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Catalytically important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17814 42698 2008 7 PDF Available
Title
Catalytically important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis
Abstract

Alginate lyase is an enzyme that degrades alginate chains via β-elimination and has been used for the production of alginate oligosaccharides and protoplasts from brown algae. Previously, we deduced the amino-acid sequence of an abalone alginate lyase, HdAly, from its cDNA sequence and, through multiple amino-acid sequence alignment, found that several basic amino-acid residues were highly conserved among the polysaccharide-lyase family 14 (PL-14) enzymes including HdAly. In the present study, we assessed the functional importance of the conserved basic amino-acid residues in HdAly by using site-directed mutants that were produced with a cold-inducible yeast expression system consisting of an expression vector pLTex321sV5H and a host Saccharomyces cerevisiae BY4743. At first, we prepared wild-type HdAly with the yeast expression system and confirmed that this recombinant possesses nearly the same properties as native HdAly with respect to specific activity (1300 U/mg), optimal pH (7.8), optimal temperature (35 °C), and protoplast-producing ability from the brown alga Laminaria japonica. Then, we prepared a series of site-directed mutants by replacing the conserved basic amino-acid residues of HdAly with Ala and determined the alginate-degrading activity of these mutants. As a result, we found that the replacement of Lys95 caused complete inactivation of HdAly and those of Arg92, Arg110, and Arg119 caused 65% or more inactivation. These results indicate that the region spanning Arg92 to Arg119 is closely related to the catalytic activity of HdAly.

Keywords
HdAly, Haliotis discus hannai alginate lyase; PL, polysaccharide-lyase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PCR, polymerase chain reactionAlginate lyase; Abalone; Haliotis discus hannai; Cold-inducible expression in yeast;
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Catalytically important amino-acid residues of abalone alginate lyase HdAly assessed by site-directed mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 43, Issue 6, 6 November 2008, Pages 396–402
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