Endo-β-1,4-d-mannanase is efficiently produced by Sclerotium (Athelia) rolfsii under derepressed conditions
Introduction
β-1,4-Mannans are substituted heteropolysaccharides that are commonly found in wood as well as in plant seeds and tubers. The hydrolysis of the backbone of these β-1,4-mannans is catalyzed by endo-β-1,4-mannanases, which are produced by a number of fungi, yeasts, bacteria, marine algae, germinating seeds of terrestrial plants, and by various invertebrates (Dekker and Richards, 1976). For the complete hydrolysis of the variously substituted and complex mannans, however, the concerted action of a number of hydrolytic enzymes, including β-mannosidase, β-glucosidase, α-galactosidase, and acetyl(mannan)esterase is necessary (Eriksson et al., 1990, Tenkanen et al., 1993). Mannanases have found several applications in food technology, where they can be used for the hydrolysis of high-molecular-weight mannans, e.g. in coffee pulp for the production of soluble coffee (Nicolas et al., 1995) or in fruit as well as in vegetables for the manufacture and clarification of juice or the extraction of oil (Dekker, 1979, Christgau et al., 1994). Mannanases can be used for the production of mannooligosaccharides from cheap agricultural by-products such as copra or konjac mannan. These oligosaccharides were reported to be excellent prebiotics stimulating growth of beneficial intestinal microorganisms and hence could be used in pharmaceuticals or in food stuffs (Akino et al., 1988, Oda et al., 1993). In the pulp and paper industry, mannanases can act synergistically with xylanases as biological prebleaching agents, allowing a significant reduction of chlorine bleaching chemicals and thereby lowering levels of hazardous halogenated organics released into the environment (Viikari et al., 1994, Cuevas et al., 1996, Gübitz et al., 1997).
In general, the endoglycanases that are involved in the degradation of lignocellulose and include mannanases, xylanases and cellulases, are inducible in microbial cells (Biely, 1993, Kubicek et al., 1993). In addition, the expression of these endoglycanases is typically controlled by carbon repression when more easily metabolizable carbon sources, e.g. glucose, are present in the culture medium together with a substrate suitable for inducing endoglycanase synthesis (de Graaff et al., 1994, Mach et al., 1996, Ruijter and Visser, 1997). Under these culture conditions, endoglycanase formation in various fungi only starts when the repressing carbohydrate glucose is completely metabolized. Furthermore, only very low levels of the endoglycanases were produced when glucose was used as the only carbohydrate substrate (Canevascini et al., 1979, Ghosh and Nanda, 1994, Piñaga et al., 1994, Kremnicky and Biely, 1997). In accordance with this mechanism of induction and carbon control, appropriate inducing C sources, e.g. various mannans, mannan-rich lignocellulosic material, or cellulose, have been reported to be employed for an efficient production of mannanases by different organisms (Reese and Shibata, 1965, Rättö and Poutanen, 1988, Araujo and Ward, 1990a, Torrie et al., 1990, Arisan-Atac et al., 1993, Stålbrand et al., 1993, Kremnicky and Biely, 1997).
In agreement with the above described mechanism, the inductive formation of endoglycanases has also been shown for the filamentous fungus Sclerotium rolfsii (Haltrich et al., 1994b, Sachslehner et al., 1997). The formation of mannanase, xylanase, and endoglucanase was found to be very closely related, with cellulose being the best inducer and resulting in highest levels of the three endoglycanases formed. However, elevated formation of mannanase activity was also observed in preliminary shaken flask cultivations when S. rolfsii was cultivated on a medium that contained glucose as the only carbohydrate substrate (Sachslehner et al., 1998). It was the objective of this study to investigate in detail the production of the industrially important enzyme mannanase under noninductive conditions using glucose-based media for the cultivation of S. rolfsii. Furthermore, it should be investigated whether by selecting appropriate culture conditions high levels of these enzyme activities with only low levels of concurrently produced cellulase could be attained, since hemicellulase preparations free of cellulase have gained significant interest during the last years because of their applications in the pulp and paper industry (Biely, 1991, Viikari et al., 1994).
Section snippets
Chemicals
α-Cellulose, p-nitrophenyl glycosides, α-naphthyl acetate, and locust bean gum (a galactomannan from Ceratonia siliqua with a mannose-to-galactose ratio of 4:1) were from Sigma (St. Louis, MO, USA); carboxymethylcellulose was from Fluka (Buchs, Switzerland); xylan from birchwood was from Roth (Karlsruhe, Germany), and peptone from meat was from Merck (Darmstadt, Germany). Azo-carob galactomannan (covalently dyed with Remazol brilliant blue) was purchased from Megazyme (Sydney, Australia).
Formation of mannanase by different Sclerotium spp.
In preliminary growth experiments performed in shaken flasks we found that S. rolfsii CBS 191.62 produces considerable activities of mannanase even in the absence of an appropriate inducer but when cultivated on a medium containing glucose as the only carbohydrate substrate (Sachslehner et al., 1998). To evaluate whether the increased formation of mannanase under these growth conditions is a peculiarity of the wild type strain S. rolfsii CBS 191.62 or whether this is a common characteristic of
Discussion
Synthesis of enzymes, which are necessary for the degradation of energy-yielding polymeric material to be used as substrates for cell growth and which include hemicellulases and cellulases, appears to be controlled by two basic regulation mechanisms (Archer and Peberdy, 1997). Formation of these glycosyl hydrolase systems is repressed in the presence of easily metabolizable substrates such as glucose, which is referred to as `the glucose effect', `glucose repression' or more generally `carbon
Acknowledgements
This work was financed by grant P10753-MOB from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung) to D.H. Marianne Prebio is kindly thanked for correction of the manuscript. We are indebted to Prof. Klaus D. Kulbe for his support and his interest in the field of fungal mannanases.
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