Challenges of steroid biotransformation with human Cytochrome P450 monooxygenase CYP21 using recombinant Schizosaccharomyces pombe

Since cytochrome P450 monooxygenases enable the regio- and stereo-selective hydroxylation of C-H bonds, they are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals. Nevertheless, for industrial applications of such enzymes, e.g., steroid hydroxylation, several challenges like cofactor and oxygen supply, limited stability and activity, or low substrate solubility have to be overcome. To identify the limiting factors in a P450 catalyzed whole cell biotransformation, 21-hydroxylation of 17-alpha-hydroxyprogesterone in Schizosaccharomyces pombe expressing human CYP21 was chosen as model reaction. We report here that resting cells of this recombinant yeast strain can be used for efficient biotransformation. In the present study, we analyzed the intracellular redox cofactor pool of S. pombe by LC-MS/MS measurements and report the first quantification of the intracellular cofactor pool during P450 hydroxylation. Thereby a limitation caused by the redox cofactor could be excluded for resting cells. In contrary, low substrate solubility and its transport into the cell affect activity. Screening for an appropriate cosolvent identified methanol as the most promising candidate, since it showed the lowest inactivation effect on the biocatalyst. Through permeabilization of the membrane with the detergent tween 80 steroid hydroxylation activity increases, leading to a productivity of 540 microM d(-1) in a final batch experiment under optimized reaction conditions

Process development for enzyme catalysed asymmetric C-C-bond formation

Benzaldehyde lyase (BAL, EC: 4.1.2.38) is a highly enantioselective enzyme, which catalyses the formation and cleavage of (R)-benzoin derivatives. The asymmetric synthesis of (R)-3, 3'-dimethoxybenzoin (2a) and (R)-3-methoxy-2'-chlorobenzoin (2b) was investigated by means of reaction engineering and a reactor concept for the preparative synthesis was developed. For the synthesis of (2b), the optimal ratio of 3-methoxybenzaldehyde (1a) and 2-chlorobenzaldehyde (1b) was found to be equimolar. An aqueous-organic two-phase system was found to be the best reaction system. The aqueous phase consists of phosphate buffer; the organic phase is formed by the substrate. As the catalyst is hydrophilic, the reaction takes place in the aqueous phase. As a result of their low solubility in the aqueous media the formed benzoins precipitate. Centrifugation and filtration were used to separate the product from the reaction media. In order to eliminate the by-products (2b) was recrystallised. The synthesis of (2a) was carried out with a high volumetric productivity of 2304gL(-1) d(-1). In a further experiment the enzyme consumption could be minimised (ttn =250,100). Twelve gram of (2a) (yield 82%, purity 97%, ee 99%) and 19.5 g of (2b) (yield 86%, purity 97%, ee 99%) were isolated. (c) 2007 Elsevier Ltd. All rights reserved

Hydrophobic Complexation Promotes Enzymatic Surfactant Synthesis from Alkyl Glucoside/Cyclodextrin Mixtures

The unique ability of cyclodextrin glycosyltransferase to form and utilize the cyclic maltooligosaccharide cyclodextrin (CD) makes this enzyme an attractive catalyst for the synthesis of alkyl glycosides. Here, we characterize the sugar headgroup elongation of alkyl glucosides (acceptor) via two transglycosylation reactions from either a linear (maltohexose) or a cyclic (CD) glycosyl donor. Inclusion complex formation overcomes both poor substrate solubility and aggregation. We have used pure alkyl glucosides and alpha CD as model compounds. The complex between CD and alkyl glucoside was efficiently used as a substrate. Kinetic and thermodynamic measurements allow the prediction of the optimal synthesis conditions. This optimum corresponds to the transition between a donor-limiting and an acceptor-limiting regime. The resulting rational design should lead to the practical development of a cost-efficient industrial synthesis. Our findings with respect to the importance of complexation should also readily apply to other enzymatic systems