The action of cellulolytic enzymes from Myrothecium verrucaria

Abstract

Although an extensive literature is available describing cell-free cellulolytic preparations which attack soluble cellulose derivatives, there is a dearth of information on the enzymic breakdown of insoluble cellulose, and virtually nothing is known about the breakdown of undegraded cellulose of the type associated with native cotton fibres [For reviews see Siu (1951), Halliwell (1959)]. Despite the failure of cell-free preparations made from them to attack undegraded cellulose, truly cellulolytic organisms can quantitatively transform all types of cellulose, undegraded or otherwise, to soluble products. The present report concerns an examination of the breakdown of an acid-swollen cellulose by culture filtrates from Myrothecium verrucaria. This substrate is an insoluble although degraded cellulose and was chosen firstly because the relatively mild method of preparation involves only small changes in the degree of polymerization (Walseth, 1952), and secondly only after having shown that our enzyme preparations produoed significant solubilization of this substrate. Conditions governing the enzymic breakdown of insoluble cellulose are described below and compared with the enzymic hydrolysis of the soluble cellulose derivative, carboxymethylcellulose. MATERIATLS AND METHODS Preparation of the culture filtrate Cultures of M. vemrcaria (I.M.I. 45541, Commonwealth Mycological Institute, Kew, Surrey), grown on the salt medium of Saunders, Siu & Genest (1948) with cellulose powder (Whatman) or de-waxed cotton fibres as substrate, were aerated at 280 by incubation in a reciprocal shaking machine operating at 60-90 strokes of 38 mm. horizontal movement per minute. After fermentation for 1-3 weeks most of the organisms and all of the residual cellulose were separated off by filtration through a sintered-glass filter (porosity 3). The filtrate was centrifuged at 54 000g for 30 min. at 10 and subsequently passed through a sinteredglass bacteriological filter (porosity 5/3). In the later work the final filtration was found to be unnecessary by inoculation on cellulose-agar slopes. The cell-free culture supernatant or filtrate fraction was used as the source of cellulolytic enzymes. For enzyme assays the following average volumes were used: for method (a) 0-2 ml. of undiluted filtrate; for method (b) 0.1 ml. of filtrate diluted 50 times.

Properties of cellulases fromSclerotium rolfsii

“…2). Halliwell (1961) observed 60% and 50% loss in cellulase activity of Myrothecium verrucaria using swollen cellulose and CMC, respectively at 54~ in 1 h. A number of workers have reported similar results wherein they concluded that the optimum temperature of incubation of cellulase from different sources varies not only with the source of enzyme but also with different components of the enzyme obtained from a single source (Gupta & Gupta 1979;Garg & Neelakantan 1982).…”

Cellulolytic enzymes of the obligately biotrophic rust fungus Uromyces viciae-fabae are regulated differentiation-specifically

“…Some of these enzymes, once separated from the functional unit, although clearly contributing to the cellulase complex, do not significantly degrade native cellulose but hydrolyse CMC at high rates. Therefore, although occasionally suggested otherwise (Reese, Siu & Levingson, 1950;Halliwell, 1961), CMC r~presents a useful substrate in biochemical studies qesigned to analyse kinetics of the formation and properties of the different cellulase components (Sharrock, 1988). The higher sensitivity of the assay with CMC as compared with non-soluble cellulose is of special importance since U. viciae-fabae, and probably the majority of obligate biotrophs, produces very low levels of cellulase activity.…”

Characteristics of Enzymes Produced by Ruminococcus flavefaciens which Degrade Plant Cell Walls

“…Cellulase and hemicellulase acthities were measured from the amount of reducing-sugar (Halliwell, 1961) released after incubation of enzyme with substrate. Activity against insoluble cellulose was assayed by incubating 1 ml enzyme with 1 mlO.25 M-MES buffer pH 6.4, 1 ml of a 3 % (w/v) suspension of Avicel or MMFP in water, and 0.1 ml toluene.…”