Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2.
A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 °C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate.
Department of Food Science and Biotechnology, Kyungsung University, Busan 608-736, Korea.
Su In
SI
Lee
Sung Hee
SH
Choi
Eun Yeol
EY
Lee
Hee Sook
HS
Kim
D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D020615:Stenotrophomonas maltophilia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase