Study On Enzymatic Production Of β-alanine

In this paper, The panD gene of E. coli BL21 (DE3)/pET28b(+) -panD, encoding the L-aspartateα-decarboxylase, was amplified from by PCR and cloned into the expression plasmid pET28c(+). The recombinant plasmid was then transformed into E. coli BL21(DE3) and screened on the resistant plate with 50μg/mL kanamycin. L-aspartateα-decarboxylase could be efficiently expressed in engineered strain E. coli BL21 (DE3) / pET28c(+)-panD by lactose induction. The value of enzymatic activity was 92.5 U/L. The PanD was correctly expressed by SDS-PAGE analysis. But it had a problem that theπ'-subunit wasn't self-cleavaged completely, the same with the strain of E. coli BL21 (DE3) / pET28b(+)-panD.The optimal condition for producing PanD were as follows: incubation at 30℃, 12 g/L lactose induction after culture for 3.5 h, incubation time was 20 h, and the optimum initial induction pH value of LB media was 5.5. Under these conditions, the recombination PanD activity was 186 U/L, 101% higher than before.The culture was freated by ultrasonic oscillation. The crude of PanD was purified by ammonium sulfate fractional precipition and colum chromatography of Ni affinity. The specific activity was 20.15 U/mg. The yield was 68.34% and the pureness of PanD was higher than 95%. Reaserch of enzymatic properties showed that the optimal temperature and pH were 45℃and 8.0. This PanD was stable between pH 5.0～8.0. Metal ion had no effect on activity of PanD. The higher temperature of water-bath could improve the self-cleavage rate. Water bath of 70℃for 12 h,the activity of PanD increased by 360%.E. coli BL21 (DE3)/pET28b(+)-panD was immobilized with calcium alginate, and the optimum embeding condition were as follows: 10% cells entrapped by 3% alginate, the optimal gelling agent and substract were CaCl2 and ASP-K. After the immobilized cells were utilized for 5 batches in continuous conversion, the immobilized particle was dissolve gradually.