Production and activity of Thermomonospora curvata cellulases on protein-extracted lucerne fibers

Summary Protein-extracted lucerne fibre (PELF) was evaluated as a carbon/energy source for cellulase production by Thermomonospora curvata and as a substrate for enzymatic conversion to soluble sugars. In shake cultures grown at 53°C, pH 8, maximal cellulase (50 endoglucanase IU and 0.7 filter paper IU/ml) was attained at a PELF concentration of 1.3% (w/v) in a vitamin-mineral salts medium buffered with 0.05 M concentrations of phosphate and N-2-hydroxyethylpiperazine-N-2-ethanesulfonate. Multiple endoglucanase enzymes were produced. The major form had an apparent molecular weight of 22 000 and K _m values of 0.23% and 0.56% for carboxymethylcellulose and PELF respectively. NaOH treatment of PELF increased its susceptibility to enzymatic hydrolysis. During enzymatic hydrolysis of NaOH-treated PELF (60°C, pH 6.1, 24h) two-thirds of total fibre carbohydrate was solubilized as cellobiose, melibiose, cellopentaose, cellotetraose, xylose and glucose in descending order of concentration.     

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.     

Cyclic AMP phosphodiesterase in Thermomonospora curvata.

Cyclic AMP phosphodiesterase (PDE; EC 3.1.4.17) in Thermomonospora curvata was purified and characterized. Fractionation of cell extracts by ion-exchange and size-exclusion chromatography revealed four PDE isozymes, which differed markedly in molecular weight, theophylline sensitivity, pH optima, and substrate affinity. Although the enzyme was labile after purification, total recovery of PDE activity was fivefold that of the crude extract. PDE biosynthesis appeared sensitive to the growth phase, growth rate, and carbon source. PDE levels in batch cultures peaked and declined rapidly during mid-exponential-phase growth. In continuous culture, maximal PDE and cellulase production occurred at dilution rates yielding mean cell generation times of about 5 and 17 h, respectively. The addition of glucose to cellulose-grown cells caused declines in both cyclic AMP and PDE levels, suggesting that the enzyme was subject to, rather than the agent of, catabolite repression.     

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.     

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.     

Effects of the detergent Tween 80 on Thermomonospora curvata

Tween 80 (0.1%, v/v) added to Thermomonospora curvata growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54°C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin.     

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.     

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 cellulases by Thermomonospora sp.

Glucose, cellulose, Avicel, and Solka Floc were utilized as substrates for growth of Thermomonospora sp in order to study the induction–repression characteristics of its associated cellulase system. While glucose proved to be an effective repressor of the cellulase enzymes, the other three substrates induced relatively high levels of enzyme activity as measured by the filter paper assay. On a unit cell mass basis the highest values of cellulase activity were obtained when Avicel was utilized as the carbon and energy source. The nature of the cellulosic material and its initial concentration were identified as two very important parameters of the induction process.     

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.     

Influence of carbon source on cell surface topology of Thermomonospora curvata.

The appearance of cell surface protuberances in Thermomonospora curvata correlated with cell-bound exoenzymes which could be removed by brief sonication. Mycelia grown on cellulose or xylan had numerous protuberances and retained 20 to 25% of endoglucanase and endoxylanase at cell surfaces, while those grown on pectin or starch had few protuberances and negligible bound pectinase or amylase.     

Selective adsorption of endoglucanases from Thermomonospora curvata on protein-extracted lucerne fibres

Cellulases of Thermomonospora curvata contain three endoglucanase components. Two of these enzymes (molecular weights of 46 and 106 kD) bound to protein-extracted lucerne fibres. Maximal adsorption occurred within 6 min, followed by a gradual desorption during fibre hydrolysis.     

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.     

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.     

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.     

Cellulase biosynthesis during degradation of cellulose derivatives by Thermomonospora curvata

A variety of commercially used cellulose derivatives were compared with crystalline cellulose as substrates for induction of cellulase biosynthesis in the actinomycete Thermomonospora curvata. Cellulase induction during growth on uncoated cellophane was as rapid as that on crystalline cellulose, but on coated cellophanes, induction was delayed. Susceptibility to enzymatic attack determined the inductive potential of the substrate. Cellulose acetate was a poor substrate because of its extreme recalcitrance to attack. With other cellulose derivatives, soluble sugar accumulation caused a transient repression of cellulase biosynthesis, but the ratio of cellobiose (a cellulase inducer) to glucose (a cellulase repressor) was not a controlling factor. Crystalline cellulose yielded the lowest inducer/repressor sugar ratio (1.1:1 compared to 3.8–4.0:1 for cellulose derivatives), but supported the highest cellulase production. Glucose could not repress cellulase biosynthesis in the presence of cellobiose due to the strong preference for uptake of the disaccharide even by glucose-grown cells.     

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.