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Additional file 1. 1. Biomass production. 2. Reduction of rac-2-phenylpropanal by isolated CtXR D51A. Time course of the reduction of 0.5 mM substrate with 240 U/mL of isolated CtXR D51A. 3. Biotransformation of rac-2-phenylpropanal. Conversions and product ee-values from the reduction of 100 mM rac-2-phenylpropanal using a lyophilized and rehydrated whole-cell catalyst. Effects of catalyst loading. Conversions and product ee-values from the reduction of 1 M rac-2-phenyl-propionaldehyde using a lyophilized and rehydrated whole-cell catalyst. Effects of catalyst loading (20 gCDW/L, 40 gCDW/L) and coenzyme concentration (NAD+ 3–14 mM). (The data are based on HPLC measurements, Table 3). 4. Reversed phase chiral HPLC. Separation of main products and by-products by HPLC. UV traces and retention times for rac-2-phenylpropanal, acetophenone, 1-phenylethanol, (R,S)-2-phenylpropanol, reaction buffer with NAD+, reaction buffer, bioreduction sample of 1 M rac-2-phenylpropanal reacted with 40 gCDW/L and 6 mM NAD+. 5. Chiral GC-FID. GC traces and retention times for main products, by-products and bioreduction replicates (N = 6) of 1 M rac-2-phenylpropanal reacted with 40 gCDW/L and 6 mM NAD+. 6. 1H-NMR. 1H-spectrum of the isolated product from a reaction with 78% analytical yield (HPLC) from 1 M rac-2-phenylpropanal reacted with 40 gCDW/L and 6 mM NAD+.

Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to “test” for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design.

D-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAO) has a long-standing history in industrial biocatalysis. The enzyme was successfully applied in the conversion of cephalosporin C to 7-aminocephalosporanic acid and has now found viable alternative uses in the preparation of chiral amino acids. TvDAO shows absolute enantioselectivity for a-amino acids in R-configuration. The specificity with respect to the structure of the amino acid side chain is however extremely relaxed, allowing a diversity of substrates to be converted. Although TvDAO is a reasonably robust catalyst, even in the presence of the oxidants present in the process (O2, H2O2), interest has been high in developing a more stable form of the enzyme that can be reused easily. Immobilization on a solid carrier was often the preferred choice. Dissociation of the cofactor FAD can limit the stability of immobilized TvDAO [1]. We have found in earlier work [1] that Cys108 of TvDAO can undergo irreversible oxidation into a stable Cys sulfinic acid. This oxidative modification leads to partial loss of enzyme activity and stability. A more rapid overall release of the cofactor FAD from oxidized compared to native enzyme is responsible for the decrease in stability [2]. Site-directed mutagenesis of Cys108 into the non-oxidized residues Ser and Asp were used in an effort to mimic the native and oxidized form of TvDAO. We present results of a characterization of C108S and C108D variants with respect to activity and stability in solution. The two mutants were immobilized on epoxy-activated Sepabeads and the stabilities of carrier-bound forms of wild-type [3] and mutated TvDAO were analyzed.