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.     

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.     

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.     

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.     

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.     

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 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.     

Component-specific stimulation of cellulase secretion in Thermomonospora curvata by the surfactant Tween 80

The non-ionic surfactant, Tween 80, stimulated the secretion of extracellular proteins by 35–140% in Thermomonospora curvata during growth on a variety of substrates. Cellulase secretion was also stimulated but fractionation of extracellular proteins by ion-exchange high performance liquid chromatography showed that this stimulation was largely confined to a single enzymatic component (or group of closely related components) active against crystalline cellulose. The surfactant's effect was more pronounced during growth on cellobiose octaacetate than on the soluble sugar, cellobiose, or on crystalline cellulose.     

Protein-extracted lucerne fibres as a carbon and energy source for cellulase production by Thermomonospora curvata

Protein-extracted lucerne fibre (PELF) was evaluated as a carbon/energy source for cellulase production by the thermophilic actinomycete, Thermomonospora curvata. Shake cultures with lucerne fibre produced only one-half the cellulase obtained from growth on cotton fibre (the most inductive substrate). Control of pH at 8·0 under fermenter conditions lowered the rate of PELF carbohydrate solubilization during early growth and raised saccharifying cellulase production by about 70% to a maximum of 0·48 filter paper units/ml.     

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%.     

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.     

Cyclic AMP levels in Drosophila during postembryonic development and in the adults.

The physiological content of Drosophila melanogaster tissues in cyclic AMP has been determined and its variations studied during postembryonic development and in the adults. Marked variations were observed, especially during metamorphosis where the ratio between the lowest and highest values (0·35 to 17·25 pmoles/mg protein) was $\text{1}\text{44}$. In larvae the variations of cyclic AMP level were not clearly related to the larval ecdyses, but the steps of metamorphosis, i.e. formation of the puparium, larval-pupal apolysis, and pupal-adult apolysis, were accompanied with rapid and drastic rises of cyclic AMP, up to the highest value mentioned. We therefore deduce that cyclic AMP is involved in the metamorphosis of D. melanogaster as a chemical signal. In adults, the cyclic AMP level was remarkably constant and was around 7 pmoles/mg protein.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Cyclic AMP levels during the maturation of Xenopus oocytes

While several studies have suggested that the induction of oocyte maturation results from a transient decrease in cAMP levels, attempts to demonstrate such a change have led to inconsistent results with respect to whether or not a decrease occurs as well as timing of the decrease. In this report the results of experiments designed to demonstrate small changes in cAMP content in Xenopus laevis oocytes are presented and a statistically significant 20% decrease in cAMP content from between 2 and 50 min postprogesterone addition is found. The cAMP content subsequently rises to 12% higher than control levels and then becomes indistinguishable from control values for the remainder of the maturation period.     

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.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

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.