open access

https://hdl.handle.net/2115/38497

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

英語

site-directed mutagenesis

2008-11-06

Elsevier

Enzyme and Microbial Technology

43(6)

396-402

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 sitedirected 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 wildtype 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℃), 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.

学術雑誌論文

Accepted Manuscript

0141-0229

AA00180542