Characterization And Engineering Of Thermstable Alcohol Dehydrogenases For Improving Oxygen-tolerance And Altering Coenzyme Specificity

Secondary alcohol dehydrogenases catalyze stereoselective reduction of ketones to chiral alcohols and are widely used in the manufacture of pharmaceuticals,chemicals,food and pesticides.Among of them,thermostable alcohol dehydrogenases have attracted great attention due to their excellent stability against heat and solvents.However,most of investigated enzymes are not robust enough to meet the industry requirements.Here,the study aims to discover more-robust alcohol dehydrogenases,and improve their catalytic capacities towards oxygen tolerance and coenzyme specificity through protein engineering.1.The allylic alcohol dehydrogenase(YsADH)from Yokenella sp.WZY002 could serve as a robust biocatalyst.The enzyme was active within a broad temperature range from 25 to 75 ?.The thermostability of YsADH was excellent and retained 54.5%of the initial activity in 65 ? after 6 h incubation.It was robust in the presence of organic solvents and retained 87.5%of the initial activity after 24 h of incubation with 20%(vol/vol)dimethyl sulfoxide.Overexpressed YsADH was prone to aggregate,and the formation of inclusion bodies could be partly avoided by lowering the induction temperature and IPTG concentration.Under the optimized over-expression conditions,the amount of YsADH in the soluble form constituted of around 5%of total heterogeneously expressed proteins.2.The alcohol dehydrogenase from hyperthermophilic archaeon Thermococcus guaymasensis(TgADH)is oxygen-sensitive,which feature constrains its practical applications.To improve the oxygen tolerance of TgADH,a high-throughput method for screening positive mutants was estabished.The results of site-directed mutagenesis indicated that the amino acid residue Cys213 was critical for the activity loss in the presence of oxygen.The alteration of Cys213 to Gly,Ala or Leu has a beneficial effect on oxygen tolerance.Particularly,the activity of the mutant Cys213Gly was 1.5 times higher than its native enzyme.On the other hand,the alterations of other amino acids such as aromatic or alkaline amino acids had no improvement of oxygen tolerance.3.The enzyme TgADH is NADP(H)-dependent,whereas NADP(H)as coenzyme is more expensive and less stable than NAD(H).To redesign the coenzyme specifity of TgADH,and the docking between TgADH and its coenzyme NADP+ was carried out,indicating that the amino acid residues Gly208,Ser209,Arg210 and Arg228 could play important roles in coenzyme binding.In the native TgADH,it was speculated that the hydroxyl group of Ser209 could form the hydrogen bonds with the oxygen atoms in 2'-phosphate group of NADP+ but not NAD+.Furthermore,the carboxyl group of the substituted Asp209 led to form two new hydrogen bonds with 2' and 3' hydroxyl groups of ribose.In addition,the alteration of Arg228Phe resulted in an pi-pi interaction in favor of coenzyme specificity towards NAD(H).The double mutant Ser209Asp/Arg228Phe was constructed,which showed good activity using both on NAD+ and NADP+ as coenzyme and exhibited the optimum temperature of 80 ?.When NADP+ was used as coenzyme,the double mutant Ser209Asp/Arg228Phe had its optimum pH of 10.5 and its Vmax/Km values were 4 times higher than those of the native TgADH.When NADP+ was used as coenzyme,the double mutant Ser209Asp/Arg228Phe had its optimum pH of 10.5 and its Vmax/Km value was 4 times higher than that of the native TgADH.However,the optimum pH using NAD+ as coenzyme was found to be pH 8.5,although its Vmax/Km values was also greater than the wild type.