胰凝乳蛋白酶SplB在枯草芽孢杆菌中的重组表达及应用

摘要/Abstract

摘要： 胰凝乳蛋白酶类蛋白酶SplB属于丝氨酸族蛋白酶，是一种具有专一性水解活性的碱性内肽酶。蛋白酶SplB无前肽，胞外表达的蛋白酶即为活性酶，且具有WELQ肽段专一性识别与切割活性。本研究通过启动子优化、宿主筛选，构建了C端带His标签的SplB表达载体，成功实现了SplB在枯草芽孢杆菌中的重组表达，对纯化的重组SplB进行了酶学性质研究，并实现了重组蛋白酶SplB在重组蛋白Prx标签切割中的应用。研究结果表明，以枯草芽孢杆菌ATCC6051Δ10为宿主，通过启动子P43介导的重组蛋白酶SplB表达活力最高（10.24 U/mL）。通过亲和层析纯化了重组表达的SplB，并进行了酶学性质研究，其最适温度为40℃，最适pH为8.5，且具有良好的温度和pH稳定性。在离子浓度较低情况下，Co2+对SplB酶活有促进作用，Mg2+、K+不影响酶活；而Cu2+、Zn2+、Ni+离子抑制SplB的活力；SDS极大抑制重组SplB的酶活力。将重组SplB应用于切割重组蛋白Prx的标签，结果表明，蛋白酶SplB具有WELQ肽段标签切割作用，并且SplB浓度越高，目标条带越浓。本研究为优化SplB的重组表达及在食品、医药领域的应用提供支持。

关键词: 胰凝乳蛋白酶SplB, 枯草芽孢杆菌, WELQ肽段标签切割

Abstract: Chymotrypsin protease SplB belongs to serine protease family and it is an alkaline endopeptidase with specific hydrolytic activity. There is no prepeptide in SplB protease. The protease expressed outside the cell is an active enzyme, and has specific recognition and cleavage activity for WELQ peptide. In this study, through promoter optimization and host screening, the SplB expression vector with C-terminal His tag was constructed, 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 and a tag. The results showed that Bacillus subtilis atcc6051 Δ 10 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 it had good temperature and pH stability. When the ion concentration was low, CO2+ promoted the activity of SplB, while Mg2+ and K+ did not affect its activity; Cu2+, Zn2+, Ni+ ions inhibited the activity of SplB; SDS greatly inhibited the enzyme activity of recombinant SplB. The recombinant SplB protease was applied in the cleavage of Prx protein and its tag. The results showed that SplB protease had the cleavage effect of WELQ tag. The higher the concentration of SplB protease, the better the cleavage effect. This study provides support for optimizing the recombinant expression of SplB and its application in food and medicine.

Key words: Chymotrypsin SplB, Bacillus subtilis, WelQ peptide tag

中图分类号:

Q939.97

潘丽洁王斌潘力. 胰凝乳蛋白酶SplB在枯草芽孢杆菌中的重组表达及应用[J]. 食品科学, 0, (): 0-0.

参考文献

[1] Saboti? J, Kos J. Microbial and fungal protease inhibitors-current and potential applications [J]. Applied Microbiology and Biotechnology, 2012, 93(4): 1351-1375.
[2] Biesebeke R, Record E, Biezen N, et al. Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates [J]. Applied Microbiology and Biotechnology, 2005, 69(1): 40-55.
[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 [J]. Applied Microbiology and Biotechnology, 2006, 72(2): 339-345.
[4] Machida M, Asai K, Sano M, et al. Genome sequencing and analysis of Aspergillus oryzae [J]. Nature, 2005, 438(7071): 1157-61.
[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 [J]. Extremophiles, 2004, 8(3): 229-35.
[6] Zugno L. The effect of trypsin on soaking of salt cured hides [J]. The Journal of the American Leather Chemists Association, 1992.
[7] 顾玉林, 牛彪, 阮俊程, 等. 重组猪胰蛋白酶在制备狂犬病疫苗（鸡胚成纤维细胞）中的应用 [J]. 中国生物制品学杂志, 2021, 34(01): 5-9.
[8] Rypniewski W R, Perrakis A, Vorgias C E, et al. Evolutionary divergence and conservation of trypsin [J]. Protein Engineering, Design and Selection, 1994, 7(1): 57-64.
[9] Page M J, Di Cera E. Serine peptidases: classification, structure and function [J]. Cellular and Molecular Life Sciences, 2008, 65(7): 1220-36.
[10] Olafson R W, Jurasek L, Carpenter M R, et al. Amino acid sequence of Streptomyces griseus trypsin. Cyanogen bromide fragments and complete sequence [J]. Biochemistry, 1975, 14(6): 1168-77.
[11] Rypniewski W, Hastrup S, Betzel C, et al. The sequence and X-ray structure of the trypsin from Fusarium oxysporum [J]. Protein Engineering, Design and Selection, 1993, 6(4): 341-8.
[12] Liu H, Zhou X, Tian S, et al. Two‐step transpeptidation of the insulin precursor expressed in Pichia pastoris to insulin ester via trypsin-catalyzed cleavage and coupling [J]. Biotechnology and Applied Biochemistry, 2014, 61(4): 408-17.
[13] Liu H, Zhou X, Xie F, et al. An efficient trypsin digestion strategy for improving desB30 productivity from recombinant human insulin precursor fusion protein [J]. Process Biochemistry, 2013, 48(5-6): 965-71.
[14] Walmsley S J, Rudnick P A, Liang Y, et al. Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics [J]. Journal of Proteome Research, 2013, 12(12): 5666-80.
[15] Yee L, Blanch H. Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli [J]. Biotechnology and Bioengineering, 1993, 41(8): 781-90.
[16] Viader-Salvadó J M, Fuentes‐Garibay J A, Castillo‐Galván M, et al. Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures [J]. Biotechnology Progress, 2013, 29(1): 11-6.
[17] Reed S B, Wesson C A, Liou L E, et al. Molecular characterization of a novel Staphylococcus aureus serine protease operon [J]. Infection and Immunity, 2001, 69(3): 1521-7.
[18] Popowicz G M, Dubin G, Stec-Niemczyk J, et al. Functional and structural characterization of Spl proteases from Staphylococcus aureus [J]. Journal of Molecular Biology, 2006, 358(1): 270-9.
[19] Pustelny K, Zdzalik M, Stach N, et al. Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation [J]. Journal of Biological Chemistry, 2014, 289(22): 15544-53.
[20] Dubin G, Stec-Niemczyk J, Kisielewska M, et al. Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln [J]. Journal of Molecular Biology, 2008, 379(2): 343-56.
[21] Pustelny K, Stach N, Wladyka B, et al. Evaluation of P1' substrate specificity of staphylococcal SplB protease [J]. Acta Biochimica Polonica, 2014, 61(1): 149-52.
[22] Yang N, Ling Z, Peng L, et al. Improvement of enzymatic stability and catalytic efficiency of recombinant Fusarium oxysporum trypsin with different N-terminal residues produced by Pichia pastoris [J]. Journal of Microbiology and Biotechnology, 2018, 28(9): 1482-92.
[23] Dubin G, Stec-Niemczyk J, Kisielewska M, et al. Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln [J]. Journal of Molecular Biology, 2008, 379(2): 343-56.
[24] Yee L, Blanch H. Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli [J]. Biotechnology and Bioengineering, 1993, 41(8): 781-90.
[25] Viader-Salvadó J M, Fuentes-Garibay J A, Castillo-Galván M, et al. Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures [J]. Biotechnology Progress, 2013, 29(1): 11-6.