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Gene Cloning,Expression And Characteristics Of Alcohol Dehydrogenase From Lactobacillus Reuteri

Posted on:2021-05-19Degree:MasterType:Thesis
Country:ChinaCandidate:Z H HuFull Text:PDF
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Alcohol dehydrogenase(ADHs,E.C. to oxidoreductase,which can use NAD(P)H as a coenzyme to catalyze the conversion of alcohols to the corresponding aldehydes or ketones.The enzyme plays an important role in the pathway of alcohol metabolism,often has high stereoselectivity and broad substrate spectrum,and is therefore widely used in pharmaceutical research,chemical production and food fields.In this paper,five ADHs from Lactobacillus reuteri DSM20016 were cloned and expressed,the induction conditions were optimized,and the enzyme properties were studied,which laid a molecular biological foundation for the in vivo verification of its effect on the aroma of cheese.The main research results were showed as follows:(1)Five genes encoding ADHs in L.reuteri DSM20016(adh-1?adh-2?adh-3?adh-4 and adh-5)were successfully cloned into plasmid pRSFDuet-1 and expressed in Escherichia coli BL21(DE3).The molecular weights calculated were 37.6kDa?37.8 kDa?35.8 kDa?36.0 kDa and 39.0 kDa,respectively.The conserved domains show that these five ADHs belong to the medium chain dehydrogenase family.Enzyme activity verification showed that five ADHs had certain enzyme activities on heptaldehyde which affected the flavor of cheese.(2)In this paper,the induction conditions of ADHs were optimized,and the activity of ADHs was improved to some extent.The optimized conditions:the initial cell concentration OD600 was 0.6,the induction temperature was 20?,the IPTG concentration was 0.4 mmol·L-1,and the culture time after induction was 16 h,shaker speed was 200 r·min-1.Under these conditions,compared with the pre-optimized E.coli BL21(DE3)/pRSFDuet-1-ADH-1 strain,the enzyme activity increased by 72%.(3)Five ADHs were purified by Ni+aff-inity,and their enzymatic properties were studied.These results were obtained:the optimum reaction pH of ADH-1 and ADH-3 is 5.0,of ADH-2 is 6.0,of ADH-5 is 7.0;the optimum reaction temperature of ADH-1 is 40?,of ADH-2 and ADH-3 are 35? and 55? respectively,ADH-4 and ADH-5 are 50?.During cheese fermentation,the core temperature can reach 40??50?,and the pH is low.In summary,the results declared that some alcohol dehydrogenases could easily adapt to the changes of environmental conditions during fermentation.(4)Substrate specificity experiments demonstrated that these five ADHs owned a broad substrate spectrum and could catalyze 11 aldehydes and alcohols related to cheese flavor.In metal ion and EDTA experiments,1 mM Zn2+promoted enzyme activity,while EDTA,Ca2+,Fe2+,and Cu2+inhibitted enzyme activity,indicating that the five ADHs were metal-dependent.The kinetic experiment results are as follows:the substrate with the highest catalytic efficiency of ADH-1 is heptaldehyde(Km=4.59×102 ?mol·L-1,Kcat/Km=039×102 s-1·mM-1),of ADH-2 is butyraldehyde(Km=0.13×102 ?mol·L-1,Kcat/Km=0.12×102s-1·mM"1),of ADH-3 and ADH-5 is 3-methylbutyraldehyde(Km=0.30×102 mol·L-1,0.14×102 mol·L-1,;Kcat/Km=4.64s-1·mM-1,0.24×102 s-1·mM-1),ofADH-4 is hexanal(Km=0.13×102 ?mol·L-1,Kcat/Km=0.18×102 s-1·mM-1).
Keywords/Search Tags:Lactobacillus reuteri, Aromatic compounds, Alcohol dehydrogenase, Enzymatic properties
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