A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0-30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0-30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5-2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.

• MeSH
• Enzyme Activation drug effects   MeSH
• Bacillus drug effects   genetics   metabolism   MeSH
• beta-Mannosidase chemistry   genetics   isolation & purification   metabolism   MeSH
• Sodium Chloride pharmacology   MeSH
• Detergents pharmacology   MeSH
• Endopeptidases metabolism   MeSH
• Gene Expression MeSH
• Genome, Bacterial MeSH
• Hydrolysis MeSH
• Ions MeSH
• Metals MeSH
• Surface-Active Agents pharmacology   MeSH
• Recombinant Proteins MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Enzyme Stability drug effects   MeSH
• Salt Tolerance * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Bacterial Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• beta-Mannosidase chemistry   genetics   isolation & purification   metabolism   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Molecular Sequence Data MeSH
• Cold Temperature MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Sphingobacterium chemistry   enzymology   genetics   MeSH
• Enzyme Stability MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

This study is focused on the analysis and mutagenesis of β-mannosidase from Bacteroides thetaiotaomicron with the aim of broadening its substrate specificity to 2-acetamido-2-deoxy-β-d-mannopyranosyl (β-ManNAc) derivatives. Various conformations ((4)C1, (4)H5 and (1)S5) of native and modified ligands were docked to the binding site of the protein to determine the most suitable conformation of sugars for further hydrolysis. Key amino acid residues were mutated in silico focusing on stabilizing the acetamido group of β-ManNAc as well as forming the oxazoline intermediate needed for hydrolysis. The results of large set of 5 ns molecular dynamic simulations showed that the majority of the active site residues are involved in substrate interaction and do not exhibit a higher flexibility except for Asn178. Mutations of Asn178 to alanine and Asp199 to serine could lead to a stabilization of the acetamido group in the binding site. So far, in vitro mutagenesis and the screen of a large variety of biological sources were unable to extend β-mannosidase's activity to include β-ManNAc derivatives.

• MeSH
• Bacteroides enzymology   MeSH
• beta-Mannosidase chemistry   genetics   metabolism   MeSH
• Biocatalysis * MeSH
• Protein Engineering * MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

β-Mannosidase (EC 3.2.1.25) is an important exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various oligomeric saccharide structures. β-Mannosidase from Aspergillus niger was expressed in Pichia pastoris and purified to clear homogeneity. β-Mannosidase was crystallized in the presence of D-mannose and the crystal diffracted to 2.41 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a=62.37, b=69.73, c=69.90 Å, α=108.20, β=101.51, γ=103.20°. The parameters derived from the data collection indicate the presence of one molecule in the asymmetric unit.

• MeSH
• Aspergillus niger chemistry   enzymology   MeSH
• beta-Mannosidase chemistry   genetics   MeSH
• Fungal Proteins chemistry   genetics   MeSH
• Crystallization MeSH
• Crystallography, X-Ray MeSH
• Mannose chemistry   MeSH
• Pichia chemistry   genetics   MeSH
• Recombinant Proteins chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

β-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various sugar chains. cDNA corresponding to the β-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The β-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44 mmol/l for p-nitrophenyl β-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).

• MeSH
• Aspergillus niger enzymology   genetics   MeSH
• beta-Mannosidase biosynthesis   chemistry   genetics   isolation & purification   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fungal Proteins biosynthesis   chemistry   genetics   isolation & purification   MeSH
• Cloning, Molecular MeSH
• Hydrogen-Ion Concentration MeSH
• Culture Media MeSH
• Pichia enzymology   genetics   MeSH
• Recombinant Proteins biosynthesis   chemistry   genetics   isolation & purification   MeSH
• Enzyme Stability MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Human beta-mannosidosis is an autosomal recessive, lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Unlike the severe clinical manifestation of the disease in ruminants, in which it leads to neonatal death, the human disease phenotype is generally milder. In addition, the phenotypic manifestation among the reported cases of human beta-mannosidosis is variable, even among members of the same family. To understand the molecular basis of the human disease and the mechanisms for such clinical variability, we sequenced the entire coding region of the human beta-mannosidase gene using a combination of cDNA library screening, RT-PCR and 5' rapid amplification of cDNA ends (RACE). The composite cDNA is 3293 nt, consisting of an 87 nt 5'-untranslated region, 2640 nt coding region and 566 nt 3'-untranslated region. The gene was localized to human chromosome 4q22-25. Analysis of a multiple tissue northern blot demonstrated a single 3.7 kb transcript. Mutation analysis of a Czech gypsy family with two siblings differently affected with beta-mannosidosis demonstrated a homozygous A-->G transition 2 bp upstream of a splice acceptor site. The associated cryptic splice site activation and exon skipping caused by this mutation resulted in two abnormally spliced mutant mRNA species in both siblings.

• MeSH
• alpha-Mannosidosis * genetics   MeSH
• beta-Mannosidase MeSH
• DNA, Complementary analysis   genetics   MeSH
• Humans MeSH
• Mannosidases * genetics   MeSH
• Molecular Sequence Data MeSH
• Mutation * MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Analysis MeSH
• Sequence Alignment MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH