Heterologous Expression and Enzymatic Properties of Fucoidanase Fcn1 from Marine <i>Flavobacterium</i> sp.(海洋黄杆菌来源岩藻多糖酶Fcn1的异源表达及酶学性质)
摘要
In order to find and explore new enzymes for degrading fucoidan, a strain of marine <i>Flavobacterium</i> 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 <i>Fcn1</i>. The full length of <i>Fcn1</i> gene is 1221bp, encoding 406 amino acids, and the molecular weight of the protein is about 46.8kDa. By primer design and PCR amplification, the <i>Fcn1</i> gene was cloned, and the heterologous expression vector <i>Fcn1</i>-pET-28a (+) was further constructed, and it was successfully induced and expressed in <i>E.coli</i> 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 332U/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 <i>K</i><sub>m</sub> of 1.17mg/mL, <i>V</i><sub>max</sub> of 10.53g·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.(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<i>Flavobacterium</i> 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<i>Fcn1</i>。<i>Fcn1</i>基因全长1221bp,编码406个氨基酸,蛋白分子质量约为46.8kDa。通过引物设计、PCR扩增,将<i>Fcn1</i>基因克隆,进一步构建了<i>Fcn1</i>基因异源表达载体<i>Fcn1</i>-pET-28a(+),并将其成