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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of carbon source on growth temperature and fatty-acid composition in Thermomonospora curvata

The growth-temperature range of the actinomycete, Thermomonospora curvata, was influenced by the nature of the soluble carbon sources used, which were derived from cellulose, pectin, starch and xylan. This thermophile had the broadest (38 to 65°C) and narrowest (42 to 59°C) temperature range during growth on cellobiose (from cellulose) and 4-deoxy-Lxxx-threo-t-hexoseulose uronic acid (from pectin), respectively. This substrate-temperature interaction was accompanied by changes in cellular fatty acids: uronic-acid-grown cells had relatively low amounts of branched chain fatty acids (particularly iso-16:0) and high amounts of monounsaturated fatty acids (particularly cis-18:1) compared with cells grown on any other substrate. Moreover, uronic-acid-grown cells could not respond to increased growth temperature by altering the ratio of branched chain fatty acids to straight chain fatty acids.F.J. Stutzenberger is with the Department of Microbiology, Clemson University, Clemson, SC 29634-1909, USA; T.C. Jenkins is with the Department of Animal, Dairy and Veterinary Sciences at the same university.     

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.     

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.     

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.     

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.     

Influence of carbon source on production of a heat stable protease from Thermomonospora fusca YX

Summary Thermomonospora fusca YX produced a very active heat stable protease when incubated in media containing cellulose as the substrate. Cultures grown on Solka-floc generated the highest amount of protease whereas the protease was produced at significantly lower levels when T. fusca YX was grown on cellobiose or glucose. Negligible growth or protease production was observed when protein was used as a carbon source. The production of the protease did not appear to be constitutive. While rapid growth was observed on either cellobiose or glucose, protease levels were at least two to fourfold lower than for the T. fusca YX cultures grown on Solka-floc wich generated 33% less cell mass. Protease production was four times lower in cultures which employed casein hydrolysate (tryptone) or xylan as carbon sources than for cellulose.     

Acetate as a carbon source for hydrogen production by photosynthetic bacteria

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.     

para-Aminobenzoic acid as a sole source of carbon and energy for Pseudomonas desmoliticum]

A strain of Psuedomonas desmoliticum has been isolated from soil. It utilizes, as a source of carbon and energy, p-aminobenozic (PABA), p-fluorobenzoic acids, and some other aromatic compounds. The strain has been isolated by inoculating soil suspensions onto Petri plates with a solid mineral medium containing 0.1% PABA as a carbon source. The preparatory metabolism of PABA was studied in this work; p-hydroxybenzoic and protocatechuic acids were found to be its intermediate products. Enzyme systems catalysing oxidation of aromatic compounds and glucose are inducible.     

Utilization of cephalothin by Yersinia enterocolitica as sole carbon and energy source

Two Yersinia enterocolitica strains were able to utilize the products of cephalothin degradation. The utilization of these products was shown by an increase of oxygen uptake by Y. enterocolitica with cephalothin as the only substrate, and by the growth of both strains with the hydrolysis products of cephalothin as sole energy and carbon sources. Nuclear magnetic resonance analysis of the cephalothin degradation reaction demonstrated the progressive disappearance of hydrolysis products. However, the products of benzylpenicillin degradation could not be utilized by Y. enterocolitica.     

Utilization of benzylpenicillin as carbon,nitrogen and energy source by a Pseudomonas fluorescens strain

A bacterium which utilizes benzylpenicillin as carbon, nitrogen and energy source was isolated from a lake sediment. The organism was identified as a strain of Pseudomonas fluorescens with a GC content of 59.71 Mol %. After growth of the organism on a mineral salts medium containing benzylpenicillin, the derivatives benzylpenicilloic acid, benzylpenilloic acid and benzylpenicillenic acid were found in culture media. There was no indication that the phenylacetate side chain of benzylpenicillin is decomposed. In uninoculated culture media benzylpenicillin, benzylpenicilloic acid and benzylpenicillenic acid were demonstrable. The following compounds were found to be absent from inoculated or uninoculated culture fluids: d-penicillamine, l-valine, l-cysteine, benzylpenillic acid and 6-aminopenicillanic acid. The organism possesses penicillinase. Penicillin acylase was not demonstrable. The reaction product of penicillinase, benzylpenicilloic acid, supports only little growth. There is no growth on 6-aminopenicillanic acid with or without NH₄Cl. Relatively little growth occurs on 6-aminopenicillanic acid in the presence of phenylacetic acid.The data indicate that the nucleus of the benzylpenicillin molecule is utilized as carbon, nitrogen and energy source. During growth a part of the substrate is destroyed into scarcely usable benzylpenicilloic acid; hereby the antibiotic is detoxified.Abbreviations TLC thin-layer chromatography - DNPH 2,4-dinitrophenylhydrazine