黑曲霉酸性蛋白酶PrA在毕赤酵母中的高效表达  

Efficient expression of the Aspergillus niger acidic protease PrA in Pichia pastoris

作　　者：李炳坤 郑毅恒 王飞[1] 成莉凤[3] 李丁 LI Bingkun;ZHENG Yiheng;WANG Fei;CHENG Lifeng;LI Ding(College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,Jiangxi,China;College of Food and Bioengineering,Henan University of Science and Technology,Luoyang 471000,Henan,China;Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha 410205,Hunan,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,Jiangsu,China)

机构地区：[1]江西农业大学生物科学与工程学院,江西南昌330045 [2]河南科技大学食品与生物工程学院,河南洛阳471000 [3]中国农业科学院麻类研究所,湖南长沙410205 [4]江苏省农业科学院动物免疫工程研究所,江苏南京210014

出　　处：《微生物学报》2024年第9期3314-3329,共16页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32002316);湖南省自然科学基金(2023JJ50315)。

摘　　要：【目的】本研究致力于获得酸性蛋白酶PrA的高产酵母菌株,以便在食品加工、饲料添加剂等领域进行应用。【方法】构建毕赤酵母表达菌株,在摇瓶水平重组表达PrA并检测其酶学性质。通过信号肽改造、基因剂量优化、共表达分子伴侣等措施逐步提高PrA产量,并利用高密度发酵进一步提高表达水平。【结果】PrA的比酶活为3974.00 U/mg,最适反应pH为3.0,最适反应温度为45℃。初始菌株的PrA产量达到738.03 U/mL。通过使用MF4I信号肽将PrA产量提高至1206.52 U/mL,增加PrA基因拷贝数导致产量提高至2406.47 U/mL。进一步共表达分子伴侣或分子伴侣组合将PrA产量提高至4091.27U/mL。经过高密度发酵,发酵168h酶活达到43088.00 U/mL,与初始产量相比提高58.4倍。【结论】在毕赤酵母中成功实现了酸性蛋白酶PrA的高效表达,为其未来的工业化应用奠定了基础。这些结果为进一步研究和开发该酶在食品加工和饲料添加剂领域的应用提供了重要参考。[Objective]To obtain a yeast strain efficiently producing the acidic protease PrA for applications in food processing,feed additives,and other related industries.[Methods]We constructed a recombinant strain of Pichia pastoris expressing PrA by fermentation in shake flasks and measured the enzymatic properties of the expressed PrA.Several strategies,such as signal peptide modification,gene dosage optimization,and co-expression with molecular chaperones,were employed to enhance the production of PrA.Additionally,high-density fermentation was employed to further improve the expression level.[Results]The expressed enzyme PrA showcased the specific activity of 3974.00 U/mg,with the optimum performance at pH 3.0 and 45℃.The production of PrA by the parental strain was 738.03 U/mL.The modification of the MF4I signal peptide increased the production of PrA to 1206.52 U/mL.Moreover,an increase in the copy number of prA further increased the PrA production to 2406.47 U/mL.Additionally,co-expression with single or combined molecular chaperones increased the PrA production to 4091.27 U/mL.After undergoing high-density fermentation,the enzyme activity reached 43088.00 U/mL within 168 h,representing a 58.4-fold increase compared with the initial production.[Conclusion]High-level expression of PrA was achieved in P.pastoris,which laid a foundation for the future industrial applications.The results provide valuable insights into the research and development of PrA for applications in food processing and feed additives.

关 键 词：毕赤酵母 酸性蛋白酶PrA 信号肽 基因剂量 分子伴侣 高密度发酵 

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