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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

Gord Noshahri, Najme; Fooladi, Jamshid; Engel, Ulrike; Muller, Delphine; Kugel, Michaela; Gorenflo, Pascal; Syldatk, Christoph; Rudat, Jens

Abstract:
ω-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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Verlagsausgabe §
DOI: 10.5445/IR/1000131117
Veröffentlicht am 08.04.2021
Originalveröffentlichung
DOI: 10.1186/s13568-021-01207-7
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Bio- und Lebensmitteltechnik (BLT)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2021
Sprache Englisch
Identifikator ISSN: 2191-0855
KITopen-ID: 1000131117
Erschienen in AMB Express
Verlag SpringerOpen
Band 11
Heft 1
Seiten 46
Schlagwörter ω-Transaminase; ortho-Xylylenediamine assay; Native PAGE; Activity staining; Growth optimization; Bacillus fermentation
Nachgewiesen in Scopus
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