白囊耙齿菌锰过氧化物酶在粟酒裂殖酵母中表达鉴定及菌酶协同发酵  

Expression and identification of manganese peroxidase of Irpexlacteus in Schizosaccharomyces pombe and synergistic fermentation of corn straw by strains and enzymes

作　　者：衡新宇 崔周磊 梁晓玉 房燕洲 武俊明[2] 陈华友[1] HENG Xinyu;CUI Zhoulei;LIANG Xiaoyu;FANG Yanzhou;WU Junming;CHEN Huayou(Jiangsu University,School of Life Sciences,Zhenjiang 212013,China;Jiangsu Zhongxing Pharmaceutical Co.,Ltd.,Zhenjiang 212000,China)

机构地区：[1]江苏大学生命科学学院,镇江212013 [2]江苏中兴药业有限公司,镇江212000

出　　处：《生物学杂志》2022年第2期63-68,共6页Journal of Biology

基　　金：江苏省镇江市重点研发计划(社会发展)项目(SH2020021);江苏省镇江市丹徒区重点研发计划(社会发展)项目(SH2018002)。

摘　　要：首次将源于白囊耙齿菌(Irpexlacteus,I.lacteus)的一种锰过氧化物酶基因mnp(GenBank:MH120207.1)经密码子优化后与无任何抗性基因且有Gfa基因的高效表达载体pREP相连,构建原核-真核穿梭表达载体pREP-mnp,再电转化导入Gfa营养缺陷型粟酒裂殖酵母(Schizosaccharomyces pombe)中,构建生物安全级粟酒裂殖酵母,SDS-PAGE验证目的蛋白表达。所构建的工程菌胞外表达MnP,酶活力在第4天达到峰值,为17.46 U/L。所构建的工程菌经混菌发酵菌种筛选和氮源优化,在菌酶协同发酵中有显著的促进玉米秸秆木质素降解的效果。玉米秸秆中添加0.5%尿素、2%(NH_(4))_(2)SO_(4)、0.02%Tween-80、使基质终浓度为0.5 mmol/L的草酸、0.5%CaCl_(2)、0.3%MnSO_(4),S.pombe-pREP-mnp与N.crassa、S.pombe-man、C.utili、1%Lac、0.4%GOD协同发酵3 d后,玉米秸秆中木质素、半纤维素和纤维素的降解率分别为60.79%、27.61%和25.49%,真蛋白含量提高了310.99%。For the first time,a manganese peroxidase gene mnp(GenBank:MH120207.1)derived from Irpexlacteus(I.lacteus)was optimized and linked to the high-efficiency expression vector pREP without any resistance gene and Gfa gene to construct a prokaryotic-eukaryotic shuttle expression vector pREP-mnp,then electro transformed into Gfa auxotrophic Schizosaccharomyces pombe(S.pombe)to construct biosafety grade S.pombe.The expression of the target protein was verified by SDS-PAGE.The constructed engineered strain expressed MnP extracellularly,and the enzyme activity reached the peak value on the 4th day,which was 17.46 U/L.After fermentation strain screening and nitrogen source optimization,the constructed engineered strain had a significant effect of promoting lignin degradation in synergistic fermentation of corn straw.Added 0.5%urea,2%(NH_(4))_(2)SO_(4),0.02%Tween-80,0.5%CaCl_(2),and 0.3%MnSO_(4)to corn straw,the final concentration of oxalic acid in the matrix 0.5 mmol/L,S.pombe-pREP-mnp,N.crassa,S.pombe-man,C.utili,1%Lac,and 0.4%GOD was co-fermented for 3 days.The degradation rates of lignin,hemicellulose and cellulose in corn straw were 60.79%,27.61%,and 25.49%,respectively,and the true protein content was increased 310.99%.

关 键 词：锰过氧化物酶 粟酒裂殖酵母 基因表达 木质素降解 真蛋白 

分 类 号：Q78[生物学—分子生物学] Q55