Establishment And Optimization Of Biocatalytic Access To ?-alanine From Fumarate By A Two-enzyme Coupling System

?-alanine is the only ?-amino acid exited in nature,and also a kind of non protein amino acid.It was used widely in many fields,such as medicine,food,chemistry and environment.It can be used as medical intermediate with high value.Enzyme-based biocatalysis is becoming an important way to produce ?-alanine,but one-enzyme way has inherented weakness because of the high price of substrate.In this study,a two-enzymatic coupling system,using combined L-Aspartase(Asp A)and L-aspartate ?-decarboxylase(ADC),was developed.Using this two-step enzymatic coupling system,?-alanine with high added value can be obtained from cheaper substrate Fumarate.Main results are as follows:(1)To establish this biosystem,Ecasp A from Escherichia coli and Cgpan D from Corynebacterium glutamicum were expressed.The optional ratio of Ec Asp A and Cg ADC was 1:80(W:W),and the concentration of Asp A was 10 ?g·m L-1.The temperature and p H was 37°C and 7.0,respectively.When the concentration of Fumarate was 100 mmol·L-1,the yield of ?-alanine reached 90 mmol·L-1(7 g·L-1)after 8 h.The yield of ?-alanine can reach 126 mmol·L-1(9.8 g·L-1)when the concentration of Fumarate was increased to 200 mmol·L-1.(2)The pan D gene from Tribolium castaneum was ligated into p ET-28a(+)vector and recombinant plasmid p ET28a-Tcpan D was constructed.Tcpan D was expressed in Escherichia coli BL21(DE3)successfully.Recombinant Tc ADC was purified to electrophoretic purity using the His-Trap and Resource Q column.The specific activity of Tc ADC was 350 mmol·g-1·h-1(350 U·g-1).Studies on enzymatic properties show that optimum temperature was 37°C and the optimum p H was 6.5.The Km value was 0.0813 mmol·L-1,the kcat value was 3.9 s-1.(3)A two-enzyme system,in which Cg ADC was replaceed with Tc ADC,was developed and optimized.The optional ratio of Ec Asp A and Tc ADC was 1:80(W:W),and the optional p H was 6.5.When the concentration of Fumarate was 200 mmol·L-1,the yield of ?-alanine reached 192 mmol·L-1(7 g·L-1)after 12 h.The yield of ?-alanine reached 340 mmol·L-1(26.5 g·L-1)when the concentration of Fumarate was increase to 400 mmol·L-1.