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

Abstract

Cellulase production was examined in two strains of Neocallimastix frontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulose (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 (1 U represents 1 μg of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. 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-1. 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-1) 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-1) 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.