title: Highly efficient expression and characterization of a β-mannanase from Bacillus subtilis in Pichia pastoris.
abstract: A β-mannanase gene (Man5) from Bacillus subtilis BS5 was cloned by PCR and integrated into the genome of Pichia pastoris GS115 via pPIC9 vector. The recombinant Man5 with a molecular mass of 43 kDa was successfully expressed and secreted into the culture medium. After methanol induction in a shake flask for 96 H, the recombinant Man5 protein reached 375 µg/mL in concentration, with an enzyme activity of 892 U/mL. The recombinant Man5 was purified 3.35-fold with 60% yield by using HiTrap DEAE FF and HiTrap Phenyl FF columns. The specific activity of the purified enzyme was 7,978 U/mg. The optimum temperature and pH of the recombinant Man5 were 50 °C and 6.0, respectively. Studies of substrate specificity showed that the optimum substrate for the Man5 was konjac flour, suggesting that it has great potential as an effective additive in the food industry.
authors.affiliation: College of Biological Science and Technology, Fuzhou University, Fujian, People's Republic of China; National Engineering Laboratory for High-Efficient Enzyme Expression, Fuzhou, People's Republic of China.
authors.forename: Ren-Kuan
authors.initials: RK
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Tzi Bun
authors.initials: TB
authors.lastname: Ng
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Juan
authors.initials: J
authors.lastname: Lin
authors.forename: Fen
authors.initials: F
authors.lastname: Yan
authors.forename: Xiu-Yun
authors.initials: XY
authors.lastname: Ye
mesh_terms: D031056:Amorphophallus / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D005433:Flour; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D044902:beta-Mannosidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: Bacillus subtilis; Pichia pastoris; highly efficient expression; β-mannanase