Enzymatic Catalysis Of D-pantoic Acid And β-alanine To Produce D-pantothenic Acid

D-pantothenic acid（vitamin B5）is widely used in feed,food,chemical,pharmaceutical and other industries,the market demand is large.At present,the production of D-pantothenic acid mostly uses chemical separation method,which is not only tedious and time-consuming but also has a great impact on the environment.In this paper,Pantothenate synthetase（PS）was used as the key enzyme for the production route of biological enzyme,which is environmentally friendly and can greatly reduce the production cost.Main research contents and results are as follows:（1）Screening of pantothenate synthetaseIn this study,pantothenate synthetase enzyme genes（panC）from 11 sources were cloned and expressed in Escherichia coli BL21（DE3）.The combinant strains were screen based on the activity level of pantothenate synthetase,and the activity was measured by High Performance Liquid Chromatography（HPLC）.（2）Property analysis of pantothenate synthetaseThe enzymatic properties of pantothenate synthetase in recombinant strain PSBb were studied.The optimum temperature and pH were first studied.It was found from the experimental results showed that the optimum temperature of PSBb was 30℃,the optimum pH was 7.0,kcat was 115.4 s^-1,and the content of ATP in cells was 1.58×10^-5μmol/mg.（3）Optimization of transformation conditions of recombinant Escherichia coli in 5L fermentation tanks while fermentationUsing D-pantoic acid and β-alanine as substrates,the medium type,inducer type,inducer optimum concentration,inducer optimum temperature and residual sugar content of D-pantothenic acid were optimized in 5L fermentation tank.It was found that the yield of D-pantothenic acid using glucose fermentation medium after the reaction was carried out for 46 h was 65.1g/L,which was 6.3g/L higher than that using TB medium.Use 0.1g/L IPTG as inducer,control the induced temperature at 30℃,control the residual sugar in the medium at 0.1-0.5g/L,the D-pantothenic acid yield can reach 86.4g/L,and the conversion rate could reach 87.8%.（4）Feeding batch fermentationIn order to reduce the toxic effect of β-alanine on cells and further improve the production of D-pantothenic acid,we tried to divide 500mL of 60g/L D-pantoic acid and 40g/L β-alanine as a substrate into three batches,five batches and eight batches for feeding.The results showed that when the substrates were put into five batches,and the D-pantothenic acid was 92.2g/L with 93.7%conversion rate.We further optimized the speed of the fermentation tank and found that the speed was related to the dissolved oxygen,and the yield of D-pantothenic acid could reach 95.7g/L,the conversion rate reached 97.2%,and the space-time yield was 50.4g/L/d at 15%（v/v）.At present,there are few data on the activity and kinetics of PS enzyme.This study lay a good foundation for the study on the activity and kinetics of pantothenic acid synthase and the production of D-pantothenic acid by enzymatic method.