马铃薯α-淀粉酶在毕赤酵母中的表达、纯化及其酶学性质的研究

doi:10.13523/j.cb.20140909

中国生物工程杂志

2014, Vol. 34

Issue (9): 56-62 DOI: 10.13523/j.cb.20140909

研究报告

马铃薯α-淀粉酶在毕赤酵母中的表达、纯化及其酶学性质的研究

王永刚¹, 马燕林¹, 马建忠¹, 马雪青², 任海伟¹

1. 兰州理工大学生命科学与工程学院兰州 730050;
2. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点试验室国家口蹄疫参考试验室兰州 730046

The Study on Expression,Purification and Characterization of α-amylase from Solanum tuberosum in P.pastoris

WANG Yong-gang¹, MA Yan-lin¹, MA Jian-zhong¹, MA Xue-qing², REN Hai-wei¹

1. College of Life Science and Engingeering, Lanzhou University of Technology, Lanzhou 730050 China;
2. State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

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摘要：

采用RT-PCR法扩增马铃薯夏波蒂的α-淀粉酶成熟肽基因，将其亚克隆至毕赤酵母表达载体pPIC9k上，Sac II 线性化重组表达载体，电击转化毕赤酵母GS115感受态细胞，构建重组酵母GS115/pPIC9k-amy，利用锥虫蓝法筛选获得高活性转化子（GSamyA5），以终浓度为0.5%甲醇诱导该重组菌表达α-淀粉酶，通过Ni²⁺-NTA agarose亲和层析纯化，并对其酶学性质进行研究。结果表明：该酶的最适反应温度为45℃，40~50℃酶活较稳定，保温50 min，残留相对活力达92.6%；最适反应pH值为6.0，并在pH 6.0~7.0范围内酶活保持稳定。Ca²⁺、K⁺可促进酶反应，以Ca²⁺影响为最，相对酶活力提高到125%；Cu²⁺，Fe²⁺，Fe³⁺，Zn²⁺对该酶有显著抑制作用；Mn²⁺，Mg²⁺对酶有微弱抑制作用，Li⁺、Na⁺对酶活影响不大。

关键词： α-淀粉酶; 马铃薯; 毕赤酵母; 表达与纯化; 酶学性质

Abstract:

The gene of α-amylase was amplified by through RT-PCR with Solanum tuberosum cv.Shepody cDNA as template, which was subcloned into vector pPIC9k and the recombinant plasmid pPIC9K-amy was linearzed with Sac II, then transformed into P.pastoris GS115 by electroporation. The high expressed amylase active strain GSamyA5 was attained by Trypan blue-BMMY medium screening and induced to express the enzyme with 0.5% methanol for 3 days under the 30℃, and fermentation broth was purified by Ni²⁺-NTA agarose. the enzyme characterization was studied. The result showed that The enzyme activity was stable between 40~50℃ with maximum activity at 45℃ and the activity hold 92.6% keeping 50min. The enzyme activity was stable between pH6.0~7.0 with maximum activity at pH6.0. Ca²⁺ and K⁺ had a stimulating effect on the recombinant amylase. The relative enzyme activity was up to 125% with Ca²⁺, Cu²⁺, Fe²⁺, Fe³⁺ and Zn²⁺ had an significantly inhibitory effect on the recombinant amylase.

Key words: α-amylase Solanum tuberosum P. pastori Expression and purification Enzyme characterization

收稿日期: 2014-02-11 出版日期: 2014-09-25

ZTFLH:

Q556.2

基金资助:

国家自然科学基金（31060041）；甘肃省自然科学基金（1212RJYA008，1212RJYA034）资助项目

通讯作者: 马建忠 E-mail: lab_ma@126.com

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引用本文:

王永刚, 马燕林, 马建忠, 马雪青, 任海伟. 马铃薯α-淀粉酶在毕赤酵母中的表达、纯化及其酶学性质的研究[J]. 中国生物工程杂志, 2014, 34(9): 56-62.

WANG Yong-gang, MA Yan-lin, MA Jian-zhong, MA Xue-qing, REN Hai-wei. The Study on Expression,Purification and Characterization of α-amylase from Solanum tuberosum in P.pastoris. China Biotechnology, 2014, 34(9): 56-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140909 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I9/56

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