Abstract
Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml−1 to 4.4 and 4.1 in presence of dithiothreitol (DTT) and β-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml−1 (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-β-d-glucopyranoside and from 14.6 U ml−1(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.
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Banik SP, Pal S, Ghorai S, Chowdhury S, Khowala S (2009) Interference of sugars in the Coomassie blue G dye binding assay of proteins. Anal Biochem 386:113–115
Banik SP, Pal S, Ghorai S, Chowdhury S, Majumder R, Mukherjee S, Khowala S (2012) In situ reversible aggregation of extracellular cellobiase in the flamentous fungus Termitomyces clypeatus. Biotechnol Bioprocess Eng 17:925–936
Banik SP, Bhattacharyya S, Ghorai S (2014) Isolation of a new Penicillium chrysogenum strain BF02 from agricultural soil of rural India producing a thermostable low Km cellobiase. J Microbiol Biotechnol Food Sci 3:322–328
Blanchette C, Lacayo CI, Fischer NO, Hwang M, Thelen MP (2012) Enhanced cellulose degradation using cellulase-nanosphere complexes. PLoS ONE 7:e42116
Chirico WJ, Brown R Jr (1987) Purification and characterization of a β-glucosidase from Trichoderma reesei. Eur J Biochem 165:333–341
Ducret A, Trani M, Lortie R (2002) Screening of various glycosidases for the synthesis of octyl glucoside. Biotechnol Bioeng 77:752–757
Ghorai S, Chowdhury S, Pal S, Banik SP, Mukherjee S, Khowala S (2010) Enhanced activity and stability of cellobiase (β-glucosidase: EC 3.2.1.21) produced in presence of 2-deoxy-d-glucose from the fungus Termitomyces clypeatus. Carbohydr Res 345:1015–1022
González-Pombo P, Pérez G, Carrau F, Guisán JM, Batista-Viera F, Brena BM (2008) One-step purification and characterization of an intracellular β-glucosidase from Metschnikowia pulcherrima. Biotechnol Lett 30:1469–1475
Gueguen Y, Chemardin P, Labrot P, Arnaud A, Galzy P (1997) Purification and characterization of an intracellular β-glucosidase from a new strain Leuconostoc mesenteroides isolated from cassava. J Appl Microbiol 82:469–476
Hsu MF, Sun SP, Chen YS, Tsai CR, Huang LJ, Tsao LT, Kuo SC, Wang JP (2005) Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca2+ signals through thiol modification in neutrophils. Biochem Pharmacol 70:1320–1329
Johansen KS, Xu F, Walton P, Mcbrayer B, Lund H, Soong C-L (2012) Methods of increasing the cellulolytic enhancing activity of a polypeptide. Intern Patent WO2012122518
Kumar S, Ravi VK, Swaminathan R (2008) How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates? Biochem J 415:275–288
Mukherjee S, Basak S, Khowala S (2001) Hetero aggregation with sucrase affects the activity, stability and conformation of extra- and intra cellular cellobiase in the filamentous fungus Termitomyces clypeatus. Enzym Microb Technol 29:213–224
Németh A, Kamondi S, Szilágyi A, Magyar C, Kovári Z, Závodszky P (2002) Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study. Biophys Chem 96:229–241
Pal S, Banik SP, Ghorai S, Chowdhury S, Khowala S (2010) Purification and characterization of a thermostable intracellular β-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus. Biores Technol 101:2412–2420
Pardo AG (1996) Effect of surfactants on cellulase production by Nectria catalinensis. Curr Microbiol 33:275–278
Reith JH, den Uil H, van Veen H, de Laat WTAM, Niessen JJ, Jong E (2002) Co-production of bioethanol, electricity and heat from biomass reidues. In: 12th European conference and technology exhibition on biomass for energy, industry and climate protection, Amsterdam, ECN-RX-02-030, pp 1–22
Smythe CV (1936) The reactions of iodoacetate and of iodoacetamide with various sulfhydryl groups, with urease, and with yeast preparation. J Biol Chem 114:601–612
Acknowledgments
The study was funded jointly by University Grants Commission and Council of Scientific and Industrial Research, Government of India. Authors also express their sincere thanks to Prof. Partha Pal, Maulana Azad College, Kolkata, India and Dr. Rituparna Ghosh, Bhairab Ganguly College, Kolkata, India for their help in carrying out the statistical analyses.
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The authors declare no conflict of interest.
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Supplementary Table 1—amino acid composition of cellobiase.
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Samudra Prosad Banik and Soumya Mukherjee have contributed equally to this work.
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Banik, S.P., Mukherjee, S., Pal, S. et al. Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus . Biotechnol Lett 37, 175–181 (2015). https://doi.org/10.1007/s10529-014-1669-0
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DOI: https://doi.org/10.1007/s10529-014-1669-0