摘要
作者从NCBI数据库中挖掘到来源于Butyrivibrio sp.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p ET-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。Mn^(2+)对其酶活力具有促进作用,SDS、Fe^(3+)、Cu^(2+)、Zn^(2+)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。
A chitosanase gene BUT from the Butyrivibrio sp. 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. Mn^2+ could significantly enhance the enzymatic activity,while SDS,Fe^3+,Cu^2+ and Zn^2+ could inhibit the activity strongly. The analysis of hydrolysates by TLC demonstrated that chitosan was degraded into(GlcN)2,(GlcN)3,and(GlcN)4.
作者
王琦
崔阳
刘进宝
孙慧慧
郭娜
孙建安
毛相朝
WANG Qi;CUI Yang;LIU Jinbao;SUN Huihui;GUO Na;SUN Jianan;MAO Xiangzhao(College of Food Science and Engineering,Ocean University of China,Qingdao 266003,China;Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Laboratory for Marine Drugs and Bioproducts,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266237,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第1期147-155,共9页
Journal of Food Science and Biotechnology
基金
宁波市科学技术局市级科技计划项目(ZD-A02-农业重大科技专项)(2017C110006)
关键词
壳聚糖酶
壳聚糖
壳寡糖
克隆表达
酶学性质
chitosanase
chitosan
chitooligosaccharide
cloning and expression
enzymatic properties
