Study On Production Of β-Alanine By L-Aspartate-α-Decarboxylase

In this paper, a new method for the determination ofβ-alanine andL-aspartatic acid by Reversed Phase High Performance LiquidChromatography with Precolumn Derivatization was established. Thesamples were derivatized with DNFB and analyzed on a 5μm Zorbax SBC18 column at 30℃using gradient elution with diode array detection at360nm. The mobile phase A was NaAc-HAc buffer(pH6.2), and the mobilephase B was acetonitrile. From 0 to 20 minutes, the percentage of mobilephase B increased from 5％to 33％. The flow rate was 1.0 mL·min^-1 andthe injection volume was 20μL.panD gene encoding L-aspartate-α-decarboxylase was cloned fromE.coli DH5αby PCR amplification. The gene was inserted into the vectorpET28b(+) to construct an expression plasmid pET28b(+)-panD, then itwas transformed into E. coli BL21(DE3), and overexpression ofL-aspartate-α-decarboxylase in E. coli BL21(DE3) was observed. TheL-aspartate-α-decarboxylase activity of recombinant strain E. coli BL21(DE3)/pET28b(+)-panD increased significantly. After induced by0.4mmol/L IPTG and 8g/L lactose, the enzyme activity were 98.00U and150.91 U, and the product yield of the recombinant strain were 14.11％and21.73％respectively, whileβ-alanine from control strains E. coli DH5α, E.coli BL21(DE3) and E. coli BL21(DE3)/pET28b(+) couldn't bedetermined by the same method.Through optimization experiments, enzyme synthesis conditions of E.coli BL21(DE3)/pET28b(+)-panD were given as follows: 50mLfermentation liquid culture was inoculated with a 1％volume of inoculumand incubated for 2h at 37℃, 200r/min; then lactose was added with finalconcentration of 10g/L and incubated for 24h at 30℃, 150r/min. Underoptimal conditions, L-aspartate-α-decarboxylase activity of E. coliBL21(DE3)/pET28b(+)-panD was 224.96U which increased 448.01％Through optimization experiments, the optimal biotransformationconditions were given as follows: the substance solution was 0.02mol/LL-aspartate sodium (pH4.49); the optimal temperature was 30℃and theoptimal time was 48h. Under optimal conditions, theβ-alanine yield of E.eoli BL21 (DE3)/pET28b(+)-panD increased 10％.