β-1,4-葡聚糖内切酶基因在大肠杆菌中分泌表达  

Research of β-1,4-endoglucanase Gene Secreting Expression in Escherichia coli

作　　者：李湘玉[1] 刘秦华[1] 李君风[1] 白晰[1] T. Desta 王思然[1] 董志浩[1] 白云峰[2] 邵涛[1] LI Xiangyu;LIU Qinhua;LI Junfeng;BAI Xi;T. Desta;WANG Siran;DONG Zhihao;BAI Yunfeng;SHAO Tao(Institute of Ensiling and Processing of Grass, Nanjing Agricultural University, Nanjing 210095 , China;Circular Agriculture Research Center, Jiangsu Province Academy of Agricultural Sciences, Nanjing 210014, China)

机构地区：[1]南京农业大学饲草调制加工与贮藏研究所,江苏南京210095 [2]江苏省农业科学院循环农业研究中心,江苏南京210014

出　　处：《食品与生物技术学报》2018年第3期309-315,共7页Journal of Food Science and Biotechnology

基　　金：江苏省自然科学青年基金项目(BK20130694);高等学校博士学科点专项科研基金项目(20130097120053);江苏省自主创新项目(CX(15)1003)

摘　　要：采用重叠延伸PCR合成技术将乳酸乳球菌(Lactococcus lactis)分泌蛋白基因序列usp45(81 bp)和瑞氏木霉(Trichoderma reesei)β-1,4-葡聚糖内切酶成熟蛋白序列egl3(657 bp)连接成融合基因usp45-egl3,构建了分泌型重组质粒pMG36e-usp45-egl3及重组大肠杆菌E.coli DH5α/pMG36e-usp45-egl3。分析重组大肠杆菌的表达效果及表达产物降解纤维素的能力。结果表明,重组大肠杆菌DH5α/pMG36e-usp45-egl3经14 h培养能分泌酶活为226 mU/mL的β-1,4-葡聚糖内切酶,并有效地降解了羧甲基纤维素钠。其中分泌的β-1,4-葡聚糖内切酶降解羧甲基纤维素钠生成还原糖的速率为2.35 mg/(h·mL)。This study constructed a secretive recombinant plasmid pMG36e-usp45-eg13 and E. coli DH5α/pMG36e-usp45-eg13 with a fusion gene usp45-egl3 connected by the secreted protein gene sequence of usp45 （81 bp） from Lactococcus lactis and the coded mature-protein gene sequence of egl3 （657 bp） from Trichoderma reesei by the method of overlapping extension PCR. After analyzing the expressive effect of recombinant Escherichia coli and evaluate the capability of secreted β-1, 4-endoglucanase degrading fiber. The results showed that the recombinant E.coli DH5α/ pMG36e-usp45-egl3 could secrete β-1,4-endoglucanase with activity of 226 mU/mL after culture 14 h and effectively degrade sodium carboxymethylcellulose. The rate of β-1,4-endoglucanase degrading sodium carboxymethylcellulose into reducing-sugar was 2.35 mg/（h· mL）.

关 键 词：瑞氏木霉 β—1 4-葡聚糖内切酶 大肠杆菌 分泌表达 

分 类 号：S816.53[农业科学—饲料科学]