Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization
Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization
复制标题
地衣芽孢杆菌耐热碱漆酶在毕赤酵母中的克隆表达及表征
DOI：
10.1016/j.biortech.2013.02.015
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发表时间：
2013-04-01
影响因子：
11.4
通讯作者：
Zhao, Min
中科院分区：
文献类型：
--
作者：
Lu, Lei;Wang, Tian-Nv;Zhao, Min
从地衣芽孢杆菌中克隆出一个热碱稳定漆酶基因，并在毕赤酵母中表达。重组漆酶分泌到培养基中，最高活性为227.9 U/L。纯化的漆酶是一个单体糖蛋白，经去糖基化后SDS-PAGE估计其分子量为65 kDa。以丁香醛嗪为底物，在pH 6.2和70℃条件下酶活性最佳。重组漆酶在30℃条件下，在7 ~ 9的pH范围内具有较高的稳定性，在50 ~ 70℃条件下表现出较好的热稳定性，70℃条件下的失活半衰期为6.9 h，与天然孢子漆酶一样，对NaCl和有机溶剂具有较高的耐受性。在乙酰丁香酮的存在下，纯化的漆酶能快速脱色活性蓝19、活性黑5和靛胭脂红。在pH为9.0的条件下，4 h脱色率超过93%。(C) 2013 Elsevier Ltd.版权所有。
A thermo-alkali-stable laccase gene from Bacillus licheniformis was cloned and expressed in Pichia pastoris. The recombinant laccase was secreted into the culture medium with a maximum activity of 227.9 U/L. The purified laccase is a monomeric glycoprotein, and its molecular weight was estimated to be 65 kDa on SDS-PAGE after deglycosylation. Optimal enzyme activity was observed at pH 6.2 and 70 degrees C with syringaldazine as substrate. The recombinant laccase was highly stable in the pH range 7-9 after 10 days at 30 degrees C. The enzyme displayed remarkable thermostability at 50-70 degrees C, with a half-life of inactivation at 70 degrees C of 6.9 h. It also exhibited high tolerance to NaCl and organic solvents like the native spore laccase. The purified laccase could rapidly decolorize reactive blue 19, reactive black 5 and indigo carmine in the presence of acetosyringone. More than 93% of the tested dyes were decolorized in 4 h at pH 9.0. (C) 2013 Elsevier Ltd. All rights reserved.