Effect of growth conditions on the activity of Michael hydratase from Rhodococcus rhodochrous ATCC 17895

Abstract

The stereoselective water addition to double bonds is chemically hard to accomplish. However, the use of a novel Michael hydratase from Rhodococcus rhodochrous ATCC 17895 gives a promising alternative route. The aim of this study is to reveal the dependency of the enzymatic activity on the culture medium and a subsequent optimisation. Focus was given to the composition of the microorganism’s culture medium due to the fact that the literature protocol from 1998 was never changed in this regard. Moreover, the use of iron sulphate and magnesium sulphate in unusually high amounts has drawn the attention to an investigation. By using a “one-variable-at-a-time” (OVAT) strategy, it was proven that the addition of iron sulphate is redundant which enhances the practical treatment of the whole cells. Via a Design of Experiments (DoE) with the support of the software Design Expert v 7.7.0 (Stat Ease, Minneapolis, MN), optimisation of the culture medium parameters’ glucose, yeast extract, peptone and magnesium sulphate was achieved. It was concluded that the addition of magnesium sulphate to the culture medium has no added value. The specific amounts of 6.59 g/L for glucose, 1.84 g/L for yeast extract and 9.20 g/L for peptone provided the best result in a restricted interval of study. Due to this culture medium optimisation, the enzymatic activity was improved by three times compared to the activity provided by the standard culture medium reported in literature. Transferring the shake-flask optimised medium compositions to a fermentor led to large amount of biomass with high Michael hydratase activity thereby saving a substantial amount of growth time. The results of this study provided a significant increase in specific Michael hydratase activity and thereby securing a sufficient amount of active whole-cells needed in the desired Mhy (Michael hydratase) isolation process.