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Occupational asthma caused by cellulase and lipase in the detergent industry.

Three employees from two different detergent companies were investigated for occupational asthma, using skin prick tests, serum specific IgE, and specific bronchial challenge. Two were challenged with lipase and one with cellulase. All three cases had immunological evidence of sensitisation to the detergent enzymes with which they worked. Bronchial challenge in each provoked a reproducible dual asthmatic response, which reproduced their work related symptoms. These are the first reported cases of occupational asthma attributable to cellulase and lipase in the detergent industry. Four of the most common enzymes used in this industry have now been reported to cause occupational asthma; continued vigilance and caution are needed when working with these or other enzymes.

Adult↗

The cellulase system of a Cytophaga species.

Cellulases (EC 3.2.1.4) of a Cytophaga species WTHC 2421 (ATCC 29474) were found in the soluble portion of the cell (the periplasm and the cytoplasm) and on the membrane. Cell-free cellulases were not found. Most of the carboxymethylcellulase activity associated with reduction of viscosity was membrane bound, whereas most of the carboxymethylcellulose (CMC) saccharifying activity was soluble. The CMC-saccharifying activity was increased 534 X by purification procedures which included ammonium sulfate precipitation and molecular exclusion chromatography with Sephadex G-75 and Biogel p-100. Periplasmic carboxymethycellulase had a molecular weight of 6250 and cytoplasmic carboxymethylcellulase had a molecular weight of 8650. Analytical ultracentrifugation of the periplasmic carboxymethylcellulase (CMCase) indicated that it had a low molecular density. The chromatographic fraction containing periplasmic CMCase also contained enzyme activity against crystalline cellulose. The activity against crystalline cellulose was 238 X higher than the activity shown by the whole cell. The reaction of the enzyme with either CMC or dewaxed cotton produced only glucose. The enzyme was slightly inhibited by the presence of 0.01% (w/v) glucose, lactose, or cellobiose, but it was not affected by sucrose, and exhibited increased activity in the presence of xylose and fructose.

Carbohydrates↗

Hyphal tip growth in Achlya. II. Subcellular localization of cellulase and associated enzymes.

The isolation and characterization of cellulase-containing membranes of Achlya ambisexualis Raper was attempted by differential and density gradient centrifugations. Maximum cellulase activity was found at an isopycnic density of 1.19 g/cm3, although some activity was found at other densities. A similar distribution of activity was shown by IDPase, ATPase, UDPG transferase, and by sedimentable carbohydrate. The coequilibration and steady enrichment of these activities during purification suggests their presence in a single type of subcellular particle. It was not possible to identify clearly the particle(s) in question from isolated fractions by electron microscopy, but when compared with the cytochemical localization of carbohydrate and IDPase in intact hypha, cytoplasmic vesicles with 150-mm diameters seem to be likely candidates.

Acid Anhydride Hydrolases↗

Production of cellulases and xylanases by low-temperature basidiomycetes.

Three of four isolates, representing phylogenetically distinct groupings of low-temperature basidiomycetes (LTB), were capable of utilizing wheat straw, and to a lesser extent conifer wood at 15 degrees C. A cottony snow mould LTB (LRS 013) and a fruit rot LTB (LRS 241) grown on straw significantly degraded filter paper, carboxymethylcellulose (CMC), p-nitrophenyl beta-glucopyranoside (i.e., beta-glucosidases), and xylan. Enzymes produced by Coprinus psychromorbidus (LRS 067) were limited to xylanases from straw and wood and beta-glucosidases from wood. A sclerotia-forming LTB (LRS 131) exhibited poor growth on both substrates, and did not produce detectable quantities of extracellular enzymes. None of the LTB isolates tested degraded avicel. The temperature optima of CMCases and xylanases in the filtrates from the straw medium ranged from 25 degrees C to 55 degrees C, and with the exception of LRS 067, significant activity was observed at 5 degrees C. Two cellulases (25 and 31 kDa) and two xylanases (24 and 34 kDa) were observed on zymograms for LRS 013 and 241. Reduction of enzymes with 2-mercaptoethanol adversely affected their activity on zymograms, and an additional cellulase band was observed for non-reduced samples. This study indicates that LTB produce an array of cellulolytic and xylanolytic enzymes, and that some of these enzymes possess low-temperature optima which may facilitate degradation of plant fibre under low-temperature conditions.

Basidiomycota↗

Cloning and overexpression of the avi2 gene encoding a major cellulase produced by Humicola insolens FERM BP-5977.

The avi2 gene encoding Avi2, which is a major cellulase produced by Humicola insolens FERM BP-5977, was cloned and sequenced. Avi2 showed high homology with other family 6 cellulases. The expression vector pNCE4 containing the avi2 gene was constructed, and this strain was transformed using a protoplast method. As a result, the pNCE4 transformant secreted 8-fold more Avi2 than the recipient strain.

Cellulases↗

Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca.

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-alpha, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.

Actinomycetales↗

Improvements in titer, productivity, and yield using Solka-floc for cellulase production.

Researchers studying cellulase enzymes for the economical production of fuel ethanol envision cellulose as the carbon source. However, submerged Trichoderma reesei cultures grown on cellulose exhibit high run-to-run variability. Thus, an investigation of 30 batch cellulase production experiments was instrumental in determining fermentation conditions that improved enzyme titers, yields, and productivities. Eighteen of the 30 batch experiments experienced minimal process upsets and were classified into eight groups based on agitation rate, gas sparge rate, and the use of oxygen supplementation. Comparing corn steep liquor with yeast extract/peptone also tested the effect of different sources of nitrogen in the media. Average 7-d enzyme titers were doubled from 4 to 8 FPU/mL primarily by increasing aeration.

Biotechnology↗

Cellulase recovery via membrane filtration.

A combined sedimentation and membrane filtration process was investigated for recycling cellulase enzymes in the biomass-to-ethanol process. In the first stage, lignocellulose particles longer than approx 50 microm were removed by means of sedimentation in an inclined settler. Microfiltration was then utilized to remove the remaining suspended solids. Finally, the soluble cellulase enzymes were recovered by ultrafiltration. The permeate fluxes obtained in microfiltration and ultrafiltration were approx 400 and 80 L/(m2 x h), respectively. A preliminary economic analysis shows that the cost benefit of enzyme recycling may be as much as 18 cents/gal of ethanol produced, provided that 75% of the enzyme is recycled in active form.

Biomass↗

The effect of additional autopolyploidization in a slow growing cellulase hyperproducer of Trichoderma.

M14-2 is a cellulase hyperproducer derived from Trichderma reesei QM 6a, but with a growth rate lower than that of the original strain. When M14-2 was autopolyploidized followed by haploidization and selection, the strain with both a higher cellulase productivity per mycelia and a higher growth rate could be obtained as M14-2B. This strain seemed to be constructed using gene sources amplified by additional autopolyploidization.

Cellulase↗

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to "fingerprint" analysis by high-resolution 2-D gel electrophoresis. Forty-five spots from 11 x 25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared to existing databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

Biotechnology↗

Use of measurement uncertainty analysis to assess accuracy of carbon mass balance closure for a cellulase production process.

Closing carbon mass balances is a critical and necessary step for verifying the performance of any conversion process. We developed a methodology for calculating carbon mass balance closures for a cellulase production process and then applied measurement uncertainty analysis to calculate 95% confidence limits to assess the accuracy of the results. Cellulase production experiments were conducted in 7-L fermentors using Trichoderma reesei grown on pure cellulose (Solka-floc), glucose, or lactose. All input and output carbon-containing streams were measured and carbon dioxide in the exhaust gas was quantified using a mass spectrometer. On Solka-floc, carbon mass balances ranged from 90 to 100% closure for the first 48 h but increased to 101 to 135% closure from 72 h to the end of the cultivation at 168 h. Carbon mass balance closures for soluble sugar substrates ranged from 92 to 127% over the entire course of the cultivations. The 95% confidence intervals (CIs) for carbon mass balance closure were typically +/-11 to 12 percentage points after 48 h of cultivation. Many of the carbon mass balance results did not bracket 100% closure within the 95% CIs. These results suggest that measurement problems with the experimental or analytical methods may exist. This work shows that uncertainty analysis can be a useful diagnostic tool for identifying measurement problems in complex biochemical systems.

Biotechnology↗

Protein disorder: conformational distribution of the flexible linker in a chimeric double cellulase.

The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178 A, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10 A and 130 A showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded.

Amino Acid Sequence↗

A new technique for localization of cellulase in situ using silver nitrate.

A new staining technique has been developed for the histochemical localization of cellulase in plant tissues by light microscopy. The products of cellulolysis are reducing sugars which can reduce the salts of heavy metals under appropriate conditions. The present technique relies on the deposition of black silver oxide due to reduction of alkaline silver nitrate to detect cellulase in tissues.

Cellulase↗

Kinetics of cell-wall digestion of orchardgrass and alfalfa silages treated with cellulase and formic acid.

The objectives of this study were to determine the effects of cellulase (from Trichoderma longibrachiatum) combined with formic acid, applied before ensiling, on the subsequent concentration and composition of the cell wall and on the extent and rate of in situ cell-wall digestion of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.). Treated and control forages of both plant species were ensiled for at least 60 d before being ruminally digested by two fistulated cows. Analyses of NDF, ADF, and acid detergent lignin were conducted sequentially on original and digested samples. Data were fitted with a first-order, nonlinear model to estimate extents and rates of digestion of potentially digestible NDF, cellulose, and hemicellulose. The concentration of indigestible residue and the discrete lag time before digestion were also determined for the cell-wall components. After ensiling, the mean NDF concentration of treated silages was 19% lower than that of control silages; the effect was greater for orchardgrass than for alfalfa. The extent of digestion of NDF, cellulose, and hemicellulose, respectively, was 33, 37, and 27% lower for treated silages than for control silages. Treatment effects on the extent of digestion varied between plant species. Cellulose from treated orchardgrass was digested 19% more slowly than cellulose from the control silage. Indigestible residue concentrations of NDF, cellulose, and hemicellulose, respectively, were 7, 8, and 7% lower in treated silages than in control silages. Thus, extensive cell-wall degradation by cellulase during ensiling resulted in less digestible cell-wall material for ruminal digestion but greater total cell-wall degradation, including that during ensiling and ruminal incubation, especially during early digestion in the rumen.

Cell Wall↗

[Localization of cellulase in the Aspergillus terreus mycelial cells].

Localization of cellulase was studied in the cells of Aspergillus terreus 17P, a thermotolerant fungus. The activity of Cx-enzyme was found in the supernatant and to be of a protein nature, as was established by means of differential centrifugation of a homogenate of the mycelium cells. Ultrathin sections of the mycelium were fixed according to Somogi and studied by electron microscopy. The cellulase was found to be located on the surface of the cell wall to which it was bound but not very firmly, and was therefore detected in the supernatant.

Aspergillus↗

Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis.

The influence of temperature and pH on the activity and stability of the cellulase system (endoglucanase, exoglucanase and cellobiase) was investigated in Nectria catalinensis. Optimal temperature for the activity of the cellulase system ranged from 50 to 55 degrees C, with an optimum for stability between 23 degrees C and 37 degrees C after a 72 h incubation period. For the different enzymes, maximal activity was registered between pH 4.2-5.8, with pH 4.8 being close to optimal for all stability studies. The activation energy was 4.97 Kcal mol-1 for endoglucanase, 4.37 Kcal mol-1 for exoglucanase and 13.73 Kcal mol-1 for cellobiase. The K(m) and Vmax values were 1.73 mg CMC ml-1 and 0.45 mumol glucose min-1 mg protein-1 for endoglucanase, 0.22 mg microcrystalline cellulose ml-1 and 57.1 nmol glucose min-1 mg protein-1 for exoglucanase, and 2.95 mM cellobiose and 0.17 mumol glucose min-1 mg protein-1 for cellobiase.

Cellulase↗

Cellulase hyperproducers constructed from polyploids of Lentinus edodes.

A mycelial mat of Lentinus edodes was treated with 0.01% (w/v) colchicine solution for 240 h at 26 degrees C and autopolyploidization occurred. The mycelia were treated subsequently with the haploidizing reagent, benomyl, and fanshaped sectors were produced from colonies. Among such sectors, cellulase hyperproducers could be selected. The cellulase productivity of the hyperproducer, L1, did not decrease through five generations.

Benomyl↗