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cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula

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Please use this identifier to cite or link to this item:http://hdl.handle.net/2115/50387

Title: cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula
Authors: Rahman, Mohammad Matiur Browse this author
Wang, Ling Browse this author
Inoue, Akira Browse this author →KAKEN DB
Ojima, Takao Browse this author →KAKEN DB
Keywords: Alginate lyase
Littorina brevicula
Marine snail
LbAly28
cDNA cloning
Issue Date: 1-Oct-2012
Publisher: Elsevier
Journal Title: Carbohydrate Research
Volume: 360
Start Page: 69
End Page: 77
Publisher DOI: 10.1016/j.carres.2012.05.019
PMID: 22940178
Abstract: Herbivorous marine snails like Littorina sp. are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, i.e., replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.
Type: article (author version)
URI: http://hdl.handle.net/2115/50387
Appears in Collections:水産科学院・水産科学研究院 (Graduate School of Fisheries Sciences / Faculty of Fisheries Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 尾島 孝男

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