Constraction Of Key Enzymes In Biotransformation Of Ursodeoxycholic Acid And Application In Large-Scale Bioconversion

Ursodeoxycholic acid(UDCA)is an effective component of traditional Chinese medicine,which has important therapeutic uses related to its better therapeutical performance in the treatment of gallstones,promoting liver transplantation,bile reflux gastritis,alcoholic liver,bile cirrhosis,and drug-induced hepatitis,market demand is large.At present,there are two main methods to prepare UDCA: Live bear bile extraction and artificial synthesis.The natural source of UDCA is live bear bile juice.Live bear is protected by animal protection law,and the source of UDCA is limited,resulting in the decrease of natural bear bile.Nowadays,most commercial UDCA is obtained from its epimer chenodeoxycholic acid(CDCA)extracted largely from bovine and goose by chemical reactions.Through the oxidation-reduction method,7-OH has configuration inversion.However,there are a series of problems such as complex reaction process,low selectivity,harsh reaction conditions,large energy consumption and pollution,especially in the process of protection and deprotection Toxic and dangerous reagents severely limit the industrial application of chemical methods.At present,UDCA produced by chemical method accounts for about 30% of the market share,and the preparation of high purity is relatively low,about 80%,which is far from meeting the market demand for UDCA quantity and quality.Compared with chemical epiisomerization,CDCA biosynthesis UDCA is more efficient and environmentally friendly.Microbial transformation or enzyme catalysis mainly revolved around 7?-hydroxysteroid dehydrogenase(7?-HSDH)and 7?-hydroxysteroid dehydrogenase(7?-HSDH).The bioconversion from CDCA to UDCA was realized by Clostridium mucosum,Clostridium disharmonious,Clostridium pasteurella and Xanthomonas maltophilia,which produced 7?-HSDH and 7?-HSDH.However,high concentrations of CDCA inhibit the accumulation of cell biomass,which is difficult in the process of product recovery and purification.In addition,previous studies showed that with the extension of culture time,the yield of intermediates increased,UDCA decreased,and industrial production could not be realized.In recent years,the combination of 7?-HSDH and 7?-HSDH to generate UDCA by two-step method with CDCA as substrate has become a research hotspot.At present,the comprehensive conversion rate of research can exceed 90%,but the reaction amount is only milliliter level,which is still a long way from industrial production.The main restrictive bottlenecks are: 1)the key enzyme 7?-HSDH in the two-step method with CDCA as the substrate is extremely unstable,although the enzyme activity is high enough,it will be rapidly inactivated in the catalytic reaction system;2)the substrate capacity of the two-step reaction system is too low(volume ratio 1%),and the system is unstable;3)the large-scale source of industrial application of two key enzymes in the twostep method;4)the cost of separation and purification of enzymatic proteins is too high.In this paper,aiming at the main restrictive bottleneck of unstable 7?-HSDH,low substrate concentration and unable to realize industrial production,based on the successful construction and heterologous expression of 7?-HSDH and 7?-HSDH,we mainly start from the construction process of genetic engineering enzyme and the construction of enzyme catalytic system to improve the catalytic performance of 7?-HSDH,improve the stability of enzyme in the system and enhance the substrate carrying capacity It can adapt to the industrial production process of enzyme catalysis;through the optimization of fermentation culture process,explore the large-scale fermentation process,the source of industrial enzyme in enzyme catalysis process;finally,the research of kilogram industrial production and purification process.In the construction process of genetic engineering enzyme 7?-HSDH,the tag had a great influence on the actual catalytic process.Although the recombinant enzyme 7?-HSDH with 6His tag had higher activity,the recombinant enzyme 7?-HSDH with GST tag had higher thermal stability and is more suitable for the actual industrial catalytic process.It was found that the heat stability of the 7?-HSDH recombinase labeled with GST was 12 times higher than that labeled with 6His.At the same time,during the construction of enzyme catalytic system,adding the composite stabilizer(0.15% EDTA + 0.5% sodium chloride + 0.2% gelatin)in the system could greatly improve the stability of the enzyme in the system,the residual rate of enzyme activity was 97.8%,improved the bearing capacity of substrate,and made the recombinant enzyme can carry out catalytic reaction continuously.The culture and induction conditions of two kinds of recombinant bacteria were optimized,and the high-density fermentation of 50 L fermentor was realized.The OD value of the recombinant bacteria reached 100,and the biological activities of 7?-HSDH and 7?-HSDH were 13.6 U/mL and 7.6 U/mL,respectively,which solved the large-scale source problem of the two key enzymes.Finally,a set of efficient UDCA enzyme catalytic production technology was constructed.After high-density fermentation,the fermentation broth was crushed and preliminarily filtered.The crude enzyme broth obtained could be put into the catalytic reaction system for catalysis,which reduced the cost of purification of enzyme and protein,increased the load of substrate,and achieved the highest yield.In the high 50 L catalytic reaction system,the loading capacity of the substrate reaches 50 g/L,the yield and efficiency of the catalytic reaction meet the requirements of industrial application.Finally,it is successfully applied to 1kg industrial production from CDCA to UDCA,and the overall conversion rate is more than 90%.