Gene Cloning,Expression And Biochemical Characteristics Of Alcohol Dehydrogenase From Proteus Mirabilis JN458

Alcohol dehydrogenases?ADHs,EC 1.1.1.1?,are the first subclass of oxidoreductases,which can typically convert aldehydes or ketones to the corresponding alcohols using NAD?P?H as a coenzyme.Due to unique physiological and biochemical properties and enzymatic properties of alcohol dehydrogenase,they are widely used in various fields,such as medical research,chemical production,food research and bioengineering research.In this paper,five genes ecoding alcohol dehydrogenase from Proteus mirabilis JN458 were successfully expressed in Escherichia coli,the expression plasmids and induction conditions were optimized,and their enzymatic properties of five dehydrogenases were characterized,and the article laid the foundation for molecular biology in the study of the aromatic odor of cheese..The main research results were showed as follows:?1?Five genes?adh1,adh2,adh3,adh4,adh5?encoding alcohol dehydrigenase derived from P.mirabilis JN458 were successfully cloned,and were ligated with pCold?to construct a recombinant plasmid which was transformed into Escherichia coli BL21?DE3?to express.The molecular weights of ADH1-5 are predicted to be 40.1 kDa,41.3 kDa,42.4 kDa,39.3 kDa and 37.3 kDa,respectively.Enzyme activity verification showed that five alcohol dehydrogenases had certain enzyme activities on phenylacetaldehyde.?2?Since the protein expression and enzyme activity of ADHs were not high,the expression plasmid and the induction conditions of the recombinant strains were optimized.The expression plasmids were optimized by pET28a,pETDuet-1,pRSFDuet-1 and pColADuet-1,andtherecombinantplasmidspET28a-adh1,pETDuet-1-adh1,pRSFDuet-1-adh1 and pColADuet-1-adh1 were constructed respectively,pETDuet-1 was finally selected as an expression plasmid.The induction conditions were optimized to obtain the optimal induction conditions,the optimized conditions were as follows:the initial pH of the medium was 6.5,the initial concentration of cell's OD₆₀₀ was 0.4,the concentration of the inducer IPTG was 0.4 mmol·L-1,the induction temperature was 15?,and the incubation time was 18 h after induction,shaker speed was 220 r·min^-1,under these conditions,the enzyme activity was increased by 77%compared to before optimization.?3?Protein purification of ADHs was carried out using Ni⁺affinity and desalting chromatograph,and the enzymatic properties of five alcohol dehydrogenases were investigated.The optimum pH of ADH-1,ADH-2 and ADH-3 is 7.0,the optimum pH of ADH-4 and ADH-5 is 8.0,and the optimum temperature of ADH-1,ADH-3 and ADH-5 is40?,the optimum temperature of ADH-2 and ADH-4 is 45?.The five alcohol dehydrogenases have broad substrate range.The kinetic parameters of the five enzymes for phenylacetaldehyde,2-methylbutyraldehyde and valeraldehyde are as follows:for ADH-1,K_m is 34.55,6.44 and 4.23mmol·L^-1,respectively;for ADH-2,K_m is 16.90,3.13 and 4.93mmol·L^-1,respectively;for ADH-3,K_m was 10.01,4.42 and 5.04mmol·L^-1,respectively;for ADH-4,K_m were 4.66,14.81 and 5.22mmol·L^-1,respectively;for ADH-5,K_m was 19.64,24.62 and 1.33mmol·L^-1,respectively.