Preparation Of DL-alanine By Alanine Racemase Method

DL-alanine was useful not only as a starting material for synthesis of pharmaceuticals,agricultural chemicals,and other products that were chiral but also in food ingredients and cosmetics.However,there's a great demand for DL-alanine all around the world.At present,the methods of industrial production DL-alanine were mainly chemical synthesis and fermentation,but there were not many enzymatic methods in vitron on research of its production.Alanine racemase(Air)could be found in prokaryotes and some eukaryotes,but it was absent in human.Alanine racemase was classified into the fold-type II group of PLP dependent enzymes and catalyzed the racemization of L-alanine and D-alanine.In this report,the full-length 1080bp Air gene of Escherichia coli K-12 was cloned into the vector of pET-28a and over expressed induced by IPTD(Isopropyl?-D-1-Thiogalactopyranoside)or lactose in Escherichia coli BL21(DE3)in LB or M9 culture medium to characterize the recombination Air and made the corresponding production of DL-alanine.The molecular mass of Air from Escherichia coli K-12 was about 39kDa,which required pyridoxal 5'-phosphate(PLP)as a coenzyme.By a series of experiments,we found that the consumption of PLP was 0.025mg per mg recombination Air.The optimum pH of the recombination Air was observed at 9.0,and its optimum temperature was 37?.The activitiy of recombinant Air was 35×10-4U.Magnesian ion(Mg2+)that the concentration was below 20mM nearly had no influence on recombinant Air,while 5mM ferrous ion(Fe2+)and 10mM two divalent manganese(Mn2+)could completely inhibit the activity of recombinant Air.In 1L aqueous solution,100g L-alanine was racemized to DL-alanine by 5mg recombination Air in twenty-four hours.Consequently,we could successfully get DL-alanine from the reaction liquid in the way of vacuum rotary evaporation crystallization.Finally,the percent recovery of DL-alanine was 90.6%,and its relative standard deviation(RSD)was 1.72%,and the optical rotation value was 0.However,We successfully got about 23mg purified recombination alanine racemase which could make the production of about 500g DL-alanine with high activity from the recombinant BL21-pET28a-Alr in 1L culture medium by genetic engineering.Enterprises could make the production of DL-alanine by recombination Air with low cost,easy way and little pollution.