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。
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.
出处
《中国农业大学学报》
CAS
CSCD
北大核心
2012年第1期125-131,共7页
Journal of China Agricultural University
基金
"863"计划课题(2011AA100905)
新世纪优秀人才支持计划课题(NCET-08-0534)
关键词
枯草芽孢杆菌
壳聚糖酶
纯化
酶学性质
Bacillus subtilis
chitosanase
purification
enzyme characterization