To date, several microorganisms that can metabolize alginate have been characterized. In these microorganisms,
a class of enzymes called alginate lyases (Alys) catalyze the depolymerization of alginate into oligomers via an
endolytic β-elimination reaction. But it is not clear how these lyases differ on their enzymological functions or their divisions
of labor. We focused on three extracellular secretory alginate lyases in the Gram-negative marine bacterium
V.alginolyticus (ATCC 17749). We found differences in lyase function and catalytic specificity, depending on substrates.
These properties apparently derived from demonstrable changes to the protein sequences. We found shared structural domains
in alginate lyases specific to the pM and pG substrates through the measurements on enzyme activities and kinetic
parameters. Substitution of hydrophobic amino acids in the isoleucine site of domain QIH could have an enormous influence
on the high-affinity to pM or pG. This isoleucine was reconfirmed to be indispensable for recognition of the pG or
G-G bond. We identified key amino acids located in the catalytic center by means of protein site-directed mutagenesis,
and found specific amino acid fragments with exclusive recognitional and catalytic activity effects for different substrates.
We reconstructed a series of proteins through splicing, with the goal of producing an engineered protein with the ability to
degrade alginate on a variety of substrates and with high enzyme activity as well as temperature stability. Our study provides
the theoretical basis for future work on alginate oligosaccharide, transport mechanisms, an area of research with potentially
large ramifications for the chemical, medical, textile, printing and agricultural industries.
Keywords: Alginate lyase, chimeric protein, ESI-MS, NMR, site-directed mutagenesis, substrate specificity.
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