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An engineered mutant, L307V of phenylalanine dehydrogenase from Bacillus sphaericus: high activity and stability in organic-aqueous solvent mixtures and utility for synthesis of non-natural l-amino acids

Paper ID Volume ID Publish Year Pages File Format Full-Text
18192 42713 2007 5 PDF Available
Title
An engineered mutant, L307V of phenylalanine dehydrogenase from Bacillus sphaericus: high activity and stability in organic-aqueous solvent mixtures and utility for synthesis of non-natural l-amino acids
Abstract

A study of an engineered phenylalanine dehydrogenase (PheDH) mutant, L307V, from Bacillus sphaericus, catalysing the oxidation of l-phenylalanine (Phe) and five non-natural p-substituted derivatives (4-F/Cl/CH3/OCH3/NO2-l-Phe) in water-miscible organic solvents, methanol, ethanol and acetonitrile, is reported. Results showed that the enzyme still had high activity with 0.2 mM 4-CH3/OCH3/NO2-l-Phe in 10% methanol. The kinetic parameters of the enzyme were determined with three substrates, 4-F/Cl/CH3-l-Phe, in the absence/presence of 10% methanol and with two less water-soluble substrates, 4-OCH3/NO2-l-Phe, only in the presence of 10% methanol. Data indicated that catalytic efficiency of the enzyme with 4-Cl/CH3-Phe as substrates was little changed in 10% methanol as compared to purely aqueous solvent. The stability of L307V, WT and N145A PheDH was assessed over 1 week at two pH values and in the presence/absence of organic solvents. In pH 8.0 buffer with either 10% methanol or 10% ethanol, L307V PheDH retained almost 85% original activity after 7 days, and WT PheDH was similarly stable. In contrast, N145A PheDH, also a versatile biocatalyst, was much less stable, the best performance being 68% retention of activity after 7 days at pH 8.0 with 10% methanol. Practical application of L307V PheDH, with baker's yeast alcohol dehydrogenase for co-enzyme recycling, to synthesise a non-natural amino acid, p-OCH3-l-Phe is also reported, illustrating the utility of this versatile biocatalyst for potential industrial uses.

Keywords
Phenylalanine dehydrogenase (PheDH) mutants; Non-natural amino acids; Biocatalysts; Organic solvents; Substrate specificity; Enzyme stability; Kinetic parameters; Co-enzyme recycling
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An engineered mutant, L307V of phenylalanine dehydrogenase from Bacillus sphaericus: high activity and stability in organic-aqueous solvent mixtures and utility for synthesis of non-natural l-amino acids
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 5, 3 April 2007, Pages 1407–1411
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