title: A Novel endo-1,4-β-mannanase from Bispora antennata with good adaptation and stability over a broad pH range.
abstract: An endo-β-1,4-mannanase encoding gene, man5, was cloned from Bispora antennata CBS 126.38, which was isolated from a beech stump. The cDNA of man5 consists of 1,299 base pairs and encodes a 432-amino-acid protein with a theoretical molecular mass of 46.6 kDa. Deduced MAN5 exhibited the highest amino acid sequence identity of 58% to a β-mannanase of glycoside hydrolase family 5 from Aspergillus aculeatus. Recombinant MAN5 was expressed in Pichia pastoris and purified to electrophoretic homogeneity. The specific activity of the final preparation towards locust bean gum was 289 U mg(-1). MAN5 showed optimal activity at pH 6.0 and 70 °C and had good adaptation and stability over a broad range of pH values. The enzyme showed more than 60% of peak activity at pH 3.0-8.0 and retained more than 80% of activity after incubation at 37 °C for 1 h in both acid and alkaline conditions (pH 4.0-11.0). The K                         (m) and V                         (max) values were 1.33 mg ml(-1) and 444 μmol min(-1) mg(-1) and 1.17 mg ml(-1) and 196 μmol min(-1) mg(-1) for locust bean gum and konjac flour, respectively. Of all tested metal ions and chemical reagents, Co(2+), Ni(2+), and β-mercaptoethanol enhanced the enzyme activity at 1 mM, whereas other chemicals had no effect on or partially inhibited the enzyme activity. MAN5 was highly resistant to acidic and neutral proteases (trypsin, α-chymotrypsin, collagenase, subtilisin A, and proteinase K). By virtue of the favorable properties of MAN5, it is possible to apply this enzyme in the paper and food industries.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Liu
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Kun
authors.initials: K
authors.lastname: Meng
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D031056:Amorphophallus / Q000737:chemistry; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D029964:Fagus / Q000382:microbiology; D005685:Galactans / Q000737:chemistry; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D008351:Mannans / Q000737:chemistry; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000494:pharmacology; D010843:Pichia / Q000235:genetics; D053149:Plant Gums / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; D011994:Recombinant Proteins; D008623:Mercaptoethanol; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase; D010447:Peptide Hydrolases; C017471:locust bean gum