一株全基因合成耐热甘露聚糖酶的表达及酶学性质分析

doi:10.13523/j.cb.20140807

中国生物工程杂志

2014, Vol. 34

Issue (8): 41-46 DOI: 10.13523/j.cb.20140807

研究报告

一株全基因合成耐热甘露聚糖酶的表达及酶学性质分析

张巍¹, 王亚伟¹, 陈丰¹, 周樱², 熊海容¹

1. 中南民族大学生命科学学院武汉 430074;
2. 武汉新华扬生物股份有限公司武汉 430074

Gene Synthesis, Expression and Characterization of a Thermostable Endo-β-1, 4-mannanase

ZHANG Wei¹, WANG Ya-wei¹, CHEN Feng¹, ZHOU Ying², XIONG Hai-rong¹

1. College of Life Science, South-central University for Nationalities, Wuhan 430074, China;
2. Wuhan Sunhy Biology Co., Ltd, Wuhan 430074, China

全文: PDF(881 KB) HTML

摘要：

根据已知耐热甘露聚糖酶ManAd3氨基酸序列与毕赤酵母密码子使用偏爱性，设计并合成了甘露聚糖酶ManA全基因（Accession No. KJ806637），与表达载体pPIC9k重组后，转化毕赤酵母GS115，筛选获得重组菌株ManA-GS115。该重组菌株发酵产物经SDS-PAGE鉴定，其中甘露聚糖酶ManA含量达到电泳纯级别，分子量大小约为30 kDa。其酶学性质检测结果显示该酶最适反应温度为75℃，最适反应pH为6.0，比活力高达3200 IU/mg，并且在75℃下处理30 min仍能维持90%以上相对酶活力。该甘露聚糖酶ManA表达量较高，在偏酸性环境下仍能够维持较高的相对酶活力，且热稳定性显著，可广泛应用于食品、酿造、饲料、纺织和医药等工业领域。

关键词： 甘露聚糖酶; 毕赤酵母; 基因合成; 耐热性

Abstract:

According to the known thermostable mannanase ManAd3 sequence and the codon bias of yeast, the optimized mannanase ManA sequence (Accession No. KJ806637) was designed and synthesized. The ManA gene was cloned into the pPIC9k vector, and then expressed in Pichia pastoris GS115 to obtain a recombinant strain ManA-GS115. The fermentation product of mannanase ManA was electrophoresis pure by SDS-PAGE identification with the molecular weight about 30 kDa. The enzymatic properties were shown that the optimal reaction temperature and pH value were 75℃ and pH6.0 respectively, and the specific activity was up to 3 200 IU/mg. Moreover, the residual activity was above 90% after 30 min treatment under 75℃, pH 6.0. With high expression, excellent thermostability, and the good thermostable tolerance under acidic condition, the mannanase ManA could be potentially applied in brewing, food, feed, textile and pharmaceuticals fields.

Key words: Mannanase Pichia pastoris Gene synthesis Thermostability

收稿日期: 2014-07-11 出版日期: 2014-08-25

ZTFLH:

Q819

基金资助:

湖北省对外科技合作项目（2013BHE016）、武汉市科技攻关项目（2013020501010180）资助项目

通讯作者: 熊海容，E-mail：xiong_hr@yahoo.com E-mail: xiong_hr@yahoo.com

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引用本文:

张巍, 王亚伟, 陈丰, 周樱, 熊海容. 一株全基因合成耐热甘露聚糖酶的表达及酶学性质分析[J]. 中国生物工程杂志, 2014, 34(8): 41-46.

ZHANG Wei, WANG Ya-wei, CHEN Feng, ZHOU Ying, XIONG Hai-rong. Gene Synthesis, Expression and Characterization of a Thermostable Endo-β-1, 4-mannanase. China Biotechnology, 2014, 34(8): 41-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140807 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I8/41

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