Study On Production β-alanine By Biotransformation

In this paper, comparing with paper chromatography, potassium permanganate titration and HPLC, a new method for the determination ofβ-alanine by HPLC-ELSD was established. Chromatography was performed on a 5μm Alltech amido column at 40℃. The mobile phase was composed of methanol - water (5 : 1) with a flow rate of 1.0 mL·min^-1. Alltech2000 evaporative light scattering detector(U.S.A) was used with a gas flow rate of 2.2L·min^-1 and tube temperature of 85℃.26 strains acrylic acid-resistant microorganism were screened from soil samples which were collected from 8 regions in country.β-alanine synthase activity of Sarcine luted was highest what in 26 strains microorganism. The specific concentration ofβ-alanine in biotransformation liquid of Sarcine luted was 0.102g·L^-1 Mutants of Sarcina lutea grew on acrylic acid medium and ferment liquid culture medium after mutation by ultraviolet and ⁶⁰Coγ-ray disposal. Theβ-alanine synthase activity of mutant strain TH0571A was 20.5U. It was increased 28.97%Through single factor and orthogonal experiment, enzyme synthesizes conditions of the mutate strain TH0517A were given. The optimal enzyme synthesizes conditions were as follows: dissolubility starch 2.3g, KNO₃0.8g, H₃BO₃ 3.4μg, ZnSO₄ 8.1μg, MgSO₄·7H₂O 0.02g, KH₂PO₄ 0.7g, NaCl 0.145g, dissolve in 100mL water, pH value 7.0; the optimal fermentation temperature was 30℃; the optimal rotating speed was 200 r/min; the optimal fermentation time was 18 hours. Under optimal conditions,β-alanine synthase activity of mutant strain TH0571A was 54.4U. It was increased 200.41%Through single factor experiment, the optimal biotransformation conditions were given. The optimal biotransformation conditions were as follows: the enzyme liquid was secondary fermentation liquid; composing of biotransformation liquid is acrylic acid 6%, 1%10^-5mol/LZnSO₄, 1%10^-5mol/L CuSO₄, 0.02%MgSO₄.7H₂O, adjust pH value to 7.0 with 0.1%Ca(OH)₂, then adjust pH value to 10.0 with ammonia; the optimal enzyme addition is 9mL enzyme liquid to 15mL biotransformation liquid; the optimal biotransformation time is 10 hours. Under optimal conditions, ratio of biotransformation of mutant strain TH0571A was 1.25%. It was increased 138%.β-alanine synthase was immobilized in Chitosan as carrier. Chitosan was first activated 3 hours by 0.4% glutaraldehyde, then the enzymes were immobilized in activated carrier. The optimal enzyme amount was 40ml/g Chitosan; the optimal immobilized was 2hours at 0℃. The recovery yield ofβ-alanine synthase was 43.16%.