On the sensitivity of isolates of Rhizobium spp. and Azotobacter chroococcum to TMTD and its degradation product NaDDC

Summary The in-vitro effect of the seed dressing fungicide tetramethyl thiuram disulphide (TMTD, Thiram) and its degradation product sodium dimethyl dithiocarbamate (NaDDC) on the sensitivity of Rhizobium spp. and Azotobacter chroococcum isolates was studied at different pH values. The Azotobacter chroococcum isolates were more sensitive than Rhizobium spp. isolates at all pH values. The sensitivity of these isolates to TMTD and NaDDC had a maximum at pH 7, and it decreased on either side of this value. The Rhizobium isolates from urid (Phaseolus radiatus) were resistant to TMTD at pH 7 but proved sensitive to its degradation product. A difference between the power of the sensitive and resistant isolates to reduce TMTD was revealed.     

Effect of Dithiocarbamates on Citric acid production byAspergillus niger

Dithiocarbamates were found to enhance the production of citric acid under solid-state fermentation conditions by Aspergillus niger. Maximum increase was observed with tetramethylthiuram disulfide (TMTD). Percent increases observed were 74.2% with 2.5 micrograms/mL of TMTD, 19.6% with 2.5 micrograms/mL of sodium dimethyldithiocarbamate and 33.1% with 0.6 micrograms/mL of zinc dimethyldithiocarbamate.     

A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars

A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase, BGL I, with high potential for application in biomass conversion. The full-length gene encoding the target enzyme was identified and cloned into Pichia pastoris KM71. Similar to the native enzyme produced by BCC2871, the recombinant beta-glucosidase showed optimal temperature at 70 degrees C and optimal pH of 5 and 6. The enzyme continued to exhibit high activity even after long incubation at high temperature, retaining almost 60% of maximal activity after 1.5h at 70 degrees C. It was also stable under basic conditions, retaining almost 100% of maximal activity after incubation for 2h at pH8. The enzyme has high activity towards cellobiose and other synthetic substrates containing glycosyl groups as well as cellulosic activity toward carboxymethylcellulose. Thermostability of the enzyme was improved remarkably in the presence of cellobiose, glucose, or sucrose. This beta-glucosidase was able to hydrolyze rice straw into simple sugars. The addition of this beta-glucosidase to the rice straw hydrolysis reaction containing a commercial cellulase, Celluclast 1.5L (Novozyme, Denmark) resulted in increase of reducing sugars being released compared to the hydrolysis without the beta-glucosidase. This enzyme is a candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

Variation in cellulase-constituting components from Trichoderma reesei with agitation intensity

Cellulase producing activity of Trichoderma reesei QM9414 was examined under various agitation intensities and at the dissolved oxygen concentration above 3 ppm. The producing activity greatly depended upon the agitation intensity, and the dependence on the agitation was different for each cellulase-constituting component. The maximum producing activities of FPA, CM Case, and beta-glucosidase were obtained under different agitation conditions, 1.0, 0.7, and 1.4 m/s in tip velocity, respectively. Intensive agitation brought about remarkable reduction in all cellulase components. The mycelial transformation through agitation intensity was also observed. Comparatively mild agitation of 0.3-1.0 m/s caused pellet formation as the culture progressed, although the pelletization was delayed with increasing agitation intensity. The behavior of the pelletization did not occur at 1.3 and 1.7 m/s throughout the course of cultivation, and under the latter agitation condition hyphae were broken up into short fragments. The cellulase producing activity is discussed in relation to such morphological changes.     

Cellulase kinetics as a function of cellulose pretreatment

Microcrystalline cellulose (Avicel) was subjected to three different pretreatments (acid, alkaline, and organosolv) before exposure to a mixture of cellulases (Celluclast). Addition of beta-glucosidase, to avoid the well-known inhibition of cellulase by cellobiose, markedly accelerated cellulose hydrolysis up to a ratio of activity units (beta-glucosidase/cellulase) of 20. All pretreatment protocols of Avicel were found to slightly increase its degree of crystallinity in comparison with the untreated control. Adsorption of both cellulase and beta-glucosidase on cellulose is significant and also strongly depends on the wall material of the reactor. The conversion-time behavior of all four states of Avicel was found to be very similar. Jamming of adjacent cellulase enzymes when adsorbed on microcrystalline cellulose surface is evident at higher concentrations of enzyme, beyond 400 U/L cellulase/8 kU/L beta-glucosidase. Jamming explains the observed and well-known dramatically slowing rate of cellulose hydrolysis at high degrees of conversion. In contrast to the enzyme concentration, neither the method of pretreatment nor the presence or absence of presumed fractal kinetics has an effect on the calculated jamming parameter for cellulose hydrolysis.     

Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover

The production of extracellular cellulases by a newly isolated thermoacidophilic fungus, Aspergillus terreus M11, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The results showed that the high-level cellulase activity was produced at 45 degrees C pH 3 and moisture 80% with corn stover and 0.8% yeast extract as carbon and nitrogen sources. 581 U endoglucanase activity, 243 U filter paper activity and 128 U beta-glucosidase activity per gram of carbon source were obtained in the optimal condition. Endoglucanase and beta-glucosidase exhibited their maximum activity at pH 2 and pH 3, respectively, and both of them showed remarkable stability in the range of pH 2-5. The activities of endoglucanase and beta-glucosidase were up to the maximum at 70 degrees C and maintained about 65% and 53% of their original activities after incubation at 70 degrees C for 6h. The enzyme preparations from this strain were used to hydrolyze Avicel. Higher hydrolysis yields of Avicel were up to 63% on 5% Avicel (w/v) for 72 h with 20 U FPase/g substrate.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Engineering cellulase mixtures by varying the mole fraction of Thermomonospora fusca E5 and E3, Trichoderma reesei CBHI, and Caldocellum saccharolyticum beta-glucosidase

In this study, different mole fractions of pure Thermomonospora fusca E(5) and E(3), plus Trichoderma reesei CBHI were studied for reducing sugar production at 2 h, degree of synergism, and cellulose binding. In addition, the effects of introducing the Caldocellum saccharolyticum beta-glucosidase into this cellulase system were investigated. The cellulases used were purified to homogeneity. Avicel PH 102 (4% w/w solution in 0.05 sodium acetate pH 5.5 buffer) was the substrate. Reactions were run at 50 degrees C for 2 h using total cellulase concentrations of 8.3 or 12.2 muM. A bimixture of T. fusca E(3) and T. reesei CBHI was very effective in hydrolyzing microcrystalline cellulose (9.1% conversion). The addition of endoglucanase E(5) to the mixture only increased conversion to 9.8%. However, when both E(5) and beta-glucosidase were added, conversion increased to 14%. It was also observed that increasing total cellulase concentration beyond 8.3 muM did little to increase percent conversion of cellulose into glucose. The results of the binding studies indicate no competition for binding sites between the endo- and exocellulases. (c) 1993 John Wiley & Sons, Inc.     

Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata

A mutant of Alternaria alternata excreted enhanced levels of carboxymethylcellulase and particularly beta-glucosidase when grown in cellulose liquid media. Both enzymes were purified two- to four-fold by ammonium sulfate precipitation and gel filtration, and the kinetic data showed K(m) values of 16.64 mg/ml of culture fluid for carboxymethylcellulase and 0.14 mM p-nitrophenyl-beta-d-glucoside and 0.81 mM cellobiose for beta-glucosidase at pH 5. Carboxymethylcellulase and extracellular beta-glucosidase functioned optimally at pH 5 to 6 and 4.5 to 5 and at temperatures of 55 to 60 and 70 to 75 degrees C, respectively. Both temperature optima and thermostability of beta-glucosidase were among the highest ever reported for the same enzyme excreted from cellulase and beta-glucosidase hyperproducing microorganisms.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.     

Purification and some properties of beta-transglycosylase of Trichoderma longibrachiatum

A beta-transglycosylase was purified to a homogeneous state from the extract of a wheat bran Koji culture of Trichoderma longibrachiatum by column chromatography. The purified enzyme showed a typical disproportionation reaction with cellopentaose as the substrate, producing a high molecular component (a water-insoluble glucan). The enzyme showed neither cellulase nor beta-glucosidase activity. The reaction was optimal at pH 6.0 and 37 degrees C. The molecular weight of the enzyme was estimated to be 11,000 by gel filtration using a TOYOPEARL HW-55F column. The amount of the glucan synthesized by the enzyme increased with prolonged incubation in a reaction with cellopentaose, and soluble cellooligosaccharides, such as cellobiose, cellotriose, cellotetraose, and cellohexaose, were also produced. No glucose was produced in the reaction even when it was carried out for a long time. The total number of molecules (cellooligosaccharides) in the reaction mixture remained at the initial substrate level during the entire reaction. The beta-transglycosylase proved to be a specific transferase showing transfer activity of glucosyl, cellobiosyl, and cellotriosyl moieties from one cellopentaose to an acceptor molecule from cellopentaose upwards with almost 100% efficiency.     

A cellulase assay coupled to cellobiose dehydrogenase

An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented. Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result. The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition. The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity. With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated. An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out.     

Productions of cellulase from Pestalotiopsis versicolor

Production of cellulase from Pestalotiopsis versicolor was studied in a shake flask culture. The cellulase system was found to be rich in beta-glucosidase. Kinetic parameters such as pH and temperature have been optimized for the various enzyme components.     

Effect of methylxanthines on production of cellulases by Penicillium echinulatum

AIM: In this work, the effect of supplementing liquid cellulase production media (CPM) with methylxanthines (aminophylline, caffeine and theophylline), with and without the addition of glucose, on the secretion of cellulases by Penicillium echinulatum strain 2HH (wild-type) and the derived mutant strain 9A02S1 was studied. METHODS AND RESULTS: When compared with unsupplemented CPM, both strains produced higher beta-glucosidase and filter paper activities (FPAs) in CPM supplemented with 1 micromol l(-1) of caffeine but lower activities with 5 micromol l(-1) of caffeine. With theophylline only, strain 9A02S1 produced higher beta-glucosidase and FPAs, while aminophylline produced no effect on the cellulase activity of either strain. Supplementation of CPM with 0.5% (w/v) of glucose plus caffeine resulted in higher beta-glucosidase and FPAs being produced by strain 2HH, but not strain 9A02S1, than in CPM supplemented with 0.5% (w/v) of glucose only. CONCLUSIONS: These results indicate that different concentrations of caffeine and theophylline can increase the beta-glucosidase and FPAs produced by P. echinulatum strains 2HH and 9A02S1. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that some methylxanthines, in adequate concentration, can be used as media components to increase cellulase production.     

Association of beta-glucosidase with intact cells of Thermoactinomyces.

The location of the B-glucosidase activity in a whole culture broth of the thermophilic organism Thermoactinomyces has been studied. Little beta-glucosidase activity was found in the culture filtrate, while the culture solids contained the major part of the activity of the whole culture broth. The activity does not appear to be adsorbed to the culture solids; rather there is evidence that it is an intracellular soluble enzyme(s). The pH and temperature optima for a crude beta-glucosidase preparation were determined to be pH 6.5 and 50--55 degrees C. Enzyme activity studies indicate that the same enzyme(s) accounts for the beta-glucosidase and the cellobiase activities. The validity of using the filter paper activity of culture filtrates from Thermoactinomyces to predict the total saccharification of cellulosic materials to glucose is discussed.     

The cellulase of Fusarium solani. Resolution of the enzyme complex

1. Culture filtrates from Fusarium solani were fractionated by ion-exchange chromatography on DEAE-Sephadex, followed by gel chromatography on Sephadex G-100, into a C(1) component, a C(x) component (CM-cellulase) and a beta-glucosidase (cellobiase) component. 2. The individual components showed little capacity for the solubilization of cotton fibre (cellulase activity), but when recombined in their original proportions 81% of the original cellulase activity was recovered. 3. The C(1) components of F. solani and Trichoderma koningii were similar in their pH optima, heat stabilities over the pH range 5-8 and elution volumes on Sephadex G-100. 4. The C(1) component of F. solani synergized with the C(x) component of T. koningii and conversely. 5. The C(1) and the beta-glucosidase components of F. solani were devoid of the swelling-factor (S-factor) activity associated with the C(x) component.     

The effect of pH on the foam fractionation of beta-glucosidase and cellulase

The surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.     

Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens

Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I (cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation.     

Addition of cloned beta-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex

A thermostable beta-glucosidase from Clostridium thermocellum which is expressed in Escherichia coli was used to determine the substrate specificity of the enzyme. A restriction map of the beta-glucosidase gene cloned in plasmid pALD7 was determined. Addition of the E. coli cell extract (containing the beta-glucosidase) to the cellulase complex from C. thermocellum increased the conversion of crystalline cellulose (Avicel) to glucose. The increase was specifically due to hydrolysis of the accumulated cellobiose. A cellulose degradation process using beta-glucosidase to assist the potent cellulase complex of C. thermocellum, as shown here can open the way for industrial saccharification of cellulose to glucose.     

Influence of enzyme loading and physical parameters on the enzymatic hydrolysis of steam-pretreated softwood

Softwood is an interesting raw material for the production of fuel ethanol as a result of its high content of hexoses, and it has attracted attention especially in the Northern hemisphere. However, the enzymatic hydrolysis of softwood is not sufficiently efficient for the complete conversion of cellulose to glucose. Since an improvement in the glucose yield is of great importance for the overall economy of the process, the influence of various parameters on the cellulose conversion of steam-pretreated spruce has been investigated. The addition of beta-glucosidase up to 50 IU g(-)(1) cellulose to the enzymatic hydrolysis process resulted in increased cellulose conversion at a cellulase loading up to 48 FPU g(-)(1) cellulose. Despite very high enzyme loading (120 FPU g(-)(1) cellulose) only about 50% of the cellulose in steam-pretreated spruce was converted to glucose when all of the material following pretreatment was used in the hydrolysis step. The influence of temperature, residence time, and pH were investigated for washed pretreated spruce at a dry matter (DM) content of 5% and a cellulase activity of 18.5 FPU g(-)(1) cellulose. The optimal temperature was found to be dependent on both residence time and pH, and the maximum degree of cellulose conversion, 69.2%, was obtained at 38 degrees C and pH 4.9 for a residence time of 144 h. However, when the substrate concentration was changed from 5% to 2% DM, the cellulose conversion increased to 79.7%. An increase from 5% to 10% DM resulted, however, in a similar degree of cellulose conversion, despite a significant increase in the glucose concentration from 23 g L(-)(1) to 45 g L(-)(1). The deactivation of beta-glucosidase increased with increasing residence time and was more pronounced with vigorous agitation.