Recombinant Expression and Application of Chymotrypsin SplB in Bacillus subtilis

被引：0
|
作者：
Pan L. [1 ]
Wang B. [1 ]
Pan L. [1 ]
机构：
[1] Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou
来源：
Shipin Kexue/Food Science | 2023年 / 44卷 / 02期
关键词：
Bacillus subtilis; Chymotrypsin SplB; WElQ peptide tag;
D O I：
10.7506/spkx1002-6630-20220122-224
中图分类号：
学科分类号：
摘要：
In this study, through promoter optimization and host screening, an SplB expression vector with His-tag at the C-terminal was constructed, and SplB was successfully expressed in Bacillus subtilis. The enzymatic properties of the purified recombinant SplB protease were studied, and the application of recombinant SplB protease in the cleavage of recombinant protein Prx with a tag was explored. The results showed that using B. subtilis ATCC6051Δ10 as the host, the recombinant expression activity of SplB mediated by promoter P43 was the highest (10.24 U/mL). The recombinant SplB was purified by affinity chromatography and its enzymatic properties were studied. The optimum temperature was 40 ℃, the optimum pH was 8.5, and the purified SplB had good temperature and pH stability. Low concentrations of Co2+ promoted the activity of SplB, while low concentrations of Mg2+ and K+ did not affect its activity; low concentrations of Cu2+, Zn2+ and Ni+ inhibited the activity of SplB, and sodium dodecyl sulfate (SDS) greatly inhibited the enzyme activity of recombinant SplB. The recombinant SplB protease could concentration-dependently cleave the WELQ peptide tag on the recombinant protein Prx. This study provides support for optimizing the recombinant expression of SplB and its application in the fields of food and medicine. © 2023, China Food Publishing Company. All right reserved.
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页码：181 / 188
页数：7
相关论文
共 28 条
  • [1] SABOTIC J, KOS J., Microbial and fungal protease inhibitorscurrent and potential applications, Applied Microbiology and Biotechnology, 93, 4, pp. 1351-1375, (2012)
  • [2] BIESEBEKE R, RECORD E, BIEZEN N, Et al., Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates, Applied Microbiology and Biotechnology, 69, 1, pp. 40-55, (2005)
  • [3] LEE Y H, TOMINAGA M, HAYASHI R, Et al., Aspergillus oryzae strains with a large deletion of the aflatoxin biosynthetic homologous gene cluster differentiated by chromosomal breakage, Applied Microbiology and Biotechnology, 72, 2, pp. 339-345, (2006)
  • [4] MACHIDA M, ASAI K, SANO M, Et al., Genome sequencing and analysis of Aspergillus oryzae, Nature, 438, pp. 1157-1161, (2005)
  • [5] OKUDA M, SUMITOMO N, TAKIMURA Y, Et al., A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp, Extremophiles, 8, 3, pp. 229-235, (2004)
  • [6] ZUGNO L A., The effect of trypsin on soaking of salt cured hides, The Journal of the American Leather Chemists Association, 87, 6, pp. 207-220, (1992)
  • [7] RYPNIEWSKI W R, PERRAKIS A, VORGIAS C E, Et al., Evolutionary divergence and conservation of trypsin, Protein Engineering, Design and Selection, 7, 1, pp. 57-64, (1994)
  • [8] PAGE M J, DI CERA E., Serine peptidases: classification, structure and function, Cellular and Molecular Life Sciences, 65, 7, pp. 1220-1236, (2008)
  • [9] OLAFSON R W, JURASEK L, CARPENTER M R, Et al., Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence, Biochemistry, 14, 6, pp. 1168-1177, (1975)
  • [10] RYPNIEWSKI W, HASTRUP S, BETZEL C, Et al., The sequence and X-ray structure of the trypsin from Fusarium oxysporum, Protein Engineering, Design and Selection, 6, 4, pp. 341-348, (1993)
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