title: Purification, characterization, and heterologous expression of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9.
abstract: Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90% activity when incubated for 2 h at 60 °C and remain about 75% and 44% activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98% with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Shurui
authors.initials: S
authors.lastname: Mao
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lu
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bie
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D006026:Glycoside Hydrolases