Bioprocess and Biosystems Engineering

, Volume 40, Issue 5, pp 651–662 | Cite as

Production and characteristics of the recombinant extracellular bifunctional endoglucanase of the polyextremophilic bacterium Bacillus halodurans and its applicability in saccharifying agro-residues

  • Ranjitha R. Prabhu
  • Deepak Parashar
  • T. SatyanarayanaEmail author
Research Paper


The recombinant alkalistable and moderately thermostable bifunctional endoglucanase gene (BhCell-Xyl) of polyextremophilic bacterium Bacillus halodurans TSLV1 has been expressed in Pichia pastoris under constitutive GAP as well as inducible AOX promoters. A higher titre of recombinant BhCell-Xyl was attained after induction (4.8 U mL−1) as compared to that of the constitutive production (2.1 U mL−1). The recombinant P. pastoris strains integrated two copies of BhCell-Xyl under AOX and GAP promoters. The pure recombinant BhCell-Xyl is a glycoprotein of 66 kDa, which is optimally active at 60 °C and pH 6.0 and 8.0. Glycosylated BhCell-Xyl exhibits higher thermostability than that of the native enzyme. The analysis of amino acids of BhCell-Xyl revealed that multiple factors are responsible for its thermostability. Kinetics and in silico analysis of the enzyme suggested that BhCell-Xyl has one active site for both endocellulase and endoxylanase activities. The BhCell-Xyl possesses a carbohydrate binding domain and saccharifies lignocellulosic agro-residues to xylo-oligosaccharides and cello-oligosaccharides, suggesting its potential application in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industries.


Bacillus halodurans Bifunctional enzyme Endoglucanase P. pastoris Polyextremophilic 



Authors gratefully acknowledge financial assistance from the Indo-US Science and Technology Forum and Department of Biotechnology, Govt. of India, New Delhi and University Grants Commission, New Delhi while carrying out the work presented in this manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

449_2016_1730_MOESM1_ESM.pdf (294 kb)
Supplementary material 1 (PDF 294 kb)


  1. 1.
    Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Biores Technol 83:1CrossRefGoogle Scholar
  2. 2.
    Khandeparker R, Numan MT (2008) Bifunctional xylanases and their potential use in biotechnology. J Ind Microbiol Biotechnol 35:635–644CrossRefGoogle Scholar
  3. 3.
    Gao D, Chundawat SPS, Krishnan C, Balan V, Dale BE (2010) Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour Technol 101:2770–2781CrossRefGoogle Scholar
  4. 4.
    Gusakov AV, Salanovich TN, Antonov AI, Ustinov BB, Okunev ON, Burlingame R, Sinitsyn AP (2007) Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. Biotechnol Bioeng 97:1028–1038CrossRefGoogle Scholar
  5. 5.
    Cheng J, Huang S, Jiang H, Zhang Y, Li L, Wang J, Fan C (2016) Isolation and characterization of a non-specific endoglucanase from a metagenomic library of goat rumen. World J Microbiol Biotechnol 32:1–8CrossRefGoogle Scholar
  6. 6.
    Qin Y, Wei X, Liu X, Wang T, Qu Y (2008) Purification and characterization of recombinant endoglucanase of Trichoderma reesei expressed in Saccharomyces cerevisiae with higher glycosylation and stability. Protein Expr Purif 58:162–167CrossRefGoogle Scholar
  7. 7.
    Sun FF, Bai R, Yang H, Wang F, He J, Wang C, Tu M (2016) Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris. Enzyme Microb Technol 92:107–116CrossRefGoogle Scholar
  8. 8.
    Zhao XH, Wang W, Wang FQ, Wei DZ (2012) A comparative study of β-1, 4-endoglucanase (possessing β-1, 4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3). Biores Technol 110:539–545CrossRefGoogle Scholar
  9. 9.
    Kumar V, Satyanarayana T (2015) Generation of xylooligosaccharides from microwave irradiated agroresidues using recombinant thermo-alkali-stable endoxylanase of the polyextremophilic bacterium Bacillus halodurans expressed in Pichia pastoris. Biores Technol 179:382–389CrossRefGoogle Scholar
  10. 10.
    Parashar D, Satyanarayana T (2016) Production of Ca2+-independent and acidstable recombinant α-amylase of Bacillus acidicola extracellularly and its applicability in generating maltooligosaccharides. Mol Biotechnol 58:707–717CrossRefGoogle Scholar
  11. 11.
    Spohner SC, Müller H, Quitmann H, Czermak P (2015) Expression of enzymes for the usage in food and feed industry with Pichia pastoris. J Biotechnol 202:118–134CrossRefGoogle Scholar
  12. 12.
    Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66CrossRefGoogle Scholar
  13. 13.
    Macauly-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270CrossRefGoogle Scholar
  14. 14.
    Rattu G, Joshi S, Satyanarayana T (2016) Bifunctional recombinant cellulase–xylanase (rBhcell–xyl) from the polyextremophilic bacterium Bacillus halodurans TSLV1 and its utility in valorization of renewable agro-residues. Extremophiles 20:831–842CrossRefGoogle Scholar
  15. 15.
    Parashar D, Satyanarayana T (2016) Enhancing the production of recombinant acidic α-amylase and phytase in Pichia pastoris under dual promoters [constitutive (GAP) and inducible (AOX)] in mixed fed batch high cell density cultivation. Process Biochem 51:1315–1322CrossRefGoogle Scholar
  16. 16.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 76:248–254CrossRefGoogle Scholar
  17. 17.
    Ribeiro LF, Furtado GP, Lourenzoni MR, Costa-Filho AJ, Santos CR, Nogueira SC, Betini JA, Maria de Lourdes TM, Murakami MT, Ward RJ (2011) Engineering bifunctional laccase–xylanase chimeras for improved catalytic performance. J Biol Chem 286:43026–43038CrossRefGoogle Scholar
  18. 18.
    Ata Ö, Boy E, Güneş H, Çalık P (2015) Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance. Bioprocess Biosyst Eng 38:889–903CrossRefGoogle Scholar
  19. 19.
    García-Fraga B, da Silva AF, López-Seijas J, Sieiro C (2015) Optimized expression conditions for enhancing production of two recombinant chitinolytic enzymes from different prokaryote domains. Bioprocess Biosyst Eng 38:2477–2486CrossRefGoogle Scholar
  20. 20.
    Várnai A, Tang C, Bengtsson O, Atterton A, Mathiesen G, Eijsink VG (2014) Expression of endoglucanases in Pichia pastoris under control of the GAP promoter. Microb Cell Fact 13:1CrossRefGoogle Scholar
  21. 21.
    Woo JH, Liu YY, Stavrou S, Neville DM (2004) Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris. Appl Environ Microbiol 70:3370–3376CrossRefGoogle Scholar
  22. 22.
    Nisha M, Satyanarayana T (2013) Characterization of recombinant amylopullulanase (gt-apu) and truncated amylopullulanase (gt-apuT) of the extreme thermophile Geobacillus thermoleovorans NP33 and their action in starch saccharification. Appl Microbiol Biotechnol 97:6279–6292CrossRefGoogle Scholar
  23. 23.
    Parashar D, Satyanarayana T (2016) A chimeric α-amylase engineered from Bacillus acidicola and Geobacillus thermoleovorans with improved thermostability and catalytic efficiency. J Ind Microbiol Biotechnol 43:473–484CrossRefGoogle Scholar
  24. 24.
    Pérez-Avalos O, Sánchez-Herrera LM, Salgado LM, Ponce-Noyola T (2008) A bifunctional endoglucanase/endoxylanase from Cellulomonas flavigena with potential use in industrial processes at different pH. Curr Microbiol 57:39–44CrossRefGoogle Scholar
  25. 25.
    Torrez M, Schultehenrich M, Livesay DR (2003) Conferring thermostability to mesophilic proteins through optimized electrostatic surfaces. Biophys J 85:2845–2853CrossRefGoogle Scholar
  26. 26.
    Strub C, Alies C, Lougarre A, Ladurantie C, Czaplicki J, Fournier D (2004) Mutation of exposed hydrophobic amino acids to arginine to increase protein stability. BMC Biochem 5:1–9CrossRefGoogle Scholar
  27. 27.
    Vinogradov AA, Kudryashova EV, Grinberg VY, Grinberg NV, Burova TV, Levashov AV (2001) The chemical modification of α-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water–organic media. Protein Eng 14:683–689CrossRefGoogle Scholar
  28. 28.
    Hall M, Rubin J, Behrens SH, Bommarius AS (2011) The cellulose binding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain. J Biotechnol 155:370–376CrossRefGoogle Scholar
  29. 29.
    Lemos MA, Teixeira JA, Domingues MR, Mota M, Gama FM (2003) The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei. Enzyme Microb Technol 32:35–40CrossRefGoogle Scholar
  30. 30.
    Galbe M, Zacchi G (2012) Pretreatment: the key to efficient utilization of lignocellulosic materials. Biomass Bioenergy 46:70–78CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ranjitha R. Prabhu
    • 1
  • Deepak Parashar
    • 1
  • T. Satyanarayana
    • 1
    Email author
  1. 1.Department of MicrobiologyUniversity of Delhi South CampusNew DelhiIndia

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