Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis

Abstract

Cellulase production was examined in two strains of Neocallimastixfrontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50°C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-' (1 U represents 1 ,Ig of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-'. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 x 103 to 10.4 x 104 U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-'. The range for PN-2 was 8.1 x 103 to 11 x 104 U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-') to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (<20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (<2 U ml-') and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity. Activity of the culture fluid towards long-chained cello-oligosaccharide, prepared by partial acid hydrolysis of cellulose, was almost sixfold higher than that for Avicel or Sigmacell, suggesting that the oligosaccharide was more suitable for the measurement of exoglucanase. Both CMCase and exoglucanase production increased when fungi were cocultured with Methanospirillum hungatei, and soluble sugars were found to be less effective than cellulose as inducers of cellulase. Yields of CMCase were higher than previously reported for some other anaerobes.

Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii

“…The other species belonging to the genus Neocallimastix being characterized by a monocentric thallus and an endogenous sporangial development. 5 Thallus expansum, polycentricus, ad rhizomycerium multa ramosa, coenocytica, multinucleatus. Zoosporangia sphaerica usque ad 150/zm diam.…”

“…There is growing interest in strictly anaerobic rumen chytridiomycetes because of their high cellulolytic activity [1][2][3][4]. These micro-organisms degrade cellulose and in addition utilize a great number of carbon substrates [1,5,6] with production of formate, acetate, lactate, ethanol, hydrogen and carbon dioxide [6][7][8][9]. Four species have so far Correspondence to: A. Breton, Groupe de Microbiologie, Uai-versit~ Blaise Pascal, 63177 Aubi~re, France.…”

The rumen anaerobic fungi

“…N. frontalis growing on Whatman No. 1 filter paper strips was shown to release relatively large amounts of carboxymethylcellulase (CMCase) into the culture fluid and activities of up to 170 U. m1-1 (IU represents 1/~g glucose equivalents released/min) were obtained for cultures grown on 2.5 g cellulose-m1-1 [28]. As much as 10.4 × 104 U of CMCase-g-1 of cellulosic substrate fermented could be obtained at full growth in cultures with an initial cellulose concentration of 0.6 mg -ml-~.…”

“…Elevation of cellulose in the culture medium led to a decline in CMCase yield which was accompanied by an accumulation of glucose. Subsequently it was demonstrated that elevated levels of glucose repressed CMCase production [28]. In contrast to the production of CMCase, that of exoglucanase, determined as the activity of culture fluid towards alkali-treated Avicel or Sigmacell micro-crystalline cellulose, was low (maximal activity about 100-fold less than CMCase) and treatment of fungal rhizoid with various solubilizing agents failed to give an increase in activity [28].…”

Effect of the carbohydrate growth substrate on polysaccharolytic enzyme formation by anaerobic fungi isolated from the foregut and hindgut of nonruminant herbivores and the forestomach of ruminants

“…frontalis isolate (Barichievich and Calza 1990;Calm 1991). However, enzyme formation by ovine Neocallimastix STA, unlike NRE7 and other species within the genus (Mountfort and Asher 1985;Williams and Orpin 1987a,b), was apparently less rigidly controlled. Similar differences in the extent of control are also apparent in the hindgut and foregut species of Piromyces studied herein and in ruminal species reported previously (Williams and Orpin 1987a,b ;Morrison et al 1990).…”