Characterization of a (R)-selective amine transaminase from Fusarium oxysporum

Gao, Shuaihua; Su, Yu; Zhao, Lu; Li, Gudong; Zheng, Guojun

doi:10.1016/j.procbio.2017.08.012

Cited by 32 publications

(15 citation statements)

References 38 publications

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“…Indeed, many R-selective TAms discovered using overall sequence homology appear to come from microorganisms other than bacteria. Gao et al (2017) characterized an Rselective TAm from the fungus Fusarium oxysporum (Gao et al 2017), while the first haloarchaeal TAm characterized for use in biocatalysis (BC61-TAm) was isolated from the genome of Halorubrum sp. CSM-61 (Kelly et al 2019a).…”

Section: Homologous Sequence Searchingmentioning

confidence: 99%

Transaminases for industrial biocatalysis: novel enzyme discovery

Kelly

Mix

Moody

et al. 2020

Appl Microbiol Biotechnol

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Transaminases (TAms) are important enzymes for the production of chiral amines for the pharmaceutical and fine chemical industries. Novel TAms for use in these industries have been discovered using a range of approaches, including activity-guided methods and homologous sequence searches from cultured microorganisms to searches using key motifs and metagenomic mining of environmental DNA libraries. This mini-review focuses on the methods used for TAm discovery over the past two decades, analyzing the changing trends in the field and highlighting the advantages and drawbacks of the respective approaches used. This review will also discuss the role of protein engineering in the development of novel TAms and explore possible directions for future TAm discovery for application in industrial biocatalysis. Key Points• The past two decades of TAm enzyme discovery approaches are explored.• TAm sequences are phylogenetically analyzed and compared to other discovery methods.• Benefits and drawbacks of discovery approaches for novel biocatalysts are discussed.• The role of protein engineering and future discovery directions is highlighted.

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Section: Homologous Sequence Searchingmentioning

confidence: 99%

Transaminases for industrial biocatalysis: novel enzyme discovery

Kelly

Mix

Moody

et al. 2020

Appl Microbiol Biotechnol

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“…The AcPhe concentration was measured via UV detection at 254 nm by mobile phase containing acetonitrile/water (50/50, v/v) at flow rate of 0.6 ml min − 1 (Gao et al 2017 ) at room temperature using a C 18 Hypersil- keystone column (250 × 4.6 mm 5 µ Hypersil). The injection volume was 1 µL.…”

Section: Methodsmentioning

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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“…KNK168, the ω-TA showed similar affinity for pyruvate, but its Km value for (R)-MBA was significantly higher than that of ATA117, which indicated that the ω-TA had slightly lower affinity for amine. However, the ω-TA showed lower Km to pyruvate and higher kcat than a transaminase from Fusarium oxysporum [38], which means higher reactivity on pyruvate (Fig. 4).…”

Section: Kinetic Parameters Of the ω-Tamentioning

confidence: 97%

Identification, Characterization and Site-Saturation Mutagenesis of a Thermostable ω-Transaminase From Chloroflexi Bacterium

Wang

Tang

Dai

et al. 2021

Preprint

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In present study, we have mined a ω-transaminase (ω-TA) from Chloroflexi bacterium from genome database by using an ω-TA sequence ATA117 Arrmut11 from Arthrobacter sp . KNK168 and an amine transaminase from Aspergillus terreus as templates in a BLASTP search and motif sequence alignment. The protein sequence of the ω-TA from C. bacterium shows 38% sequence identity to ATA117 Arrmut11. The gene sequence of the ω-TA was inserted into pRSF-Duet1 and functionally expressed in E. coli BL21(DE3). Results showed that the recombinant ω-TA has a specific activity of 1.19 U/mg at pH 8.5, 40 °C. The substrate acceptability test showed that ω-TA has significant reactivity to aromatic amino donors and amino receptors. More importantly, the ω-TA also exhibited a good affinity towards some cyclic substrates such as 1-Boc-3-piperidone. The homology model of the ω-TA was built by Discovery Studio and docking was performed to describe the relative activity towards some substrates. The ω-TA was evolved by site-saturation mutagenesis and found that the Q192G mutant increased the activity to the (R)-α-methylbenzylamine (MBA) by around seven-fold. The Q192G mutant was then used to convert two cyclic ketones, N -Boc-3-pyrrolidinone and N -Boc-3-aminopiperidine, and the conversions were both improved compared to the parental ω-TA.

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Characterization of a (R)-selective amine transaminase from Fusarium oxysporum

Cited by 32 publications

References 38 publications

Transaminases for industrial biocatalysis: novel enzyme discovery

Transaminases for industrial biocatalysis: novel enzyme discovery

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

Identification, Characterization and Site-Saturation Mutagenesis of a Thermostable ω-Transaminase From Chloroflexi Bacterium

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Characterization of a (R)-selective amine transaminase from Fusarium oxysporum

Cited by 32 publications

References 38 publications

Transaminases for industrial biocatalysis: novel enzyme discovery

Transaminases for industrial biocatalysis: novel enzyme discovery

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

Identification, Characterization&nbsp;and Site-Saturation Mutagenesis&nbsp;of a&nbsp;Thermostable ω-Transaminase From Chloroflexi&nbsp;Bacterium

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Identification, Characterization and Site-Saturation Mutagenesis of a Thermostable ω-Transaminase From Chloroflexi Bacterium