Cloning, sequence analysis and expression of gene alyVI encoding alginate lyase from marine bacterium Vibrio sp. QY101.
The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp. QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli. Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa. AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography. AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl2. A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI. However, AlyVI could degrade both M block and G block. These results indicate that a novel alginate lyase-encoding gene has been cloned.
Han
Feng
F
Key Laboratory of Marine Drugs, Ocean University of China, Ministry of Education, P.R. China.
Gong
Qian-Hong
QH
Song
Kai
K
Li
Jing-Bao
JB
Yu
Wen-Gong
WG
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007795:Laminaria / Q000382:microbiology*; D008969:Molecular Sequence Data; D010140:Pacific Ocean; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial; D014733:Vibrio / Q000235:genetics*
D017931:DNA Primers; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase