等离子诱变Trametes sanguinea WTFA5产漆酶培养基优化及酶学性质研究  

Medium Optimization and Characterization of Laccase Production by Trametes sanguinea WTFA5 Mutated via Plasma Mutagenesisi

作　　者：杜杰克 张川川 李沙 袁海波 刘洪玲 王腾飞[1] DU Jie-ke;ZHANG Chuan-chuan;LI Sha;YUAN Hai-bo;LIU Hong-ling;WANG Teng-fei(State Key Laboratory of Biomaterial and Green Papermaking,Qilu University of Technology(Shandong Academy of Sciences),Jinan 250353,China;Shandong Fullsail Biotechnology Co.,Ltd,Zibo 256300,China)

机构地区：[1]齐鲁工业大学(山东省科学院)生物基材料与绿色造纸国家重点实验室,济南250353 [2]山东富欣生物科技股份有限公司,淄博256300

出　　处：《齐鲁工业大学学报》2021年第3期19-27,共9页Journal of Qilu University of Technology

基　　金：山东省自然科学基金(No.ZR2020QB041);生物基材料与绿色造纸国家重点实验室开放基金(No.ZZ20200119)。

摘　　要：以愈创木酚-YPD培养基筛选产漆酶菌株,用等离子诱变、单因素和正交实验法提高其产漆酶效率,以活性电泳法纯化漆酶。筛选到一株产漆酶的血红栓菌Trametes sanguinea WTFA5;等离子诱变后获得遗传稳定的突变株T.sanguinea WTFS4,使发酵时间由10 d缩短至9 d,并且漆酶酶活由123.2 U/L提高至228.5 U/L;培养基优化后的最优组合为:葡萄糖40 g/L、豆粕粉20 g/L、铜离子6 mmol/L、2,5-二甲基苯胺20μmol/L,以此培养基进行发酵,S4菌发酵周期缩短至5 d,酶活提高至40000 U/L。通过对漆酶进行纯化,测得漆酶最适pH为3.0,最适温度为25℃,Km为(58.76±5.33)μmol/L,Kcat为(13.89±0.01)s^(-1)。等离子诱变和培养基优化可以显著提高T.sanguinea产漆酶效率。Guaiacol-YPD medium was used to screen laccase-producing strains.Plasma mutagenesis,single factor and orthogonal experimental methods were used to improve the efficiency of laccase production.Laccase was purified by native gel electrophoresis.A laccase-producing strain named Trametes sanguinea WTFA5 was obtained.After plasma mutagenesis,a genetically stable mutant strain T.sanguinea WTFS4 was obtained,which shortened the fermentation time from 10 days to 9 days.The laccase activity increased from 123.2 U/L to 228.5 U/L.Through the medium optimization,the laccase fermentation time was further shortened to 5 days,and the laccase activity increased to 40000 U/L under the optimal medium(glucose 40 g/L,soybean meal 20 g/L,copper ion 6 mmol/L,2,5-dimethylaniline 20μmol/L).By purifying the laccase,the optimum pH of the laccase is 3.0,the optimum temperature is 25℃,the Km is(58.76±5.33)μmol/L,and the Kcat is(13.89±0.01)s^(-1).Plasma mutagenesis and medium optimization can significantly improve the efficiency of laccase production by T.sanguinea.

关 键 词：血红栓菌 漆酶 等离子诱变 培养基优化 

分 类 号：Q814.1[生物学—生物工程]