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 com...

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Bibliographic Details
Published in:DNA sequence 2004-10, Vol.15 (5-6), p.344-350
Main Authors: Han, Feng, Gong, Qian-hong, Song, Kai, Li, Jing-bao, Yu, Wen-gong
Format: Article
Language:English
Subjects:
Alginate lyase
Amino Acid Sequence
AY221030
Base Sequence
Cloning
Cloning, Molecular
DNA Primers
Escherichia coli
Escherichia coli - metabolism
Expression
Gene Expression
Hydrogen-Ion Concentration
Laminaria
Laminaria - microbiology
Molecular Sequence Data
Pacific Ocean
Plasmids - genetics
Polysaccharide-Lyases - genetics
Polysaccharide-Lyases - metabolism
Sequence Alignment
Sequence Analysis, DNA
Transformation, Bacterial
Vibrio
Vibrio - genetics
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Summary: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°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.
ISSN:1042-5179
1029-2365
DOI:10.1080/10425170400019300