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张水龙,陈东,曹树威,吴仁智,黄日波.黑曲霉木聚糖酶基因xynB的克隆及在酿酒酵母中的表达[J].广西科学,2013,20(2):148-151,157. [点击复制]
- ZHANG Shui-long,CHEN Dong,CAO Shu-wei,WU Ren-zhi,HUANG Ri-bo.Cloning of xynB Gene Encoding Xylanase B from Aspergillus niger and Expression in Sacharomyces cerevisiae[J].Guangxi Sciences,2013,20(2):148-151,157. [点击复制]
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| 黑曲霉木聚糖酶基因xynB的克隆及在酿酒酵母中的表达 |
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张水龙1, 陈东2, 曹树威1, 吴仁智2, 黄日波1,2
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| (1.广西大学生命科学与技术学院, 广西南宁 530004;2.广西科学院非粮生物质酶解国家重点实验室, 国家非粮生物质能源工程技术研究中心, 广西生物炼制重点实验室, 广西南宁 530007) |
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| 摘要: |
| 从黑曲霉(Aspergillus niger)克隆木聚糖酶基因xynB,利用重叠延伸PCR法去除其中的内含子。将该基因与质粒pYES2连接,构建真核表达载体,用醋酸锂化转法导入酿酒酵母(Sacharomyces cerevisiae)INVSC1菌株表达。利用α信号肽替换基因原信号肽,以考察信号肽对表达蛋白分泌的影响。结果获得2株重组菌株,分别为xynB连接原信号肽的pYES2-xynB菌株和xynB连接α信号肽的pYES2-xynB-α菌株。木聚糖酶B成功表达,SDS-PAGE检测到约24kDa目的蛋白质条带。木聚糖酶B最适催化温度为50℃,最适催化pH值为5.0,Mn2+对酶活具有强烈的抑制作用。将α信号肽取代原信号肽使酶活达到最高的诱导时间由72h缩短为48h,但是置换信号肽使最高酶活由7.59U降低为3.97U。 |
| 关键词: 木聚糖酶 黑曲霉 酿酒酵母 |
| DOI： |
| 投稿时间：2013-03-06修订日期：2013-03-28 |
| 基金项目:国家973项目(2010CB736209,2012CB723605),国家863项目(2012AA022106,2012AA023406,2012AA022302,2013AA050701),国家国际合作项目(2010DFB63590,2011DFA61910),广西科学研究与技术开发计划项目(桂科合10100019-21,桂科攻1099071,桂科合1140010-15),广西科技创新能力与条件建设计划项目(桂科能12237022),广西自然科学基金项目(2012GXNSFAA053062),广西科学院基本科研业务费项目(10YJ25SW15,12YJ25SW04),八桂学者建设工程专项经费项目资助。 |
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| Cloning of xynB Gene Encoding Xylanase B from Aspergillus niger and Expression in Sacharomyces cerevisiae |
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ZHANG Shui-long1, CHEN Dong2, CAO Shu-wei1, WU Ren-zhi2, HUANG Ri-bo1,2
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| (1.Life Science and Technology College, Guangxi University, Nanning, Guangxi, 530004, China;2.State Key Laboratory of Non-Food Biomass and Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Science, Guangxi Key Laboratory of Biorefinery, Nanning, Guangxi, 530007, China) |
| Abstract: |
| The xylanase xynB gene from Aspergillus niger was cloned and its introns were trimmed off by overlap extension PCR. An expression vector was successfully constructed by ligating the xynB with vector pYES2 and transformed into Saccharomyces cerevisiae INVSC1 using Lithium Acetate method. The signal peptide of xylanase B was replaced with α signal peptide to identify the effect of signal peptide on the secretion of expressed protein,recombinant S.cerevisiae pYES2-xynB with xylanase B original signal peptide and pYES2-xynB-α with α-signal peptide were obtained.The result showed that the xylanase B was expressed successfully. A target protein band of approximately 24kDa was identified on SDS-PAGE gel map. The optimal action temperature of expressed xylanase B was 50℃ and the optimal action pH value was 5.0,Mn2+ inhibited the activity of xylanass B strongly.The induction time of maximum activity of xylanase B decreased from 72h to 48h by replacing the xylanse B original signal peptide with α-signal peptide.However,the replacement of signal peptide caused the decrease of the maximum activity of xylanas B from 7.59U to 3.97U. |
| Key words: xylanase B Aspergillus niger Saccharomyces cerevisiae |
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