CLONING, SEQUENCING AND OVEREXPRESSION IN ESCHERICHIA-COLI OF THE ALGINATE-LYASE-ENCODING ALY GENE OF PSEUDOMONAS-ALGINOVORA - IDENTIFICATION OF 3 CLASSES OF ALGINATE LYASES

A gene of Pseudomonas alginovora, called aly, has been cloned in Esche richia coli using a battery of PCR techniques and sequenced. It encode s 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 condi tions in which the formation of inclusion bodies is avoided. His-tagge d P. alginovora Aly has the same enzymic properties as the wild-type e nzyme and has the specificity of a mannuronate lyase. It can be purifi ed in a one-step procedure by affinity chromatography on Ni2+-nitriloa cetate 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 str ucture fall into three distinct classes, one of which comprises the pa ir P. alginovora Aly and Klebsiella pneumoniae Aly.