Research On Alginate Lyase From Marine Vibrio Sp. QY105

With the growing demand for resources, caused the drying up of earth’s energysources, the governments pay attentions to the development and utilization of newenergy. Biomass energy has attracted much attention in these new energy. Brownalgae exhibit several key features of an ideal feedstock for production of biofuels andrenewable commodity chemical compounds. Requiring no arable land, fertilizer, orfresh water resources. Large-scale cultivation is practiced in several countries,yielding15million metric tons per year, Rong Cheng yielding1million metric tons（wet weight） per year, which affords abundant raw materials.Alginates are acidic heteropolysaccharides consisting of β-D-mannuronate （M） and itsC5epimer α-L-guluronate （G） organized by the1,4glycosidic bond. Which areorganized in three ways: homopolymeric G blocks （polyG）; homopolymeric M blocks（polyM）; and heteropolymeric M/G blocks （polyMG）. Alginates have been widelyused in the food, textile, printing, and chemical industries since they are founded byStanford from sea-tangles in1881.Alginate lyase catalyzes a β-elimination of glycosidic bonds and produces unsaturatedoligosaccharides with double bonds at the non-reducing end. And yield a productcontaining4-deoxy-L-erythro-hex-4-enopyranosyluronic acid as the nonreducingterminal moiety. According to their substrate specificity, alginate lyases are generallyclassified as polyM lyase （EC4.2.2.3）, polyG lyase （EC4.2.2.11） and alginate lyasecan degrade both of polyG and polyM. Most of the alginate lyases degrade polyMmore efficiently. The combination of alginate lyases with different substratespecificities could facilitate the degradation of alginate. So the enzymes whichdegrade polyG more efficiently are imminently needed. In order to better use brownalgae, the clarification of metabolic pathway for transport and metabolism of alginateoligomers is significant.In this study, an alginate lyase-producing bacterium, QY105, was screened andidentified. Alignment of16S rRNA gene sequences showed that strain QY105had99%identity to many Vibrio strains. Therefore, QY105is a strain of genus Vibrio. Strain QY105secreted90%of the enzyme activity within the first15h offermentation. The polyG-preference alginate lyase, AlyV5, with an apparentmolecular weight of37kDa and a specific activity of2152U/mg was purified fromthe culture supernatant of strain QY105. AlyV5was most active at38°C and pH7.0in20mM Tris-HCl. The enzyme was stable over a broad pH range （6.0-9.0） andremained nearly40%of the activity after incubation at90°C for10min. NaCl hadthe largest stimulatory effect on AlyV5. AlyV5detected1.25%activity with out NaCl.Another monovalent metal ion had no evident effect on its activity. All other cations,such as Cu²⁺, Ni²⁺, Zn²⁺, Ba²⁺, Fe³⁺, Al³⁺showed a significantly inhibitory effectexcept for Mg²⁺, Ca²⁺, Mn²⁺, The chelating agent EDTA inhibited the activity ofAlyV5. Detergent SDS had no effect on the activity of AlyV5.AlyV5showed activities towards both polyG and polyM, but degraded the formermore efficiently. AlyV5mainly produced disaccharide, trisaccharide andtetrasaccharide from polyG, trisaccharide, tetrasaccharide and pentasaccharide frompolyM. Indicating that AlyV5uses pentasaccharide as a minimum substrate of polyG,hexose as a minimum substrate of polyM and splits their central glycosidic bond.Combinate AlyV5with polyM-preference alginate lyases can degrade alginateefficiently.In order to research alginate metabolic systems, three of the encoding genes ofoligoalginate lyases were cloned using degenerate PCR and SiteFinding-PCR. Theamino acid sequence of this three oligoalginate lyases deduced from the full-lengthgene was aligned using NCBI Basic Local Alignment Search Tool （BLAST）. Thesequence of this three oligoalginate lyases exhibited more than90%homology witholigoalginate lyases from Vibrio.The found these lyases accelerating the process of using alginate to productebioethanol and providing a new method to solve the energy crisis.