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cDNA cloning and bacterial expression of an endo-β-1,4-mannanase, AkMan, from Aplysia kurodai

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Title: cDNA cloning and bacterial expression of an endo-β-1,4-mannanase, AkMan, from Aplysia kurodai
Authors: Zahura, Umme Afsari Browse this author
Rahman, Mohammad Matiur Browse this author
Inoue, Akira Browse this author →KAKEN DB
Tanaka, Hiroyuki Browse this author →KAKEN DB
Ojima, Takao Browse this author →KAKEN DB
Keywords: Aplysia
mollusks
gastropod
β-1,4-mannanase
mannan
bacterial expression
Issue Date: Aug-2011
Publisher: Elsevier
Journal Title: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Volume: 159
Issue: 4
Start Page: 227
End Page: 235
Publisher DOI: 10.1016/j.cbpb.2011.05.001
PMID: 21601647
Abstract: Previously we isolated an endo-β-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1,392 bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96 Da. Comparison of the primary structure of AkMan with other β-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 β-1,4-mannanases indicated that AkMan together with other molluscan β-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21(DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells.
Type: article (author version)
URI: http://hdl.handle.net/2115/46815
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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