A method for rapid screening of ketone biotransformations: Detection of whole cell Baeyer–Villiger monooxygenase activity

Linares-Pastén, Javier A.; Chávez-Lizárraga, Georgina Aurelia; Villagomez, Rodrigo; Mamo, Gashaw; Hatti‐Kaul, Rajni

doi:10.1016/j.enzmictec.2011.10.004

Cited by 9 publications

(10 citation statements)

References 28 publications

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“…A screening method for ketone biotransformations was also presented, which was used successfully for the detection of BVMO activity. 14 This method is based on the reaction between alicyclic ketones and 3,5-dinitrobenzoic acid under alkaline conditions, resulting in the formation of a purple-colored product. Despite being fast and simple, this method requires relatively high activities, and it is limited to a certain class of compounds.…”

Section: Introductionmentioning

confidence: 99%

A Generic, Whole-Cell–Based Screening Method for Baeyer-Villiger Monooxygenases

et al. 2013

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Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell-based screening method was validated by screening site-saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.

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Section: Introductionmentioning

confidence: 99%

A Generic, Whole-Cell–Based Screening Method for Baeyer-Villiger Monooxygenases

et al. 2013

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“…strains are known to have a variety of oxygenases [17][18][19]. This work presents cloning, expression and substrate scope of a BVMO isolated from Dietzia sp.…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of a Baeyer–Villiger monooxygenase oxidizing linear aliphatic ketones from Dietzia sp. D5

Bisagni¹,

Smuś²,

Chávez³

et al. 2014

Journal of Molecular Catalysis B: Enzymatic

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“…7,122,123 S. glaucescens GLA.0 cells were primarily screened for BVMO activity using cyclohexanone as a model substrate. Qualitative analysis of cyclohexanone consumption by the resting bacterial cells, by a method based on the reaction of an enolizable ketone (i.e., cyclohexanone) with dinitrobenzoic acid in an alkaline solution leading to the formation of a purple coloured complex, 124 showed total disappearance of color within 24-48 hours. Monitoring the bioconversion of cyclohexanone by the bacterial cells over time revealed 6-carbon and 7-carbon esters and traces of 3-caprolactone by GC-MS analysis.…”

Section: Methodsmentioning

confidence: 99%