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

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 1 221 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.