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Screening Identification Of L-lactate Dehydrogenase From Lactobacillus Fermentum And Its Application In The Synthesis Of L-Danshensu

Posted on:2020-05-21Degree:MasterType:Thesis
Country:ChinaCandidate:H LuFull Text:PDF
GTID:2370330578464257Subject:Fermentation engineering
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L-Danshensu?L-DSS?is a water-soluble phenolic acid compound with great physiological activity and medicinal value.It has significant pharmacological effects,such as antithrombosis,antioxidation,antisepsis and antiinflammation,attenuating microcirculatory disturbance and so on,which provides a wide application prospect in prevention and treatment of cardiovascular and cerebrovascular diseases.Beyond that,it is an important intermediate for the synthesis of a variety of chiral and bioactive substances.There is no L-Danshensu in nature,only its enantiomer D-Danshensu.Pharmacological studies have shown that D-and L-Danshensu have the same pharmacological activity and the efficacy of L-Danshensu is more prominent.In recent years,there have been reports of chemical synthesis of L-Danshensu.However,due to its serious pollution,cumbersome operation,high cost,poor optical purity of the product and low synthesis efficiency,it is considerably limited by industrial application.Therefore,exploring a biosynthesis pathway of optical pure L-Danshensu with environmentally friendly,moderate conditions,primitive operation,low cost and high efficiency is absolutely necessary.Furthermore,Lactate dehydrogenase can produce?-hydroxycarboxylic acid by stereoselective reduction of?-ketoic acid,providing a valuable reference for the biosynthesis of L-Danshensu.In this research,the genes of l-lactate dehydrogenase and glucose dehydrogenase were introduced into Escherichia coli BL21?DE3?for heterogenous expression,and then L-Danshensu was synthesized with whole-cell by catalyzing substrate 3,4-dihydroxyphenylpyruvate and glucose.The main results of this study are showed as follows:?1?The strain Lactobacillus fermentum JN248 with high reduction activity of3,4-dihydroxyphenylpyruvate was screened from 20 strains of lactobacillus preserved in the laboratory.The whole genome sequences of Lactobacillus fermentum F-6 was analyzed,and 5l-lactate dehydrogenase genes?lf-l-ldh0845?lf-l-ldh1566?lf-l-ldh1732?lf-l-ldh0611 and lf-l-ldh0372,protein molecular weight ranged from 32 to 34 kDa?were cloned from L.fermentum JN248.They were respectively connected to plasmid pETDuet-1 and introduced into E.coli BL21for induced expression.SDS-PAGE analysis and enzyme activity assay were carried out on the enzyme solution before and after purification,and it was found that the five enzymes revealed obvious bands with theoretical molecular mass,and LF-L-LDH0845 showed the highest activity.?2?The enzymatic properties of LF-L-LDH0845 were studied.The results showed that LF-L-LDH0845 was most active and stable at pH 6.0;the optimum temperature was 25°C and the stability is excellent in the range of 10-25°C;there is a wide substrate coverage wherein the optimum substrate is pyruvic acid followed by phenylpyruvate;values of Km,Kcat and Kcat/Km for3,4-dihydroxyphenylpyruvate were 11.3709 mM,0.2931 s-1,0.0258 mM-1·s-1,respectively.?3?Inorder to regenerating coenzyme NAD?P?H,the strainE.coli BL21?DE3?/pETDuet-1-ldh-gdh was constructed by co-expressing glucose dehydrogenase derived from Bacillus subtilis and LF-L-LDH0845.The induction conditions were optimized,results showed that the optimized induction conditions were as follows:the concentration of IPTG,0.4 mmol·L-1;induction time,25 h;induction temperature,25?;cell concentration,OD600=0.5.?4?The whole cell transformation conditions were further optimized.The optimal transformation conditions were as follows:transformation temperature,25°C;The PH of the conversion system,pH=6.5;dry weight of the cells,20 g·L-1;concentration of3,4-dihydroxyphenylpyruvate,70 mmol·L-1.The final yield of L-Danshensu reached 15.91 g·L-1.
Keywords/Search Tags:L-Danshensu, L-lactate dehydrogenase, Enzymatic properties, Glucose dehydrogenase, Whole cells biotransformation
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