海洋黄杆菌来源岩藻多糖酶Fcn1的异源表达及酶学性质  

Heterologous Expression and Enzymatic Properties of Fucoidanase Fcn1 from Marine Flavobacterium sp.

作　　者：王立平 武俊义 王莹[1] 秦敏 陈芊汝 陈铁军 WANG Liping;WU Junyi;WANG Ying;QIN Min;CHEN Qianru;CHEN Tiejun(College of Food Science and Engineering,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学食品科学与工程学院,山东青岛266109

出　　处：《食品科学技术学报》2024年第4期135-144,共10页Journal of Food Science and Technology

基　　金：山东省青年自然科学基金资助项目(ZR2021QC042);青岛农业大学人才引进项目(665/1120037)。

摘　　要：为了寻找和挖掘降解岩藻多糖的新型酶,以从海带中筛选获得的一株可以降解岩藻多糖的海洋黄杆菌Flavobacterium sp.RC2-3为研究对象,对其进行了全基因组测序。通过与已报道的岩藻多糖酶氨基酸序列比对,并进行转录组学分析及实时荧光定量PCR验证,挖掘了1个可能的岩藻多糖酶基因,并将其命名为Fcn1。Fcn1基因全长1221 bp,编码406个氨基酸,蛋白分子质量约为46.8 kDa。通过引物设计、PCR扩增,将Fcn1基因克隆,进一步构建了Fcn1基因异源表达载体Fcn1-pET-28a(+),并将其成功地在大肠杆菌BL21(DE3)表达宿主中进行了诱导表达。所得重组酶Fcn1通过带有His标签的镍柱进行分离纯化,采用铁氰化钾法测定纯化酶酶解岩藻多糖的比酶活(332 U/mg),纯化倍数为2.25。酶学性质研究表明,重组酶Fcn1最适反应温度为50℃,最适反应pH值为8.0,在20~30℃和pH值为7.0~8.0时表现出较好的稳定性。结合酶学性质研究结果,确定酶的最适反应条件为30℃,pH值为8.0,并测定了岩藻多糖酶水解反应的动力学参数,K m为1.17 mg/mL,V_(max)为10.53 g·L^(-1)·min^(-1)。该酶降解岩藻多糖的酶活力较高,在岩藻多糖资源的开发领域具有较大应用潜力。In order to find and explore new enzymes for degrading fucoidan,a strain of marine Flavobacterium sp.RC2-3 which can degrade fucoidan was screened from kelp as the research object,and its whole genome was sequenced.By comparing with the amino acid sequence of fucoidanase reported,transcriptomic analysis and real-time fluorescence quantitative PCR verification,a possible fucoidanase gene was discovered and named Fcn1.The full length of Fcn1 gene is 1221 bp,encoding 406 amino acids,and the molecular weight of the protein is about 46.8 kDa.By primer design and PCR amplification,the Fcn1 gene was cloned,and the heterologous expression vector Fcn1-pET-28a(+)was further constructed,and it was successfully induced and expressed in E.coli BL21(DE3)expression host.The recombinant enzyme Fcn1 was separated and purified by a nickel column with His tag.The specific enzyme activity of the purified enzyme in hydrolyzing fucoidan was 332 U/mg determined by potassium ferricyanide method,and the purification multiple was 2.25.Enzymatic property study showed that the optimal reaction temperature of Fcn1 was 50℃,the optimal reaction pH was 8.0,and the stability of Fcn1 was better at 20-30℃ and pH 7.0-8.0.Combined with the results of enzymatic properties,the optimum reaction conditions were determined to be 30℃ at pH 8.0,and the kinetic parameters of enzymatic hydrolysis of fucoidanase were determined,with K m of 1.17 mg/mL,V max of 10.53 g·L-1·min-1.The enzyme has high activity in degrading fucoidan,and has great application potential in the development and utilization of fucoidan resources.

关 键 词：海洋黄杆菌 岩藻多糖酶 基因克隆 异源表达 蛋白纯化 酶学性质 

分 类 号：TS201.4[轻工技术与工程—食品科学]