Study On The ι-carrageenases Of Marine Bacterium Cellulophaga Sp.QY3

Carrageenases are kinds of glycoside hydrolases which could cleave the internalβ (1â†'4) linkages of carrageenans. Based on the substrate specificity, the enzymes areclassified into three groups, namely, κ-, ι-, and λ-carrageenases. ι-Carrageenases havebeen classified into family GH82according to the sequence and structure. Up to now,nine gene sequences of family GH82ι-carrageenases have been found, but only fourof them have been characterized. The relationship between structures and functions ofGH82is still in its infancy. Only the catalytic residues have been identified. Therefore,the discovery and study of the new ι-carrageenases are important not only for the richof GH82but also for the interpretation of the relationship between structures and theirfunctions.The study on ι-carrageenase CgiA from Cellulophaga sp. QY3was continued onthe basis of the preliminary work of our lab. At first, we studied the degradationmethods of CgiA on substrates in different conformations. The degradation of solubleι-carrageenans was by random-type cleaving. In contrast, the degradation of gelsubstrates was by processive-type. It was the first time to report that GH82ι-carrageenase showed different types of degradations which depended on the formsof substrates.Secondly, the three-dimensional structure of CgiA was obtained byhomology modeling. Combined the structure and the electrophoresis analysis, wefound that CgiA cleaved ι-carrageenan yielding neo-ι-carrabiose andneo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was theminimum substrate. The number of subsites for CgiA was eight (-4~+4) and thehexasaccharide bound to subsites4to+2.The sequence logo of GH82showed the amino acid residues in catalyticcavity which closed to the substrate within6. The amino acid residues at subsite-1and+1were more conserved than those at other subsites. Based on the conservation and position in three-dimensional structure, we chose six residues(G228, Y229, R254, R314, K335and K480) for site-directed mutagenesis.Based on the site-directed mutagenesis of the substrate-binding residues infamily GH82, we could infer that the conservations of residues were positivecorrelation to the the importance of them. Site-directed mutagenesis followed bykinetic and structure analyses identified four residues at subsite-1and+1of CgiA,G228, Y229, R254and R314, which played important roles for substrate binding.G228, Y229and R254were strictly conserved in family GH82ι-carrageenases,therefore we inferred they were the important substrate binding residues for GH82.The most important binding residues (Y229and R254in CgiA) were interactspecifically with the sulfate groups of the sugar substrate located at subsite-1and+1and this may shed light on the mechanism of the substrate specificity for family GH82toward ι-carrageenan. It was the first time to identify the substrate binding residuesand explain the mechanism of the substrate specificity for GH82.The gene for a new ι-carrageenase, CgiB, from Cellulophaga sp. QY3wascloned by degenerate PCR and Sitefinding PCR. It comprised an open readingframe of1386bp, which encodes a protein consisting of461amino acid residues.According to the sequence analysis, CgiB was categorized as a new member ofGH family82and shared the highest identity of32%in amino acids withι-carrageenase CgiA2from Zobellia galactanovorans except an uncharacterizedι-carrageenase.The recombinant CgiB showed maximum specific activity at45°C and pH6.5.The enzyme retained70%of the original activity after incubation at the temperaturesbelow40°C for1h. It was stable between pH6.0-9.0. Cations Na+and K+increasedthe activity of CgiB significantly. CuCl2, SDS and EDTA reduced the enzyme activitysignificantly, while most divalent metal cations, such as Mg2+, Fe2+and Mn2+,increased the enzyme activity notably.According to the size-exclusion chromatography analysis on the degradationproducts, CgiB was an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages ofι-carrageenan, yielding tetrasaccharide as the main product (more than80%of the total product). By the secondary mass spectrometry analysis, we identified the mainproduct was neo-ι-carratetraose showing CgiB cleaved the internal β (1â†'4) linkagesof ι-carrageenan. The characterization of CgiB was different from other reportedι-carrageenases, which showing great benefit not only for the rich of ι-carrageenasesbut also for the production and purification of carrageenan oligosaccharides.