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

• Published in: Carbohydrate research 2001-09, Vol.335 (1), p.11-21
• Main Authors: Sawabe, Tomoo, Takahashi, Hiromasa, Ezura, Yoshio, Gacesa, Peter
• Format: Article
• Language: English
• Subjects: Alginate lyase; Amino Acid Sequence; Bacterial enzymes; Base Sequence; Cloning; Cloning, Molecular; Gammaproteobacteria - enzymology; Gammaproteobacteria - genetics; Molecular Sequence Data; Polysaccharide-Lyases - chemistry; Polysaccharide-Lyases - genetics; Polysaccharide-Lyases - metabolism; Protein Structure, Tertiary; Pseudoalteromonas elyakovii; Restriction Mapping; Sequence analysis; Sequence Homology, Amino Acid; Substrate Specificity; Transformation, Bacterial
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/S0008-6215(01)00198-7

A gene ( alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia 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 p I 5.29. A region G 165 to V 194 in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (p I 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G 165 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 region 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 of Streptomyces chitinases, but did not exhibit any catalytic activity. The putative domain structure of the gene alyPEEC encoding the novel extracellular alginate lyase with broad substrate specificity of Pseudoalteromonas elyakovii was proposed to be comprised of three parts: (1) signal sequence; (2) function unknown-chitin binding domain-like domain A32-G164; (3) mature enzyme domain, G165-N398.