Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases.
A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.
Laboratoire de Biochimie Analytique et Synthèse Bioorganique, Université Claude Bernard, Villeurbanne, France.
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F
Chavagnat
C
C
Duez
M
M
Guinand
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P
Potin
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T
Barbeyron
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B
Henrissat
J
J
Wallach
J M
JM
Ghuysen
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017421:Sequence Analysis
D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase