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Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material


Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K m and V max values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 μmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.

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  1. 1.

    Barr B, Hsieh YL, Ganem B, Wilson DB (1996) Identification of two functionally different classes of exocellulases. Biochemistry 35:586–592

  2. 2.

    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

  3. 3.

    Mateo C, Palomo JM, Lorente GF, Guisan JM, Lafuente RF (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym Microb Technol 40:1451–1463

  4. 4.

    Cao L (2005) Immobilized enzymes: science or art? Curr Opin Chem Biotechnol 9(2):217–226

  5. 5.

    Kumar A, Srivastava A, Galaev IY, Mattiasson B (2007) Smart polymers: physical forms and bioengineering applications. Prog Polym Sci 32:1205–1237

  6. 6.

    Busto MD, Ortega N, Mateos MP (1997) Stabilization of cellulase by cross-linking with glutaraldehyde and soil humates. Bioresour Technol 60:33–37

  7. 7.

    Yuan X, Shen N, Sheng J, Wei X (1999) Immobilization of cellulas using acrylamide grafted acrylonitride copolymer membranes. J Membr Sci 155:101–106

  8. 8.

    Ohison I, Tragardh G, Hahn-Hagerdal B (1984) Enzymatic hydrolysis of sodium hydroxide pretreated sallow in an ultrafiltration membrane reactor. Biotechnol Bioeng 26:647–653

  9. 9.

    Henley RG, Yang RYK, Greenfield PF (1980) Enzymatic saccharification of cellulose in membrane reactors. Enzym Microbiol Technol 2:206–208

  10. 10.

    Tjerneld F, Persson I, Albertsson PA, Lee JM (1991) Enzyme cellulose hydrolysis in anattrition bioreactor combined with an aqueous two-phase system. Biotechnol Bioeng 37:876–882

  11. 11.

    Afsahi B, Kazemi A, Kheirolomoom A, Nejati S (2007) Immobilization of cellulase on non-porous ultrafine silica particles. Sci Iranica 14(4):379–383

  12. 12.

    Fraser JE, Bickerstaff GF (1997) Entrapment of enzymes and cells in calcium alginate. In: Immobilization of enzymes and cells. Humana Press, Totowa, pp 61–66

  13. 13.

    Blandino A, Macias M, Cantero D (1999) Formation of calcium alginate gel capsules: influence of sodium alginate and CaCl2 concentration on gelation kinetics. J Biosci Bioeng 88:686–689

  14. 14.

    Blandino A, Macias M, Cantero D (2000) Glucose oxidase release from calcium alginate gel capsules. Enzym Microb Technol 27:319–324

  15. 15.

    Andre B, Bioencapsulation encyclopedia.

  16. 16.

    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268

  17. 17.

    Abida A, Qader SA, Aliya R, Samina I, Abid A (2009) Calcium alginate: a support material for immobilization of proteases from newly isolated strain of Bacillus subtilis KIBGE-HAS. World Appl Sci J 7(10):1281–1286

  18. 18.

    Roig MG, Rashid DH, Kennedy JF (1995) High-alkaline protease from Bacillus PD92 entrapped in calcium alginate gel: physicochemical and microscopic study. Appl Biochem Biotechnol 55:95–121

  19. 19.

    Norouzian D (2003) Enzyme immobilization, and the state of art in biotechnology: a review. Iran J Biotechnol 1:197–206

  20. 20.

    Sartoglu K, Demir N, Acar J, Mutlu M (2001) The use of commercial pectinase in the fruit industry, part 2: determination of kinetic behaviour of immobilized commercial pectinase. J Food Eng 47(4):271–274

  21. 21.

    Buga ML, Ibrahim S, Nok AJ (2010) Physico-chemical characteristics of immobilized polygalacturonase from Aspergillus niger (SA6). Afr J Biotechnol 9(52):8934–8943

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It is the outcome of a Manpower Development Program for Energy & Resources Supported by The Ministry of Knowledge and Economy (MKE).

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Correspondence to Don-Hee Park.

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Andriani, D., Sunwoo, C., Ryu, H. et al. Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material. Bioprocess Biosyst Eng 35, 29–33 (2012).

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  • Immobilization
  • Cellulase
  • Calcium alginate
  • Bacillus subtilis TD6
  • Takifugu rubripes