Summary
The activity of ten actinomycete strains against p-nitrophenyl β-d-cellobioside (pNPC) was investigated. Intra- and extracellular activities were detected in all strains, although activities were found to vary between strains. Extracellular pNPC activity, detected in the thermophilic Streptomycete EC22 was six times greater than that of the next best strain. Culture supernatant studies on Streptomyces strain EC22 revealed that pNPC activity was optimal at 65° C and between pH 6.0 and pH 8.0, although this temperature value represented a compromise between increased reaction rate and thermal denaturation. At 65° C, the half-life of activity against pNPC was 150 min. The K m value of pNPC activity was found to be 3.33 mm. Enzyme activity was subject to competitive inhibition by both glucose and cellobiose with substrate inhibition constant (K i) values of 2.2 mm and 1.4 mm respectively. Fast protein liquid chromatograph (FPLC) of culture supernatants from EC22 using anion exchange chromatography revealed the presence of two proteins with pNPC activity. This result was confirmed using gel-filtration chromatography which estimated the molecular mass of the two proteins to be approximately 48 and 33 kDa, with the specific activity of the 48 kDa enzyme being eight times greater than that of the 33 kDa protein.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ball AS, McCarthy AJ (1988) Saccharification of straw by actinomycete enzymes. J Gen Microbiol 134:2139–2147
Ball AS, McCarthy AJ (1989) Purification and properties of actinomycete xylanases. J Appl Bacteriol 66:439–444
Ball AS, Betts WB, McCarthy AJ (1989) Lignocarbohydrate solubilisation from straw by actinomycetes. Appl Environ Microbiol 56:3017–3022
Bartley T, Waldron C, Eveleigh D (1984) A cellobiohydrolase from a thermophilic actinomycete, Microbispora bispora. Appl Biochem Biotechnol 9:337–338
Bisaria VS, Ghose TK (1981) Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products. Enzyme Microb Technol 3:90–104
Desphande MV, Peterson LG, Eriksson LG (1988) A selective assay for exo 1,4 β-glucanases. Methods Enzymol 160:126–130
Fennington G, Neubauer D, Stutzenberger F (1984) Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata. Appl Environ Microbiol 47:201–204
Ferchak JD, Hagerdal B, Pye EK (1980) Saccharification of cellulose by the cellulolytic system of Thermomonospora sp. II. Hydrolysis of cellulosic substrates. Biotechnol Bioeng 22:1427–1542
Gilkes NR, Langsford ML, Kilburn DJ, Miller RC, Warren RAJ (1984) Mode of action and substrate specification of cellulases from cloned bacterial genes. J Biol Chem 259:10455–10459
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith C, Ward JM, Schrempf H (1985) Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation, Norwich
Johnson WS, Lindsey AJ (1939) An improved universal buffer. Analyst 64:490–492
Kluepfel D, Shareck F, Mondou F, Morosoli R (1986) Characterisation of cellulase and xylanase activities of Streptomyces lividans. Appl Microbiol Biotechnol 24:230–234
Ladisch MR, Lin KW, Voloch M, Tsao GT (1983) Process considerations in the enzymatic hydrolysis of biomass. Enzyme Microb Technol 5:82–102
Lowry OH, Rosebrough NJ, Fall AL, Randall AL (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
MacKenzie CR, Bilous D, Johnson KG (1984) Purification and characterization of an exoglucanase from Streptomyces flavogriseus. Can J Microbiol 30:1171–1178
McCarthy AJ (1987) Lignocellulose-degrading actinomycetes. FEMS Microbiol Rev 46:145–163
McCarthy AJ, Peace E, Broda P (1985) Studies on the extracellular xylanase activity of some thermophilic actinomycetes. Appl Microbiol Biotechnol 21:238–244
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:238–244
Moreira AR, Phillips JA, Humphrey AE (1981) Utilization of carbohydrates by Thermomonospora sp. grown on glucose, cellobiose and cellulose. Biotechnol Bioeng 23:1325–1328
Ramachandra M, Crawford DL, Pometto III AL (1987) Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp: a comparative study of wild type and genetically manipulated strains. Appl Environ Microbiol 53:2754–2760
Wachinger G, Bronnenmeier K, Staudenbauter WL, Schrempf H (1989) Identification of mycelium-associated cellulase from Streptomyces reticuli. Appl Environ Microbiol 55:2653–2657
Waldron CR, Becker-Vallone CA, Eveleigh DE (1986) Isolation and characterization of a cellulolytic actinomycete Microbispora bispora. Appl Microbiol Biotechnol 24:477–486
Wilson DB (1988) Cellulases of Thermomonospora fusca. Methods Enzymol 160:314–323
Author information
Authors and Affiliations
Additional information
Offprint requests to: A. S. Ball
Rights and permissions
About this article
Cite this article
Ball, A.S., Brabban, A. & McCarthy, A.J. Studies on the extracellular p-nitrophenyl β-d-cellobiosidase activity of a thermophilic streptomycete. Appl Microbiol Biotechnol 36, 473–477 (1992). https://doi.org/10.1007/BF00170186
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00170186