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 which 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 (kcat/KM) of the 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 V115A/C295A/I86A mutant in asymmetric reduction. The 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 M151A/C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8°C and +2.4°C, respectively, with thermal deactivation constant (kd) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 °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 V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.
ID: ARyouSQr
Submitter: Shu-Ching Ou
Submission Date: March 25, 2019, 1:21 p.m.
Version: 1
For V115A+C295A+I86A, A85G+C295A+I86A variants, protein concentration in the kinetic experiments is halved.
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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Percent Identity | Matching Chains | Protein | Accession | Entry Name |
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400.0 | A,B,C,D | NADP-dependent isopropanol dehydrogenase | P14941 | ADH_THEBR |