壳聚糖酶的基因克隆表达及酶学性质研究

Cloning，Expression，and Characterization of Chitosanase

DOI：10.3969/j.issn.1673-1689.2019.01.022

中文关键词: 壳聚糖酶 壳聚糖 壳寡糖 克隆表达 酶学性质

英文关键词: chitosanase,chitosan,chitooligosaccharide,cloning and expression,enzymatic properties

基金项目:

摘要点击次数: 78

全文下载次数: 253

中文摘要:

作者从NCBI数据库中挖掘到来源于Butyrivibriosp. MC2013的壳聚糖酶基因，命名为BUT，该壳聚糖酶基因大小为903 bp，编码301个氨基酸。通过NCBI数据库和进化树比对发现，该壳聚糖酶（BUT）属糖苷水解酶46家族（后简称GH-46），与其它壳聚糖酶相似度为59%，是一种新型的壳聚糖酶。序列经密码子优化后进行全基因序列合成，与表达载体pET21a（+）连接构建重组质粒pET-21a-BUT，转入大肠杆菌E. coli BL21（DE3）表达宿主，进行诱导表达。所得重组壳聚糖酶通过Ni-NTA亲和层析进行纯化，SDS-PAGE确定其蛋白分子量为35 kDa，DNS法测定其酶活为146.0 U/mg。对BUT酶的酶学性质进行探究，结果表明BUT酶最适温度和pH分别为45 ℃、8.0，在中性偏碱性条件下稳定性较强。Mn2+对其酶活力具有促进作用，SDS、Fe3+、Cu2+、Zn2+等的抑制作用极强。通过TLC对其水解产物分析发现，BUT水解壳聚糖的产物是壳二糖、壳三糖和壳四糖。

英文摘要:

A chitosanase gene BUT from theButyrivibriosp. MC2013 was selected from the NCBI database，which contains 903 bp and encodes a protein with 301 animo acids. Research has suggested that the chitosanase belongs to the GH-46 by sequence alignment in NCBI datebase and phylogenetic analysis. It is a novel chitosanase which shares 59% identities with any other chitosanases. The genes weresynthesized after codon optimization and inserted into pET21a（+），then the recombinant plasmid，pET-21a-BUT，was transformed into E. coli BL21（DE3）. The chitosanase was induced in ZYP-5052 and purified with the affinity chromatography of Ni-NTA. The molecular weight was estimated to be 35 kDa by SDS-PAGE. The maximum activity of purified enzyme was 146.0 U/mg. The enzymatic properties of recombinant chitosanase showed that the optimal temperature and pH of the BUT were 45 ℃ and 8.0，respectively. The stability in alkaline conditions was higher than that in acidic conditions. Mn2+ could significantly enhance the enzymatic activity，while SDS，Fe3+，Cu2+ and Zn2+ could inhibit the activity strongly. The analysis of hydrolysates by TLC demonstrated that chitosan was degraded into（GlcN）2，（GlcN）3，and（GlcN）4.

查看全文 查看/发表评论 下载PDF阅读器