Abstract
Gene coding for a putative endoglucanase was isolated and characterized from a disk abalone Haliotis discus discus cDNA library and was named as hdEndgI. The full length (1,908 bp) abalone cDNA contained a 1,824 bp open reading frame (ORF), encoding 608 amino acids (aa). The predicted molecular mass and isoelectric point were 65 kDa and 4.5, respectively. The C-terminal region showed conserved catalytic site regions, with the catalytic site residues responsible for the endoglucanase activity. RT–PCR results revealed greater hdEndgI mRNA expression in the hepatopancreas than in the digestive tract, suggesting the key site at which the enzyme is produced. hdEndgI mRNA expression decreased from baseline to the 8th week of starvation in the hepatopancreas; and once re-feeding was initiated, mRNA transcription increased to normal levels within 14 days. This suggests that gene transcription is not totally disturbed due to starvation-induced stress, showing the potential of hdEndgI as a molecular tool for identification of food availability. It further suggests that abalone can survive for 8 weeks without food. The purified hdEndgI recombinant fusion protein was shown to have in vitro activity against carboxymethylcellulose. The enzyme exhibits a broad range of pH stability, the optimal temperature and pH being 40°C and 4.5, respectively.
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Bernfeld P (1955) Amylases α and β. Methods Enzymol 1:149–155
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brummell DA, Lashbrook CC, Bennett AB (1994) Plant endo-1,4-β-d-glucanases. ACS Symp Ser 566:100–129
Byrne KA, Lehnert SA, Johnson SE, Moore SS (1999) Isolation of cDNA encoding a putative cellulase in the red claw crayfish Cherex quadricarinatus. Gene 239:317–324
Chauvaux S, Beguin P, Aubert JP (1992) Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD. J Biol Chem 267:4472–4478
Chauvaux S, Souchons H, Alzari PM, Chariot P, Beguin P (1995) Structural and functional analysis of the metal binding sites of Clostridium thermocellum Endoglucanase CelD. J Biol Chem 270(17):9757–9762
Crawford AC, Kricker JA, Anderson AJ, Neil RH, Mather PB (2004) Structure and function of a cellulase gene in redclaw crayfish Cherax quadricarinatus. Gene 340(2):267–274
Gilkes NR, Henrissat B, Kilburn DG, Miller RC, Warren RAJ (1991) Domains in microbial β-1,4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev 55:303–315
Henry M, Boucaud-Camou E, Lefort Y (1991) Functional micro anatomy of the digestive gland of the scallop Pecten maximus (L.). Aquat Living Resour 4:191–202
Khademi S, Guarino LA, Watanabe H, Tokuda G, Meyer EF (2002) Structure of an endoglucanase from termite, Nasutitermes takasagoensis. Acta Crystallogr D 58:653–659
Kumar S, Tamura K, Nei M (2004) MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
Lee SJ, Kim SR, Yoon HJ, Kim I, Lee KS, Je YH, Lee SM, Seo SJ, Sohn HD, Jin BR (2004) cDNA cloning, expression, and enzymatic activity of a cellulose from the mulberry longicorn beetle, Apriona germari. Comp Biochem Physiol Part B 139:107–116
Lo N, Watanabe H, Sugimura M (2003) Evidence for the presence of a cellulase gene in the last common ancestor of bilaterian animals. Proc R Soc Lond B 270:S69–S72 (Suppl)
Loya-Javellana GN, Fielder DR, Thorne MJ (1993) Food choice by free-living stages of the tropical freshwater crayfish. Cherax quadricarinatus (Parastacidae: Decapoda). Aquaculture 118:299–308
Nielsen H, Engelbrecht J, Brunak S, Heijne GV (1997) A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8(5–6):581–599
Nikapitiya C, Oh C, Whang I, Kim CG, Lee YH, Kim SJ, Lee J (2009) Molecular characterization, gene expression analysis and biochemical properties of α-amylase from the disk abalone, Haliotis discus discus. Comp Biochem Physiol Part B 152:271–281
Nishida Y, Suzuki K, Kumagai Y, Tanaka H, Inoue A, Ojima T (2007) Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus. Biochimie 89:1002–1011
Saha BC (2004) Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides. Process Biochem 39:1871–1876
Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucl Acids Res 31:3381–3385
Sheehan J, Himmel M (1999) Enzymes, energy, and the environment: a strategic perspective on the US Department of Energy, Research and Development Activities for Bioethanol. Biotechnol Prog 15:817–827
Smant G, Stokkermans JPWG, Yan Y, JMDe Boer, Baum TJ, Wang X, Hussey RS, Gommers FJ, Henrissat B, Davis EL, Helder J, Schots A, Bakker J (1998) Endogenous cellulases in animals: isolation of β-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proc Natl Acad Sci USA 95:4906–4911
Sugimura M, Watanabe H, Lo N, Saito H (2003) Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow spotted longicorn beetle, Psacothea hilaris. Eur J Biochem 270:3455–3460
Suzuki K, Ojima T, Nishita K (2003) Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai. Eur J Biochem 270:771–778
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680
Tomme P, Warren RAJ, Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. Adv Microbiol Physiol 37:1–81
Watanabe H, Tokuda G (2001) Animal cellulases. Cell Mol Life Sci 58:1167–1178
Watanabe H, Noda H, Tokuda G, Lo N (1998) A cellulase gene of termite origin. Nature 394:330–331
Wong WKR, Gerhard B, Guo ZM, Kilburn DG, Warren AJ, RC Miller Jr (1986) Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi. Gene 44:315–324
Xu B, Hellman U, Ersson B, Janson JC (2000) Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-β-1,4-glucanase from blue mussel, Mytilus edulis. Eur J Biochem 267:4970–4977
Xu B, Janson JC, Sellos D (2001) Cloning and sequencing of a molluscan endo-beta-1,4-glucanase gene from the blue mussel, Mytilus edulis. Eur J Biochem 268(13):3718–3727
Xue XM, Anderson AJ, Richardson NA, Anderson AJ, Xue GP, Mather PB (1999) Characterization of cellulase activity in the digestive system of the redclaw crayfish (Cherax quadricarinatus). Aquaculture 180:373–386
Yan Y, Smant G, Stokkermans J, Qin L, Helder J, Baum T, Schots A, Davis E (1998) Genomic organization of four β-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications. Gene 220:61–70
Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microb Biotechnol 56:17–34
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This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea.
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Nikapitiya, C., Oh, C., De Zoysa, M. et al. Characterization of beta-1,4-endoglucanase as a polysaccharide-degrading digestive enzyme from disk abalone, Haliotis discus discus . Aquacult Int 18, 1061–1078 (2010). https://doi.org/10.1007/s10499-010-9324-9
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DOI: https://doi.org/10.1007/s10499-010-9324-9