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Effective production of Pro–Gly by mutagenesis of l-amino acid ligase

Paper ID Volume ID Publish Year Pages File Format Full-Text
19994 43152 2016 5 PDF Available
Title
Effective production of Pro–Gly by mutagenesis of l-amino acid ligase
Abstract

l-Amino acid ligase (Lal) catalyzes dipeptide synthesis from unprotected l-amino acids by hydrolysis ATP to ADP. Each Lal displays unique substrate specificity, and many different dipeptides can be synthesized by selecting suitable Lal. We have already successfully synthesized Met–Gly selectively by replacing the Pro85 residues of Lal from Bacillus licheniformis (BL00235). From these results, we deduced that the amino acid residue at position 85 had a key role in enzyme activity, and applied these findings to other Lals. When Pro and Gly were used as substrates, TabS from Pseudomonas syringae, synthesized the salt taste enhancing dipeptide Pro–Gly and other three dipeptides (Gly–Pro, Pro–Pro, and Gly–Gly) was hardly synthesized from its substrate specificity. However, the amount of Pro–Gly was low. Therefore, to alter the substrate specificity and increase the amount of Pro–Gly, we selected amino acid residues that might affect the enzyme activity, Ser85 corresponding to Pro85 of BL00235, and His294 on the results from previous studies and the predicted structure of TabS. These residues were replaced with 20 proteogenic amino acids, and Pro–Gly synthesizing reactions were conducted. The S85T and the H294D mutants synthesized more Pro–Gly than wild-type. Furthermore, the S85T/H294D double mutant synthesized considerably more Pro–Gly than the single mutant did. These results showed that the amino acid position 85 of TabS affect the enzyme activity similarly to BL00235. In addition, replacing the amino acid residue positioning around the N-terminal substrate and constructing the double mutant led to increase the amount of Pro–Gly.

Keywords
Dipeptide; l-Amino acid ligase; Site-directed mutagenesis; TabS; Salt taste enhancer
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Effective production of Pro–Gly by mutagenesis of l-amino acid ligase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 122, Issue 2, August 2016, Pages 155–159
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