Abstract
Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K m of the enzyme changed from 5.9 mg ml–1 to 9.1 mg ml–1 upon immobilization. The V max of the immobilized enzyme showed an increase from 90.9 µmol ml–1 min–1 (for the free enzyme) to 111.1 µmol ml–1 min–1. The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.
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References
Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257–270
Bajpai P (1997) Microbial xylanolytic enzyme system: properties and applications. In: Neidleman S, Laskin A (eds) Advances in applied microbiology. Academic Press, New York, pp 141–194
Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microb Technol 56:326–338
Bisaria VS, Mishra S, Sahai V, Jain JK, Mathur RM (2003) Pre-bleaching of kraft pulp with xylanase enzyme for paper manufacture. In: Proceedings of the International Symposium on "New Horizons in Biotechnology", Trivandrum, India, April 2001 (in press)
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 72:248–254
Cong L, Kaul R, Dissing U, Mattiasson B (1995) A model study on Eudragit and polyethyleneimine as soluble carriers for α-amylase for repeated hydrolysis of starch. J Biotechnol 42:75–84
Cornish-Bowden A (1995) Analysis of enzyme kinetic data. Oxford University Press, Oxford, pp 27–36
Dinnella C, Lanzarini G, Ercolessi P (1995) Preparation and properties of an immobilized soluble-insoluble pectinylase. Process Biochem 30:151–157
Dumitriu S, Popa M, Artenie V, Dan F (1989) Bioactive polymers. 56: Urease immobilization on carboxymethylcellulose. Biotechnol Bioeng 34:283–290
Fujimura M, Mori T, Tosa T (1987) Preparation and properties of soluble-insoluble immobilized proteases. Biotechnol Bioeng 29:747–752
Gray CG (1993) Stabilization of enzymes with soluble additives. In: Gupta MN (ed) Thermostability of enzymes. Springer, Berlin Heidelberg New York, pp 124–143
Gupta MN, Mattiasson B (1992) Unique applications of immobilized proteins in bioanalytical systems. In: Suelter CH, Kricka, L (eds) Bioanalytical applications of enzymes, vol 36. Wiley, New York, pp 1–34
Hoshino K, Taniguchi M, Netsu Y, Fujii M (1989) Repeated hydrolysis of raw starch using amylase immobilized on a reversibly soluble-insoluble carrier. J Chem Eng Jpn 22:54–59
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Roy I, Gupta M N (2002) Macroaffinity ligands in bioseparation. In: Gupta MN (ed) Methods for affinity-based separation of proteins/enzymes, Birkhauser, Basel, pp 130–147
Roy I, Sharma S, Gupta MN (2002) Smart biocatalysts: design and applications. Adv Biochem Eng Biotechnol (in press)
Saraswat V, Bisaria VS (2000) Purification, characterization and substrate specificities of xylanase isoenzyme from Melanocarpus albomyces IIS 58. Biosci Biotechnol Biochem 64:1167–1180
Sardar M, Roy I, Gupta MN (2000) Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer EudragitTM L-100. Enzyme Microb Technol 27:672–679
Suh WC, Lim BS, Chun M, Sernetz M (1987) Immobilization of nuclease P1 from Penicillium citrinum and production of 5-nucleotides by bioreactor. Korean Biochem J 20:17–23
Tischer W, Kasche V (1999) Immobilized enzymes: crystals or carriers? Trends Biotechnol 17:326–335
Tyagi R, Roy I, Agarwal R, Gupta MN (1998) Carbodiimide coupling of enzymes to the reversibly soluble-insoluble polymer Eudragit S-100. Biotechnol Appl Biochem 28:201–206
Viikari L, Suurnakki A, Buchert J (1996) Enzyme-aided bleaching of kraft pulps: fundamental mechanisms and practical applications. In: Jeffries TW, Viikari L (eds) Enzymes for pulp and paper processing. ACS Symposium series 665, Oxford University Press, Oxford, pp 15–24
Williams RA, Blanch HW (1994) Covalent immobilization of protein monolayers for biosensor applications. Biosens Bioelectron 9:159–167
Acknowledgements
The partial supports provided by the Council for Scientific and Industrial Research (CSIR) (Extramural Division and Technology Mission on Oilseeds, Pulses and Maize) and the Department of Science and Technology, both Government of India organizations, are gratefully acknowledged. A.G. is grateful to IIT Delhi for financial support.
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I. Roy and A. Gupta contributed equally to the work described here.
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Roy, I., Gupta, A., Khare, S.K. et al. Immobilization of xylan-degrading enzymes from Melanocarpus albomyces IIS 68 on the smart polymer Eudragit L-100. Appl Microbiol Biotechnol 61, 309–313 (2003). https://doi.org/10.1007/s00253-002-1213-3
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DOI: https://doi.org/10.1007/s00253-002-1213-3