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Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase

  • Dwamena, Amos K. (Department of Chemical & Biological Engineering, Hanbat National University) ;
  • Phillips, Robert S. (Department of Chemistry, University of Georgia) ;
  • Kim, Chang Sup (Department of Chemical & Biological Engineering, Hanbat National University)
  • 투고 : 2018.12.11
  • 심사 : 2019.01.01
  • 발행 : 2019.03.28

초록

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency ($k_{cat}/K_M$) of the ${\underline{M151A}}$/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using ${\underline{V115A}}$/C295A/I86A mutant in asymmetric reduction. The ${\underline{A85G}}$/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the ${\underline{M151A}}$/C295A/I86A and ${\underline{V115A}}$/C295A/I86A mutants significantly increased ${\Delta}T_{1/2}$ by $+6.8^{\circ}C$ and $+2.4^{\circ}C$, respectively, with thermal deactivation constant ($k_d$) close to the wild-type enzyme. The ${\underline{M151A}}$/C295A/I86A mutant reacts optimally at $70^{\circ}C$ with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by ${\underline{V115A}}$/C295A/I86A, and (R)-1-phenylethanol from acetophenone by ${\underline{M151A}}$/C295A/I86A mutant, in large-scale bioreduction processes.

키워드

참고문헌

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