毕赤酵母Kex2蛋白酶的同源表达及酶学性质
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摘要
Kex2蛋白酶是一种来源于酵母的前体加工蛋白酶。利用毕赤酵母(Pichia pastoris)同源表达来源于毕赤酵母的Kex2蛋白酶(PPKex2),研究其表达特性和酶学性质,同时与毕赤酵母表达的酿酒酵母(Saccharomyces cerevisiae) Kex2蛋白酶(SCKex2)进行比较。首先,分别从毕赤酵母和酿酒酵母基因组中获得Kex2基因,将其插入到表达载体p PIC9K中,并转化毕赤酵母菌株GS115。重组菌株经甲醇诱导表达后,结果表明PPKex2的发酵上清液比活是SCKex2的7倍。Kex2蛋白酶经Q-FF强阴离子交换柱纯化后进行酶学性质研究。酶学性质研究结果表明,PPKex2的最适反应pH是8. 0～9. 0,最适反应温度是37℃,与SCKex2性质相近。在稳定性方面,PPKex2在pH 7. 0时最稳定,在碱性条件下的稳定性高于SCKex2,在酸性条件下的稳定性低于SCKex2,另外PPKex2的温度稳定性略低于SCKex2。酶促反应动力学研究表明,PPKex2的kcat和kcat/Km值分别为SCKex2的4. 8倍和3. 3倍。首次报道了同源表达毕赤酵母Kex2蛋白酶的表达特性及酶学性质,为其今后的研究及应用奠定了基础。
Kex2 protease is a precursor-processing protease from yeast. Kex2 protease from Pichia pastoris( PPKex2) was homologous expressed in Pichia pastoris. Then the expression and enzymatic characteristics of PPKex2 were studied and further compared with the Kex2 protease from Saccharomyces cerevisiae( SCKex2)expressed in Pichia pastoris. Firstly,these Kex2 genes were cloned from the genomes of Pichia pastoris and Saccharomyces cerevisiae,inserted into pPIC9 K expression vector,and then transformed into Pichia pastoris GS115. The expression of PPKex2 and SCKex2 were induced by methanol and purified with anion exchange chromatography( Q-FF). Finally,the enzymatic characteristics of these Kex2 protease were characterized. The specific enzyme activity of PPKex2 in the supernatant was seven times higher than that of SCKex2. Enzymatic characterization showed that PPKex2 was similar to SCKex2 and had the optimal activity at p H 8. 0-9. 0 and37℃. In terms of stability,PPKex2 was most stable at pH 7. 0. PPKex2 was more stable than SCKex2 in alkaline pH and less stable than SCKex2 in acidic pH. Meanwhile,the thermostability of PPKex2 is lower compared with that of SCKex2. The kinetic analysis showed that the kcatand kcat/Kmof PPKex2 was 4. 8-and 3. 3-fold higher than that of SCKex2,respectively. The first time report the expression and enzymatic characteristics of PPKex2 homologous expressed in Pichia pastoris,which shows potential application in the future.
Kex2 protease is a precursor-processing protease from yeast. Kex2 protease from Pichia pastoris( PPKex2) was homologous expressed in Pichia pastoris. Then the expression and enzymatic characteristics of PPKex2 were studied and further compared with the Kex2 protease from Saccharomyces cerevisiae( SCKex2)expressed in Pichia pastoris. Firstly,these Kex2 genes were cloned from the genomes of Pichia pastoris and Saccharomyces cerevisiae,inserted into pPIC9 K expression vector,and then transformed into Pichia pastoris GS115. The expression of PPKex2 and SCKex2 were induced by methanol and purified with anion exchange chromatography( Q-FF). Finally,the enzymatic characteristics of these Kex2 protease were characterized. The specific enzyme activity of PPKex2 in the supernatant was seven times higher than that of SCKex2. Enzymatic characterization showed that PPKex2 was similar to SCKex2 and had the optimal activity at p H 8. 0-9. 0 and37℃. In terms of stability,PPKex2 was most stable at pH 7. 0. PPKex2 was more stable than SCKex2 in alkaline pH and less stable than SCKex2 in acidic pH. Meanwhile,the thermostability of PPKex2 is lower compared with that of SCKex2. The kinetic analysis showed that the kcatand kcat/Kmof PPKex2 was 4. 8-and 3. 3-fold higher than that of SCKex2,respectively. The first time report the expression and enzymatic characteristics of PPKex2 homologous expressed in Pichia pastoris,which shows potential application in the future.
引文
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[10]Brenner C,Fuller R S. Structural and enzymatic characterization of a purified prohormone-processing enzyme:secreted,soluble Kex2 protease. Proc Natl Acad Sci USA,1992,89(3):922-926.
[11] Holyoak T,Wilson M A,Fenn T D,et al. 2. 4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor.Biochemistry,2003,42(22):6709-6718.
[12]刘颖颖,王之可,李素霞. Kex2蛋白酶在毕赤酵母中的表达、纯化和性质研究.中国生化药物杂志,2015,35(1):37-42.Liu Y Y,Wang Z K,Li S X. Expression,purification and properties of recombinant Kex2 in Pichia pastoris. Chinese Journal of Biochemical Pharmaceutics,2015,35(1):37-42.
[13]Brennan S O,Peach R J. Calcium-dependent Kex2-like protease found in hepatic secretory vesicles converts proalbumin to albumin. FEBS Lett,1988,229(1):167-170.
[14]Bader O,Schaller M,Klein S,et al. The Kex2 gene of Candida glabrata is required for cell surface integrity. Mol Microbiol,2001,41(6):1431-1444.
[15]Bader O,Krauke Y,Hube B. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans,C. glabrata,Saccharomyces cerevisiae and Pichia pastoris. BMC Microbiol,2008,8:116.
[16] Wheatley JL,Holyoak T. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2. Proc Natl Acad Sci USA,2007,104(16):6626-6631.
[17]Vogl T,Sturmberger L,Kickenweiz T,et al. A toolbox of diverse promoters related to methanol utilization:functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synth Biol,2016,5(2):172-186.
[18] Fang C,Wang Q,Selvaraj J N,et al. High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by r DNA-mediated integration. Sci Rep,2017,7(1):8747.
[19] Fang Z, Xu L, Pan D, et al. Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies. J Ind Microbiol Biotechnol,2014,41(10):1541-1551.
[2] Mizuno K,Nakamura T,Ohshima T,et al. Yeast Kex2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun,1988,156(1):246-254.
[3]Fuller R S,Brake A,Thorner J. Yeast prohormone processing enzyme(Kex2 gene product)is a Ca2+-dependent serine protease. Proc Natl Acad Sci USA,1989,86(5):1434-1438.
[4]Sreenivas S,Krishnaiah S M,Govindappa N,et al. Enhancement in production of recombinant two-chain insulin glargine by overexpression of Kex2 protease in Pichia pastoris. Appl Microbiol Biotechnol,2015,99(1):327-336.
[5] Cunningham K W,Wickner W T. Yeast Kex2 protease and mannosyltransferase I are localized to distinct compartments of the secretory pathway. Yeast,1989,5(1):25-33.
[6]Staniszewska M,Bondaryk M,Kazek M,et al. Effect of serine protease Kex2 on Candida albicans virulence under halogenated methyl sulfones. Future Microbiol,2017,12(4):285-306.
[7]Antunes A A,Jesus L O P,Manfredi M A,et al. Thermodynamic analysis of Kex2 activity:The acylation and deacylation steps are potassium-and substrate-dependent. Biophys Chem,2018,235:29-39.
[8]冯兴军,李静,宋雪莹,等.抗菌肽FAPs在毕赤酵母中的重组表达研究.东北农业大学学报,2013,44(9):68-72.Feng X J,Li J,Song X Y,et al. Study on recombinant expression of antimicrobial peptides FAPs in Pichia pastoris. Journal of Northeast Agricultural University,2013,44(9):68-72.
[9] Thomas G, Thorne B A, Thomas L, et al. Yeast Kex2endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells. Science,1988,241(4862):226-230.
[10]Brenner C,Fuller R S. Structural and enzymatic characterization of a purified prohormone-processing enzyme:secreted,soluble Kex2 protease. Proc Natl Acad Sci USA,1992,89(3):922-926.
[11] Holyoak T,Wilson M A,Fenn T D,et al. 2. 4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor.Biochemistry,2003,42(22):6709-6718.
[12]刘颖颖,王之可,李素霞. Kex2蛋白酶在毕赤酵母中的表达、纯化和性质研究.中国生化药物杂志,2015,35(1):37-42.Liu Y Y,Wang Z K,Li S X. Expression,purification and properties of recombinant Kex2 in Pichia pastoris. Chinese Journal of Biochemical Pharmaceutics,2015,35(1):37-42.
[13]Brennan S O,Peach R J. Calcium-dependent Kex2-like protease found in hepatic secretory vesicles converts proalbumin to albumin. FEBS Lett,1988,229(1):167-170.
[14]Bader O,Schaller M,Klein S,et al. The Kex2 gene of Candida glabrata is required for cell surface integrity. Mol Microbiol,2001,41(6):1431-1444.
[15]Bader O,Krauke Y,Hube B. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans,C. glabrata,Saccharomyces cerevisiae and Pichia pastoris. BMC Microbiol,2008,8:116.
[16] Wheatley JL,Holyoak T. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2. Proc Natl Acad Sci USA,2007,104(16):6626-6631.
[17]Vogl T,Sturmberger L,Kickenweiz T,et al. A toolbox of diverse promoters related to methanol utilization:functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synth Biol,2016,5(2):172-186.
[18] Fang C,Wang Q,Selvaraj J N,et al. High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by r DNA-mediated integration. Sci Rep,2017,7(1):8747.
[19] Fang Z, Xu L, Pan D, et al. Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies. J Ind Microbiol Biotechnol,2014,41(10):1541-1551.