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.
Submitter: Shu-Ching Ou
Submission Date: March 25, 2019, 1:21 p.m.
For V115A+C295A+I86A, A85G+C295A+I86A variants, protein concentration in the kinetic experiments is halved.
|Number of data points||189|
|Proteins||NADP-dependent isopropanol dehydrogenase|
|Assays/Quantities/Protocols||Experimental Assay: T1/2;T50 ; Experimental Assay: Relative Activity ; Experimental Assay: Km ; Experimental Assay: kcat ; Experimental Assay: kcat/Km ; Experimental Assay: kd at 60 °C: Thermo-deactivation Constant ; Experimental Assay: kd at 70 °C: Thermo-deactivation Constant ; Experimental Assay: kd at 80 °C: Thermo-deactivation Constant ; Experimental Assay: kd at 90 °C: Thermo-deactivation Constant ; Derived Quantity: SD of Relative Activity ; Derived Quantity: SD of Km ; Derived Quantity: SD of kcat ; Derived Quantity: SD of kcat/Km|
|Libraries||Variants for TeSADH_Substrate|