Comparative studies on enzyme preparations and role of cell components for (R)‐phenylacetylcarbinol production in a two‐phase biotransformation

Abstract

Whole cell pyruvate decarboxylase (PDC) from Candida utilis enhanced the enzymatic production of (R)-phenylacetylcarbinol (PAC) in an aqueous/octanol biotransformation compared to the partially purified PDC especially for a lower range of initial activities (0.3-2.5 U/mL). With an initial activity of 1.1 U/mL and at a 1:1 phase volume ratio, whole cell PDC achieved a maximum specific PAC production of 42 mg/U (2.8 g/L/h) in comparison to 13 mg/U (0.9 g/L/h) for partially purified PDC. The enhanced performance of whole cell PDC was associated with high stability towards the substrate benzaldehyde. The strong PDC inactivation by benzaldehyde was minimal even when whole cells were broken as long as cell debris was not removed from the broken cells. Biotransformations with various cellular components added to partially purified PDC revealed that membrane components especially 2 mg/mL phosphatidylcholine enhanced PAC concentrations. The role of surfactants was further confirmed from the results with synthetic surfactant sodium bis(2-ethyl-1-hexyl)sulfosuccinate (AOT). It was apparent that the membrane components in whole cells were sufficient for optimal PAC production and no further surfactant addition is required for optimal performance.

Enzymatic Dynamic Reductive Kinetic Resolution Towards 115 g/L (S)-2-Phenylpropanol

“…Use of the whole cells provided an extreme case of catalyst stabilization in the presence of 2-phenylpropanal: The isolated enzyme was deactivated by 0.5 mM aldehyde whereas the whole-cell catalyst and the supernatant thereof were able to tolerate and convert 1 M of the substrate. Stabilization of the catalytic enzyme by whole-cells and cell debris was previously reported for the synthesis of (R)phenylacetylcarbinol from benzaldehyde and pyruvate by a Candida utilis pyruvate decarboxylase (35). The stabilization was ascribed to membrane components that form a microenvironment around the enzyme and thereby decrease aldehyde transfer to the enzyme and protect the enzyme from deactivation at the aqueous/organic interphase.…”

“…phenylacetylcarbinol from benzaldehyde and pyruvate by a Candida utilis pyruvate decarboxylase (35). The stabilization was ascribed to membrane components that form a microenvironment around the enzyme and thereby decrease aldehyde transfer to the enzyme and protect the enzyme from deactivation at the aqueous/organic interphase.…”

Modeling of biocatalytic reactions: A workflow for model calibration, selection, and validation using Bayesian statistics

“…The theory that the enzyme BAL is inactivated by the substrate 3,5-DMBA is in accordance with previous studies, in which an inactivation of a decarboxylase by benzaldehydes has also been reported. [16][17][18]…”

Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation

“…From a process perspective, the use of intact whole microbial cells that over-express specific recombinant ω -TAs may be advantageous, since it offers a more simple overall process configuration with a reduced number of upstream unit operations and less generation of waste material [9, 10]. Additionally, cell metabolism can be exploited for the (re)-generation of co-factors and co- substrates provided that there is an attendant assimilation of a carbon and energy source during the reaction [11].…”