Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase

A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Esche richia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A r egion G(165) to V-194 in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alg inate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) a nd isoelectric point (pI 4.78) of the region resembled those of the purifie d enzyme. Expression of enzymically-active alginate lyase from alyPEEC requ ired growth of recombinant E. coli in LB broth containing 50% (v/v) artific ial seawater (ASW). Alginate lyase activity with broad substrate specificit y was detected in both 42 and 30 kDa products. Subcloning of the region G(1 65) to N-398 of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A re gion A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain o f Streptomyces chitinases, but did not exhibit any catalytic activity. (C) 2001 Elsevier Science Ltd. All rights reserved.