title: Efficient Secretory Overexpression of Endoinulinase in Escherichia coli and the Production of Inulooligosaccharides.
abstract: Endoinulinase production was achieved by heteroexpression of endoinulinase-encoding gene from Aspergillus ficuum which is an eukaryotic organism in Escherichia coli BL21 (DE3). Further analysis demonstrated that the native signal peptide existed in inu2 gene lowered the enzyme expression level. To realize extracellular accumulation of target protein and improve its expression level, native signal peptide was substituted with pelB, ompC, and pelB fusing with the native signal peptides; then, the effects on endoinulinase production were investigated. As a result, E. coli A606-3, with replacement of pelB as its signal peptide, showed the highest endoinulinase enzyme activity (75.22 U/mg). Also, it suggested that eukaryotic signal peptides have an inhibition on enzyme expression in prokaryotic organism. Moreover, the condition for inulooligosaccharide (IOS) production from inulin was optimized, and an IOS yield of 94.41 % was achieved under the condition of 15 % (w/v) inulin, purified endoinulinase dosage of 5 U/g inulin, 55 °C, and pH 4.6 for 24 h. The major products of hydrolysis of inulin were identified as DP3 to DP7.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Peipei
authors.initials: P
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China. majiangfeng@njtech.edu.cn.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing, 211816, People's Republic of China. majiangfeng@njtech.edu.cn.
authors.forename: Jiangfeng
authors.initials: J
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Mingke
authors.initials: M
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Zhongxue
authors.initials: Z
authors.lastname: Dai
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China. bioengine@njtech.edu.cn.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing, 211816, People's Republic of China. bioengine@njtech.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Jiang
mesh_terms: D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D006868:Hydrolysis; D007444:Inulin / Q000737:chemistry; D060847:Metabolic Engineering; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics
chemical_list: D009844:Oligosaccharides; D021382:Protein Sorting Signals; D007444:Inulin; D006026:Glycoside Hydrolases
keywords: Aspergillus ficuum; Endoinulinase; Escherichia coli; Inulooligosaccharides; Signal peptide