| Alcohol dehydrogenases(ADH),which is a member of the oxidation-reduction enzyme family,can reversibly catalyze the oxidation of alcohols and the reduction of aldehydes by using cofactors.ADH also has a wide range of substrate specificity and high enantioselectivity and its catalytic substrates include normal and branched fatty alcohols,aromatic alcohols,primary alcohols,secondary alcohols,and corresponding aldehydes and ketones,etc.Therefore,it has great research value in the study of metabolic interaction in the cells,disease diagnosis,medical analysis,organic synthesis and biopharmaceutical and other fields.However,room temperature ADH is often limited by the high temperature requirements of the production process,resulting in its inability to play a role in large-scale organic synthesis.The ADH encoded by hyperthermophilic microorganisms possesses thermophilicity and thermostablilty,which can precisely meet the requirements of high-temperature production processesin the large-scale application of ADH in organic synthesis.The hyperthermophilic and barophilic archaeon Thermococcus barophilus Ch5 was isolated from a hydrothermal vent in the mid-Atlantic ridge and could withstand 95℃ and 40 MPa.At present,the genome of T.barophilus Ch5 was sequenced,encoding two NAD+-dependent Ⅲ type alcohol dehydrogenases:Tba-ADH641 and TbaADH547.In this thesis,we characterized the biochemical characteristics of ethanol oxidation and acetaldehyde reduction catalyzed by Tba-ADH641,inculding optimal reaction temperature and pH value,metal ion preference,substrate specificity and thermostability,and the effect of amino acid residues in its conserved domains on the enzyme activity by studying its mutants.The first part of this thesis is about the gene cloning,protein expression and purification of Tba-ADH641.We first amplified the Tba-ADH641 gene fragment by PCR and then constructed the expression vector Tba-ADH641-pET30a(+)by molecular cloning methods such as enzyme digestion,ligation and transformation.After confirming the positive clone with the target gene through phenol chloroform extraction method and sequencing,the validated Tba-ADH641-pET30a(+)plasmid was transformed into the expressing cells for induction of expression.The cells collected by centrifugation were disrupted by ultrasonic,and some of the E.coli impurity proteins were removed by heat treatment.Finally,Tba-ADH641 protein(44.66 kDa)was obtained by Ni column affinity purification.Additionally,the gel-fitration experiments were conducted on the purified Tba-ADH641 protein,confirming that the enzyme is a monomer protein at pH 8.0.The second part of this thesis is about the biochemical characteristics of ethanol oxidation and acetaldehyde reduction catalyzed by Tba-ADH641.The biochemical data showed that the optimal temperature of Tba-ADH641 for ethanol oxidation and acetaldehyde reduction was 65℃ and 80℃,respectively.However,the oxidation and reduction activities of Tba-ADH641 had the same optimal pH value of 8.0.The thermostability datat showed that the activity of Tba-ADH641 abolished about 50%after heating at 70℃for 20 min and 94%oxidation activity of the enzyme was lost after heat treatment at 90℃ for 20 min.However,Tba-ADH641 maintained 21%reduction activity after heating at 100℃ for 20 min,thus suggesting that the thermostability of oxidizing activity of the enzyme is lower than that of its reducing activity.Additionally,the oxidation activity of Tba-ADH641 was independent of metal ions.While Cu2+,Mn2+ and Mg2+inhibited the oxidation activity of Tba-ADH641,Zn2+ and Fe2+ stimulated the oxidation activity of Tba-ADH641,among which Fe2+ displayed the best effect.By contrast,the reducing activity of Tba-ADH641 depends on divalent metal ions,showing that Ca2+,Fe2+ and Mg2+ can promote the reducing activity of Tba-ADH641,and Mg2+ is the best metal ion.However,Cu2+,Ni2+,Zn2+ and Mn2+ can inhibit the reducing activity of TbaADH641.Furthermore,the substrate specificity experiment showed that the catalytic oxidation efficiency of Tba-ADH641 in the following order from high to low was:ethanol>n-butanol>1-hexanol.Tba-ADH641 catalyzed aldehyde reduction efficiency in the following order from high to low:acetaldehyde>acetone≈ 1-butanal>hexal.The kinetic experiment results showed that the catalytic efficiency of Tba-ADH641 for acetaldehyde reduction is higher than that for ethanol oxidation.The third part of this thesis is about the catalytic mechanism of Tba-ADH641 and the influence of cofactor types on its catalytic efficiency.The experimental data showed that Tba-ADH641 preferred NADP(H)to NAD(H)as the cofactor of the catalytic reaction.Mutation data showed that the Tba-ADH641 D115A mutant completely abolished its oxidation and reduction activities,suggesting that residue D115 is necessary for TbaADH641 to catalyze ethanol oxidation and acetaldehyde reduction.Furthermore,the K118A,EI59A,D190A and E215A mutants only retained part of their oxidation and reduction activities,suggesting that resdiues K118,E159,D190 and E215 are involved in ethanol oxidation and acetaldehyde reduction catalyzed by Tba-ADH641.Interestingly,the relative catalytic activities of E159 A mutant for ethanol oxidation and acetaldehyde reduction were increased by 43.5-fold and 15.2-fold with NADP(H)compared to the reactions with NAD(H),suggesting that residue E159 might be related to cofactor binding and participate in partial catalysis.In conclusion,this thesis revealed the biochemical characteristics and catalytic mechanism of Tba-ADH641 by using molecular biology and biochemical techniques,which has provided a theoretical basis for revealing the mechanism of alcohol oxidation and aldehyde reduction catalyzed by hyperthermophilic archaea.In addition,TbaADH641 can catalyze the oxidation of ethanol and the reduction of acetaldehyde under high temperature conditions,and possesses the characteristics of high temperature resistance,which can provide thermostable ethanol dehydrogenase for biotransformation.Therefore,the research results in this thesis have important application value. |
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