Cellulase Regulation Research Articles

Cellulase in Tobacco Callus: Regulation and Purification

Summary Cellulase, 1,4-β-D-glucan 4-glucanohydrolase (EC 3.2.1.4), was purified 1500-fold from callus of Nicotiana tabacum L. cv. Petit Havana SR1 by differential extraction and by hydrophobic affinity, and gel permeation chromatography. The enzyme has a broad pH optimum between 5.5 and 6.5 and a pI of ca. 8.2. SDS-PAGE showed two polypeptides of 50 and 52 kDa, respectively. The enzyme was obviously isolated as a macromolecular aggregate. It degraded hydroxyethylcellulose, carboxymethylcellulose, Avicel, 1,3; 1,4-β-D-glucan and tobacco xyloglucan, but Tropaeolum xyloglucan, xylan and polygalacturonic acid were not degraded by the purified enzyme. The cellulase activity in tobacco tissue was rapidly regulated in response to a morphogenetic hormone treatment. Following transfer of callus tissue to a shoot-inducing medium the enzyme activity decreased during the first 4 days to ca. 25% of the value in tissue on callus-maintaining medium. The involvement of the downward regulation of cellulase in the early events of morphogenesis is discussed.

Regulation of protein and cellulase excretion in the ruminal fungusNeocallimastix frontalis EB188

Extracellular protein and cellulase excretion secretions were studied in the ruminal fungusNeocallimastix frontalis EB188. Cellulase assay and polyacrylamide gel electrophoresis was used to characterize protein excretion patterns caused by media switches of established cultures. Glucose switch medium caused the excretion of low levels of cellulase and increased amounts of low-molecular-weight proteins. Cellulose switch medium caused the excretion of high levels of cellulase and increased amounts of high-molecular-weight proteins. Several proteins were excreted uniquely or in greater amounts in cellulose cultures than in glucose cultures. Distinct protein excretion patterns suggested that regulation of cellulase was a closely controlled process in ruminal fungi.

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.

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

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, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram 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 10(3) to 10.4 X 10(4) 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 10(3) to 11 X 10(4) 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 (less than 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 (less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and regulation of cellulases in Trichoderma harzianum

The highest production of extracellular cellulases (Filter paper activity, Carboxymethyl cellulase and β-glucosidase) by Trichoderma harzianum was achieved on delignified wheat straw. Glycerol at a concentration of one per cent strongly repressed the formation of all the cellulolytic components. Addition of cyclic AMP did not relieve the repression caused by glycerol but lowered the level of existing cellulase enzymes.

Cellulase production and regulation byAgaricus bisporus

Cellulose is degraded during the growth of the cultivated mushroomA. bisporus on composted straw. At the time of sporophore enlargement, a marked increase in extracellular endocellulase activity occurs. A high level of enzyme activity is maintained during subsequent cropping cycles.

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.

Regulation of cellulase from Ruminococcus.

The regulation of cellulase was examined in Ruminococcus albus and R. flavefaciens. Hydrolysis of cellulose, as shown by the formation of clear zones around the colonies of bacteria grown in cellulose-agar roll tubes, was inhibited by moderate levels of cellobiose. An intermediate in the metabolism of cellobiose may be responsible for the inhibition since strains which can use either sucrose or lactose were similarly inhibited by these energy sources. The inhibition of cellulase was examined in relation to either repression of enzyme synthesis or product inhibition of the enzyme activity. There was no inhibition by cellobiose added either to the routine enzymatic assay or to assays using low concentrations of carboxymethylcellulose. A repression mechanism was indicated by the decrease in specific activity of cultures grown in higher concentrations of cellobiose. The specific activity was calculated as the enzymatic activity on carboxymethylcellulose with respect to cell growth. The mechanism of repression was not distinguished between the model proposed by Jacob and Monod and catabolite repression. The growth of R. albus cultured in cellobiose–cellulose liquid medium exhibited a diauxic pattern similar to that described by Monod.

Regulation of cellulase and cellobiase in [formula omitted

Regulation of cellulase and cellobiase in [formula omitted