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Directed evolution of tyrosinase for enhanced monophenolase/diphenolase activity ratio

Paper ID Volume ID Publish Year Pages File Format Full-Text
17532 42675 2010 5 PDF Available
Title
Directed evolution of tyrosinase for enhanced monophenolase/diphenolase activity ratio
Abstract

Tyrosinases are copper-containing enzymes which perform two reactions, the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (l-DOPA) and the subsequent oxidation to form dopaquinone. The latter diphenolase activity is much faster than the monophenolase activity, limiting the use of tyrosinases for diphenol synthesis. The key residues influencing tyrosinase specificity are not well known. Random mutagenesis was performed on tyrosinase from Bacillus megaterium in order to find variants with altered selectivity. A high throughput colorimetric screening assay in 96-well plates was used to evaluate separately the monophenolase and diphenolase activity. Variant R209H exhibited a 1.7-fold increase in monophenolase activity accompanied by a 1.5-fold decrease in diphenolase activity, resulting in a 2.6-fold improvement in the monophenolase/diphenolase activity ratio. In silco modeling suggested that the imidazole group of the histidine residue obstructs the entrance to the active site thus interfering with l-DOPA binding to CuB. This work highlights a new amino acid residue that is important for catalysis of monophenols and supports the hypothesis that tyrosine and l-DOPA bind differently in the active site.

Keywords
Tyrosinase; Directed evolution; Selectivity; Catechol
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Directed evolution of tyrosinase for enhanced monophenolase/diphenolase activity ratio
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 47, Issue 7, 8 December 2010, Pages 372–376
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us