葡萄穗霉中β-葡萄糖苷酶的基因克隆、表达及酶学性质分析

生物技术通报 ›› 2013, Vol. 0 ›› Issue (6): 128-132.

葡萄穗霉中β-葡萄糖苷酶的基因克隆、表达及酶学性质分析

于海玲^{1, 2, 4}, 李树伟^{1, 2}, 王华明^{3, 4}

(1. 新疆塔里木大学新疆生产建设兵团塔里木盆地生物资源保护利用重点实验室, 阿拉尔 843300;2. 新疆塔里木大学生命科学学院, 阿拉尔 843300;3. 工业酶国家工程实验室, 天津 300308;4. 中国科学院天津工业技术研究所, 天津 300308)

收稿日期:2013-06-20 修回日期:2013-06-20 出版日期:2013-06-20 发布日期:2013-06-20
作者简介:于海玲, 女, 硕士研究生, 研究方向: 生物化学与分子生物学;E-mail: 472097056@qq.com
基金资助:
天津科委真菌-淀粉酶高温活性改造(102CKFSY06200), 天津市工业微生物基因组解析(11ZCZDSY08300)

Cloning, Expression and Characterization of β-glucosidase from Stachybotrys chartarum

Yu Hailing^{1, 2, 4} Li Shuwei^{1, 2} Wang Huaming^{3, 4}

(1. Xinjiang Production & Construction Corps, Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Tarim University, Alar 843300;2. College of Life Science, Tarim University, Alar 843300;3. National Engineering Laboratory for Industrial Enzymes, Tianjin 300308;4. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308)

Received:2013-06-20 Revised:2013-06-20 Published:2013-06-20 Online:2013-06-20

摘要/Abstract

摘要： 以黑曲霉(Aspergillus niger) G1为宿主菌表达葡萄穗霉(Stachybotrys chartarum) β-葡萄糖苷酶。对葡萄穗霉进行全基因组分析得到编码β-葡萄糖苷酶的基因, 设计引物, 通过PCR扩增得到g10158, 将其克隆到载体pGm上, 并转化到黑曲霉G1中, 经amdS二次平板筛选, PCR方法验证, 获得高效表达该基因的生物工程菌株G1-pGm-g10158-13。SDS-PAGE检测结果显示, 表达蛋白的分子大小为87.9 kD, 阴性对照中无此条带。粗酶液酶学性质表明, 该β-葡萄糖苷酶的最适反应温度为50℃, 最适pH为4.8。在最适pH和最适温度下, 粗酶液活性达1 856.48 U/mL。葡萄穗霉β-葡萄糖苷酶在黑曲霉G1中可表达, 并具有较高的活性。

关键词: β-葡萄糖苷酶, 葡萄穗霉, 黑曲霉G1, 原核表达

Abstract: To express β-glucosidase from Stachybotrys chartarum using Aspergillus niger G1 as host. Through genome sequencing and analysis, the β-glucosidase gene(g10158)was isolated from the genome of Stachybotrys chartarum through PCR. The coding region of the gene was inserted to the vector pGm. The expression plasmid was transformed into A. niger G1 strain. An β-glucosidase producing strain(G1-pGm-g10158-13)were selected after screening several transformants using amdS selection plates and confirmed by PCR validation. Results showed that the molecular mass of the enzyme was 87.9 kD by SDS-PAGE gel analysis. Our results indicated that the recombinant enzyme exhibited optimum activity at pH4.8 and 50℃. The β-glucosidase activity was 1 856.48 U/mL under the optimized conditions. The β-glucosidase from Stachybotrys chartarum was expressed in A. niger G1 strain and it was proved to have a high activity.

Key words: β-Glucosidase, Stachybotrys chartarum, Aspergillus niger, G1 Prokaryotic expression

于海玲, 李树伟, 王华明. 葡萄穗霉中β-葡萄糖苷酶的基因克隆、表达及酶学性质分析 [J]. 生物技术通报, 2013, 0(6): 128-132.

Yu Hailing, Li Shuwei, Wang Huaming. Cloning, Expression and Characterization of β-glucosidase from Stachybotrys chartarum[J]. Biotechnology Bulletin, 2013, 0(6): 128-132.

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