黑曲霉高耐热葡聚糖酶的分子截短与制备条件优化  

Molecular truncation of a hyperthermophilic glucanase from Aspergillus niger and optimization of its preparation conditions

作　　者：吴尚武 杨梦莲 沈微[1] 单逸蓝 王施岚 杨海泉[1] 夏媛媛 陈献忠[1] WU Shangwu;YANG Menglian;SHEN Wei;SHAN Yilan;WANG Shilan;YANG Haiquan;XIA Yuanyuan;CHEN Xianzhong(School of Biotechnology,Key Laboratory of Industrial Biotechnology,Jiangnan University,Wuxi 214122,China;Wuxi AppTec New Drug Development Co.Ltd.,Suzhou 215000,China)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,江苏无锡214122 [2]苏州药明康德新药开发有限公司,江苏苏州215000

出　　处：《食品与发酵工业》2023年第4期216-223,共8页Food and Fermentation Industries

摘　　要：该文旨在通过分子截短的方式提高高耐热葡聚糖酶AnEglA6的表达水平并针对重组菌建立相应的发酵方法。高耐热β-葡聚糖酶在禽类饲料生产中有着重要作用。黑曲霉来源的葡聚糖酶AnEglA6具有优越的耐热性能,适合在禽类饲料中使用。采用C端截短方式对该葡聚糖酶进行分子精简,构建了去除连接桥和纤维素结合结构域的截短体基因eglA6c,分别在乳酸克鲁维酵母和巴斯德毕赤酵母中进行异源表达获得重组酶AnEglA6cK和AnEglA6cP。乳酸克鲁维酵母表达的重组酶AnEglA6cK最适温度为75℃,在80℃下酶活力半衰期达55 min,能短时耐受85℃高温,应用性能显著优于AnEglA6cP。针对截短体酶AnEglA6cK在发酵后期酶活力快速下降的问题,对发酵条件进行了优化。通过以大豆蛋白胨替代酪蛋白胨的方式延长产酶时间、大幅度提高了摇瓶发酵水平。This paper aims to increase the expression level of hyperthermophilicβ-glucanase AnEglA6 from Aspergillus niger molecular truncation,and establish the fermentation conditions for the recombinant strain.Hyperthermophilicβ-1,4-glucanase plays an important role in poultry feed production.The glucanase AnEglA6 from Aspergillus niger has superior heat resistance,and it is suitable for use in the poultry feed industry.The molecular weight of glucanase AnEglA6 was reduced by C-terminal truncation,and the gene segment,which encoded the cellulose-binding domain and linker peptide,was removed from the gene eglA6.The truncated gene eglA6c was expressed in Pichia pastoris and Kluyveromyces lactis,and the expression products were designated as AnEglA6cP and AnEglA6cK,respectively.The recombinant enzyme AnEglA6cK,which was expressed by K.lactis,showed highest activity at 75℃,and its half-life reached 55 min at 80℃.In addition,the enzyme can withstand high temperature of 85℃for a short time.The application performance of AnEglA6cK was evidently better than that of AnEglA6cP.The enzymatic activity of the truncated enzyme AnEglA6cK decreased rapidly in the later stage of fermentation;thus,the organic nitrogen source of the culture medium was optimized.The growth time of the enzymatic activity was prolonged by replacing casein peptone with soybean peptone,and the highest enzymatic activity of fermentation broth in a shake flask was greatly improved.

关 键 词：高耐热β-1 4-葡聚糖酶 AnEglA6 分子截短 乳酸克鲁维酵母 大豆蛋白胨 

分 类 号：TQ925[轻工技术与工程—发酵工程]