纤维微菌β-1,3-葡聚糖酶的克隆表达及对灵芝细胞壁的水解作用  

Cloning and expression of Cellulosimicrobium β-1,3-glucanase and its hydrolysis on the cell wall of Ganoderma lucidum

作　　者：杨梦莲 单逸蓝 沈微[1] 朱新文 杨海泉[1] 陈献忠[1] 陈磊 夏媛媛 YANG Menglian;SHAN Yilan;SHEN Wei;ZHU Xinwen;YANG Haiquan;CHEN Xianzhong;CHEN Lei;XIA Yuanyuan(School of Biotechnology,Key Laboratory of Industrial Biotechnology,Jiangnan University,Wuxi 214122,China;Agricultural and Rural Bureau of Dong’e County,Shandong Province,Liaocheng 252200,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]山东省东阿县农业农村局,山东聊城252200

出　　处：《食品与发酵工业》2023年第1期10-18,共9页Food and Fermentation Industries

摘　　要：该文旨在研究一种酶解灵芝(Ganoderma lucidum)菌丝体从而提高其胞内蛋白提取效率的方法。通过PCR方法扩增获得纤维微菌(Cellulosimicrobium sp.)CICIM6906的β-1,3-葡聚糖酶编码基因,该基因命名为bgl6906并与表达载体连接,构建重组质粒pET28a-bgl6906,重组质粒转化大肠杆菌BL21(DE3),获得重组大肠杆菌BL21(DE3)/pET28a-bgl6906。重组菌在TB培养基中进行诱导表达,表达产物在自身信号肽引导下大部分分泌到细胞外。以茯苓多糖为底物时,重组酶BGL6906纯酶比酶活力为567.8 U/mg,最适pH为5.5,最适温度为50℃。重组菌在15 L发酵罐中发酵72 h胞外酶活力达到67 U/mL。重组酶BGL6906与果胶酶交替水解灵芝菌丝体培养物,可以有效水解细胞壁,获得部分原生质体。进一步辅助短时超声波破碎可以大幅度提高灵芝胞内产物的释放。This paper aims to study the enzymatic hydrolysis of Ganoderma lucidum mycelium to improve the protein extraction efficiency.The gene bgl6906 encodingβ-1,3-glucanase was amplified from the genome DNA of Cellulosimicrobium sp.CICIM B6906.The recombinant plasmid pET28a-bgl6906 was constructed and transformed into E.coli BL21(DE3),the recombinant strain Escherichia coli BL21(DE3)/pET28a-bgl6906 was constructed to express bgl6906.The recombinant strain was cultured in TB medium and induced gene expression.Most of the expression products of the gene bgl6906 was secreted outside the cell under the guidance of its own signal peptide.The recombinant enzyme BGL6906 showed the highest activity when pachyman was used as a substrate,and the specific activity of the pure enzyme was 567.8 U/mg.The optimum temperature and pH of BGL6906 was 50°C and 5.5,respectively.The recombinant strain was used in a fermentation experiment using a 15 L fermenter.The highest enzyme activity was 67 U/mL,which was reached after 72 h of fermentation.BGL6906 and pectinase were used alternatively for the cell wall hydrolysis of the G.lucidum mycelium.The cell wall was successfully degraded,and a small amount of protoplast was observed.The mycelium cells treated with BGL6906 and pectinase alternatively could be disrupted effectively by short-time ultrasonic treatment.About 76%of the total protein could be extracted to the alkaline solution.

关 键 词：纤维微菌 Β-1 3-葡聚糖酶 分泌表达 灵芝 细胞破碎 

分 类 号：S567.31[农业科学—中草药栽培]