Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

Liu, Yan; Tang, Tuo-Xian; Pei, Xiaokun; Zhang, Chao; Wu, Zhong‐Liu

doi:10.1016/j.molcatb.2014.01.009

Cited by 56 publications

(28 citation statements)

References 37 publications

(58 reference statements)

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“…is the anti-Prelog SDR that shares the highest sequence identity with ChKRED20 [24]. The specific activity of LSADH toward phenyl trifluromethyl ketone was reported to be 10.3 U/mg at 25 • C [25], which was comparable to the specific activity of ChKRED20 at 30 • C (10.7 U/mg) toward substrate 1a.…”

Section: Effects Of Ph and Temperaturementioning

confidence: 95%

“…The plasmid, pET-28a-chKRED20, encoding ChKRED20 [24] was transformed into E. coli BL21(DE3) (Novagen, Madison, WI, USA) competent cells and selected on Luria-Bertani (LB) agar plates containing 50 g kanamycin/ml. Single colonies were grown overnight at 37 • C in LB medium containing 50 g kanamycin/ml.…”

Section: Expression and Purification Of Chkred20 In Escherichia Coli mentioning

confidence: 99%

“…ChKRED20 was routinely purified to homogeneity using Ni-NTA resin [24], and applied in all the characterizations. We first investigated the substrate scope and limitation of ChKRED20 by subjecting a panel of acetophenone derivatives to the bioreduction reaction (Table 2).…”

Section: Activity and Stereoselectivity Of Chkred20 Toward Various Kementioning

confidence: 99%

“…1). The identification of the key enzyme, ChKRED20, has been accomplished through draft genome sequence analysis followed by heterologous expression, biotransformation and transcriptional analysis [24]. ChKRED20 is a short-chain dehydrogenase/reductase (SDR) sharing the highest sequence identity with LSADH from Leifsonia sp.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

Tang

Liu

2014

Journal of Molecular Catalysis B: Enzymatic

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Section: Effects Of Ph and Temperaturementioning

confidence: 95%

Section: Expression and Purification Of Chkred20 In Escherichia Coli mentioning

confidence: 99%

Section: Activity and Stereoselectivity Of Chkred20 Toward Various Kementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

Tang

Liu

2014

Journal of Molecular Catalysis B: Enzymatic

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“…ChKRED03 was a putative ketoreductase mined from the genome of Chryseobacterium sp. CA49 (Liu et al 2014). In this cascade reaction, ChKRED03 performed the efficient bioreduction of the substrates to provide (S)-allylic alcohols in the presence of the GDH-catalyzed NADPH recycling system, followed by SMO-catalyzed epoxidation to achieve glycidol derivatives (Fig.…”

Section: Cascade Reactions Involving Smo and Ketoreductasementioning

confidence: 99%

Asymmetric bio-epoxidation catalyzed with the styrene monooxygenase from Pseudomonas sp. LQ26

Liu

2016

Bioresour. Bioprocess.

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Styrene monooxygenases (SMOs) can catalyze the asymmetric epoxidation of alkenes to obtain optically active epoxides. This account describes a series of work of our group on the isolation, application, and improvement of an SMO from Pseudomonas sp. LQ26. The strain was isolated from an active sludge sample based on indigo-forming capacity. Then the gene encoding SMO was expressed in Escherichia coli, and the whole cells were applied in biocatalytic reactions. The substrate spectrum of SMO was successfully expanded from conjugated styrene derivatives to non-conjugated alkenes, especially α-substituted secondary allylic alcohols, affording enantiopure epoxides. Most significantly, cascade reactions involving ketoreductase and SMO were designed, which resulted in glycidol derivatives or epoxy ketones with excellent enantio-and diastereo-selectivity using α,β-unsaturated ketones as the substrates. In addition, mutants of SMO with altered substrate preference and enhanced activity were constructed, which indicated great potential of SMO for further improvement.

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Fluorescence‐based screening for engineered aldo‐keto reductase KmAKR with improved catalytic performance and extended substrate scope

Qiu

et al. 2021

Biotechnology Journal

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Background Aldo‐keto reductases‐catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. Previously, we have discovered an aldo‐keto reductase (AKR) from Kluyveromyces marxianus that is active to the aliphatic tert‐butyl 6‐substituted (5R/S)‐hydroxy‐3‐oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent KmAKRmutant for ensuring the simultaneous improvement of activity‐thermostability toward tert‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐1) and broadening the universal application prospects toward more substrates covering both aliphatic and aromatic ketones, a fluorescence‐based high‐throughput (HT) screening technique was established. Main Methods and Major Results The directed evolution of KmAKR variant M5 (KmAKR‐W297H/Y296W/K29H/Y28A/T63M) produced the “best” variant M5‐Q213A/T23V. It exhibited enhanced activity‐thermostability toward (5R)‐1, improved activity toward all 18 test substrates and strict R‐stereoselectivity toward 10 substrates in comparison to M5. The enhancement of enzymatic activity and the extension of substrate scope covering aromatic ketones are proposed to be largely attributed to pushing the binding pocket of M5‐Q213A/T23V to the enzyme surface, decreasing the steric hindrance at the entrance and enhancing the flexibility of loops surrounding the active center. In addition, combined with 0.94 g dry cell weight (DCW) L−1 glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) for NADPH regeneration, 2.81 g DCW L−1 M5‐Q213A/T23V completely converted (5R)‐1 of up to 450 g L−1 at 120 g g–1 substrates/catalysts (S/C), yielding the corresponding optically pure tert‐butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐2, > 99.5% d.e.p) with a space‐time yield (STY) of 1.08 kg L−1 day−1. Conclusions A fluorescence‐based HT screening system was developed to tailor KmAKR's activity, thermostability and substrate scope. The “best” variant M5‐Q213A/T23V holds great potential application for the synthesis of aliphatic/aromatic R‐configuration alcohols.

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Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

Cited by 56 publications

References 37 publications

Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

Asymmetric bio-epoxidation catalyzed with the styrene monooxygenase from Pseudomonas sp. LQ26

Fluorescence‐based screening for engineered aldo‐keto reductase KmAKR with improved catalytic performance and extended substrate scope

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