Cloning Expression And Characteristic Studies On Fucoidan Degradation Enzyme

The application prospect of low molecular weight fucoidan?LMWF,low molecular weight fucoidan?has attracted wide attention.However,the high molecular weight of natural fucoidan and lacking in commercial fucoidan degrading enzyme preparations limit their large-scale applications in many fields,for instance medicine,food and cosmetics,etc.Microorganisms are an important source of fucoidan degrading enzymes.It is essential to screening new strains for the discovery of fucoidan-degrading enzymes of potential industrial value.Eight strains with productivity of fucoidan degrading enzymes were obtained from samples collected from Changxing Island,Dalian taking fucoidan as the sole carbon source.Among them,the enzymatic activity produced by strain 2-8 was higher?0.15 U/mL?than others.Strain 2-8 was classified as Bacillus cereus based on the morphological properities and phylogenetic analysis.According to the information of fucoidan degrading enzymes in NCBI database,degenerate primers of fucoidase from Bacillus cereus were designed.Prokaryotic expression vector of BcFucA was constructed then expressed in E.coli BL21?DE3?.BcFucA with 6×His tag was obtained,and the crude enzyme was purified by nickel column affinity chromatography.In order to investigate the effect of the linker?Lys261-Thr681?on the enzyme activity of BcFucA,truncated protein BcFucA_?linkerlinker was further obtained by RF-Cloning technique.BcFucA was constituted by 3411 bases encoding 1136 amino acids with a theoretical molecular weight of 125.35 kDa and an isoelectric point of 5.87.The purified BcFucA enzyme activity was 11.655 U.The optimum temperature and pH were 50°C and 4.0,respectively.It was also stability at a broad range of temperature20-50?and pH 3.0-7.0.The enzymatic activity of BcFucA can significantly promote by 1 mM Fe³⁺,but strongly inhibited by 1 mM Zn²⁺.Production analysis detected by MALDI-TOF-MS showed that the main products were monosaccharides,disaccharides and pentasaccharides.The study of the truncated body protein BcFucA_?linker demonstrates the importance of the linker for BcFucA activity...