The Gene Analysis,Food-grade Expression And Enzymatic Properties Of L-Arabinose Isoserase From Lactic Acid Bacteria With High D-tagatose Yield

L-arabinose isomerase is the key enzyme that catalyze the reversible isomerization of D-galactose to D-tagatose.Paper chromatography and modified cysteine carbazole sulphuric-acid method were used to screen lactic acid bacteria with high D-tagatose yield from the separation of a number of lactic acid bacteria that existed in pickled vegetables and pickled cabbages.As a result,a strain of lactic acid bacteria with high D-tagatose yield was isolated from a number of lactic acid bacteria.Based on the sequence analysis of 16 S rDNA and biochemical characteristics analysis,the strain was identified as Lactobacillus plantarum and was named L.plantarum WU14.Partial sequence of 16 S rDNA of the strain was uploaded to the NCBI,obtained Genbank accession number: KJ918744.The gene primers of L-Arabinose Isomerase was designed by the strain specificity and gene conservation.Meanwhile,the gene of L-Arabinose Isomerase of the strain was amplified,analysed,TA cloned and uploaded sequence.In addition,the structure and specific of L-Arabinose Isomerase protein of Lactobacillus plantarum were depth researched by study of the gene of L-Arabinose Isomerase of Lactobacillus plantarum WU14 with bioinformatics and enzymatic properties.The results showed that L-arabinose isomerase was mainly composed of alpha helix and random coil.It was a kind of endoenzyme without signal peptide and transmembrane protein.The optimal reaction temperature,pH and substrate concentration for L-arabinose isomerase were 60?,7.17 and 0.8mol/L separately.Under those conditions,the conversion rate reached 56.12% after 28 hours.The Genbank accession number of L-AI gene was KJ778785.In the premise of unchange the amino acid primary structure of L-AI,we designed inner and outer specific primers of L-AI gene of Lactobacillus plantarum WU14,recombinanted PCR technology to remove the Nco?sites of L-AI gene of L.plantarum WU14,inserted the recombinant L-AI gene into the food-grade expression vector pRNA48 that contain Nco ? and Hind ? Restriction enzyme sites,electro transformed the recombinant plasmid into L.lactis NZ9000,and the recombinant L.lactis NZ9000/pRNA48-L-AI was obtained finally.Sulphate-polyacrylamide gel electrophoresis and cysteine carbazole sulphuric-acid method were used to analyze the expression of target protein and determined crude enzyme activity after extracted intracellular protein that were obtained by nisin induced.The results showed that the target protein expression reached the maximum amount when the induced concentration of nisin reached 30ng/m L with 12 h induction,and crude enzyme acticity of recombinant bacteria reached 6.21U/m L under 60 ?.The enzymatic properties of suspended bactreia liquid and crushing coarse enzyme liquid for L.lactis NZ9000/pRNA48-L-AI were analyzed at the same time,the results showed that the optimal conversion rate reached 38.31% for recombinant L.lactis NZ9000/pRNA48-L-AI under the condition of 50?,pH 7.17,D-galactose concentration was 0.6 mol/L separately.