Molecular Identification of Sphingomonas sp.A1 Alginate Lyase(Al-IV')as a Member of Novel Polysaccharide Lyase Family 15 and Implications in Alginate Lyase Evolution

摘要

Sphingomonas sp.Al(strain Al)produces three endotypes(Al-I [65 kDa],Al-II [25 kDa],and A1-III [40 kDa])and an exotype(Al-IV [86 kDa])alginate lyases in cytoplasm.These four enzymes cooperatively depolymerize alginate into constituent monosaccharides.In addition to the genes for these lyases,novel genes encoding hypothetical proteins homologous with Al-IV were found in the genomes of many bacteria including strain Al.One such protein,Al-IV(90 kDa)of strain Al,was overexpressed in Escherichia coli cells,purified,and characterized.Al-IV catalyzed the cleavage of glycosidic bonds in alginate through a beta-elimination reaction and released unsaturated di- and trisaccharides as main products,thus indicating that the enzyme is an endo-type alginate lyase.Al-IV,which differed from Al-IV in some enzymatic properties,was not expressed in strain Al,suggesting that Al-IV has no significant role in alginate metabolism.Al-IV and other Al-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures,implying the evolution route of alginate lyases in family PL-15.