Impact of Expanded Small Alkyl-Binding Pocket by Triple Point Mutations on Substrate Specificity of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase
| Autor: | Robert S. Phillips, Amos K. Dwamena, Chang Sup Kim |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Models
Molecular 0106 biological sciences Stereochemistry Mutant Molecular Conformation Thermoanaerobacter 01 natural sciences Applied Microbiology and Biotechnology Substrate Specificity Thermoanaerobacter ethanolicus Bacterial Proteins Catalytic Domain 010608 biotechnology Enzyme Stability Point Mutation Enzyme kinetics Enzyme Assays Alcohol dehydrogenase chemistry.chemical_classification biology Chemistry Mutagenesis Alcohol Dehydrogenase Wild type Acetophenones Substrate (chemistry) Sequence Analysis DNA General Medicine Ketones biology.organism_classification Alcohol Oxidoreductases Kinetics Enzyme Alcohols Mutagenesis Site-Directed biology.protein Genetic Engineering Biotechnology |
| Zdroj: | Journal of Microbiology and Biotechnology. 29:373-381 |
| ISSN: | 1738-8872 1017-7825 |
| DOI: | 10.4014/jmb.1812.12016 |
| Popis: | 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 (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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|