Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima,protease and ion sensitivities

Summary The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono--lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to -endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values

Summary Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastix frontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel R _f values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Media carbon induction of extracellular cellulase activities inNeocallimastix frontalis isolate EB188

Extracellular cellulase induction in the ruminal fungusNeocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5.     

Carbon source,cyclic nucleotide,and protein inhibitor effects on protein and cellulase secretions inNeocallimastix frontalis EB188

Protein and cellulase secretion inNeocallimastix frontalis EB188 was studied. Induction of secretion in this ruminal fungus was dependent on the amount of medium cellulose and on de novo protein synthesis. Medium glucose repressed secretions and diminished the utilization of medium cellulose. Exogenously added cyclic nucleotides failed to derepress glucose repression. Medium glycerol had no effect on protein and cellulase secretion, would not support cell growth, and was nontoxic.     

The effect of Aspergillus oryzae fermentation extract on the anaerobic fungus Neocallimastix frontalis EB 188: effects on physiology

 Aspergillus oryzae fermentation extract (Amaferm) was used to stimulate the in vitro growth of the cellulolytic fungus Neocallimastix frontalis EB 188. Soluble and filter-sterilized extract was added either at the start or throughout culture growth. Culture mass, protein secretion and cellulase secretion were measured in stationary test-tubes or round-bottom flasks and a stirred (desktop) fermenter. The soluble extract increased culture mass and protein and cellulase secretion in a dose-dependent manner. Maximum stimulation caused the supernatant cellulase to nearly double (i.e., 87% over controls; P<0.05), cell mass increased by 27% (P<0.05) over controls and secreted protein increased 37% (P<0.05) over controls. The timing of extract addition did not alter the culture response and suggested a recycling of components. The robustness of fungal zoospores used as inoculum, however, greatly influenced the effectiveness of the extract to stimulate secretions. Extracts did not directly influence the pH of the culture medium or the endogenous levels of enzymes. The rate of carbon source utilization and morphology of the fungus were unchanged by soluble-extract additions at any level tested. The extract was inhibitory when added to concentrations exceeding an amount equivalent to 20 g animal^-1 day^-1. Received: 6 December 1995/Received revision: 7 February 1996/Accepted: 4 March 1996     

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 g/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 g/ml and 4.5 ± 1.5 g/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 10⁹ cfu/ml and decreased to 1.5 (± 0.2) × 10⁹ cfu/ml and 0.5 (± 0.3) × 10⁹ cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Nascent synthesis and secretion of cellobiase inNeocallimastix frontalis EB188

De novo synthesis and the secretion of cellobiase fromNeocallimastix frontalis EB188 were studied. Cellobiase was secreted rapidly in cellulose switch cultures. Chemical inhibition of protein synthesis and radiotracer studies suggested secretion was dependent on nascent protein synthesis. Inhibitors of protein glycosylation also inhibited secretion. An 85,000 dalton protein (and several others) represented the principal differences in de novo synthesis between cellulose- and glucose-switched cultures. Concentrations of the 85,000 daltons protein increased with culture incubation time and ultimately accounted for 7% of the total protein secreted. This protein was purified by gel electrophoresis separations and was determined to be a cellobiase. Secretion of this cellobiase was correlated with its radiolabeling. The possibility of cellobiase induction representing a specialized gene control system inNeocallimastix frontalis EB188 is discussed.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

The effect of Aspergillus oryzae fermentation extract on the anaerobic fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206: generalized effects and component analysis

Three fungi Neocallimastix frontalis EB 188, Piromyces communis DC 193 and Orpinomyces ssp. RW 206, representing the predominant cultures isolated from cattle, were shown to respond to the addition of Aspergillus oryzae fermentation extract (i.e., Amaferm; BioZyme Inc., St. Joseph, Mo.) stimulation. Growth rates, protein and cellulase secretion and fungal mass production were all accelerated in the presence of the extract. Analysis of volatile fatty acids produced by these three species suggested that extract addition increased and altered gas production. Fractionation and preliminary analysis of the components present in the soluble extract, which stimulated the growth of the cellulolytic fungus N. frontalis EB 188, were also attempted. Soluble and filtered, sterilized extract was treated prior to use as a stimulant. Pretreatments included dialysis, ultraviolet irradiation, freeze thaw cycling, boiling, autoclaving, digestion with protease, autodigestion, organic extraction, decolorizing-carbon binding and polyethylene glycol concentration. Boiling, protease treatment, organic extraction, freeze thaw cycling and decolorizing-carbon binding reduced the ability of the extract to stimulate fungal cultures. Gel electrophoresis methods demonstrated that protein- and cellulasesecretion profiles were not identical in control and stimulated cultures. High-performance liquid chromatography methods allowed the separation of the extract into a limited number of ultraviolet-absorbing peaks, of which several stimulated the physiology of the fungus. Received: 6 December 1995/Received revision: 7 February 1996/Accepted: 4 March 1996     

Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities

Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml⁻¹, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran.     

The effect of pH on the production of cellulases in Aspergillus terreus

Summary The optimal pH for the production of extracellular cellulolytic enzymes in the wild strain of Aspergillus terreus was shown to be pH 5.0. After 160 h of cultivation, carboxymethyl cellulase reached 9.0 IU/ml, filterpaper, cellulase 0.5 IU/ml and -glucosidase 0.9 IU/ml. The rate of synthesis of CM- and FP-cellulases decreased after 90 h of cultivation but -glucosidase was produced linearly for 160 h. Some of the enzymes produced were released into the medium during the fungal growth while others remained bound. The binding of enzymes to cells and residual crystalline cellulose was strongly affected by the pH of the medium. FP-cellulase and particularly -glucosidase were bound more effectively, at lower pHs. Cold shock treatment of the cell suspension increased the activities of FP- and CM-cellulases but -glucosidase activity was not affected.     

Biostoning of denims by Penicillium occitanis (Pol6) cellulases

The Pol6 mutant of Penicillium occitanis, secreting a large quantity of cellulases, was cultivated in fermentor using a local paper pulp as an inducer substrate. A high titer of extracellular cellulase activity was reached after a fed batch process: 23 IU ml⁻¹ filter paper activity, 21 IU ml⁻¹ CMCases activity (endoglucanase units) and 25 mg ml⁻¹ of proteins. Various tests were done to compare the action of the P. occitanis cellulases with those commercially available and with the traditional stonewashing process. This cellulase preparation was successfully applied in a biostoning process at an industrial scale. The abrasive effect of the P. occitanis cellulases was very uniform and with an efficiency comparable to that obtained by the commercial ones.     

A solid-state bioreactor coupled with forced aeration and pressure oscillation

A novel design of a solid-state bioreactor, operated with periodic pressure oscillation coupled with forced aeration through the medium, gave efficient control of temperature. The evaluation of the bioreactor assembly with respect to temperature and cellulase production by Penicillium decumbens JUA10 showed that, at 4 atm and the bed depth of 6 cm, the maximal temperature variation in the reactor was +1.5 °C at a set value of 30 °C compared with +6.8 °C in a static tray system. The highest cellulase and -glucosidase activities were 15 IU g^–1 and 51 IU g^–1 substrate dry matter at 96 h, respectively, while only 10 IU g^–1 and 24 IU g^–1 were obtained in the static tray culture system.     

Disruption of a filamentous fungal organism (N. sitophila) using a bead mill of novel design II. Increased recovery of cellulases

A native strain of Neurospora sitophila was disrupted using enzymatic pretreatment combined with mechanical disruption in order to facilitate recovery of constitutive cellulases. Exceptional disruption (approaching 100%) was achieved when the enzymatic pretreatment protocol was used prior to mechanical disruption at a low rotor speed via a new bead mill (the Annu Mill). Further, increased recovery of cellulases (ca. two-fold increases in cellulase activity per unit biomass) appears attainable when this disruption protocol is employed. The enzyme preparation employed was Zymolyase, which lyses the walls of viable fungi. Combined disruption of the mycelial biomass appears to provide a secondary source of cellulases from Neurospora sitophila in addition to the extracellular primary source derived from the filtered (unprocessed) fermentation broth.Nomenclature CMCase carboxymethyl cellulase - FPase filter paper'ase - IU international unit (mol liberated hydrolysis product/min.) - N number of passes through the bead mill (–) - R total fraction of cells disrupted (–) - Ro fraction of cells disrupted after enzymatic pretreatment alone (–) - X cell concentration (dry weight) (gDW/L) Abbreviations DW dry weight     

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.     

Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation

Summary Six thermophilic fungi were examined for their ability to produce cellulolytic enzymes in liquid (LF) and solid-state fermentation (SSF). The best cellulase activities were achieved by Thermoascus aurantiacus and Sporotrichum thermophile. Taking into consideration that solid-state medium obtained from 100 g of dry sugar-beet pulp occupies about 11 of fermentor volume equivalent to 11 of LF, it was confirmed that enzyme productivity per unit volume from both fungi was greater in SSF than in LF. The cellulase system obtained by SSF with T. aurantiacus contained 1.322 IU/1 of exo--d-glucanase, 53.269 IU/1 of endo--d-glucanase and 8.974 IU/1 of -d-glucosidase. The thermal and pH characteristics of cellulases from solid-state fermentation of T. aurantiacus and S. thermophile are described.     

Characterization of cellulase and xylanase activities of Streptomyces lividans

Summary The production of cellulases and of xylanase by Streptomyces lividans 1326 was studied under different growth conditions. The strain grew between 18°C and 46°C and is therefore thermotolerant. Submerged cultures of the microorganism, when grown on a defined salt medium containing xylan as main carbon source, exhibited an overall cellulolytic activity as determined by the filter paper test. S. lividans produced optimal levels of extracellular -1,4-glucan-glucanohydrolase (1 IU/ml) and large amounts of -1,4-xylanxylanohydrolase (50 IU/ml) at 40°C. A better production of both enzymes was observed when xylan instead of cellulose was used as substrate.The stability of the enzyme was found to be significantly greater than those of the cellulases and xylanases produced by other streptomycetes. The optimal incubation temperatures for the enzyme assays were 55°C and 60°C for CM-cellulase and xylanase respectively and optimal pH values were found in the range of pH 6–7.     

Thermostable cellulases from <Emphasis Type="Italic">Streptomyces</Emphasis>sp.: scale-up production in a 50-l fermenter

Thermostable cellulase was produced by Streptomyces sp. T3-1 grown in a 50-l fermenter. Maximum cellulase activity was attained on the fourth day when agitation speeds and aeration rates were controlled at 300 rpm and 0.75 vvm, respectively. Maximum enzyme activities were: 148 IU CMCase ml^–1, 45 IU Avicelase ml^–1, and 137 IU -glucosidase ml^–1 with productivity of 326 IU l^–1 h^–1, which were 10--32% higher than the values obtained in shake-flask culturesRevisions requested 12 October 2004/1 November 2004; Received received 1 November 2004/14 December 2004