Whole cell application of Lactobacillus paracasei BD101 to produce enantiomerically pure (S)‐cyclohexyl(phenyl)methanol

In this study, a total of 10 bacterial strains were screened for their ability to reduce cyclohexyl(phenyl)methanone 1 to its corresponding alcohol. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful biocatalyst to reduce the ketones to the corresponding alcohols. The reaction conditions were systematically optimized for the reducing agent L paracasei BD101, which showed high enantioselectivity and conversion for the bioreduction. The preparative scale asymmetric reduction of cyclohexyl(phenyl)methanone ( 1 ) by L paracasei BD101 gave (S)‐cyclohexyl(phenyl)methanol ( 2 ) with 92% yield and >99% enantiomeric excess. The preparative scale study was carried out, and a total of 5.602 g of (S)‐cyclohexyl(phenyl)methanol in high enantiomerically pure form (>99% enantiomeric excess) was produced. L paracasei BD101 has been shown to be an important biocatalyst in asymmetric reduction of bulky substrates. This study demonstrates the first example of the effective synthesis of (S)‐cyclohexyl(phenyl)methanol by the L paracasei BD101 as a biocatalyst in preparative scale.     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.     

Production of (R)‐1‐(1,3‐benzodioxol‐5‐yl)ethanol in high enantiomeric purity by Lactobacillus paracasei BD101

Piperonyl ring is found in a number of naturally occurring compounds and possesses enormous biological activities. There are many studies in the literature with compounds containing a piperonyl ring, but there are very few studies on the synthesis of chiral piperonyl carbinol. The objective of this study was to determine the microbial reduction ability of bacterial strains and to reveal the effects of different physicochemical parameters on this reduction ability. A total of 15 bacterial isolates were screened for their ability to reduce 1‐(benzo[d][1,3]dioxol‐5‐yl) ethanone 1 to its corresponding alcohol. Among these isolates Lactobacillus paracasei BD101 was found to be the most successful biocatalyst to reduce the ketone containing piperonyl ring to the corresponding alcohol. The reaction conditions were systematically optimized for the reducing agent L paracasei BD101, which showed high enantioselectivity and conversion for the bioreduction. The preparative scale study was performed, and a total of 3.72 g of (R)‐1‐(1,3‐benzodioxol‐5‐yl) ethanol in high enantiomeric form (>99% enantiomeric excess) was produced in a mild, cheap, and environment‐friendly process. This study demonstrates that L paracasei BD101 can be used as a biocatalyst to obtain chiral carbinol with excellent yield and selectivity.     

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone ( 1m ) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol ( 2m ) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.     

Highly Enantioselective Production of Chiral Secondary Alcohols Using Lactobacillus paracasei BD101 as a New Whole Cell Biocatalyst and Evaluation of Their Antimicrobial Effects

Chiral secondary alcohols are valuable intermediates for many important enantiopure pharmaceuticals and biologically active molecules. In this work, we studied asymmetric reduction of aromatic ketones to produce the corresponding chiral secondary alcohols using lactic acid bacteria (LAB) as new biocatalysts. Seven LAB strains were screened for their ability to reduce acetophenones to their corresponding alcohols. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful at reducing the ketones to the corresponding alcohols. The reaction conditions were further systematically optimized for this strain and high enantioselectivity (99%) and very good yields were obtained. These secondary alcohols were further tested for their antimicrobial activities against important pathogens and significant levels of antimicrobial activities were observed although these activities were altered depending on the secondary alcohols as well as their enantiomeric properties. The current methodology demonstrates a promising and alternative green approach for the synthesis of chiral secondary alcohols of biological importance in a cheap, mild, and environmentally useful process.     

Production of enantiomerically pure (S)-phenyl(pyridin-2-yl)methanol with Lactobacillus paracasei BD101

Abstract

Asymmetric reduction studies of heteroaryl ketones, including phenyl(pyridin-2-yl)methanone in enantioselective form with biocatalysts are very few, and chiral heteroaryl alcohols have been synthesized generally in the small scale. In this study, seven bacterial strains have been used to produce the (S)-phenyl(pyridin-2-yl)methanol in high enantiomeric excess and yield. Among the tested strains, Lactobacillus paracasei BD101, was found to be the best biocatalyst for the reducing phenyl(pyridin-2-yl)methanone to the (S)-phenyl(pyridin-2-yl)methanol at gram scale. The asymmetric bioreduction conditions were systematically optimized using L. paracasei BD101, which demonstrated excellent enantioselectivity and high level of conversion for the bioreduction reaction. (S)-phenyl(pyridin-2-yl)methanol, which is an analgesic, was produced enantiomerically pure form in the first time on gram scale using a biocatalyst. In total, 5.857?g of (S)-phenyl(pyridin-2-yl)methanol in enantiomerically pure form (>99% enantiomeric excess) was obtained in 52?h with 93% yield using whole cells of L. paracasei BD101. Enantiomerically pure (S)-phenyl (pyridin-2-yl)methanol, which is an analgesic, was first produced in the gram scale using a biocatalyst with excellent ee (>99%) and yield (93%).     

Production of enantiomerically enriched chiral carbinols using Weissella paramesenteroides as a novel whole cell biocatalyst

Abstract

In this study, four bacterial strains were tested for their ability to reduce acetophenones to its corresponding alcohol. Among these strains Weissella paramesenteroides N7 was found to be the most successful biocatalyst to reduce the ketones to the corresponding alcohols. The reaction conditions were systematically optimized for W. paramesenteroides N7 that resulted in high enantioselectivity and conversion rates for the bioreduction. The scale-up asymmetric reduction of 1-(4-methoxyphenyl) propan-1-one (1r) by W. paramesenteroides N7 gave (R)-1-(4-methoxyphenyl) propan-1-ol (2r) with 94% yield and >99% enantiomeric excess. This is the first report showing the synthesis of (R)-1-(4-methoxyphenyl) propan-1-ol (2r) in enantiopure form using a biocatalyst on a gram scale. The whole cell catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that W. paramesenteroides N7 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest as a promising and alternative green approach.     

Synthesis of enantiopure (S)-6-chlorochroman-4-ol using whole-cell Lactobacillus paracasei biotransformation

Chromane, which has a fused cyclic structure, is a significant molecule that can be found in the structure of many important compounds. Lactobacillus paracasei BD101 was demonstrated as whole-cell biocatalyst for the synthesis of (S)-6-chlorochroman-4-ol with immense enantioselectivity. The conditions of asymmetric reduction were optimized one factor by one factor using L paracasei BD101 to achieve enantiomerically pure product and complete conversion. Using these obtained optimization conditions, asymmetric reduction of 6-chlorochroman-4-one was performed under environmentally friendly conditions; 6-chlorochroman-4-one, having a fused cyclic structure as previously noted to be difficult to asymmetric reduction with biocatalysts, was enantiomerically reduced to (S)-6-chlorochroman-4-ol with an enantiomeric excess >99% on a high gram scale. This study is the first example in the literature for the enantiopure synthesis of (S)-6-chlorochroman-4-ol using a biocatalyst. Also notably, the optical purity of (S)-6-chlorochroman-4-ol obtained in this study through asymmetric bioreduction using whole-cell biocatalyst is the highest value in the literature. In this study, (S)-6-chlorochroman-4-ol was produced on a gram scale by an easy, inexpensive, and environmentally friendly method, which has shown the production of valuable chiral precursors for drug synthesis and other industrial applications. This study provides a convenient method for the production of (S)-6-chlorochroman-4-ol, which can meet the industrial green production demand of this chiral secondary alcohol.     

Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of substituted acetophenones

Chiral secondary alcohols are convenient mediator for the synthesis of biologically active compounds and natural products. In this study fifteen yeast strains belonging to three food originated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to 1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to the corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high conversion rates. This is the first report on the enantioselective reduction of acetophenone by D. hansenii P1 from past?rma, a fermented Turkish meat product. The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess. Compound 2g can be used for the synthesis of (+)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced acetophenone derivatives. This study showed that this yeast can be used industrially to produce enantiomerically pure chiral secondary alcohols, which can be easily converted to different functional groups.     

First green synthesis of (R)-2-methyl-1-phenylpropan-1-ol using whole-cell Lactobacillus paracasei BD101 biotransformation

Abstract

Green chemistry includes a novel process in the production of drugs precursors and biological active molecules using biocatalysts, so reducing the threats for human sanitary and ecological pollutions. Asymmetric bioreduction of prochiral ketones by biocatalysts is one of the best prevalent used methods in synthetic organic chemistry due to the production of enantiopure chiral carbinols. This study emphasizes the application biocatalyst L paracasei BD101 for enantioselective bioreduction of 2-methyl-1-phenylpropan-1-one ketone, which contain branched alkyl chain, to (R)-2-methyl-1-phenylpropan-1-ol ((R)-2) in high yields and excellent enantiomeric excess (>99%). The scale-up production was performed, and 4.61?g of (R)-2 in enantiopure form was synthesized. L paracasei BD101 was proved to be a substantial biocatalyst in asymmetric bioreduction of a ketone which contains a branched alkyl chain. There is not any work in the literature similar to our study. Hence, it is important to work on filling this gap. This study is the first example for an enantiopure synthesis of (R)-2 by a biocatalyst. The new green method was developed for bioreduction of bulky ketones, which contains a branched alkyl chain, and it approves the synthesis of novel chiral carbinols in an easy, cheap, and environmentally friendly condition using L paracasei BD101.     

Green synthesis of enantiopure quinoxaline alcohols using Daucus carota

Green chemistry comprises a new approach in the synthesis of biologically active compounds using biocatalysts, thus diminishing the hazards for human health and environmental pollution. Asymmetric bioreduction is one of the most widely employed strategies in chemoenzymatic synthesis to produce enantiomerically pure chiral alcohols. The present study highlights the use biocatalyst Daucus carota for selective bioreduction of quinoxaline ketones 1a‐6a to their corresponding optically pure alcohols 1b‐6b in high yields (up to 84%) and good enantioselectivity (up to 98%). The absolute configuration of the chiral product (R)‐1‐(3‐methyl 7‐nitroquinoxalin‐2‐yl) ethan‐1‐ol 2b was confirmed by X‐ray crystallography studies. The chiral R‐configuration of the products obtained was confirmed by absolute configuration studies and was assigned following anti‐Prelogs rule. Quinoxaline pharmacophores form a part of well‐known potent drug molecules; hence, the chiral products were studied for determination of their molecular properties using SwissADME property analyser. All the chiral products show no Lipinski rule violations and are expected to have good oral bioavailability. As per the molecular properties prediction studies, the compound 6b (R)‐1‐(6,7‐dichloro‐3‐ methylquinoxalin‐2‐yl) ethanol is observed to show the best physicochemical properties to be a good lead molecule. Thus, the sustainable methodology was developed, and it confirms the synthesis of novel quinoxaline chiral alcohols in a simple, inexpensive, and eco‐friendly condition using D carota.     

Utilization of deep‐sea microbial esterase PHE21 to generate chiral sec‐butyl acetate through kinetic resolutions

We previously identified and characterized 1 novel deep‐sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)‐3‐hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)‐sec‐butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)‐sec‐butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)‐sec‐butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)‐sec‐butyl acetate reached 64% and 43%, respectively, after process optimization. Deep‐sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate

The two enantiomers of ethyl 3‐hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)‐3‐hydroxybutyrate. Herein, we also functionally characterized one novel salt‐tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)‐3‐hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio‐selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.     

Effect of riboflavin‐producing bacteria against chemically induced colitis in mice

Aim

To assess the anti‐inflammatory effect associated with individual probiotic suspensions of riboflavin‐producing lactic acid bacteria (LAB) in a colitis murine model.

Methods and Results

Mice intrarectally inoculated with trinitrobenzene sulfonic acid (TNBS) were orally administered with individual suspensions of riboflavin‐producing strains: Lactobacillus (Lact.) plantarum CRL2130, Lact. paracasei CRL76, Lact. bulgaricus CRL871 and Streptococcus thermophilus CRL803; and a nonriboflavin‐producing strain or commercial riboflavin. The extent of colonic damage and inflammation and microbial translocation to liver were evaluated. iNOs enzyme was analysed in the intestinal tissues and cytokine concentrations in the intestinal fluids. Animals given either one of the four riboflavin‐producing strains showed lower macroscopic and histologic damage scores, lower microbial translocation to liver, significant decreases of iNOs+ cells in their large intestines and decreased proinflammatory cytokines, compared with mice without treatment. The administration of pure riboflavin showed similar benefits. Lact. paracasei CRL76 accompanied its anti‐inflammatory effect with increased IL‐10 levels demonstrating other beneficial properties in addition to the vitamin production.

Conclusion

Administration of riboflavin‐producing strains prevented the intestinal damage induced by TNBS in mice.

Significance and Impact of the Study

Riboflavin‐producing phenotype in LAB represents a potent tool to select them for preventing/treating IBD.     

Enzymatic hydrogen transfer reduction of α-chloro aromatic ketones catalyzed by a hyperthermophilic alcohol dehydrogenase

An alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (PFADH) effectively catalyzed the reductions of various substituted α-chloroacetophenones to furnish the corresponding (R)-configurated α-chlorohydrins with excellent enantiomeric purity. The co-factor NADH could be recycled with d-glucose dehydrogenase/d-glucose system or in a coupled substrate approach using iso-propanol as the hydrogen donor. The hydrogen transfer mode should be more cost-effective. Thus, the PFADH-catalyzed hydrogen transfer reductions of some substrates were carried out on the preparative scale, demonstrating that this enzyme would be a valuable biocatalyst for the preparation of chiral chlorohydrins of pharmaceutical interest.     

Bio-catalytic asymmetric synthesis of β-adrenergic receptor blocker precursor: (R)-2-bromo-1-(naphthalen-2-yl)ethanol

Abstract

Aromatic α-halohydrins, particularly 2-haloethanols as significant precursor of drugs, can easily be converted to chiral β-adrenergic receptor blockers. Eight strains of Lactobacillus curvatus were tested as biocatalysts for asymmetric reduction of 2-bromo-1-(naphthalen-2-yl)ethanone 1 to 2-bromo-1- (naphthalen-2-yl) ethanol 2. The parameters of the bioreduction were optimized using L. curvatus N4, the best biocatalyst found. As a result, (R)-2-bromo-1-(naphthalen-2-yl)ethanol 2, which can be β-adrenergic receptor blocker precursor, was produced for the first time in high yield and enantiomerically pure form using biocatalysts. Moreover, the gram scale synthesis was performed and 7.54?g of (R)-2 was synthesized as enantiopure form (enantiomeric excess >99%) in 48?h. The important advantages of this process are that it produces of (R)-2 for the first time in enantiopure form, in excellent yield and under environmentally friendly and moderate reaction conditions. This system is of the potential to be applied at a commercial scale.     

The effect of growth media and physical treatments on the adhesion properties of canine probiotics

Aims

The manufacturing processes have been reported to influence the properties of probiotics with potential impact on health properties. The aim was to investigate the effect of different growth media and inactivation methods on the properties of canine‐originated probiotic bacteria alone and in combination mixture.

Methods and Results

Three established dog probiotics, Lactobacillus fermentum VET9A, Lactobacillus plantarum VET14A and Lactobacillus rhamnosus VET16A, and their combination mixture were evaluated for their adhesion to dog mucus. The effect of different growth media, one reflecting laboratory and the other manufacturing conditions, and inactivation methods (95°C, 80°C and UV irradiation) on the mucus adhesion of the probiotic strains was characterized. Evaluation of dog probiotics was supported by cell visualization using transmission electron microscopy (TEM). Higher adhesion percentage was reported for probiotic strains growing in laboratory rather than in manufacturing conditions (P < 0·05). Inactivation by heat (95°C, 80°C) decreased the adhesion properties when strains were cultivated in soy‐based growth media compared with those grown in MRS broth (P < 0·05). TEM observations uncovered differences in cell‐surface components in nonviable forms of probiotic strains as compared with their viable forms.

Conclusions

Manufacturing process conditions such as growth media and pretreatment methods may significantly affect the adhesive ability of the tested strains.

Significance and Impact of the Study

Growth conditions, growth media, pretreatment methods and different probiotic combinations should be carefully considered for quality control of existing probiotics and for identification of new probiotics for dogs. These may also have an impact on health benefits for the host.     

Evaluation of acetophenone monooxygenase and alcohol dehydrogenase activities in different fungal strains by biotransformation of acetophenone derivatives

Experimental conditions using whole cells to select fungal strains for specific bioreduction of ketones and formation of Baeyer–Villiger oxidation products were studied. Epicoccum nigrum SSP 1498 was effective in the bioreduction leading to the chiral alcohols in up to 98% enantiomeric excess. High acetophenone monooxygenase activity was observed by the use of the fungus Emericella nidulans CCT 3119 as biocatalyst.     

A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

Abstract

This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.