Use of hydrophobic bacterium Rhodococcus rhodochrous NBRC15564 expressed thermophilic alcohol dehydrogenases as whole-cell catalyst in solvent-free organic media

Abstract

The hydrophobic bacterium Rhodococcus rhodochrous NBRC15564 was employed as a whole-cell biocatalyst to examine its potential for bioconversion in solvent-free organic media. The genes encoding two different thermostable alcohol dehydrogenases (ADHTt1 and ADHTt2) of Thermus thermophilus HB27 were expressed in R. rhodochrous cells. To inactivate indigenous mesophilic enzymes in R. rhodochrous, transformant cells were heated at 70°C for 10min. Heat-treated hydrophobic wet cells were used for the bioconversion of 2,2,2-trifluoroacetophenone (TFAP) to α-(trifluoromethyl) benzyl alcohol (TFMBA) as a model reaction with ADHTt1. NADH, which was supplied in aqueous solution, was regenerated by converting cyclohexanol to cyclohexanone by ADHTt2. All reactions were performed by suspending heat-treated cells in solvent-free organic media consisting of 3.7M TFAP and 4.8M cyclohexanol (1:1, v/v ratio) at 60°C. When 800mg heat-treated R. rhodochrous cells were dispersed in 2mL of solvent-free organic media (400mgcells/mL), the product concentration reached about 3.6M TFMBA by 48h with a total NADH turnover number of approximately 900. The overall productivity was 190molTFMBA/kgcells/h.