Screening Of D-lactate Dehydrogenase And Its Application In Producing D-Danshensu

D-danshensu?D-DSS?,a water-soluble phenolic acid compound of Chinese herbal medicine Salvia miltiorrhiza.Numerous studies have shown that it has a wide range of physiological pharmacological activities,clinically applied to dilate coronary arteries,inhibits platelet aggregation and cardiomyocyte apoptosis,antithrombotic and anti-atherosclerosis,liver protection,anti-inflammatory and enhances immunity.At present,the yield of D-DSS is far from meeting the clinical needs,so the preparation of high-yield,high-purity D-DSS has become the focus of this study.In this thesis,D-DSS was prepared by enzymatic conversion method,D-lactate dehydrogenase?D-LDH?with higher yield of D-DSS was screened,and the enzymatic properties were studied.At the same time,the co-expression of D-LDH and glucose dehydrogenase?GDH?was studied to realize the regeneration of coenzyme,and D-DSS was produced by whole cell catalysis.The main results are as follows:?1?Reduction of 3,4-dihydroxyphenylpyruvic acid?DPA?to D-DSS catalyzed by 19strains of lactic acid bacteria preserved in laboratory,and L.reuteri JN516 with the highest yield was found.?2?The D-lactate dehydrogenase of L.reuteri JN516 was analyzed,and the predicted four D-lactate dehydrogenase genes were d-ldh83871,d-ldh82948,d-ldh82319 and d-ldh83931,respectively.These genes were subjected to primer design and molecular cloning,ligated to the vector pColdII,and then induced to express at low temperature in E.coli BL21?DE3?.D-LDH82319 was found to have a higher DPA reduction activity.By predicting the molecular weight of D-LDH82319 is 35 kDa,and the optical purity of the catalyzed D-DSS is 100%.The results of enzymatic properties were as follows:the optimum pH was 8.0 and the optimum reaction temperature was 25°C.It has high catalytic activity for pyruvic acid.Ca²⁺and Ni²⁺promoted its enzyme activity,but Cu²⁺inhibited enzyme activity.When NADH was used as coenzyme,D-LDH82319 had the greatest affinity for DPA(K_m=0.09 mmol·L^-1),and when NADPH was used as coenzyme,D-LDH82319 had the greatest affinity for DPA(K_m=0.10 mmol·L^-1),which indicated that D-LDH82319 had non-coenzyme specificity.?3?Construction of D-LDH82319 and GDH?28 kDa,from Bacillus subtilis ATCC 13952?co-expression strains E.coli BL21?DE3?/pColdII-d-ldh82319-T7-gdh and E.coli BL21?DE3?/pColdII-gdh-T7-d-ldh82319,the regeneration of coenzyme is achieved.Comparing the yields of D-DSS produced by the two co-expressing strains,the results showed that the yield of D-DSS was higher in E.coli BL21?DE3?/pColdII-d-ldh82319-T7-gdh.?4?The optimum conditions for the whole cell catalytic synthesis of D-DSS were as follows:the concentration of DPA was 2 g·L^-1,the concentration of glucose was 1.5 g·L^-1,the temperature was 25°C,the pH was 8.0,and the conversion rate could reach 95.93%for 4hours.