Study On The Enzymatic Synthesis Of Alanine Series Product From The Raw Material Maleic Anhydride

L-alanine,β-alanine and DL-alanine are important kind of chiral drug intermediates,it was widely used in many industries,such as pharmaceutical chemicals,food,health products and so on.At present,the methods of synthesis the chiral amino acids in the world divided into two kinds:chemical synthesis and enzymatic.Because of short reaction time,mild conditions,the enzyme specificity,high catalytic efficiency,less environmental pollution and many other advantages,enzyme catalysis had exhibited a broad development prospects in the industry.In this paper,widely sources and cheap of maleic anhydride is a starting material,It is synthesize β-alanine,DL-alanine and L-malic acid by maleate cis-trans isomerase,aspartate enzyme,aspartate-α-decarboxylase,aspartate-β-decarboxylase,alanine racemase and fumarase.Works are as follows:1.enzymatic synthesize DL-alanine by aspartate-β-decarboxylase and alanine racemase with the material of L-aspartic acid.Aspartate-β-decarboxylase and alanine racemase was built in this paper,two plasmids of pETDuet and pGEX-KG were used in this study,to build four genes engineered bacteria altogether.Through the initial activity experiment,to select pGEX-KG plasmid vector in the engineered bacteria activity higher than pET-Duet plasmid vector activity.The optimum reaction conditions of pGEX-KG-Asd is 45℃ and pH 6.0,The optimal reaction conditions of pGEX-KG-Alr were 40℃ and pH 7.0.200 g/L aspartic acid was converted into 132 g/L L-alanine in the catalytic reaction of pGEX-KG-Asd at 16 hours,the molar conversion rate can reach 99%;200 g/L L-alanine was completely converted into DL-alanine by GEX-KG-Alr at 6 hours.Finally,aspartate-β-decarboxylase and alanine racemase coupling reaction was carried out,In 30 mL reaction system,pH 7.0,40℃,the ratio of enzyme to Asd:Alr=1:2,1.2 mmol/L PLP,150 g/L L-alanine was completely converted into DL-alanine at 10 hours,the conversion rate of 99%above.2.enzymatic synthesize β-alanine by maleate cis-trans isomerase,aspartate enzyme and aspartate-α-decarboxylase with the material of maleic anhydride.Maleate cis-trans isomerase engineering Strain constructed for this study,the optimum temperature and pH were 35℃-40℃and pH 7.0 respectively.pETDuet-maiA-PanD double enzyme co-expression engineering bacteria is built,the optimum temperature is 45℃ and optimum pH is 8.0.Finally,under respective optimum conditions,comparing the multi-enzyme coupling and single strain of the double enzyme catalytic efficiency and yield of β-alanine,The results showed that,a multi-enzyme coupling system,the reaction after 12 h the production of β-alanine was 29 g/L in 40 g/L ammonium maleic substrate,the conversion rate to 97%;the single strain and double enzyme reaction system the reaction after 12 h the production amount ofβ-alanine was 17 g/L and a production yield of 57%.3.fumaric acid was converted into malic acid by fumarase,This paper selected fumaric genes from two different sources,eventually,their expressed in E.coli cells.Fumarase was built with two sources of C.glutamicum and E.coli.They were called fumCc and fumCe respectively.At the respective optimum conditions,1.5 g sodium fumarate catalyzed by fumCc and fumCe,L-malic acid yield reached the highest,the yield rate of was 85%and 34%respectively.