title: High level extracellular production of a truncated alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli by the optimization of induction condition and fed-batch fermentation.
abstract: To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01-0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.|Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China.
authors.forename: Hongchen
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.|Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China.
authors.forename: Zhenxiao
authors.initials: Z
authors.lastname: Yu
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.|Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Fu
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.|Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China.
authors.forename: Shufang
authors.initials: S
authors.lastname: Li
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.|Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China.
authors.forename: Jianyong
authors.initials: J
authors.lastname: Xu
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. song_h@tib.cas.cn.|Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 XiQiDao, Tianjin Airport Economic Park, Tianjin, 300308, China. song_h@tib.cas.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Song
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D019149:Bioreactors; D003080:Cold Temperature; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D007218:Industrial Microbiology; D017830:Octoxynol / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D044902:beta-Mannosidase / Q000378:metabolism*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D017830:Octoxynol; D044902:beta-Mannosidase
keywords: Alkaline β-mannanase; Escherichia coli; One-time feeding; Triton X-100; Truncation