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Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

Paper ID Volume ID Publish Year Pages File Format Full-Text
17649 42686 2010 6 PDF Available
Title
Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity
Abstract

Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity kcat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant Km values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.

Keywords
FAGLA, N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide; FM, l-phenylalanine methyl ester; HPLC, high performance liquid chromatography; ZD, N-carbobenzoxy-l-aspartic acid; ZDFM, N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl esterAspartame; Enzymatic pep
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Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 46, Issues 3–4, 5 March 2010, Pages 320–325
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