Expression And Modification Of L-aspartate α-decarboxylase For The Whole-cell Transformation Of β-alanine

β-Alanine is the natural β amino acid.It is an important pharmaceutical precondition and has an important application in both health food and chemical products.Compared with the chemical synthesis for β-alanine,the bioconversion method has the advantages of cleanliness and friendly environment,high conversion rate,mild conditions,and less by-products.But L-aspartate α-decarboxylase(ADC)catalyzing the synthesis of β-alanine,has the low enzymatic activity and the serious mechanism-based inactivation phenomenon.Those flaws make the relatively low utilization of enzyme.So it will help to expand the industrial production and application of β-alanine by the screening of higher enzymatic activity,attenuating mechanism-based inactivation and improvement of the properties of L-aspartate α-decarboxylase.In this work,to realize the efficient production of β-alanine,we expressed ADC proteins,and resigned their enzyme properties using site-directed mutagenesis to weaken the mechanism-based inactivation of ADC protein.L-aspartate α-decarboxylase gene(PanD)from Bacillus subtilis,Corynebacterium glutamicum and Lactobacillus plantarum,was constructed to the p ET-28 a vector,respectively.Then the expression vectors were transfected into E.coli BL21.The recombinant strains were induced and expressed at 30℃ for 12 h.SDS-PAGE analysis showed that two evident bands with different molecular weight were observed.The results suggested that the successful expression and self-cleavage and self-maturation of ADC protein.The whole-cell transformation of E.coli BL21/p ET28a-Bs Pan D confirmed that ADC could catalyze Asp to β-alanine.Three ADC proteins were purified by affinity chromatography.SDS-PAGE analysis showed that the purity of the purified protein was over 90%.The optimum temperatures of Bs ADC,Cg ADC and Lp ADC were 65,70 and 70℃,respectively.The optimum p H of Bs ADC,Cg ADC and Lp ADC were 7.0,6.5 and 6.5 respectively.Among three ADC enzymes,ADC from B.subtilis presented higher specific activity and more stable.Therefore,it was selected for the further experiments.The five amino residuals adjacent to its catalytic site of Bs ADC,Tyr58,Ile60,Glu56,Asn41 and Lys63,were selected for site-directed mutagenesis.Seven mutations of conservative Tyr58 and the saturation mutations of Ile60 lost their enzyme activity because they failed in their self-cleavage and self-maturation.The three mutations,Asn41 Gly,Glu56Ser and Lys63 Glu,did not affect the self-cleavage and self-maturation.They showed 1.3-1.6 folds higher activity than the wild-type at 37℃.The E56 S mutant presented slight thermostability,while N41 G and K63 E showed no change compared to the wild-type.The E56S showed 1.4-fold higher residual activity after the reaction for 2 h,suggesting E56 S mutation cloud attenuate the mechanism-based inactivation,while N41 G and K63 E had almost no effect on enhancing mechanism-based inactivation.The E56 S mutant was constructed to p ET-28 a,p MA-5 and p XMJ-19,and then transfected into E.coli,B.subtilis and C.glutamicum,respectively.The recombinant cells were cultured in 5-L bioreactor,and the corrected was used for the transformation of β-alanine.The E.coli BL21/p ET28a-E56 S strain produced 215.3 g·L-1 β-alanine in a yield of 94% at 37℃,p H 7.0 with a shake of 600 r·min-1.