“…Nie and coworkers reported the stereoinversion process to product (S)-PED catalyzed by Candida parapsilosis [13,14]. It was found that it got high optical purity of 99% and yield of 83% at substrate concentration of 15 g/l and low optical purity of 35.5% and yield of 67.7% at high substrate concentration of 30 g/l [15]. In further researches, two key enzymes, NAD + -linked (R)-specific alcohol dehydrogenase (CPADH) and NADPH-linked (S)-specific carbonyl reductase (CPCR) were purified and characterized [16,17].…”
“…Nie and coworkers reported the stereoinversion process to product (S)-PED catalyzed by Candida parapsilosis [13,14]. It was found that it got high optical purity of 99% and yield of 83% at substrate concentration of 15 g/l and low optical purity of 35.5% and yield of 67.7% at high substrate concentration of 30 g/l [15]. In further researches, two key enzymes, NAD + -linked (R)-specific alcohol dehydrogenase (CPADH) and NADPH-linked (S)-specific carbonyl reductase (CPCR) were purified and characterized [16,17].…”
“…It was reported that wild-type (WT) C . parapsilosis and recombinant Escherichia coli containing RCR and SCR biotransform ( R )-PED to the corresponding ( S )-isomer with low yields, most likely because of the unbalanced efficiency of turnover of the enzymes [ 8 , 9 ]. Fig.…”
BackgroundCandida parapsilosis (R)-carbonyl reductase (RCR) and (S)-carbonyl reductase (SCR) are involved in the stereoconversion of racemic (R,S)-1-phenyl-1,2-ethanediol (PED) to its (S)-isomer. RCR catalyzes (R)-PED to 2-hydroxyacetophenone (2-HAP), and SCR catalyzes 2-HAP to (S)-PED. However, the stereoconversion efficiency of racemic mixture to (S)-PED is not high because of an activity imbalance between RCR and SCR, with RCR performing at a lower rate than SCR. To realize the efficient preparation of racemic mixture to (S)-PED, an in situ expression of RCR and a two-stage control strategy were introduced to rebalance the RCR- and SCR-mediated pathways.ResultsAn in situ expression plasmid pCP was designed and RCR was successfully expressed in C. parapsilosis. With respect to wild-type, recombinant C. parapsilosis/pCP-RCR exhibited over four-fold higher activity for catalyzing racemic (R,S)-PED to 2-HAP, while maintained the activity for catalyzing 2-HAP to (S)-PED. The ratio of kcat/KM for SCR catalyzing (R)-PED and RCR catalyzing 2-HAP was about 1.0, showing the good balance between the functions of SCR and RCR. Based on pH and temperature preferences of RCR and SCR, a two-stage control strategy was devised, where pH and temperature were initially set at 5.0 and 30 °C for RCR rapidly catalyzing racemic PED to 2-HAP, and then adjusted to 4.5 and 35 °C for SCR transforming 2-HAP to (S)-PED. Using these strategies, the recombinant C. parapsilosis/pCP-RCR catalyzed racemic PED to its (S)-isomer with an optical purity of 98.8 % and a yield of 48.4 %. Most notably, the biotransformation duration was reduced from 48 to 13 h.ConclusionsWe established an in situ expression system for RCR in C. parapsilosis to rebalance the functions between RCR and SCR. Then we designed a two-stage control strategy based on pH and temperature preferences of RCR and SCR, better rebalancing RCR and SCR-mediated chiral biosynthesis pathways. This work demonstrates a method to improve chiral biosyntheses via in situ expression of rate-limiting enzyme and a multi-stage control strategy to rebalance asymmetric pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0539-y) contains supplementary material, which is available to authorized users.
“…To date, there are no reports available for biocatalytic transformation of Felodipine into its active S(−)-Felodipine. The microorganisms selected for the biotransformation study include Bacillus subtilis [9], Escherichia coli [10], Pseudomonas putida [11], Sphingomonas paucimobilis [12], Rhodococcus erythropolis [13], Streptomyces halstedii [14], Aspergillus niger [15], Geotricum candidum [16], Rhizopus oryzae [17], Candida parapsilosis [18], Cunninghamella elegans [19], Cunninghamella blakesleeana [20] and enzyme triacylglycerol lipase from Aspergillus niger (Lipase AP6) [21].…”
The present study involves the enantioselective resolution of racemic Felodipine by using free and immobilized forms of microbial cultures as well as an enzyme (Lipase AP6). Among the microbial cultures employed in the present study, Aspergillus niger, Sphingomonas paucimobilis, Cunninghamella elegans, Escherichia coli, Pseudomonas putida and Cunninghamella blakesleeana were found to possess capability of enantioselective resolution of racemic Felodipine. The enantiomeric excess (ee%) of Felodipine after reaction catalyzed by whole-cell A. niger and S. paucimobilis was found as 81.59 and 71.67%, respectively. Immobilization enhanced the enantioselectivity (enantiomeric ratio (E)) of the biocatalysts and hence this led to enhanced enantiomeric purity of the drug. The ee% values were found to be enhanced in reactions catalyzed by A. niger and S. paucimobilis cultures after immobilization as 98.27 and 93.56%, respectively. Enantiomeric ratio (E) of the reactions catalyzed by all the biocatalysts has been improved after immobilization. E value of the reaction catalyzed by immobilized A. niger was found to be excellent (E > 100) and hence the drug showed high enantiomeric purity. In lipase AP6 catalyzed study, the enantioselectivity was enhanced after immobilization with excellent E value, which led to enhanced enantiomeric purity of the drug (99.21% ee%).
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