分子伴侣增强蛋白酶K在毕赤酵母中的表达及对羊毛鳞片层的作用分析  

Enhanced Expression of Protease K in Pichia pastoris through Molecular Chaperones and Analysis of Its Effect on Wool Scale Layer

作　　者：蔡逸安 张轶群 杨子璇 刘业学 刘文龙 路福平[1] 李玉[1] CAI Yi-an;ZHANG Yi-qun;YANG Zi-xuan;LIU Ye-xue;LIU Wen-ong;LU Fu-ping;LI Yu(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457;Shandong Longkete Enzyme Preparation Co.Ltd.,Yishui 276400)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]山东隆科特酶制剂有限公司,沂水276400

出　　处：《生物技术通报》2024年第7期307-313,共7页Biotechnology Bulletin

基　　金：国家重点研发计划(2021YFC2104002)。

摘　　要：【目的】通过分子伴侣共表达增强毕赤酵母中异源蛋白酶K的分泌水平,并对其的羊毛无氯剥鳞作用机制进行解析,旨在提高蛋白酶K的表达量并为高效酶法剥鳞技术的应用奠定基础。【方法】利用毕赤酵母表达系统对tprK基因进行异源表达,首次分析了影响蛋白质折叠和质量控制的分子伴侣Ssa1、Erj5、Sil1、Hac1、Kar2、Lhs1和Ydj1分别过表达对TPRK的表达量和酶活力的作用,并对TPRK处理的羊毛纤维效果进行分析。【结果】TPRK在毕赤酵母GS115中表达,其最适反应条件为65℃、pH 9.0,且具有良好的热稳定性和pH稳定性。过表达ssa1、hac1、erj5和sil1基因的重组菌株酶活分别提升了36.8%、20.0%、17.7%和14.8%。5 L发酵罐进行高密度发酵,诱导72 h后,TPRK的酶活达到77471.99 U/mL。在羊毛水解应用中TPRK可水解羊毛鳞片内层使鳞片逐渐剥落,起到剥鳞效果,并且最适水解条件为:TPRK添加量为300 U/mL、反应温度55℃、pH 9.0和反应时间2 h。【结论】过表达分子伴侣Ssa1能够有效提升TPRK的表达量,利用该TPRK处理羊毛纤维,可有效去除羊毛鳞片层,而对羊毛核心皮质层造成的损伤较小。【Objective】To augment the expression level of protease K and pave the way for the implementation of efficient enzymatic scaling technology,the co-expression of molecular chaperones was employed to enhance the secretion of the heterologous protease K in Pichia pastoris,and the mechanism underpinning its chlorine-free wool scale stripping efficacy has been meticulously investigated.【Method】We used the Pichia pastoris expression system to have the tprK gene heterologously expressed.Then at the first time we analyzed the effects of overexpressions of molecular chaperone Ssa1,Erj5,Sil1,Hac1,Kar2,Lhs1,and Ydj1,which affect protein folding and quality control,on the expressions and enzymatic activities of TPRK.Also we analyzed the effect of TPRK treatment on wool fibers.【Result】TPRK is expressed in P.pastoris GS115,and its optimal reaction conditions are 65℃and pH 9.0,with good thermal stability and pH stability.The enzyme activities of recombinant strains overexpressing the ssa1,hac1,erj5,and sil1 genes increased by 36.8%,20.0%,17.7%,and 14.8%,respectively.High density fermentation was carried out in a 5 L fermentation tank,and after 72 h of induction,the TPRK enzyme activity reached 77471.99 U/mL.In the application of wool hydrolysis,TPRK hydrolyzed the inner layer of wool scales to gradually peel them off,achieving a peeling effect.The optimal hydrolysis conditions are:TPRK addition of 300 U/mL,reaction temperature of 55℃,pH 9.0,and reaction time of 2 h.【Conclusion】Overexpression of molecular chaperone Ssa1 can effectively increase the expression of TPRK.Using this TPRK to process wool fibers can effectively remove the wool scale layer,while causing less damage to the core cortex layer of wool.This lays the foundation for the promotion and application of protease K in wool scale removal technology.

关 键 词：蛋白酶K 分子伴侣 毕赤酵母 生物酶处理 防毡缩 

分 类 号：TQ925[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]