| Title | High-level expression and characterization of the Bacillus subtilis subsp subtilis str. BSP1 YwaD aminopeptidase in Pichia pastoris |
| Authors | Tang, Wei Li, Zhezhe Li, Chunhua Yu, Xianhong Wang, Fei Wan, Xin Wang, Yaping Ma, Lixin |
| Affiliation | Hubei Univ, Coll Life Sci, Hubei Key Lab Ind Biotechnol, Hubei Collaborat Innovat Ctr Green Transformat Bi, Wuhan 430062, Peoples R China. Peking Univ, Inst Mol Med, Beijing 100871, Peoples R China. Wuhan Inst Biol Prod Co Ltd, Wuhan 430062, Peoples R China. |
| Keywords | Amino acid mutation Carbamide Glycosylation High activity High yield RECOMBINANT LEUCINE AMINOPEPTIDASE SODIUM DODECYL-SULFATE DOUBLE-ZINC AMINOPEPTIDASE DENATURATION INACTIVATION SPECIFICITY STABILITY MECHANISM |
| Issue Date | 2016 |
| Publisher | PROTEIN EXPRESSION AND PURIFICATION |
| Citation | PROTEIN EXPRESSION AND PURIFICATION.2016,122,23-30. |
| 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. (C) 2016 Elsevier Inc. All rights reserved. |
| URI | http://hdl.handle.net/20.500.11897/434808 |
| ISSN | 1046-5928 |
| DOI | 10.1016/j.pep.2016.02.009 |
| Indexed | SCI(E) PubMed |
| Appears in Collections: | 分子医学研究所 |
