, N340R, and S354R) displayed a good balance between high activity and stability at higher temperature and broader pH. This quartet variant was characterized by an almost threefold increased activity and a sevenfold increased stability compared to native mannanase Man23. Our results suggest that such work is safe to increase our target protein stability with no loss of activity.

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are important virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of diffusible signal factor, DSF) regulate the production of these determinants. Addition of DSF or Xcc extracellular protein containing partially purified mannanase (EC 3.2.1.78, encoded by manA) can disperse the cell aggregates formed by rpfF mutant. In this study, nucleotide G 64 nt upstream of the manA translation start codon was determined as the transcription initiation site by the 5' RACE technique. Transcriptional fusion assays showed that manA transcription is positively regulated by Clp and RpfF and induced by locust bean gum. The manA coding region was cloned and expressed in E. coli as recombinant ManA (rManA). The rManA was purified by affinity chromatography, and its biochemical properties were characterized. The rManA had a pH optimum at 7.0 (0.1 M Hepes) and a temperature optimum at about 37 degrees C. Sequence and mutational analyses demonstrated that Xcc manA encodes the major mannanase, a member of family 5 of glycosyl hydrolases. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of manA in Xcc, but it also for the first time characterizes Xanthomonas mannanase at the protein level.

Beta-mannanases (beta-1,4-D-mannanase, EC 3.2.1.78), as a hemicellulose hydrolase, are widely distributed in bacteria, fungi, plants and even animals. They can randomly hydrolyze the beta-1,4-mannosidic linkages in mannan and heteromannan and have great potential in the food/feed, pulp/paper, medicine, oil exploitation and detergent industries. Most beta-mannanases often display a modular organization and usually contain structurally discrete catalytic and non-catalytic modules. Catalytic domains of these enzymes share a (beta/alpha)8-barrel fold, which play important roles in substrate binding and catalysis. Carbohydrate binding modules, as the most common non-catalytic modules, fold as beta-sandwich and facilitate the targeting of these enzymes to polysaccharide. In this review, a brief introduction is given concerning structural characteristics and function of these beta-mannanase modules.

MAN5, the main extracellular saccharide hydrolase from Bacillus sp. MSJ-5, is an endo-beta-mannanase with a demand of at least five sugar moieties for effective cleavage. It has a pH optimum of 5.5 and a temperature optimum of 50 degrees C and is stable at pH 5-9 or below 65 degrees C. MAN5 has a very high ability to hydrolyze konjac flour, 10 U/mg of which could completely liquefy konjac flour gum in 10 min at 50 degrees C. HPLC analysis showed that most glucomannan in the konjac flour was hydrolyzed into a large amount of oligosaccharides with DP of 2-6 and a very small amount of monosaccharide. With the culture supernatant as enzyme source, the optimum condition to prepare oligosaccharides from konjac flour was obtained as 10 mg/ml konjac flour incubated with 10 U/mg enzyme at 50 degrees C for 24 h. With this condition, more than 90% polysaccharides in the konjac flour solution were hydrolyzed into oligosaccharides and a little monosaccharide (2.98% of the oligosaccharides). Konjac flour is an underutilized agricultural material with low commercial value in China. With MAN5, konjac flour can be utilized to generate high value-added oligosaccharides. The high effectiveness and cheapness of this technique indicates its potential in industry.

BCman, a beta-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-beta-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the beta4 and beta7 strands, respectively. The 1.45-A crystal structure reveals that BCman is a typical (beta/alpha)(8) folding type. One large difference from the saddle-shaped active center of other endo-beta-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+1 and -1) within the active site of BCman. These two sites are involved in the enzyme's mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzyme's stability.

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.

The C-terminal 176 amino acids of a Thermotoga maritima mannanase (Man5) constitute a carbohydrate binding module (CBM) that has been classified into CBM family 27. The isolated CBM27 domain, named TmCBM27, binds tightly (K(a)s 10(5)-10(6) M(-1)) to beta-1, 4-mannooligosaccharides, carob galactomannan, and konjac glucomannan, but not to cellulose (insoluble and soluble) or soluble birchwood xylan. The X-ray crystal structures of native TmCBM27, a TmCBM27-mannohexaose complex, and a TmCBM27-6(3),6(4)-alpha-D-galactosyl-mannopentaose complex at 2.0 A, 1.6 A, and 1.35 A, respectively, reveal the basis of TmCBM27's specificity for mannans. In particular, the latter complex, which is the first structure of a CBM in complex with a branched plant cell wall polysaccharide, illustrates how the architecture of the binding site can influence the recognition of naturally substituted polysaccharides.