2019 ; Vol.29-3: 373~381
|Author||Amos K. Dwamena, Robert S. Phillips, Chang Sup Kim|
|Place of duty||Hanbat National University, Republic of Korea,South Dakota State University, USA|
|Title||Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase|
J. Microbiol. Biotechnol.2019 ;
|Abstract||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
M) 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 +6.8ºC and
+2.4ºC, respectively, with thermal deactivation constant (k
d) close to the wild-type enzyme.
The M151A/C295A/I86A mutant reacts optimally at 70 ºC 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.|
|Key_word||Computer-aided modeling, alcohol dehydrogenase, substrate specificity, asymmetric reduction, thermostability|
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