Studies On The Enzymatic Properties And Immobilization Conditions Of L-arabinose Isomerase From Lactobacillus Plantarum

D-tagatose is a kind of rare sugar,which has many physiological effects such as inhibiting hyperglycemia and improving intestinal flora.It is a low-energy sweetener with similar physical and chemical properties to sucrose.L-arabinose isomerase?L-AI?is an effective enzyme for the production of D-tagatose.Lactose is usually used as a cheap raw material in the industrial production of D-tagatose,which is hydrolyzed to D-galactose under acidic conditions,and then D-tagatose is produced by L-AI.At present,there are few reports on L-AI adapting to acidic conditions.In this paper,the L-arabinose isomerase gene?araA?fromLactobacillus plantarum WCFS1?gene number:1061986?was selected and synthesized,which is 1425 bp in length.The restriction site is Bam H?and Xho?,the vector is pET-28a?+?,and the host is E.coli TOP10.The plasmid was extracted and transformed into E.coli BL21?DE3?.After adding IPTG with a final concentration of 0.7 m M,L-AI can be efficiently expressed after induction at 16?for 16 h.The eluted protein was eluted four times with 100 m M imidazole eluent,and then eluted with 250 m M imidazole to obtain the target protein with higher purity.Enzyme activity was measured by resorcinol method,using D-galactose as substrate,the optimum reaction conditions of D-galactose were as follows:p H 7.0,temperature 55?,reaction time 36 h,and substrate concentration 150 g/L.The final concentration of Mn²⁺is5 m M.The recombinant cells were embedded with sodium alginate:the final concentration of sodium alginate was 3%?m/v?,the concentration of CaCl₂ solution was 0.3 M,and the dosage of recombinant bacteria is 30 g/L and hardened at 4?for 6 hours.The results showed that the optimum reaction conditions of free cells and immobilized cells were as follows:p H7.0,temperature 55?,reaction time 36 h,substrate concentration 150 g/L and final concentration 3 m M of Mn²⁺.The enzyme activities of enzyme solution,free cells and immobilized cells under acidic conditions were compared,all using 0.03 g of recombinant bacteria,and the corresponding enzyme solution concentration was about 0.27 mg/mL,the results showed that all of them could maintain high enzyme activity under the condition of p H 6.5,and the enzyme activity of the latter two was much higher than that of the former,among which the immobilized cells had the best stability,the highest enzyme activity was29 U/mL,and the enzyme activity could still maintain about 27 U/mL after three reaction batches.After four reaction batches,the enzyme activity decreased somewhat,but it could still maintain 21 U/mL,only 0.03 g recombinant bacteria with 1mL concentration of 30 g/L were used in the experiment.In this paper,taking the araA gene fromLactobacillus plantarum WCFS1 as the research object,the induction conditions and enzymatic properties of L-AI were studied.The immobilized cell method was used to further improve the enzyme activity and stability under acidic conditions and meet the actual production requirements of D-tagatose.