Recombinant Expression Of The Key Gene In The Enzymatic Synthesis Of β-Alanine And Its Transformation Research

The L-aspartate α-decarboxylase （PanD） catalyzes L-aspartate to generate β-alanine andCO2. β-alanine is one of the most important materials in the production of many types of dugsand drug intermesiates, such as pantothenate, carnosine, pamidronate disodium andbalsalazide, so it is widely used in the pharmaceutical, food and chemical industries. Atpresent, the β-alanine is synthesized by chemical method in factories, and there are still manyproblems in this process, such as difficulties in the purification caused by by-products, highdemand on the conditions, environmental pollution because of the exist of nitrile material.The enzymatic synthesis of β-alanine which is characterized by simple process, easypurification, non-pollution becomes a research hotspot currently, however, many problems areremained to be resolved in this method, such as low enzyme activity, low coversion rate,substrate inhibition, etc. In this research, the panD from Escherichia coli andCorynebacterium glutamicum were cloned and expressed, the recombinant enzyme was usedin the enzymatic synthesis of β-Ala. The main results were described as follows:（1） The L-aspartate α-decarboxylase genes from Escherichia coli str. K-12substr.MG1655and Corynebacterium glutamicum ATCC13032were cloned and high-efficientlyexpressed in Escherichia coli BL21（DE3）. Through the comparation of the two recombinants,the PanD expression level of the recombinant whose panD comes from E. coli was higherthan the other one due to the homologous expression, but the capability of bioconversion wasopposite. Based on all of this, the recombinant whose panD came from C. glutamicum wasdetermined to be studied by the follow-up experiment.（2） Then, the growth of the recombinant whose panD came from C. glutamicum and theexpression of the PanD were investigated. The enzyme activity reached123.5U/mL afterunivariate optimization in shake flask, which increased2.21-fold compared to the initialactivity55.76U/mL. On a preliminary exploration of the fermentor cultivation, the amount ofthe bacterias and the enzyme activity reached32.44g/L,684.85U/mL respectively with theuse of synthetic medium.（3） To explore the enzymatic characteristics of PanD, high purity of PanD was obtainedby ammonium sulfate precipitation, DEAE ion exchange chromatography and G-75chromatography. The reaction temperature of the purified PanD was optimal at55oC andstable below37oC. The reaction pH of PanD was optimal at6.0and stable at4.0-7.0.Km=8.995×10^-3mol/L, Vmax=0.0189mol·L^-1·h^-1. Ca²⁺actived the PanD while Pb inhibitedenzyme activity intensely.（4） The process of biotransformation was investigated. The enzymatic synthesis wasinhibited by L-aspartate and β-alanine. And the inhibition of the L-aspartate did not belongto the common types of reversible and irreversible inhibition. Compared with others, the typeof adding solid L-aspartate in batches could make the most effective use in reducing theinhibition of the substrate to improve the coversion rate in the conversion, adding the batchesof the substrate could relieve the inhibition of the substrate further, so the amount of enzymerequired for the reaction could be reduced. When L-aspartate was added in batches with3000 U PanD per gram L-Asp, the conversion ratio of100g/L L-Asp reached97.8%.