β-葡萄糖苷酶的枯草芽孢杆菌表达、酶学性质分析及其在宝藿苷Ⅰ制备中的应用  

Expression of Recombinant β-Glucosidase in Bacillus subtilis and Its Enzymatic Characterization and Application in Preparation of Icariside Ⅱ

作　　者：裴雯雯 曾艳[2] 刘娟娟 张建刚 王敏[1] 孙媛霞[2] PEI Wenwen;ZENG Yan;LIU Juanjuan;ZHANG Jiangang;WANG Min;SUN Yuanxia(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457,China;National Engineering Laboratory for Industrial Enzymes,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所工业酶国家工程实验室,天津300308

出　　处：《食品工业科技》2022年第6期133-140,共8页Science and Technology of Food Industry

基　　金：国家重点研发计划(2021YFC2101403);天津市合成生物技术创新能力提升行动项目(TSBICIP-KJGG-004);中国科学院科技服务网络计划(KFJ-STS-QYZD-200)。

摘　　要：为获得安全高效的β-葡萄糖苷酶用于生物催化淫羊藿苷水解制备宝藿苷Ⅰ,通过大肠杆菌-枯草芽孢杆菌穿梭载体pMA5在食品安全菌株枯草芽孢杆菌WB600中异源表达Thermotoga petrophila来源的耐热β-葡萄糖苷酶TpBgl3,研究了所表达重组酶的酶学性质及其水解宝藿苷制备宝藿苷Ⅰ的工艺条件。结果表明,菌株在30℃、SR培养基中发酵培养48 h后,上清液中的β-葡萄糖苷酶酶活力达到69.68 U/mL。重组表达的TpBgl3最适温度为85℃,最适pH为4.0,在65℃、pH4.0下放置3 h仍能保持85%以上的相对酶活。在65℃、pH4.0、酶用量0.16 U/mg、反应时间20 min的优化条件下,其能将10 g/L的淫羊藿苷水解转化为7.50 g/L的宝藿苷Ⅰ,摩尔转化率为98.67%。β-葡萄糖苷酶的枯草芽孢杆菌分泌表达为宝藿苷Ⅰ及其他高附加值苷元类化合物的工业化安全高效生物制备提供了新途径。To obtain a safe and efficient β-glucosidase for preparing icariside Ⅱ from icariin by biocatalytic hydrolysis, a GRAS(generally recognized as safe) strain Bacillus subtilis WB600 was used as the host to produce a thermophilic β-glucosidase from Thermotoga petrophila, by construction of a Escherichia coli-Bacillus subtilis shuttle vector pMA5.Enzymatic properties of the recombinant enzyme and its process conditions of hydrolyzing icariin to prepare icariside Ⅱ were studied. The result showed that after cultivated in SR culture for 48 h at 30 ℃, the activity of β-glucosidase in the fermented broth reached to 69.68 U/mL. TpBgl3 exhibited the maximal activity at 85 °C and pH4.0. More than 85% of the maximum activity was retained after incubation at 65 °C and pH4.0 for 3 h. Under optimized conditions of 65 °C, pH4.0,and 0.16 U/mg enzyme dosage, the recombinant enzyme could transform 10 g/L icariin into 7.50 g/L icariside II in 20 min with a molar conversion of 98.67%. The successful secretory expression of β-glucosidase in Bacillus subtilis could provide new ways to the industrial safe and efficient biopreparation of icariside Ⅱ and other high-value-added aglycon compounds.

关 键 词：Β-葡萄糖苷酶 枯草芽孢杆菌 异源表达 酶学性质 淫羊藿苷水解 宝藿苷Ⅰ 

分 类 号：TS201.3[轻工技术与工程—食品科学]