Kinetic Resolution of Racemic Amines to Enantiopure (S)-amines by a Biocatalytic Cascade Employing Amine Dehydrogenase and Alanine Dehydrogenase

Amine dehydrogenases (AmDHs) efficiently catalyze the NAD(P)H-dependent asymmetric reductive amination of prochiral carbonyl substrates with high enantioselectivity. AmDH-catalyzed oxidative deamination can also be used for the kinetic resolution of racemic amines to obtain enantiopure amines. In the present study, kinetic resolution was carried out using a coupled-enzyme cascade consisting of AmDH and alanine dehydrogenase (AlaDH). AlaDH efficiently catalyzed the conversion of pyruvate to alanine, thus recycling the nicotinamide cofactors and driving the reaction forward. The ee values obtained for the kinetic resolution of 25 and 50 mM rac-α-methylbenzylamine using the purified enzymatic systems were only 54 and 43%, respectively. The use of whole-cells apparently reduced the substrate/product inhibition, and the use of only 30 and 40 mgDCW/mL of whole-cells co-expressing AmDH and AlaDH efficiently resolved 100 mM of rac-2-aminoheptane and rac-α-methylbenzylamine into the corresponding enantiopure (S)-amines. Furthermore, the applicability of the reaction protocol demonstrated herein was also successfully tested for the efficient kinetic resolution of wide range of racemic amines.

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The Kinetic Resolution of Racemic Amines Using a Whole-Cell Biocatalyst Co-Expressing Amine Dehydrogenase and NADH Oxidase

Catalysts ◽

10.3390/catal7090251 ◽

2017 ◽

Vol 7 (9) ◽

pp. 251 ◽

Cited By ~ 8

Author(s):

Hyunwoo Jeon ◽

Sanghan Yoon ◽

Md Ahsan ◽

Sihyong Sung ◽

Geon-Hee Kim ◽

...

Keyword(s):

Kinetic Resolution ◽

Nadh Oxidase ◽

Whole Cell ◽

Whole Cell Biocatalyst ◽

Amine Dehydrogenase ◽

Racemic Amines

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A multi-enzyme cascade reaction for the production of 6-hydroxyhexanoic acid

Zeitschrift für Naturforschung C ◽

10.1515/znc-2018-0216 ◽

2019 ◽

Vol 74 (3-4) ◽

pp. 71-76 ◽

Cited By ~ 13

Author(s):

Vishnu S.T. Srinivasamurthy ◽

Dominique Böttcher ◽

Uwe T. Bornscheuer

Keyword(s):

Escherichia Coli ◽

Cascade Reactions ◽

Product Inhibition ◽

Stirred Tank ◽

Fed Batch ◽

Batch Mode ◽

Whole Cells ◽

Tank Reactor ◽

Enzyme Cascade

Abstract Multi-enzyme cascade reactions capture the essence of nature’s efficiency by increasing the productivity of a process. Here we describe one such three-enzyme cascade for the synthesis of 6-hydroxyhexanoic acid. Whole cells of Escherichia coli co-expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase (CHMO) for internal cofactor regeneration were used without the supply of external NADPH or NADP+. The product inhibition caused by the ε-caprolactone formed by the CHMO was overcome by the use of lipase CAL-B for in situ conversion into 6-hydroxyhexanoic acid. A stirred tank reactor under fed-batch mode was chosen for efficient catalysis. By using this setup, a product titre of >20 g L−1 was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.

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Bioencapsulation in Sol-Gel Glasses

MRS Proceedings ◽

10.1557/proc-519-171 ◽

1998 ◽

Vol 519 ◽

Cited By ~ 9

Author(s):

L. Bergogne ◽

S. Fennouh ◽

J. Livage ◽

C. Roux

Keyword(s):

Sol Gel ◽

Porous Silica ◽

Silica Matrix ◽

Whole Cells ◽

The Past ◽

Wide Range ◽

Activity Of Enzymes ◽

Gel Glasses ◽

Micro Organisms

AbstractBioencapsulation in sol-gel materials has been widely studied during the past decade. Trapped species appear to retain their bioactivity in the porous silica matrix. Small analytes can diffuse through the pores allowing bioreactions to be performed in-situ, inside the sol-gel glass. A wide range of biomolecules and micro-organisms have been encapsulated. The catalytic activity of enzymes is used for the realization of biosensors or bioreactors. Antibody-antigen recognition has been shown to be feasible within sol-gel matrices. Trapped antibodies bind specifically the corresponding haptens and can be used for the detection of traces of chemicals. Even whole cells are now encapsulated without any alteration of their cellular organization. They can be used for the production of chemicals or as antigens for immunoassays.

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Development of Anti-Infectives Using Phage Display: Biological Agents against Bacteria, Viruses, and Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00567-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4569-4582 ◽

Cited By ~ 50

Author(s):

Johnny X. Huang ◽

Sharon L. Bishop-Hurley ◽

Matthew A. Cooper

Keyword(s):

Phage Display ◽

Significant Contribution ◽

Biological Agents ◽

Preclinical Development ◽

Phage Display Technology ◽

Whole Cells ◽

Drug Candidates ◽

Phage Display Libraries ◽

Wide Range ◽

Display Technology

ABSTRACTThe vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.

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A General Fitting Function to Estimate Apparent Reaction Orders of Kinetic Profiles

10.26434/chemrxiv.7885760 ◽

2019 ◽

Author(s):

Robert Pollice

Keyword(s):

Chemical Reactions ◽

Catalyst Deactivation ◽

Rapid Development ◽

Product Inhibition ◽

Time Profile ◽

Time Data ◽

Concentration Time ◽

Wide Range ◽

Reaction Orders

The rapid development of analytical methods in recent decades has resulted in a wide range of readily available and accurate reaction-monitoring techniques, which allow for easy determination of high-quality concentration-time data of chemical reactions. However, while the acquisition of kinetic data has become routine in the development of new chemical reactions and the study of their mechanisms, not all the information contained therein is utilized because of a lack of suitable analysis tools which unnecessarily complicates mechanistic studies. Herein, we report on a general method to analyze a single concentration-time profile of chemical reactions and extract information regarding the reaction order with respect to substrates, the presence of multiple kinetic regimes, and the presence of kinetic complexities, such as catalyst deactivation, product inhibition, and substrate decomposition.<br>

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Exploring the Glycans of Euglena Gracilis

10.20944/preprints201711.0051.v1 ◽

2017 ◽

Author(s):

Ellis C. O'Neill ◽

Sakonwan Kuhaudomlarp ◽

Martin Rejzek ◽

Jonatan U. Fangel ◽

Kathirvel Alagesan ◽

...

Keyword(s):

Euglena Gracilis ◽

High Capacity ◽

Surface Protein ◽

Protein Glycosylation ◽

Plant Cell Walls ◽

Beta Glucan ◽

Whole Cells ◽

Wide Range ◽

Glycan Profiling ◽

Complex Glycan

Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single celled organism. Here we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its’ protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to production of pharmaceutical glycoproteins.

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Enantioselective Transesterification of Allyl Alcohols with (E)-4-Arylbut-3-en-2-ol Motif by Immobilized Lecitase™ Ultra

Catalysts ◽

10.3390/catal10070798 ◽

2020 ◽

Vol 10 (7) ◽

pp. 798

Author(s):

Aleksandra Leśniarek ◽

Anna Chojnacka ◽

Radosław Drozd ◽

Magdalena Szymańska ◽

Witold Gładkowski

Keyword(s):

High Activity ◽

Cyanogen Bromide ◽

Kinetic Resolution ◽

Negative Effects ◽

Subsequent Reaction ◽

Wide Range ◽

Allyl Alcohols ◽

Reaction Cycles ◽

Acyl Donors ◽

First Time

Lecitase™ Ultra was immobilized on four different supports and tested for the first time as the biocatalyst in the kinetic resolution of racemic allyl alcohols with the (E)-4-arylbut-3-en-2-ol system in the process of transesterification. The most effective biocatalyst turned out to be the enzyme immobilized on agarose activated with cyanogen bromide (LU-CNBr). The best results (E > 200, ees and eep = 95–99%) were obtained for (E)-4-phenylbut-3-en-2-ol and its analog with a 2,5-dimethylphenyl ring whereas the lowest ee of kinetic resolution products (90%) was achieved for the substrate with a 4-methoxyphenyl substituent. For all substrates, (R)-enantiomers were esterified faster than their (S)-antipodes. The results showed that LU-CNBr is a versatile biocatalyst, showing high activity and enantioselectivity in a wide range of organic solvents in the presence of commonly used acyl donors. High operational stability of LU-CNBr allows it to be reused in three subsequent reaction cycles without negative effects on the efficiency and enantioselectivity of transesterification. This biocatalyst can become attractive to the commercial lipases in the process of the kinetic resolution of allyl alcohols.

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NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1821233116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16448-16453 ◽

Cited By ~ 6

Author(s):

Julia M. Diaz ◽

Sydney Plummer ◽

Colleen M. Hansel ◽

Peter F. Andeer ◽

Mak A. Saito ◽

...

Keyword(s):

Sequence Similarity ◽

Cell Types ◽

Redox Balance ◽

Superoxide Production ◽

Marine Phytoplankton ◽

Marine Diatom ◽

High Sequence Similarity ◽

Whole Cells ◽

Wide Range ◽

Diphenylene Iodonium

Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.

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Unprecedented, Fully Recyclable, Solid-Supported Reagent for the Kinetic Resolution of Racemic Amines Through Enantioselective N-Acetylation.

ChemInform ◽

10.1002/chin.200545087 ◽

2005 ◽

Vol 36 (45) ◽

Author(s):

Stellios Arseniyadis ◽

Pithani V. Subhash ◽

Alain Valleix ◽

Alain Wagner ◽

Charles Mioskowski

Keyword(s):

Kinetic Resolution ◽

Racemic Amines

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Immobilized Whole-Cell Transaminase Biocatalysts for Continuous-Flow Kinetic Resolution of Amines

Catalysts ◽

10.3390/catal9050438 ◽

2019 ◽

Vol 9 (5) ◽

pp. 438 ◽

Cited By ~ 14

Author(s):

Zsófia Molnár ◽

Emese Farkas ◽

Ágnes Lakó ◽

Balázs Erdélyi ◽

Wolfgang Kroutil ◽

...

Keyword(s):

Continuous Flow ◽

Kinetic Resolution ◽

Sol Gel ◽

Chiral Amines ◽

Flow Mode ◽

Immobilized Cell ◽

Hollow Silica ◽

E Coli ◽

Sol Gel Process ◽

Racemic Amines

Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a–d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d.

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