Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic Library
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A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.
Abstract
A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-β-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.
참고문헌 (33)
- Amann RI, "Phylogenetic identification and in situ detection of individual microbial cells without cultivation", Microbiol. Rev, 59, pp.143~169 (1995)
- Balachandrababu Malini Asha, "Purification and Characterization of a Thermophilic Cellulase from a Novel Cellulolytic Strain, Paenibacillus barcinonensis", Journal of Microbiology and Biotechnology, 22(11), pp.1501~1509 (2012), 한국미생물·생명공학회[DOI]
- Beguin P, "The biological degradation of cellulose", FEMS Microbiol. Rev, 13, pp.25~58 (1994)
- Bhat MK, "Cellulose degrading enzymes and their potential industrial applications", Biotechnol. Adv, 15, pp.583~620 (1997)
- B olam DN, "Pseudomonas cellulosebinding domains mediate their effects by increasing enzyme substrate proximity", Biochem. J, 331, pp.775~781 (1998)
- Ca ntarel BL, "The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics", Nucleic Acids Res, 37, pp.233~238 (2009)
- Deka D, "Purification and characterization of an alkaline cellulase produced by Bacillus subtilis (AS3)", Prep. Biochem. Biotechnol, 43, pp.256~270 (2013)
- Diener S, "Conversion of organic material by black soldier fly larvae: establishing optimal feeding rates", Waste Manag. Res, 27, pp.603~610 (2009)
- Junli Dong, "Molecular Cloning, Purification, and Characterization of a Novel, Acidic, pH-Stable Endoglucanase from Martelella mediterranea", The Journal of Microbiology, 48(3), pp.393~398 (2010), 한국미생물학회
- Doukyu N, "Organic solvent-tolerant enzymes", Biochem. Eng. J, 48, pp.270~282 (2010)
- Duan CJ, "Mining metagenomes for novel cellulase genes", Biotechnol. Lett, 32, pp.1765~1775 (2010)
- Gon g X, "Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene", BMC Res. Notes, 5, pp.566~ (2012)
- Ha ndelsman J, "Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products", Chem. Biol, 5, pp.245~249 (1998)
- Jagtap SS, "Characterization of a novel endo-beta-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis", Appl. Microbiol. Biotechnol, 98, pp.661~669 (2014)
- Jeon JH, "Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome", Appl. Microbiol. Biotechnol, 81, pp.865~874 (2009)
- Ki m SJ, "Characterization of a gene encoding cellulase from uncultured soil bacteria", FEMS Microbiol. Lett, 282, pp.44~51 (2008)
- KIM, SOO-JIN, "Screening and Characterization of an Enzyme with β-Glucosidase Activity from Environmental DNA", Journal of Microbiology and Biotechnology, 17(6), pp.905~912 (2007), 한국미생물·생명공학회
- Wontae Kim, "Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae", BMB Reports, 44(6), pp.387~392 (2011), 생화학분자생물학회
- Ko KC, "A novel bifunctional endo-", exo-type cellulase from an anaerobic ruminal bacterium, 2011, pp.89, 1453~1462
- L ee JK, "Wohlfahrtiimonas larvae sp. nov., isolated from the larval gut of Hermetia illucens (Diptera: Stratiomyidae)", Antonie Van Leeuwenhoek, 105, pp.15~21 (2014)
- L i Q, "Bioconversion of dairy manure by black soldier fly (Diptera: Stratiomyidae) for biodiesel and sugar production", Waste Manag, 31, pp.1316~1320 (2011)
- Li X, "Purification and characterization of an organic solvent-tolerant alkaline cellulase from a halophilic isolate of Thalassobacillus", Biotechnol. Lett, 34, pp.1531~1536 (2012)
- Linder M, "The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose", Proc. Natl. Acad. Sci. USA, 93, pp.12251~12255 (1996)
- Liu J, "Cloning and functional characterization of a novel endobeta- 1,4-glucanase gene from a soil-derived metagenomic library", Appl. Microbiol. Biotechnol, 89, pp.1083~1092 (2011)
- Lynd LR, "Microbial cellulose utilization: fundamentals and biotechnology", Microbiol. Mol. Biol. Rev, 66, pp.506~577 (2002)
- Mar geot A, "New improvements for lignocellulosic ethanol", Curr. Opin. Biotechnol, 20, pp.372~380 (2009)
- Nacke H, "Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes", Biotechnol. Lett, 34, pp.663~675 (2012)
- Streit WR, "Metagenomics - the key to the uncultured microbes", Curr. Opin. Microbiol, 7, pp.492~498 (2004)
- Tringe SG, "Comparative metagenomics of microbial communities", Science, 308, pp.554~557 (2005)
- Turner P, "Potential and utilization of thermophiles and thermostable enzymes in biorefining", Microb. Cell Fact, 6, pp.9~ (2007)
- Voget S, "Characterization of a metagenome-derived halotolerant cellulase", J. Biotechnol, 126, pp.26~36 (2006)
- Wu J, "Enhancing enzymatic s accharification of waste newsprint by surfactant addition", Biotechnol. Prog, 14, pp.649~652 (1998)
- Zaldivar J, "Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration", Appl. Microbiol. Biotechnol, 56, pp.17~34 (2001)