Abstract
Plate screening tests were designed for the selection and isolation of mutant strains of the fungus Aspergillus awamori CMI 142717 showing over-production and constitutive synthesis of xylanase and β-xylosidase. Following mutation by N-methyl-N-nitro-N-nitrosoguanidine, nitrous acid and UV (254 nm), two generations of mutants were isolated and cultured in shake fiasks containing glucose, ball-milled oat straw or oat speit xylan as carbon source. Growth of a number of selected mutants in shake flask culture on medium containing oat spelt xylan produced the highest titres of xylanase and β-xylosidase. Thus, xylanase producton by mutant AANTG43 was 132 U/ml when the Somogyl-Nelson (alkaline copper) method of measuring reducing sugar released was used, or 1160 U/ml using the dinitrosalicylic acid method of reducing sugar analysis. These values were 8-fold higher than those produced by the wild type. A 20-fold improvement in β-xylosidase production was produced by mutant AANO19 (3.51 U/ml). The titres for these two enzyme activities are the highest recorded so far in the literature. Mutant AANTG43 also produced high levels of xylanase (49.8 U/ml) in submerged culture in a fermenter and showed a substantial improvement in the overall productivity of enzyme compared to the wild type strain.
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References
Aspinall, G.O. 1959 Structural chemistry of the hemicelluloses. Advances in Carbohydrate Chemistry 14, 429–468.
Biely, P. 1985 Microbial xylanolytic systems. Trends in Biotechnology 3, 286–290.
Biely, P., Mackenzie, C., Puls, J. & Schneider, H. 1986 Induction of acetyl xylan esterase by Trichoderma reesei and Schizophyllum commune. Biotechnology 4, 731–737.
Bradford, M.M. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principles of protein dye binding. Analytical Biochemistry 72, 248–254.
Breuil, C. & Saddler, J.N. 1985 Comparison of the 3,5,dinitrosalicilic and Nelson— Somogyi methods of assaying for reducing sugars and determining cellulase activity. Enzyme and Microbial Technology 7, 327–332.
Brown, J.A., Collin, S.A. & Wood, T.M. 1987 Development of a medium for high cellulase, xylanase and β-glucosidase production by a mutant strain of the fungus Penicillium pinophilum. Enzyme and Microbiol Technology 9, 176–180.
Carter, B.L.A. & Bull, A.T. 1969 Studies of fungal growth and intermediary metabolism under steady and non-steady conditions. Biotechnology and Bioengineering 11, 785–804.
Conrad, D. 1981 Enzymatic hydrolysis of xylans. I. A high xylanase and β-xylosidase-producing strain of Aspergillus niger. Biotechnology Letters 3, 345–350.
Cuskey, S.M., Schamhard, D.H.J., Chase, T.Jr, Montenecourt, B.S. & Eveleigh, D.E. 1980 Screening for β-glucosidase mutants of Trichoderma reesei with resistance to end products inhibition. Developments in Industrial Microbiology 21, 471–480.
DeCosta, M.B. & Van Uden, N. 1980 Use of 2-deoxyglucose in the selective isolation of mutants of Trichoderma reesei with enhanced β-glucosidase production. Biotechnology and Bioengineering 22, 2429–2432.
Dekker, R.F.H. 1983 Bioconversion of hemicellulose: aspects of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose. Biotechnology and Bioengineering 15, 1127–1146.
Dubeau, H., Chahal, D.S. & Ishaque, M. 1987 Xylanase of Chaetomium cellulolyticum: its nature of production and hydrolytic potential. Biotechnology Letters 4, 275–280.
Eberhart, B., Cross, D.F. & Chase, L. 1964 β-Glucosidase systems of Neurospora crassa I. β-Glucosidase and cellulase activities of mutant and wild-type strains. Journal of Bacteriology 87, 761–770.
Farkas, V., Liskova, M. & Biely, P. (1985) Novel medium for the detection of microbial producers of cellulase and xylanase. FEMS Microbiology Letters 28, 137–140.
Flannigan, B. & Gilmour, J.E.M. 1980 A simple plate test for xylanolytic activity in wood rotting Basidiomycetes. Mycologia 72, 1219–1221.
Hartley, R.D. & Jones, E.C. 1977 Phenolic components and degradability of cell walls of grass and legume species. Phytochemistry 16, 1531–1534.
Hoffman, R.M. & Wood, T.M. 1985 Isolation and partial characterization of C mutant of Penicillium funiculosum for the saccharification of straw. Biotechnology and Bioengineering 27, 81–85.
Johnson, K.G., Harrison, B.A., Schneider, H., Mackenzie, C. & Fontana, J.D. 1988 Xylan hydrolysis enzymes from Streptomyces spp. Enzyme and Microbial Technology 10, 403–409.
Leathers, T.M. 1986 Color variants of Aureobasidium pullulans overproduce xylanase with extremely high specific activities. Applied and Environmental Microbiology 52, 1026–1030.
Mandels, M. 1982 Cellulases. Annual Report on Fermentation Processes 5, 35–77.
Miller, G.L. 1959 Dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry 31, 426–428.
Montenecourt, B.S. & Eveleigh, D.E. 1979 Preparation of mutants of Trichoderma reesei with enhanced cellulase production. Applied and Environmental Microbiology 34, 777–782.
Montenecourt, B.S. & Eveleigh, D.E. 1979 Selective screening methods for the isolation of high yielding mutants of Trichoderma reesei. Advances in Chemistry Series 181, 289–301.
Perkins, D.D. 1962 Preservation of Neurospora stock cultures in anhydrous silica gel. Canadian Journal of Microbiology 8, 591–594.
Reese, E.T., Maguire, A. & Parrish, F.W. 1973 Glucosidases and exoglucanases. Canadian Journal of Microbiology 19, 1065–1074.
Robyt, J.F. & Whelan, W.J. 1972 Reducing value methods for maltodextrins I. Chain-length dependence of alkaline 3,5,dinitro-salicylate and chain length idependence of alkaline copper. Analytical Biochemistry 45, 510–516.
Royer, J.C. & Nakas, J.P. 1989 Xylanase production by Trichoderma longibranchiatum. Enzyme and Microbial Technology 11, 405–410.
Sokai, R. & Rohlf, F.J. 1981 Biometry. 2nd edn. San Francisco: Freeman.
Somogyi, M.J. 1952 Notes on sugar determination. Journal of Biological Chemistry 195, 19–23.
Teather, R.M. & Wood, P.J. 1982 Use of Congo red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Applied and Environmental Microbiology 43, 777–780.
Warzywoda, M., Ferre, V. & Pourquie, J. (1983) Development of a culture medium for large-scale production of cellulolytic enzymes by Trichoderma reesei. Biotechnology and Bioengineering 15, 3005–3010.
Wilkie, K.C.B. 1979 The hemicelluloses of grasses and cereals. Advances in Carbohydrate Research 148, 71–85.
Yu, E.K.C., Tan, L.U.L., Chan, M.K.-H., Deschatelets, L. & Saddler, J.N. 1987 Production of thermostable xylanase by Thermoascus aurantiacus. Enzyme and Microbial Technology 9, 16–24.
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The authors are with the Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK.
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Smith, D.C., Wood, T.M. Isolation of mutants of Aspergillus awamori with enhanced production of extracellular xylanase and β-xylosidase. World Journal of Microbiology and Biotechnology 7, 343–354 (1991). https://doi.org/10.1007/BF00329401
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DOI: https://doi.org/10.1007/BF00329401