title: Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp. CY24.
abstract: The alginate lyase encoding gene (alyPI) of marine bacterium Pseudoalteromonas sp. CY24 was cloned using a battery of PCR techniques. Gene alyPI was composed of a 1575 bp open reading frame encoding a protein of 57.4 kDa containing 524 amino acid residues with a signal peptide of 23 amino acids. The AlyPI protein was expressed in Escherichia coli with a His-tag sequence fused at the C-terminal end and purified to electrophoretic homogeneity using Ni-sepharose affinity chromatography. AlyPI was most active at 40 degrees C and pH 7.0 in the presencce of 0.1 mol/L NaCl and stable over a broad range of pH, 6.0-10.6. The presence of Na+, K+, Mn2+, Ca2+, and Fe3+ can enhance the enzyme activity. The alginate lyase consensus region YFKAGXYXQ, regarded as a striking feature at the C termini of several alginate lyase of ~30 kDa, was found in AlyPI, which belongs to the ~60 kDa group. Another nine amino acid consensus region, YXRSELREM, only found in G-specific alginate lyases previously existed in AlyPI, which could degrade sodium alginate, M blocks, and G blocks and appeared to be a broad substrate-specific alginate lyase.
authors.affiliation: Department of Molecular Biology, Ocean University of China, Qingdao, China.
authors.forename: Gaofei
authors.initials: G
authors.lastname: Duan
authors.forename: Feng
authors.initials: F
authors.lastname: Han
authors.forename: Wengong
authors.initials: W
authors.lastname: Yu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D020536:Enzyme Activators; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase