Enzyme identification and development of a whole‐cell biotransformation for asymmetric reduction ofo‐chloroacetophenone

Abstract

Chiral 1-(o-chlorophenyl)-ethanols are key intermediates in the synthesis of chemotherapeutic substances. Enantioselective reduction of o-chloroacetophenone is a preferred method of production but well investigated chemo- and biocatalysts for this transformation are currently lacking. Based on the discovery that Candida tenuis xylose reductase converts o-chloroacetophenone with useful specificity (kcat/Km=340 M(-1) s(-1)) and perfect S-stereoselectivity, we developed whole-cell catalysts from Escherichia coli and Saccharomyces cerevisiae co-expressing recombinant reductase and a suitable system for recycling of NADH. E. coli surpassed S. cerevisiae sixfold concerning catalytic productivity (3 mmol/g dry cells/h) and total turnover number (1.5 mmol substrate/g dry cells). o-Chloroacetophenone was unexpectedly "toxic," and catalyst half-life times of only 20 min (E. coli) and 30 min (S. cerevisiae) in the presence of 100 mM substrate restricted the time of batch processing to maximally ∼5 h. Systematic reaction optimization was used to enhance the product yield (≤60%) of E. coli catalyzed conversion of 100 mM o-chloroacetophenone which was clearly limited by catalyst instability. Supplementation of external NAD+ (0.5 mM) to cells permeabilized with polymyxin B sulfate (0.14 mM) resulted in complete conversion providing 98 mM S-1-(o-chlorophenyl)-ethanol. The strategies considered for optimization of reduction rate should be generally useful, however, especially under process conditions that promote fast loss of catalyst activity.

Granzyme B Cleaves Decorin, Biglycan and Soluble Betaglycan, Releasing Active Transforming Growth Factor-β1

“…In the context of chronic diseases such as aneurysms and skin aging, this rate is likely significant as GrB has a high affinity for PGs leading to its increased accumulation and cleavage would occur over a prolonged period of time in areas of extracellular GrB accumulation, as suggested in previous publications [9], [10]. In addition, several other proteases have been characterized to cleave substrates as similar rates, and have been determined to be catalytically efficient [38], [39], [40], [41], [42]. Nonetheless, future studies are necessary to examine GrB-mediated proteoglycan cleavage in vivo and to identify GrB-derived cleavage fragments in chronic human disease.…”

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant

“…It has been demonstrated that about 20% total cellular NAD(H) in wild-type cells is freely accessible and the (4). In several studies, the cellular cofactor concentration has been increased through manipulating the NAD ϩ biosynthetic pathway (12,16) or feeding permeabilized cells (18,36,37). The NAD(H) level was increased up to 6-fold previously (12).…”

Synthesis of a chiral alcohol using a rationally designed Saccharomyces cerevisiae reductase and a NADH cofactor regeneration system

“…Although many reductases efficiently mediate a variety of enantioselective bioconversion, the cofactor is too expensive for the enzymatic bioconversion to be performed economically [1,3]. To alleviate this cost factor, a cofactor recycling system is usually introduced to the reductase bioconversion: namely, the enzyme coupling reaction of glucose dehydrogenase (GDH) [4][5][6] or formate dehydrogenase (FDH) [7][8][9].…”