Study On Oligoalginate Lyases From Marine Bacterium Shewanella Sp. Kz7

Alginate is an acidic hetero-polysaccharide consisting of β-D-mannuronate (M)and α-L-guluronate (G) arranged as a poly M-block, a poly G-block, and analternating or random poly MG-block. As the most abundant carbohydrate in brownseaweed, alginate is a commercially useful polysaccharide that is widely used infood and pharmaceutical industry due to its high viscosity and gelling properties.Alginate lyase is an enzyme that catalyzes the degradation of alginate by aβ-elimination mechanism, introducing a double bond between C4and C5at thenonreducing end showing strong absorption at235nm. In the Carbohydrate-ActiveenZYmes (CAZy) database, alginate lyases belong to the polysaccharide lyasefamilies (PL). Among the total of21PL families, the PL families containing alginatelyases are only PL-5,6,7,14,15,17, and18. The mode of degradation of alginateby alginate lyase can be classified into endo-and exo-lytic fashion. Endotypealginate lyases give rise to unsaturated di-, tri-, and tetra-saccharides as thedegradation products. Exotype oligoalginate lyase can degrade both alginate polymerand oligomeric alginate mainly into unsaturated monomers that nonenzymaticallytransformed to4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The greatmajority of alginate lyases have been reported to be endotype enzymes; while onlyfive oligoalginate lyases have been reported.In this study, two new oligoalginate lyases OalS6and OalS17from the marinederived bacteria Shewanella sp. Kz7were cloned, purified and characterized. Thenovel oligoalginate lyase encoded by oalS6gene has no significant sequencesimilarity with other known oligoalginate lyase. OalS6belonged to PL-6based onthe results of multiple sequence alignments between OalS6and other known PL-6lyases. Based on the conserved domains analysis, it contained a chondroitinase-likedomain structurally different from other known oligoalginate lyases. OalS17exhibited the highest sequence identity of49%with oligoalginate lyase fromStenotrophomonas maltophilia KJ-2which was classified into the PL-17family. OalS6is a poly G specific enzyme that depolymerizes poly G-block andoligoalginate into an unsaturated monomeric sugar acid with an exolytic pattern. Therecombinant oligoalginate lyase exhibited the highest activity at pH7.3and40°C.OalS6remained80%and60%of activity after incubation at40°C and45°C for1h,respectively; and it was stable at acidities ranging from pH6.2to pH8.2. NaCl andKCl could enhance the activity of OalS6markedly, while its activity was notdependent on them.The recombinant oligoalginate lyase OalS17consisting of729amino acids waspurified on Ni-Sepharose column and exhibited the highest activity at pH6.8and50°C. OalS17remained88%of activity after incubation at40°C1h, and it was stableat acidities ranging from pH6to pH9. In the test of substrate specificity, OalS17preferentially degraded the glycosidic bond of poly M-block than poly G-block intoa monomeric sugar acid. OalS17could be used in combination with other alginatelyases for a synergistic saccharification of alginate.The main products of OalS6were assigned from ESI-MS, one-dimensional1H-NMR,13C-NMR and two-dimensional1H-1H TOCSY NMR spectra. Thestructure was2,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (TPC),in contrast to the main products of other known oligoalginate lyases that are4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). TPC were mixtures of twoconformational isomers in C2and C6, and they might be formed by the hydrationand cyclization reactions of DEH.In this study, an oligoalginate lyase-producing bacterial strain, Shewanella sp.Kz7, was isolated from sea mud of Jiaozhou Bay. Two oligoalginate lyase genesoalS6and oalS17were cloned from strain Kz7and expressed in Escherichia coli. Toour knowledge, OalS6is the first oligoalginate lyase belonging to PL-6. OalS6andOalS17could be used in combination with other alginate lyases for a synergisticsaccharification of alginate.