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重组枯草芽孢杆菌壳聚糖酶的纯化和性质研究
引用本文:张舒平,周鹏,苏春元,江正强,闫巧娟.重组枯草芽孢杆菌壳聚糖酶的纯化和性质研究[J].中国农业大学学报,2012,17(1):125-131.
作者姓名:张舒平  周鹏  苏春元  江正强  闫巧娟
作者单位:1. 中国农业大学食品科学与营养工程学院,北京,100083
2. 中国农业大学工学院,北京,100083
基金项目:“863”计划课题(2011AA100905);新世纪优秀人才支持计划课题(NCET-08-0534)
摘    要:研究嗜热枯草芽孢杆菌壳聚糖酶基因在大肠杆菌中的克隆、表达及其重组酶的纯化和性质。该基因序列全长723bp,编码240个氨基酸。根据基因同源性分析,该壳聚糖酶与枯草芽孢杆菌Bacillus subtilis 168的壳聚糖酶前体基因的同源性最高,为98%。粗酶液经Ni-IDA亲和层析得到电泳级纯酶,比活力高达1 051.8U/mg。经测定,该酶反应最适温度为45℃,最适pH为6.0,在40℃和pH 4.5~8.0下稳定。该酶为内切壳聚糖酶,能够高效降解壳聚糖,生成一系列的壳寡糖,在壳寡糖的制备生产方面具有广阔的应用前景。

关 键 词:枯草芽孢杆菌  壳聚糖酶  纯化  酶学性质
收稿时间:2011/7/9 0:00:00

Purification and characterization of a recombinantchitosanase from Bacillus subtilis
ZHANG Shu-ping,ZHOU Peng,SU Chun-yuan,JIANG Zheng-qiang and YAN Qiao-juan.Purification and characterization of a recombinantchitosanase from Bacillus subtilis[J].Journal of China Agricultural University,2012,17(1):125-131.
Authors:ZHANG Shu-ping  ZHOU Peng  SU Chun-yuan  JIANG Zheng-qiang and YAN Qiao-juan
Institution:1.College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China; 2.College of Engineering,China Agricultural University,Beijing 100083,China)
Abstract:A chitosanase gene from thermophilic Bacillus subtilis WY34 was cloned and expressed in E.coli.The recombinant chitosanase was purified and characterized.The chitosanase gene consisted of 723 bp and encodes a polypeptide of 240 amino acids residues.Homology analysis of gene sequences showed that the enzyme shared 98% identity with the chitosanase gene from Bacillus subtilis 168.Crude enzyme was purified to homogeneity by Ni-IDA affinity chromatography.The specific activity of the purified chitosanase was up to 1 051.8 U/mg.The optimal pH and temperature for the purified chitosanase were 6.0 and 45 ℃,respectively.The enzyme was stable below 40 ℃ and from pH 4.5 to 8.0.The chitosanase showed excellent hydrolysis ability towards chitosan and produced a mixture of chitobiose,chitotetraose and some chitooligosaccharides with a longer chain length.These results indicated that the enzyme was an endo-type chitosanase and might be a good candidate for biotechnological application to produce chitooligosaccharides.
Keywords:Bacillus subtilis  chitosanase  purification  enzyme characterization
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