title: A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic Bacillus subtilis (TBS2) Expressed in Pichia pastoris and Its pH and Temperature Stability.
abstract: A gene encoding a highly thermostable β-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable β-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0-8.0 and was active at 20-100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other β-mannanase reported up to now, suggesting its potential for industrial applications.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, No. 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Zhangcai
authors.initials: Z
authors.lastname: Luo
authors.affiliation: School of Biological Sciences, Ludong University, Yantai, 264025, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Miao
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, No. 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Guoying
authors.initials: G
authors.lastname: Li
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, No. 1800 Lihu Avenue, Wuxi, 214122, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Du
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, No. 1800 Lihu Avenue, Wuxi, 214122, China. xbyu@jiangnan.edu.cn.
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005285:Fermentation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: Expression; Highly thermostable β-mannanase; Pichia pastoris; Stability; Thermophilic Bacillus subtilis