title: High-level expression and characterization of the Bacillus subtilis subsp. subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris.
abstract: Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: wei521903@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: 598392746@qq.com.
authors.forename: Zhezhe
authors.initials: Z
authors.lastname: Li
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: 351145317@qq.com.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Li
authors.affiliation: Institute of Molecular Medicine, Peking University, Beijing 100871, PR China. Electronic address: kukuyu2012@hotmail.com.
authors.forename: Xianhong
authors.initials: X
authors.lastname: Yu
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: fhda142@163.com.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Wuhan Institute of Biological Product Co., Ltd., Wuhan 430062, PR China. Electronic address: 924253948@qq.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Wan
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: 545080994@qq.com.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, PR China. Electronic address: malixing@hubu.edu.cn.
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D005285:Fermentation; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D000626:Aminopeptidases
keywords: Amino acid mutation; Carbamide; Glycosylation; High activity; High yield