Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases

摘要: A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery PCR techniques and sequenced. It encodes 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, the form 233-amino-acid precursor. P. alginovora Aly overproduced E. with His-tag sequence fused at C-terminal end under conditions which formation inclusion bodies is avoided. His-tagged same enzymic properties as wild-type enzyme specificity mannuronate lyase. can be purified one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin. The yield 5 mg per litre culture. amplification factor 12.5 compared level production alginovora. six lyases known primary structure fall into three distinct classes, one comprises pair Klebsiella pneumoniae Aly.