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Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome

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Abstract

A cellulase encoding gene, Cel PRII, was identified from Mehsani buffalo rumen metagenome, and cloned and expressed in Escherichia coli BL21(DE3)pLysS. The 1170 bp full length gene encodes a 389 residue polypeptide (Cel PRII) containing a catalytic domain belonging to glycosyl hydrolase (GH) 5 family. The fusion protein consisting of the Cel PRII, thioredoxin tag and 6x Histidine tag with predicted molecular weight of 63 kDa when recovered from inclusion bodies under denaturing conditions, exhibited cellulolytic activity against carboxymethyl cellulose (CMC). Recombinant Cel PRII was stable in the pH range 4.0–10.0 with pH optima 6.0. The optimal reaction temperature of Cel PRII was 30 °C with more than 50% of its activity retained at the temperatures ranging from 0 to 50 °C. Cel PRII exhibited enhanced enzymatic activity in the presence of Mn2+ ions and was inhibited in the presence of chelating agent EDTA. The K m and V max values for CMC were found to be 166 mg/mL and 1292 IU/mg, respectively. Cel PRII identified in the present study may act as an excellent candidate for industrial applications, and may aid in lignocellulosic biomass conversion because of its potential cellulolytic activity, thermostability, and excellent pH stability.

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Abbreviations

GH:

Glycosyl hydrolase

CMC:

Carboxymethyl cellulose

ORF:

Open reading frame

DNS:

3,5-Dinitrosalicylic acid

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

EDTA:

Ethylene diamine tetraacetic acid

References

  • Andreaus J, Azevedo H, Cavaco-Paulo A (1999) Effects of temperature on the cellulose binding ability of cellulase enzymes. J Mol Catal B Enzym 7:233–239

    Article  CAS  Google Scholar 

  • Anwar Z, Gulfraz M, Irshad M (2014) Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review. J Radiat Res Appl Sci 7:163–173

    Article  CAS  Google Scholar 

  • Aspeborg H, Coutinho PM, Wang Y, Brumer H, Henrissat B (2012) Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol 12:186

    Article  CAS  Google Scholar 

  • Bao L, Huang Q, Chang L, Zhou J, Lu H (2011) Screening and characterization of a cellulase with endocellulase and exocellulase activity from yak rumen metagenome. J Mol Catal B Enzym 73:104–110

    Article  CAS  Google Scholar 

  • Bashir Y, Pradeep Singh S, Kumar Konwar B (2014) Metagenomics: an application based perspective. Chin J Biol 2014. doi:10.1155/2014/146030

  • Bhat M, Bhat S (1997) Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv 15:583–620

    Article  CAS  Google Scholar 

  • Brulc JM, Antonopoulos DA, Miller MEB, Wilson MK, Yannarell AC, Dinsdale EA, Edwards RE, Frank ED, Emerson JB, Wacklin P (2009) Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases. Proc Natl Acad Sci 106:1948–1953

    Article  CAS  Google Scholar 

  • Cheema TA, Jirajaroenrat K, Sirinarumitr T, Rakshit SK (2012) Isolation of a gene encoding a cellulolytic enzyme from swamp buffalo rumen metagenomes and its cloning and expression in Escherichia coli. Anim Biotechnol 23:261–277

    Article  CAS  Google Scholar 

  • Daniel R (2005) The metagenomics of soil. Nature Rev Microbiol 3:470–478

    Article  CAS  Google Scholar 

  • Duan CJ, Xian L, Zhao GC, Feng Y, Pang H, Bai XL, Tang JL, Ma QS, Feng JX (2009) Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens. J Appl Microbiol 107:245–256

    Article  CAS  Google Scholar 

  • Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  CAS  Google Scholar 

  • Feng Y, Duan C-J, Pang H, Mo X-C, Wu C-F, Yu Y, Hu Y-L, Wei J, Tang J-L, Feng J-X (2007) Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl Microbiol Biotechnol 75:319–328

    Article  CAS  Google Scholar 

  • Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A (2005) Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ Microbiol 7:1996–2010

    Article  CAS  Google Scholar 

  • Ferrer M, Martínez-Martínez M, Bargiela R, Streit WR, Golyshina OV, Golyshin PN (2016) Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. Microb Biotechnol 9:22–34

    Article  CAS  Google Scholar 

  • Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA (2008) Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nature Rev Microbiol 6:121–131

    Article  CAS  Google Scholar 

  • Franco-Cirigliano MN, Rezende RdC, Gravina-Oliveira MP, Pereira PHF, Do Nascimento RP, Bon EPdS, Macrae A, Coelho RRR (2013) Streptomyces misionensis PESB-25 produces a thermoacidophilic endoglucanase using sugarcane bagasse and corn steep liquor as the sole organic substrates. BioMed Res Int 2013. doi:10.1155/2013/584207

  • Godfrey T, West S (1996) Textiles. In: Industrial enzymology. Macmillan Press, London, pp 360–371

  • Gong X, Gruniniger R, Forster R, Teather R, McAllister T (2013) Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library. Appl Microbiol Biotechnol 97:2423–2431

    Article  CAS  Google Scholar 

  • Gullert S, Fischer MA, Turaev D, Noebauer B, Ilmberger N, Wemheuer B, Alawi M, Rattei T, Daniel R, Schmitz RA, Grundhoff A, Streit WR (2016) Deep metagenome and metatranscriptome analyses of microbial communities affiliated with an industrial biogas fermenter, a cow rumen, and elephant feces reveal major differences in carbohydrate hydrolysis strategies. Biotechnol Biofuels 9:121. doi:10.1186/s13068-016-0534-x

    Article  Google Scholar 

  • Healy F, Ray R, Aldrich H, Wilkie A, Ingram L, Shanmugam K (1995) Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose. Appl Microbiol Biotechnol 43:667–674

    Article  CAS  Google Scholar 

  • Hess M, Sczyrba A, Egan R, Kim T-W, Chokhawala H, Schroth G, Luo S, Clark DS, Chen F, Zhang T (2011) Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331:463–467

    Article  CAS  Google Scholar 

  • Isikgor FH, Becer CR (2015) Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym Chem 6:4497–4559

    Article  CAS  Google Scholar 

  • Jørgensen H, Kristensen JB, Felby C (2007) Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod Biorefin 1:119–134

    Article  Google Scholar 

  • Juturu V, Wu JC (2014) Microbial cellulases: engineering, production and applications. Renew Sustain Energy Rev 33:188–203

    Article  CAS  Google Scholar 

  • Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J (2012) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7:1511–1522

    Article  Google Scholar 

  • Kim SC, Kang SH, Choi EY, Hong YH, Bok JD, Kim JY, Lee SS, Choi YJ, Choi IS, Cho KK (2016) Cloning and characterization of an endoglucanase gene from Actinomyces sp. Korean native goat 40. Asian Australas J Anim Sci 29:126

    Article  CAS  Google Scholar 

  • Krause DO, Denman SE, Mackie RI, Morrison M, Rae AL, Attwood GT, McSweeney CS (2003) Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics. FEMS Microbiol Rev 27:663–693

    Article  CAS  Google Scholar 

  • Kuhad RC, Gupta R, Singh A (2011) Microbial cellulases and their industrial applications. Enzyme Res 2011. doi:10.4061/2011/280696

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  Google Scholar 

  • Liu L, Feng Y, Duan C-J, Pang H, Tang J-L, Feng J-X (2009) Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World J Microbiol Biotechnol 25:1035–1042

    Article  Google Scholar 

  • Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nature Rev Microbiol 3:510–516

    Article  CAS  Google Scholar 

  • Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577

    Article  CAS  Google Scholar 

  • Mackie RI (1997) Gut environment and evolution of mutualistic fermentative digestion. In: Mackie RI, White BA (eds) Gastrointestinal microbiology. Springer, New York, pp 13–35

    Chapter  Google Scholar 

  • Miller G (1959) Modified DNS method for reducing sugars. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  • Palackal N, Lyon CS, Zaidi S, Luginbühl P, Dupree P, Goubet F, Macomber JL, Short JM, Hazlewood GP, Robertson DE (2007) A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut. Appl Microbiol Biotechnol 74:113–124

    Article  CAS  Google Scholar 

  • Patel DD, Patel AK, Parmar NR, Shah TM, Patel JB, Pandya PR, Joshi CG (2014) Microbial and carbohydrate active enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet. Gene 545:88–94

    Article  CAS  Google Scholar 

  • Pirzadah TB, Malik B, Kumar M, Rehman RU (2014) Lignocellulosic biomass: as future alternative for bioethanol production. In: Hakeem K, Jawaid M, Rashid U (eds) Biomass Bioenergy. Springer, Switzerland, pp 145–163

    Google Scholar 

  • Pol D, Laxman RS, Rao M (2012) Purification and biochemical characterization of endoglucanase from Penicillium pinophilum MS 20. Indian J Biochem Biophys

  • Rashamuse K, Visser DF, Hennessy F, Kemp J, Roux-van der Merwe M, Badenhorst J, Ronneburg T, Francis-Pope R, Brady D (2013) Characterisation of two bifunctional cellulase–xylanase enzymes isolated from a bovine rumen metagenome library. Curr Microbiol 66:145–151

    Article  CAS  Google Scholar 

  • Rondon MR, Goodman RM, Handelsman J (1999) The Earth’s bounty: assessing and accessing soil microbial diversity. Trends Biotechnol 17:403–409

    Article  CAS  Google Scholar 

  • Tomme P, Warren R, Gilkes N (1995) Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol 37:1–81

    Article  CAS  Google Scholar 

  • Voget S, Steele H, Streit W (2006) Characterization of a metagenome-derived halotolerant cellulase. J Biotechnol 126:26–36

    Article  CAS  Google Scholar 

  • Wong DD, Chan VJ, McCormack AA, Batt SB (2010) A novel xyloglucan-specific endo-β-1,4-glucanase: biochemical properties and inhibition studies. Appl Microbiol Biotechnol 86:1463–1471

    Article  CAS  Google Scholar 

  • Yang J, Dang H (2011) Cloning and characterization of a novel cold-active endoglucanase establishing a new subfamily of glycosyl hydrolase family 5 from a psychrophilic deep-sea bacterium. FEMS Microbiol Lett 325:71–76

    Article  CAS  Google Scholar 

  • Zhao S, Wang J, Bu D, Liu K, Zhu Y, Dong Z, Yu Z (2010) Novel glycoside hydrolases identified by screening a Chinese Holstein dairy cow rumen-derived metagenome library. Appl Environ Microbiol 76:6701–6705

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are thankful to the supporting staff at Department of Animal Nutrition, Anand Agricultural University, Anand, for their support in sample collection. This work was supported by Niche Area of Excellence project funded by Indian Council of Agricultural Research, New Delhi. The first author is also thankful to UGC-CSIR (New Delhi) for providing financial support in the form of fellowship.

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Authors and Affiliations

  1. Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, 388110, India

    Ravi K. Shah, Amrutlal K. Patel, Deepti M. Davla, Ishan K. Parikh, Subhash J. Jakhesara & Chaitanya G. Joshi

  2. P. G. Department of Biosciences, Sardar Patel University, Bakrol, 388315, India

    Ravi K. Shah, Ramalingam B. Subramanian & Kamlesh C. Patel

  3. Hester Biosciences Ltd., Merda-Adraj, Kadi, 382728, India

    Amrutlal K. Patel

  4. Zydus Vaccine Technology Centre, Changodar, Ahmedabad, 382213, India

    Ishan K. Parikh

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  1. Ravi K. Shah
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Correspondence to Chaitanya G. Joshi.

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Shah, R.K., Patel, A.K., Davla, D.M. et al. Molecular cloning, heterologous expression, and functional characterization of a cellulolytic enzyme (Cel PRII) from buffalo rumen metagenome. 3 Biotech 7, 257 (2017). https://doi.org/10.1007/s13205-017-0895-2

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