Thermostability Modification Of L-aspartate ?-decarboxylase For The Whole-cell Biocatalysis Of ?-alanine

L-aspartate?-decarboxylase catalyzes the?-decarboxylation of L-aspartate to?-alanine.?-alanine has a mutilpurpose in food,medicine,chemical and other fields.The synthesis methods of?-alanine mainly include chemical synthesis and bioconversion method.Compared with the chemical synthesis,bioconversion method has many advantages such as less by-products,easily operated,environmentally friendly.However,the ADC enzyme activity for catalyzing the production of?-alanine was at a comparatively low level,which limits the large-scale preparation of?-alanine.In this study,molecular modification was applied on ADC to improve its catalytic performance and establishment of bioprocess for synthesis of?-alanine.The main results are as follows:?1?Based on the evolutionary information from thermophilic bacteria,Lysine and Glycine located on the surface of L-aspartate?-decarboxylase were mutated to Arginine and Alanine,respectively,which successfully improved the thermal stability.Twenty-two variants were constructed in this study.Three variants showed better catalytic performance while the others showed similar performance with the wide type.The specific activity of K221R increased by20.3%;The residual activity of K49R,K221R and G369A were increased 0.7,1.2 and 1.0 times compared with wide type after treated at 50°C for 30 min.Analysis of variant structures demonstrated that increased thermostability was largely attributed to interaction formed with surrounding amino acids and reduction of flexible regions.?2?The process of one-time addition and substrate fed-batch reaction were compared and the latter method was better.After feeding for 3 times,the?-alanine production of wide type was 1079.9 mmol·L^-1,and the molar conversion rate was 90.0%;The optimal feeding times of K221R strain and G369A strain were 4 times,and the?-alanine production were 1512.2mmol·L^-1 and 1509.6 mmol·L^-1,while the molar conversion rate were 94.5%and 94.4%,respectively.?3?High cell-density cultivation using K221R was applied in 5 L bioreactor.Fed-batch cultivation was performed.After optimization of inducer types and concentrations,the optimal condition was as follows:the inducer IPTG was added to fermentation when cell density reached OD₆₀₀=60,and the final concentration was 0.8 mmol·L^-1.After fermentation for about53 h,the maximal cell density can reached to OD₆₀₀=130 and cell activity was 851.6 U·mL^-1.An analysis of the suitability of the catalytic process showed that up to 1820.0 mmol·L^-1?-alanine could be produced using cells expressing the recombinant K221R variant,and the molar conversion rate was 91.0%.