Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Cellulase Production by Thermomonospora curvata Isolated from Municipal Solid Waste Compost

A cellulolytic, thermophilic actinomycete (previously isolated from municipal refuse compost samples) was identified as Thermomonospora curvata. A determination was made of the optimal conditions for cellulase production by T. curvata when grown at 55 C in a medium containing mineral salts, cellulose, and yeast extract. The pH and temperature optima (pH 6.0 and 65 C) for the cellulase produced by T. curvata were identical to those previously observed for the cellulase extracted from crude compost samples. Such similarities, together with the prevalence of T. curvata in compost samples and its ability to grow at composting temperatures, indicate that this actinomycete could possibly be considered as a major cellulose decomposer in the municipal refuse composting process.     

Temperature-dependent patterns of exoenzyme biosynthesis in Thermomonospora curvata

Production of depolymerlzing exoenzymes in the thermophilic actinomycete, Thermomonospora curvata, grown at 40°, 50° and 61°C, were compared. Cellulase-specific activities were similar at the three growth temperatures. Amylase-and pectinase-specific activities decreased with increasing growth temperature, while xylanase had the reverse pattern. This pattern of thermodependence correlated with ability of the actinomycete to use the product of each exoenzyme as sole carbon source. Therefore the activities of depolymerizing enzymes produced by the actinomycete during the composting temperature ascent are influenced by its ability to utillize each depolymerization product at that temperature.F. Stutzenberger is with the Department of Microbiology and T. Jenkins is with the Department of Animal, Dairy and Veterinary Science, Clemson University, Clemson, SC 29634-1909 USA.     

Xylanase production by Thermomonospora curvata

F.J. STUTZENBERGER AND A.B. BODINE. 1992. The thermophilic actinomycete, Thermomonospora curvata , produced <1 xylanase U/mg dry cell weight during growth in minimal medium with soluble sugars, 3–7 U/mg on purified xylan or cellulose and 28 U/mg on cotton fibres. The optimal growth temperature for xylanase production was 55°C. Cell-bound xylanase decreased from about 30% of total activity in early culture to about 2% in stationary phase. Fractionation of extracellular proteins by isoelectric focusing and size exclusion chromatography yielded three endoxylanases (XI, X2 and X3) with pI and mol. wts of pH 4.2, 7.1 and 8.4 and 36, 19 and 15 kDa respectively. X1, X2 and X3 had similar pH optima (7.8, 7.2 and 6.8) and K_m for xylan (2.5, 1.4 and 2.0 mg/ml) respectively, but differed in their thermostability; half-lives at 75°C were 21 h for X1, 151 h for X2 and 302 h for X3.     

Cellulolytic Activity of Thermomonospora curvata: Nutritional Requirements for Cellulase Production

The use of a minimal medium for cellulase (C(1) and C(x)) production by Thermomonospora curvata increased extracellular C(1) activity (measured by rate of cotton fiber hydrolysis) 11-fold compared with the previously used yeast extract medium. Ground cotton fibers supported the highest cellulase production when compared to other soluble and insoluble carbohydrate sources. Maximal cellulase production occurred at 45 C, slightly less at 55 C, and was insignificant at 65 C (the highest temperature at which cellulase activity appeared stable). At a temperature of 55 C, an optimal pH of 8.0, and a cotton fiber concentration of 8 mg/ml, shake cultures of T. curvata degraded about 75% of the cellulose during the 10-day period.     

Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata.

Specific cellulase production rates (SCPR) were compared with intracellular cyclic AMP (cAMP) levels in the thermophilic actinomycete, Thermomonospora curvata, during growth on several carbon sources in a chemically defined medium. SCPR and cAMP levels were 0.03 U (endoglucanase [EG] units) and 2 pmol per mg of dry cells, respectively, during exponential growth on glucose. These values increased to about 6 and 25, respectively, during growth on cellulose. Detectable EG production ceased when cAMP levels dropped below 10. Cellobiose (usually considered to be a cellulase inducer) caused a sharp decrease in cAMP levels and repressed EG production when added to cellulose-grown cultures. 2-deoxy-D-glucose, although nonmetabolizable in T. curvata, depressed cAMP to levels observed with glucose, but unlike glucose, the 2DG effect persisted until cells were washed and transferred to fresh medium. SCPR values and cAMP levels in cells grown in continuous culture under conditions of cellobiose limitation were markedly influenced by dilution rate (D). The maxima for both occurred at D = 0.085 (culture generation time of 11.8 h). When D was held constant and cellobiose concentration was increased over a 14-fold range to support higher steady state population levels, SCPR values decreased about fivefold, indicating that extracellular catabolite accumulation may be a factor in EG repression. The role of cAMP in the mechanism of this repression appears to be neither simple nor direct, since large changes (up to 200-fold) in SCPR accompany relatively small changes (10-fold) in cellular cAMP levels.     

Preferential Utilization of Cellobiose by Thermomonospora curvata

Thermomonospora curvata was cultivated on mineral salts medium containing glucose and cellobiose under conditions that increasingly favored the uptake of glucose. In each case cellobiose was utilized in preference to glucose and induced β-glucosidase and endoglucanase activity. [¹⁴C]glucose metabolism studies indicated that cellobiose was not cleaved by extracellular β-glucosidase and transported as glucose. No evidence of cellobiose phosphorylase or a cellobiose-specific phosphoenolpyruvate-phosphotransferase system was observed.     

Changes in Endoglucanase Patterns during Growth of Thermomonospora curvata on Cellulose

The endoglucanases of the thermophilic actinomycete Thermomonospora curvata were characterized. Early-exponential-phase culture fluid contained at least three endoglucanases, with molecular weights of 23,000, 46,000, and 146,000 and K_m values of 1.54, 3.60, and 1.32% carboxymethyl cellulose, respectively. The stationary-phase pattern was altered to include three enzymes with molecular weights of 52,000, 114,000, and 106,000, with respective K_m values of 1.77, 8.30, and 1.91%.     

Production of extracellular enzymes by Thermomonospora curvata during growth on protein-extracted lucerne fibres

After extraction of food protein from lucerne, the residual fibre was used as a carbon and energy source by the thermophilic actinomycete, Thermomonospora curvata. Induction of catabolic exoenzymes during growth for 7 d on the fibre at 53°C in a mineral salts minimal medium was compared with that on a variety of other inductive substrates. A fibre concentration of 1.5% (w/v) was optimal for total protein secretion. The fibre was a poor substrate for amylase production due to lack of inducer rather than to catabolite repression by soluble sugars released during degradation. β-Glucosidase release during growth on the fibre was about 10 times that observed in cultures grown on cellobiose or cellulose, but production of other cellulolytic enzymes was about one-half that produced on cellulose. Pectinolytic activity (measured as polygalacturonate lyase) was equal to that produced on pectin. Cells grown on the fibre released about eight times as much proteinase as those grown on cellulose, but proteolytic activity was transient and decreased rapidly during later growth. Xylanase appeared to be co-ordinately induced with cellulolytic enzymes; comparable maximal activities, observed during growth on either the fibre or cellulose, were three times that produced on xylan or xylose.     

Cell surface changes in Thermomonospora curvata during cellulase induction and repression

Cellulosomes are cell surface protuberances which contain cellulases functional in substrate adherence and hydrolysis. The mycelia of Thermomonospora curvata , which adhere to and grow on native cellulose fibres, formed cellulosomal structures during cellulase induction, but did not when cellulase biosynthesis was repressed. Cell-bound enzyme accounted for about 5% of total culture cellulase activity.     

Induction of α-amylase by maltooligosaccharides in Thermomonospora curvata

The action pattern of the α-amylase produced by Thermomonospora curvata is unique. Maltooligosaccharides (maltose to maltopentaose) were tested individually for their ability to induce α-amylase in this thermophilic actinomycete. Maltotetraose was the most inductive followed by maltotriose. Maltose was a good inducer of amylase production when used as sole carbon source, but had relatively little inductive capacity in the presence of either glucose or cellobiose. When cellobiose was added during exponential growth on maltose, maltose utilization and extracellular α-amylase accumulation were transiently inhibited. With maltotriose as the initial carbon source, addition of cellobiose did not inhibit the utilization of the trisaccharide; however, cellobiose, whether added during exponential growth or stationary phase, resulted in the rapid degradation of amylase when maltotriose was depleted from the medium. This inactivation did not appear to be a growth phase-induced phenomenon because stationary phase cells in the absence of cellobiose maintained their peak extracellular amylase level. This cellobiose-mediated α-amylase inactivation would be particularly important during production of the enzyme on a complex lignocellulosic substrate.     

Cellulolytic Activity of Thermomonospora curvata: Optimal Assay Conditions, Partial Purification, and Product of the Cellulase

Thermomonospora curvata produces cellulases active against both cotton fibers (designated C(1) activity) and carboxymethylcellulose (C(x) activity). In reaction systems employing optimal substrate concentration, pH, and temperature, hydrolysis rates (measured by the release of soluble reducing sugars) were initially linear and decreased on prolonged incubation, although only a small amount of substrate (1 to 2%) had been hydrolyzed. Persistence of this lower rate, even after addition of fresh enzyme (in the C(1) assay system), indicated alteration of cellulose susceptibility to hydrolysis rather than enzyme inactivation. Partial purification by (NH(4))(2)SO(4) precipitation and exclusion chromatography resolved cellulase activity into two fractions. The sole product of purified cellulase activity on ground cotton fibers appears to be cellobiose.     

Adsorption of Thermomonospora curvata cellulases on insoluble substrates

Cellulosic substrates were tested for their ability to bind the cellulases of Thermomonospora curvata . Protein-extracted lucerne fibres had the highest adsorptive capacity while native cotton fibres had the least. Analysis of binding as a Langmuir adsorption isotherm yielded maximal binding capacities of 0.012 filter paper units and 0.51 endoglucanase units/mg lucerne fibre at enzyme saturation. This capacity was increased about 12-fold by fibre pre-treatment in hot NaOH solution.     

Enhanced cellulase production in mutants of Thermomonospora curvata

Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta 1-4-glucanase (EG)when grown on cellulose-mineral salts liquid medium. The EG activity(measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differ in their kinetic properties. Exposure of Thm. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plant containing 2-deoxtglucose of glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures.     

Stabilization of Thermomonospora curvata beta-glucosidase by a low molecular weight intracellular factor

The beta-glucosidase in Thermomonspora curvata was stabilized against thermal denaturation by a soluble intracellular factor. This factor had an apparent molecular weight of 6kD, was resistant to proteolytic digestion, stable to boiling but was inactivated by autoclaving. It had little effect on beta-glucosidase maximal velocities or substrate affinity but it reduced the enzyme inactivation rate five-fold at 63°C.     

Effect of municipal refuse metals on cellulase production by Thermomonospora curvata.

The high-concentration metals in municipal refuse compost were tested for effects on cellulase production and activity in Thermomonospora curvata. Although none altered cellulase reaction rates, both Al and Ca appeared to specifically inhibit cellulase production.     

Extracellular and cell-associated forms of beta-glucosidase in Thermomonospora curvata

The thermophilic actinomycete, Thermomonospora curvata , released 16-times the beta-glucosidase when grown on protein-extracted lucerne fibre compared with growth on cellobiose or purified cellulose. The intracellular and extracellular betaglucosidases had the same mol. wts (66 kD), but the extracellular enzyme had higher affinities for both p -nitrophenyl glucoside and cellobiose and was more resistant to thermal inactivation.