Study On Alginate Lyase AlyV4 From Marine Bacterium Vibrio Sp.QY104

Alginate, a major structural material in cell-wall matrices of brown algae and in biofilms of certain bacteria, is a linear polysaccharide composed of P-D-mannuronic acid (M) and a-L-guluronic acid (G). The two monomers are linked together by a-/β-1,4-linkages arranged in three types of block structures:poly-a-L-guluronate (polyG), poly-β-Dmannuronate (polyM), and heteropolymeric (polyMG) regions.Alginate has been recognized as a useful polysaccharide in various industrial fields, and alginate oligosaccharides are also shown to exhibit some biological activities.Alginate lyases catalyze the depolymerization of alginates by β-elimination reaction to produce oligosaccharides possessing an unsaturated sugar at the new non-reducing terminus. There are several potential applications for alginate lyase, such as producing alginate-derived oligosaccharides, treating the lungs of CF sufferers and preparing protoplasts of marine algae.In this study,13 strains with high production of alginate lyase have been isolated from seawater, mud and rotten algae. The substrate specificity results demonstrated that only the culture supernatant of strain QY014 had the specificity for polyG block. Strain QY104 was identified to belong to genus Vibrio based on its 16S rRNA gene sequence.An alginate lyase was purified from the culture supernatant of strain QY104 by ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatograpy. SDS-PAGE analysis showed that it had an apparent molecular mass of 33 kDa. The alginate lyase degraded alginate at an optimum pHof 7.6 and optimum temperature of 45℃. The enzyme was stable over pH7.0-10.6 and at temperatures below 45℃. The addition of NaCl enhanced its activity and thermostability markedly. In addition, the enzymatic activity was significantly inhibited by Cu2+, Fe3+, EDTA and somewhat inhibited by Fe2+,Al3+,Ba2+, Ni+, Zn2+, Mn2+ and SDS. The FACE analyses showed that alginate lyase mainly released DP 2-4 oligosaccharides from polyG and DP 2-5 oligosaccharides from alginate.According to the partial amino acids sequence of the alginate lyase, its encoding gene alyV4 was cloned using degenerate PCR and sitefinding PCR. The gene alyV4 consisted of a 1359bp open reading frame, encoding 452 amino acid residues. The deduced protein has a theretical molecular mass of 50 kDa and a theoretical isolectric point of 4.6. Sequence analysis revealed that alginate lyase AlyV4 belongs to polysaccharide lyase 7 (PL-7), and it shared 63% identity with the alginate lyase from Saccharophagus sp. Myt-1. AlyV4 has three domains:a singal peptide (S), a N-terminal carbohydrate-binding module (N) and a C-terminal alginate lyase sequence(C). The alginate lyase purifed from the culture supernatant of strain QY104 is the C-terminal alginate lyase of AlyV4.The alginate lyase gene alyV4 is subcloned into pET24a (+). The sesualting plasmid was transformed into E.coli BL21 (DE3). The cells were grown at 37℃ in Luria-Bertani (LB) broth containing 30 μg/ml kanamycin until mid-growth then induced at 25℃ with 0.1 mM IPTG for 20 h. Harvested cells were sonicated, and the released recombinant protein was purified using a Histrap column. The properties of recombinant protein AlyV4-C and alginate lyase AlyV4 are consistent, and there are some differences between the characterization of recombinant Aly V4-NC and that of AlyV4-C. The recombinant AlyV4-NC was cleaved after two weeks at 4℃.