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Isolation of anaerobic cellulolytic thermophiles and production and purification of the cellulase from Clostridium thermocellulaseum M-7 Lee, Byong Hoon

Abstract

An enrichment procedure led to the isolation, by the cellulose roll tube method, of a number of actively cellulolytic anaerobic thermophilic bacteria. Two isolates were terminally sporing rods and were tentatively identified as Clostridium thermocellulaseum (Enebo, 1951). Strain M-7 (0.6 μm x 4.0 μm) from manure grew optimally at 58°C to 63°C, pH 6.0 to 6.5 and did not require organic nitrogen. Strain C-19 (0.3 μm x 4.5 μm) from compost was similar but grew optimally at 50°C to 68°C, pH 7.5. Both utilized cellobiose and a wide range of other sugars. Strain C-19 did not utilize glucose, raffinose and inositol but did use inulin. The mean generation times in a rich nutrient medium containing cellobiose were 35 min for strain M-7 and 25 min for strain C-19. Strain M-7 had a mean generation time of 2 hr when grown on cellulose. Yeast extract (0.5%) stimulated growth and cellulase production by strain M-7 but was inhibitory at higher concentrations. Other organic nitrogen sources acted similarly. Cellulose at 1.0% gave maximum cellulase production after 72 hr incubation of strain M-7. Higher concentrations of cellulose were not completely degraded in 72 hr. Strain M-7 did not produce cellulase when grown on any carbon source other than cellulose substrates. The addition of cellobiose (0.3%) and glucose (0.4%) prevented cellulose hydrolysis in cellulose medium. This may have been repression of synthesis but cellulase was inhibited by both sugars. Both C₁, cellulase (degrades native cellulose) and Cx cellulase (β-1,4-glucanase) activities in strain M-7 cultures were assayed by measuring the liberation of reducing sugars, using dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67°C. Cx cellulase could conveniently be assayed by a new automated procedure. Strain M-7 was very actively cellulolytic when compared to previously microbial species. The 48 hr culture contained Cx activity (56 μg glucose/min/ml from carboxymethyl cellulose) and C₁ activity (8 μg glucose/min/ml from cotton fibres); the ratio of C₁,:Cx was 1:7. The cellulase(s) from strain M-7 were extra-cellular, produced during exponential growth but were not free in the growth medium until 50% of the cellulose was hydrolyzed. Glucose and cellobiose were the only soluble products liberated by the cellulase from cellulose. ZnCl₂ precipitation appeared to be a good method for the concentration of cellulase activity but subsequent purification was not successful. Isoelectric focusing indicated the presence of four Cx cellulases (pI 4.5, 6.3, 6.8, and 8.7). DEAE-Sephadex chromatography indicated three Cx components. It is concluded that C. thermocellulaseum M-7 produces cellulase(s) capable of rapidly hydrolyzing native cellulose. The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of cellulose is possible at elevated temperature.

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