Transaminases and their Applications

Dold, Sarah-Marie; Syldatk, Christoph; Rudat, Jens

doi:10.1002/9781118828083.ch29

Cited by 13 publications

(8 citation statements)

References 152 publications

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“…Conducting Coomassie blue staining indicates that this assay appears sensitive without any background color and a need for removing by-product. In particular, several α-transaminases (α-TA) are constitutively expressed enzymes in Bacillus as well as in Escherichia as in every living cell (Dold et al 2016 ) and should have been stained with an unspecific staining method. So the OXD assay appears to be specific for ω-TAs.…”

Section: Discussionmentioning

confidence: 99%

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

et al. 2021

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Abstractω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

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Section: Discussionmentioning

confidence: 99%

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

et al. 2021

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“…Over the last 20 years, a significant number of enzymatic routes have been developed for the synthesis of chiral amines, among which are enzymes that catalyze the reductive amination of prochiral ketones into amines: transaminases (TAs), amine dehydrogenases (AmDHs), and imine reductases (IREDs) have attracted considerable interest ( Schrittwieser et al, 2015 ; Sharma et al, 2017 ; D Patil et al, 2018 ; Grogan, 2018 ; Liu et al, 2020 ; Montgomery et al, 2020 ). Both TAs and AmDHs are currently limited to the synthesis of primary amines, necessitating subsequent alkylation chemistry for the synthesis of chiral secondary and tertiary amines ( Dold et al, 2016 ; Knaus et al, 2017 ). In particular, IREDs can catalyze the NAD(P)H-dependent reduction of prochiral imines to chiral amines ( Mitsukura et al, 2010 ) and prefer the reduction of cyclic imine substrates but lead to poor conversion with the amination of prochiral ketones ( Huber et al, 2014 ; Wetzl et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Engineering of Reductive Aminases for Asymmetric Synthesis of Enantiopure Rasagiline

Zhang

Zhu

et al. 2021

Front. Bioeng. Biotechnol.

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Reductive aminases (RedAms) for the stereoselective amination of ketones represent an environmentally benign and economically viable alternative to transition metal–catalyzed asymmetric chemical synthesis. Here, we report two RedAms from Aspergillus calidoustus (AcRedAm) and bacteria (BaRedAm) with NADPH-dependent features. The enzymes can synthesize a set of secondary amines using a broad range of ketone and amine substrates with up to 97% conversion. To synthesize the pharmaceutical ingredient (R)-rasagiline, we engineered AcRedAm through rational design to obtain highly stereoselective mutants. The best mutant Q237A from AcRedAm could synthesize (R)-rasagiline with >99% enantiomeric excess with moderate conversion. The features of AcRedAm and BaRedAm highlight their potential for further study and expand the biocatalytic toolbox for industrial applications.

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“…[8][9][10][11][12][13] In addition, in the past decades the scientific advances in protein engineering techniques have provided a better access to specifically improved enzymes for tailor-made bioprocesses, also in the chiral amine synthesis. 14,15 Especially amine transaminases, [16][17][18][19] amine dehydrogenases, 20,21 and imine reductases (IREDs) have recently attracted a lot of attention for synthesizing a wide selection of chiral amines. A special case is IREDs, which allow a direct synthesis of enantiopure secondary amines from prochiral imines.…”

Section: Introductionmentioning

confidence: 99%

Integration of ion exchange resin materials for a downstream‐processing approach of an imine reductase‐catalyzed reaction

Meyer

Brundiek²,

Langermann

2020

Biotechnology Progress

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In this study, an ion exchange resin-based downstream-processing concept for imine reductase (IRED)-catalyzed reactions was investigated. As a model reaction, 2-methylpyrroline was converted to its corresponding product (S)-2-methylpyrrolidine with >99% of conversion by the (S)-selective IRED from Paenibacillus elgii B69. Under optimized reaction conditions full conversion was achieved using a substrate concentration of 150 and 500 mmol/L of D-glucose. Seven commercially available cation-and anion-exchange resins were studied with respect to their ability to recover the product from the reaction solution. Without any pretreatment, cation-exchange resins Amberlite IR-120(H), IRN-150, Dowex Monosphere 650C, and Dowex Marathon MSC showed high recovery capacities (up to >90%). A 150-ml preparative scale reaction was performed yielding 1 g hydrochloride salt product with >99% purity. Any further purification steps, for example, by column chromatography or recrystallization, were not required.

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Transaminases and their Applications

Cited by 13 publications

References 152 publications

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil

Engineering of Reductive Aminases for Asymmetric Synthesis of Enantiopure Rasagiline

Integration of ion exchange resin materials for a downstream‐processing approach of an imine reductase‐catalyzed reaction

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