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.
Chavagnat
F
F
Laboratoire de Biochimie Analytique et Synthèse Bioorganique, Université Claude Bernard, Villeurbanne, France.
Duez
C
C
Guinand
M
M
Potin
P
P
Barbeyron
T
T
Henrissat
B
B
Wallach
J
J
Ghuysen
J M
JM
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