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Cellulase
Terms modified by Cellulase Selected AbstractsOptimization of extraction of bulk enzymes from spent mushroom compostJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2003Avneesh D Singh Abstract The profiling of ligninase, hemicellulase and cellulase of Pleurotus sajor-caju after inoculation of spawn in bags containing sawdust was done at monthly intervals for a period of 6 months. Xylanase (EC 3.2.1.8) was produced throughout the 6 months studied with the productivity range from 5.60 to 7.51 U g,1. Cellulase (EC 3.2.1.4) and ,-glucosidase (EC 3.2.1.21) productivities were highest at 4 months, producing 3.31 U g,1 and 121.13 U g,1 respectively. Laccase (EC 1.10.3.2) productivity was highest at 2 months with a value of 7.59 U g,1. Lignin peroxidase (EC 1.11.1.14) productivity was highest at 5 months with a value of 206.20 U g,1. Total soluble proteins were highest at 4 months with a value of 0.139 mg cm,3. The profiling of lignin peroxidase in 5-month-old spent mushroom compost was monitored over a period of 10 months. It was observed that lignin peroxidase was produced throughout the period but productivity was variable. The average lignin peroxidase productivity ranged from 30 to 110 U g,1. The activities of the enzymes extracted in tap water at pH 8.4 were comparable to that extracted in 50 mmol sodium citrate buffer at pH 4.8 and distilled water at pH 5.2 at 4 °C using an incubator shaker at 200 rpm for 18 h. The optimum extraction time was 1 h using an incubator shaker at 4 °C. When an incubator shaker was used, there was no significant difference in the recovery of xylanase, cellulase and laccase at different pH values at 4 °C and 28 °C. No significant difference was observed in the recovery of ,-glucosidase using an incubator shaker at different pH values at 4 °C although the enzyme recovery was slightly higher at pH 8.12, with a value of 29.27 U g,1. The optimum extraction of ,-glucosidase was at pH 4 at room temperature using an incubator shaker. For the lignin peroxidase enzyme, the optimum pH for extraction was 6 at 4 °C and pH 7 at room temperature using an incubator shaker at 200 rpm for 1 h. Homogenization for 8 min at 8000 rpm using tap water at pH 4 had an advantage over the use of the incubator shaker for the extraction as high titers of enzymes were recovered. Copyright © 2003 Society of Chemical Industry [source] Challenge of synthetic celluloseJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 4 2005Shiro Kobayashi Abstract This article focuses on why and how the chemical synthesis of cellulose was accomplished. The synthesis of cellulose was an important, challenging problem for half a century in polymer chemistry. For the synthesis, a new method of enzymatic polymerization was developed. A monomer of ,- D -cellobiosyl fluoride (,-CF) was designed and subjected to cellulase catalysis, which led to synthetic cellulose for the first time. Cellulase is a hydrolysis enzyme of cellulose; cellulase, inherently catalyzing the bond cleavage of cellulose in vivo, catalyzes the bond formation via the polycondensation of ,-CF in vitro. It is thought that the polymerization and hydrolysis involve a common intermediate (transition state). This view led us to a new concept, a transition-state analogue substrate, for the design of the monomer. The preparation of cellulase proteins with biotechnology revealed the enzymatic catalytic functions in the hydrolysis and polymerization to cellulose. High-order molecular structures were in situ formed and observed as fibrils (cellulose I) and spherulites (cellulose II). In situ small-angle neutron scattering measurements suggested a fractal surface formation of a synthetic cellulose assembly. The principle of cellulose synthesis was extended to the synthesis of other natural polysaccharides, such as xylan and amylose, and unnatural polysaccharides. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 693,710, 2005 [source] Shear Deactivation of Cellulase, Exoglucanase, Endoglucanase, and ,-Glucosidase in a Mechanically Agitated ReactorBIOTECHNOLOGY PROGRESS, Issue 6 2001Tejas P. Gunjikar Shear deactivation of cellulase and its major component enzymes, viz., exoglucanase (exo -1,4-,- D -glucan-4-cellobiohydrolase), endoglucanase (endo -1,4-,- D -glucanhydrolase), and 1,4-,-glucosidase, was carried out by exposing cellulase to shear in a mechanically agitated reactor in the presence as well as in the absence of the substrate cellulose. Cellulase was found to undergo deactivation when subjected to shear, and the extent of deactivation increased with increasing speed of agitation. Among the three major component enzymes of cellulase, exoglucanase showed rapid deactivation and contributed the most to cellulase deactivation. The presence of a substrate did not affect the deactivation of cellulase. [source] Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresisELECTROPHORESIS, Issue 12 2004Mikael Nilsson Abstract A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using ,-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors. [source] A multivariate biomarker-based model predicting population-level responses of Daphnia magnaENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2003Wim M. De Coen Abstract A multivariate model is proposed relating short-term biomarker measurements in Daphnia magna to chronic effects (21-d exposure) occurring at the population level (time to death, mean brood size, mean total young per female, intrinsic rate of natural increase, net reproductive rate, and growth). The results of the short-term exposure (48h-96 h) to eight model toxicants (cadmium, chromium, mercury, tributyl tin, linear alkylsulfonic acid, sodium pentachlorophenolate, lindane, and 2,4-dichloro-phenoxyacetic acid) on the following biomarkers were used for the multivariate model: digestive enzymes (amylase, cellulase, ,-galactosidase, trypsin, and esterase), enzymes of the intermediary metabolism (glycogen phosphorylase, glucose-6-phosphate de-hydrogenase, pyruvate kinase, lactate dehydrogenase, and isocitrate dehydrogenase), cellular energy allocation (CEA) (protein, carbohydrate, and lipid content and electron transport activity), and DNA damage and antioxidative stress activity. Using partial least squares to latent structures (PLS), a two-component model was obtained with R2 of 0.68 and a Q2 value of 0.60 based on the combined analysis of a limited number of the 48- and 96-h biomarker responses. For the individual population-level responses, the R2 values varied from 0.66 to 0.77 and the Q2 values from 0.52 to 0.69. Energy-related biomarkers (cellular energy allocation, lipid contents, anaerobic metabolic activity,pyruvate kinase, and lactate dehydrogenase), combined with parameters related to oxidative stress (catalase) and DNA damage measured after 48 and 96 h of exposure, were able to predict long-term effects at higher levels of biological organization. [source] Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilarisFEBS JOURNAL, Issue 16 2003Masahiro Sugimura A cellulase (endo-,-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min,1·(mg protein),1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min,1·(mg protein),1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods. [source] Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-,-1,4-glucanase from blue mussel, Mytilus edulisFEBS JOURNAL, Issue 16 2000Bingze Xu A cellulase (endo-,-1,4- d -glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55,60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication). [source] Preparation of phosphorus and carbohydrate microcapsules for manipulating dietary C : P ratio for aquatic suspension-feedersFRESHWATER BIOLOGY, Issue 2 2003Daniel A. Kreeger SUMMARY 1.,Dietary phosphorus can be limiting for aquatic animals such as suspension-feeders. However, our understanding has been limited by the difficulty of manipulating dietary P without altering other aspects of food quality. We microencapsulated various forms of bioavailable P with carbohydrate to manipulate dietary C : P ratio for suspension-feeders. 2.,Calcium phosphate, sodium hexametaphosphate, sodium tripolyphosphate and tetrasodium pyrophosphate were each mixed with a concentrated solution of a carbohydrate base (either maltodextrin or potato starch) and microencapsulated using an interfacial polymerisation technique. Each of the 10 types of capsules produced had a particle size ideal for suspension-feeders (3,10 ,m). 3.,Leakage rates were low (<12% of capsule weight per day). Relative enzymatic breakdown in vitro by carbohydrases (amylase or cellulase) was similar among the 10 capsule types and was always at least 15 times the comparable leakage rate. 4.,Release of dissolved P from enzyme-treated capsules varied depending on capsule P content. Liberation of P from capsules prepared from 20% w/w sodium hexametaphosphate in maltodextrin (molar C : P = 1.8) was three times greater than all other types, and this combination appears most suitable as a dietary supplement for zooplankton. 5.,Although P content and capsule integrity were greatly influenced by choice of carbohydrate, choice of P compound, and the mixing ratio of the two, P-rich artificial microparticles can be produced that have low leakiness, high digestibility, and a physical size suitable for aquatic suspension-feeders. Therefore, microcapsules represent promising tools for manipulating dietary C : P for suspension feeders. [source] In-situ oxygen profiling and lignin modification in guts of wood-feeding termitesINSECT SCIENCE, Issue 3 2010Jing Ke Abstract, Reports on the capability of wood-feeding termites (WFTs) in degrading wood particles and on the existence of aerobic environment in the localized guts suggest that their high efficiency of cellulose utilization is not only caused by cellulase, but also by biochemical factors that pretreat lignin. We thus extend the hypothesis that for highly efficient accessibility of cellulose, there should be direct evidence of lignin modification before the hindgut. The lignin degradation/modification is facilitated by the oxygenated environment in intestinal microhabitats. To test our hypothesis, we conducted experiments using a dissolved oxygen microelectrode with a tip diameter < 10 ,m to measure oxygen profiles in intestinal microhabitats of both Coptotermes formosanus (Shiraki) and Reticulitermes flavipes (Kollar). Lignin modification during passage through their three gut segments was also analyzed with pyrolysis gas chromatography/mass spectrometry. The data showed relatively high levels of oxygen in the midgut that could have promoted lignin oxidation. Consistent with the oxygen measurements, lignin modifications were also detected. In support of previously proposed hypotheses, these results demonstrate that lignin disruption, which pretreats wood for cellulose utilization, is initiated in the foregut, and continues in the midgut in both termites. [source] Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystemJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2002A. Santra Aims:,To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. Methods and Results:,Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg,1 body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3 -N were lower (P < 0·01) while TVFA, total-N and TCA-ppt-N were higher (P < 0·01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0·01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. Conclusions:,Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3 -N concentration, indicating better energy and protein utilization in defaunated lambs. Significance and Impact of the Study: Defaunation improved energy and protein utilization in lambs. [source] Isolation and chemical structure characterization of enzymatic lignin from Populus deltoides woodJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2010Ali Abdulkhani Abstract Cellulytic enzymes were used for the isolation and structural characterization of Populus deltoides wood lignin as a fast growing and important species in wood processing technology. The isolation was based on the hydrolysis and partial solubilization of wood xylan and cellulose using combination of Thricoderma lanuginosus xylanase, Aspergillus sp. plus, A. niger cellulase, and almond glycosidase, followed by lignin purification using Bacillus licheniformis alkaline protease (for hydrolysis of cellulase contamination). The structure of enzymatic lignin (EL) was elucidated using chemical analysis, Py-GC/MS, FTIR, and quantitative 13C-NMR techniques. Different lignin structures of acetylated and nonacetylated lignin preparation were calculated. P. deltoides EL has been determined to have an h : g : s ratio of 5 : 60 : 35. Also, P. deltoides EL contained 0.59/Ar of ,-O-4 moieties with small amounts of other structural units such as pino/syringyresinol (0.05/Ar), phenylcoumaran (0.05/Ar), and spirodienone (0.01/Ar). The degree of condensation was estimated at 20%. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] Xylanolytic complex from Aspergillus giganteus: production and characterizationJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2003Glauciane Danusa Coelho An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 °C and under shaking at 120 rpm during 84h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 °C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T50 of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 °C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg+2, Cu+2 and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and ,-xylosidase activity in assay conditions. [source] Enzymatic hydrolysis of sugarcane bagasse for bioethanol production: determining optimal enzyme loading using neural networksJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2010Elmer Ccopa Rivera Abstract BACKGROUND: The efficient production of a fermentable hydrolyzate is an immensely important requirement in the utilization of lignocellulosic biomass as a feedstock in bioethanol production processes. The identification of the optimal enzyme loading is of particular importance to maximize the amount of glucose produced from lignocellulosic materials while maintaining low costs. This requirement can only be achieved by incorporating reliable methodologies to properly address the optimization problem. RESULTS: In this work, a data-driven technique based on artificial neural networks and design of experiments have been integrated in order to identify the optimal enzyme combination. The enzymatic hydrolysis of sugarcane bagasse was used as a case study. This technique was used to build up a model of the combined effects of cellulase (FPU/L) and ,-glucosidase (CBU/L) loads on glucose yield (%) after enzymatic hydrolysis. The optimal glucose yield, above 99%, was achieved with cellulase and ,-glucosidase concentrations in the ranges of 460.0 to 580.0 FPU L,1 (15.3,19.3 FPU g,1 bagasse) and 750.0 to 1140.0 CBU L,1 (2,38 CBU g,1 bagasse), respectively. CONCLUSIONS: The dynamic model developed can be used not only to the prediction of glucose concentration profiles for different enzymatic loadings, but also to obtain the optimum enzymes loading that leads to high glucose yield. It can promote both a successful hydrolysis process control and a more effective employment of enzymes. Copyright © 2010 Society of Chemical Industry [source] Effect of adsorption characteristics of a modified cellulase on indigo backstainingJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 6 2004Diomi Mamma Abstract The effect of limited proteolysis (digestion) of a commercial cellulase preparation (Ecostone® L350) on backstaining with indigo was investigated. The influence of protease (papain) concentration on limited proteolysis of cellulase preparation was studied, applying different ratios of papain/cellulase (w/w). Changes in adsorption on Avicel cellulose of the non-digested compared with the papain-digested Ecostone® L350 were examined using the Langmuir adsorption isotherm. The non-digested Ecostone® L350 exhibited stronger interaction to Avicel cellulose compared with the digested form, while the maximum efficiency of cellulase adsorption to Avicel cellulose decreased after digestion. When papain-digested Ecostone® L350 was applied on cotton fabrics during the dyeing procedure with indigo, a reduction of indigo backstaining was obtained compared with the non-digested Ecostone® L350. Copyright © 2004 Society of Chemical Industry [source] Optimization of extraction of bulk enzymes from spent mushroom compostJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2003Avneesh D Singh Abstract The profiling of ligninase, hemicellulase and cellulase of Pleurotus sajor-caju after inoculation of spawn in bags containing sawdust was done at monthly intervals for a period of 6 months. Xylanase (EC 3.2.1.8) was produced throughout the 6 months studied with the productivity range from 5.60 to 7.51 U g,1. Cellulase (EC 3.2.1.4) and ,-glucosidase (EC 3.2.1.21) productivities were highest at 4 months, producing 3.31 U g,1 and 121.13 U g,1 respectively. Laccase (EC 1.10.3.2) productivity was highest at 2 months with a value of 7.59 U g,1. Lignin peroxidase (EC 1.11.1.14) productivity was highest at 5 months with a value of 206.20 U g,1. Total soluble proteins were highest at 4 months with a value of 0.139 mg cm,3. The profiling of lignin peroxidase in 5-month-old spent mushroom compost was monitored over a period of 10 months. It was observed that lignin peroxidase was produced throughout the period but productivity was variable. The average lignin peroxidase productivity ranged from 30 to 110 U g,1. The activities of the enzymes extracted in tap water at pH 8.4 were comparable to that extracted in 50 mmol sodium citrate buffer at pH 4.8 and distilled water at pH 5.2 at 4 °C using an incubator shaker at 200 rpm for 18 h. The optimum extraction time was 1 h using an incubator shaker at 4 °C. When an incubator shaker was used, there was no significant difference in the recovery of xylanase, cellulase and laccase at different pH values at 4 °C and 28 °C. No significant difference was observed in the recovery of ,-glucosidase using an incubator shaker at different pH values at 4 °C although the enzyme recovery was slightly higher at pH 8.12, with a value of 29.27 U g,1. The optimum extraction of ,-glucosidase was at pH 4 at room temperature using an incubator shaker. For the lignin peroxidase enzyme, the optimum pH for extraction was 6 at 4 °C and pH 7 at room temperature using an incubator shaker at 200 rpm for 1 h. Homogenization for 8 min at 8000 rpm using tap water at pH 4 had an advantage over the use of the incubator shaker for the extraction as high titers of enzymes were recovered. Copyright © 2003 Society of Chemical Industry [source] Enhanced ethanol production from enzymatically treated steam-exploded rice straw using extractive fermentationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2001Yoshitoshi Nakamura Abstract Alcohol fermentation of an enzymatic hydrolyzate of exploded rice straw was studied experimentally. Rice straw was treated under variable conditions, such as steam pressure and steaming time. The exploded rice straw was separated into water-soluble material, methanol-soluble lignin, Klason lignin, and a mixture of cellulose and a low molecular weight substance. The effects of steam explosion on the characteristics of the exploded rice straw were clarified from the point of view of the amounts of extractive components. Steam explosion was found to be effective for the delignification of rice straw and for increasing its susceptibility to enzyme hydrolysis and alcohol fermentation. The polysaccharides (cellulose and hemicellulose) in the rice straw treated at a steam pressure of 3.5,MPa with a steaming time of 2,min were hydrolyzed almost completely into monosaccharides, (ie glucose and xylose) by a mixture of Trichoderma viride cellulase (Meicelase) and Aspergillus aculeatus cellulase (Acucelase). The enzymatic hydrolyzate of exploded rice straw was converted into ethanol efficiently by Pichia stipitis and the ethanol yield from sugar was about 86%(w/w) of the theoretical value. The ethanol concentration in a membrane bioreactor coupled with a pervaporation system reached 50,gdm,3 and was about five times higher than that in the culture broth. The energy efficiency (ratio of combustion energy of ethanol produced to energy for steam explosion) reached a maximum value at a pressure of 3.5,MPa for 2,min. © 2001 Society of Chemical Industry [source] Development of a Sensitive Serological Method for Specific Detection of Latent Infection of Macrophomina phaseolina in CowpeaJOURNAL OF PHYTOPATHOLOGY, Issue 1 2009Leonard Afouda Abstract A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific detection and quantification of Macrophomina phaseolina in plant tissue. Both polyclonal antisera produced against immunogens from mycelium and culture filtrate of M. phaseolina detected the fungus in mycelial and plant extracts, although the antibodies raised against mycelium were more sensitive. No cross-reaction occurred with Rhizopus stolonifer, Pythium ultimum, Mucor hiemalis, Fusarium oxysporum, Septoria nodorum, Rhizoctonia solani, Sclerotinia sclerotiorum, Phytophthora infestans and Aspergillus niger. In enzyme assays, activity of the endo-acting hydrolytic enzymes 1,3-,-glucanase and, less, cellulase, but not xylanase was detected in infected plants. DAS-ELISA was more sensitive than the 1,3-,-glucanase assay. In polyacrylamide gel electrophoresis (PAGE) up to 18 protein bands were observed, with four bands occurring in the 12 tested isolates deriving from various geographical origin in Niger and Nigeria. The enzyme assays and protein patterns were considered not suitable for specific M. phaseolina detection. Macrophomina phaseolina was essentially located in the roots and hypocotyls, and less in epicotyls and leaves of infected plants. The antibodies were also useful to detect latent infection and the infection of cowpea seeds. [source] Challenge of synthetic celluloseJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 4 2005Shiro Kobayashi Abstract This article focuses on why and how the chemical synthesis of cellulose was accomplished. The synthesis of cellulose was an important, challenging problem for half a century in polymer chemistry. For the synthesis, a new method of enzymatic polymerization was developed. A monomer of ,- D -cellobiosyl fluoride (,-CF) was designed and subjected to cellulase catalysis, which led to synthetic cellulose for the first time. Cellulase is a hydrolysis enzyme of cellulose; cellulase, inherently catalyzing the bond cleavage of cellulose in vivo, catalyzes the bond formation via the polycondensation of ,-CF in vitro. It is thought that the polymerization and hydrolysis involve a common intermediate (transition state). This view led us to a new concept, a transition-state analogue substrate, for the design of the monomer. The preparation of cellulase proteins with biotechnology revealed the enzymatic catalytic functions in the hydrolysis and polymerization to cellulose. High-order molecular structures were in situ formed and observed as fibrils (cellulose I) and spherulites (cellulose II). In situ small-angle neutron scattering measurements suggested a fractal surface formation of a synthetic cellulose assembly. The principle of cellulose synthesis was extended to the synthesis of other natural polysaccharides, such as xylan and amylose, and unnatural polysaccharides. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 693,710, 2005 [source] Effect of fibrolytic enzymes and an inoculant on in vitro degradability and gas production of low-dry matter alfalfa silageJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2008Lazar K Kozelov Abstract BACKGROUND: The objective of this study was to investigate the effect of polysaccharide-degrading enzymes (a cellulase and a xylanase) alone or in a combination with a bacterial inoculant on fermentation parameters and in vitro degradability and gas production of low-dry matter (DM) alfalfa silage. First cut alfalfa (Medicago sativa L.), harvested at about 5% bloom stage [260 g kg,1 dry matter (DM)] was ensiled in laboratory-scale silos without preservatives or preserved with formic acid, a cellulase (Cell), a xylanase, a cellulose/xylanase enzyme combination (Cell/Xyl), a lactic acid bacteria-based inoculant (Inoc), and a mix of Inoc and Cell (Inoc/Cell). Triplicate silos were opened on days 1, 3, 7, 15 and 60. RESULTS: Silage pH and ammonia N and total free amino acids concentrations were the lowest (P < 0.05) for the formic acid silage. Inoc and Inoc/Cell produced the highest (P < 0.05) lactate concentration in the 60-day silage. In vitro degradability of silage DM was not affected (P = 0.998) by treatment, but amylase-treated neutral detergent fiber degradability was increased (P < 0.05) by formic acid. Compared with the control (51.3 mL 100 mg,1 silage DM), all treatments except Cell/Xyl increased (P < 0.001) the 24 h cumulative gas production. CONCLUSIONS: Overall, enzyme and lactic acid bacteria-based preparations had minor effects on silage fermentation in this experiment. The increased cumulative gas production indicates some preservation or liberation of fermentable organic matter with most treatments tested. It is not clear, however, to what extent this effect would impact silage ruminal degradability in vivo. Copyright © 2008 Society of Chemical Industry [source] Effect of adding an anaerobic fungal culture isolated from a wild blue bull (Boselophus tragocamelus) to rumen fluid from buffaloes on in vitro fibrolytic enzyme activity, fermentation and degradation of tannins and tannin-containing Kachnar tree (Bauhinia variegata) leaves and wheat strawJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2006Shyam S Paul Abstract The study investigated the effects of adding an anaerobic fungus (Piromyces sp FNG5; isolated from the faeces of a wild blue bull) to the rumen fluid of buffaloes consuming a basal diet of wheat straw and concentrates on in vitro enzyme activities, fermentation and degradation of tannins and tannin-rich tree leaves and wheat straw. In experiment 1, strained rumen fluid was incubated for 24 and 48 h, in quadruplicate, with or without fungal culture using condensed tannin-rich Bauhinia variegata leaves as substrates. In experiment 2, in vitro incubation medium containing wheat straw and different concentrations of added tannic acid (0,1.2 mg mL,1) were incubated for 48 h, in quadruplicate, with strained buffalo rumen fluid with or without fungal culture. In experiment 3, tolerance of the fungal isolate to tannic acid was tested by estimating fungal growth in pure culture medium containing different concentrations (0,50 g L,1) of tannic acid. In in vitro studies with Bauhinia variegata tree leaves, addition of the fungal isolate to buffalo strained rumen liquor resulted in significant (P < 0.01) increase in neutral detergent fibre (NDF) digestibility and activities of carboxymethyl cellulase (P < 0.05) and xylanase (P < 0.05) at 24 h fermentation. There was 12.35% increase (P < 0.01) in condensed tannin (CT) degradation on addition of the fungal isolate at 48 h fermentation. In in vitro studies with wheat straw, addition of the fungus caused an increase in apparent digestibility (P < 0.01), true digestibility (P < 0.05), NDF digestibility (P < 0.05), activities of carboxymethyl cellulase (P < 0.001), ,-glucosidase (P < 0.001), xylanase (P < 0.001), acetyl esterase (P < 0.001) and degradation of tannic acid (P < 0.05). Rumen liquor from buffaloes which had never been exposed to tannin-containing diet had been found to have substantial inherent tannic acid-degrading ability (degraded 55.3% of added tannic acid within 24 h of fermentation). The fungus could tolerate tannic acid concentration up to 20 g L,1 in growth medium. The results of this study suggest that introduction of an anaerobic fungal isolate with superior lignocellulolytic activity isolated from the faeces of a wild herbivore may improve fibre digestion from tannin-containing feeds and degradation of tannins in the rumen of buffaloes. Copyright © 2005 Society of Chemical Industry [source] Belowground carbon allocation by trees drives seasonal patterns of extracellular enzyme activities by altering microbial community composition in a beech forest soilNEW PHYTOLOGIST, Issue 3 2010Christina Kaiser Summary ,Plant seasonal cycles alter carbon (C) and nitrogen (N) availability for soil microbes, which may affect microbial community composition and thus feed back on microbial decomposition of soil organic material and plant N availability. The temporal dynamics of these plant,soil interactions are, however, unclear. ,Here, we experimentally manipulated the C and N availability in a beech forest through N fertilization or tree girdling and conducted a detailed analysis of the seasonal pattern of microbial community composition and decomposition processes over 2 yr. ,We found a strong relationship between microbial community composition and enzyme activities over the seasonal course. Phenoloxidase and peroxidase activities were highest during late summer, whereas cellulase and protease peaked in late autumn. Girdling, and thus loss of mycorrhiza, resulted in an increase in soil organic matter-degrading enzymes and a decrease in cellulase and protease activity. ,Temporal changes in enzyme activities suggest a switch of the main substrate for decomposition between summer (soil organic matter) and autumn (plant litter). Our results indicate that ectomycorrhizal fungi are possibly involved in autumn cellulase and protease activity. Our study shows that, through belowground C allocation, trees significantly alter soil microbial communities, which may affect seasonal patterns of decomposition processes. [source] Herbicidal cyanoacrylates with antimicrotubule mechanism of actionPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 11 2005Stefan Tresch Abstract The herbicidal mode of action of the new synthetic cyanoacrylates ethyl (2Z)-3-amino-2-cyano-4-ethylhex-2-enoate (CA1) and its isopropyl ester derivative CA2 was investigated. For initial characterization, a series of bioassays was used indicating a mode of action similar to that of mitotic disrupter herbicides such as the dinitroaniline pendimethalin. Cytochemical fluorescence studies including monoclonal antibodies against polymerized and depolymerized tubulin and a cellulose-binding domain of a bacterial cellulase conjugated to a fluorescent dye were applied to elucidate effects on cell division processes including mitosis and microtubule and cell wall formation in maize roots. When seedlings were root treated with 10 µM of CA1 or CA2, cell division activity in meristematic root tip cells decreased within 4 h. The chromosomes proceeded to a condensed state of prometaphase, but were unable to progress further in the mitotic cycle. The compounds caused a complete loss of microtubular structures, including preprophase, spindle, phragmoplast and cortical microtubules. Concomitantly, in the cytoplasm, an increase in labelling of free tubulin was observed. This suggests that the herbicides disrupt polymerization and microtubule stability, whereas tubulin synthesis or degradation appeared not to be affected. In addition, cellulose labelling in cell walls of root tip cells was not influenced. The effects of CA1 and CA2 were comparable with those caused by pendimethalin. In transgenic Arabidopsis plants expressing a green fluorescent protein-microtubule-associated protein4 fusion protein, labelled arrays of cortical microtubules in living epidermal cells of hypocotyls collapsed within 160 min after exposure to 10 µM CA1 or pendimethalin. Moreover, a dinitroaniline-resistant biotype of goosegrass (Eleusine indica (L) Gaertn) with a point mutation in ,-tubulin showed cross-resistance against CA1 and CA2. The results strongly indicate that the cyanoacrylates are a new chemical class of herbicide which possess the same antimicrotubule mechanism of action as dinitroanilines, probably including interaction with the same binding site in ,-tubulin. Copyright © 2005 Society of Chemical Industry [source] Sensitization to wheat flour and enzymes and associated respiratory symptoms in British bakersAMERICAN JOURNAL OF INDUSTRIAL MEDICINE, Issue 2 2009Joanne Harris-Roberts PhD Abstract Rationale Current literature suggests that flour exposed workers continue to be at risk of allergic sensitization to flour dust and respiratory ill health. Objectives A cross-sectional study of 225 workers currently potentially exposed to flour dust in British bakeries was performed to identify predictors of sensitization to wheat flour and enzymes. Results Work-related nasal irritation was the most commonly reported symptom (28.9%) followed by eye irritation (13.3%) and work-related cough or chest tightness (both 10.2%). Work-related chest tightness was significantly associated (OR 7.9, 1.3,46.0) with co-sensitization to wheat flour and any added enzyme. Working at a bakery with inadequate control measures was not a risk factor for reporting work-related respiratory symptoms (OR 1.3, 0.4,3.7). Fifty-one workers were atopic and 23 (14%) were sensitized to workplace allergens. Atopy was the strongest predictive factor (OR 18.4, 5.3,64.3) determining sensitization. Current versus never smoking (OR 4.7, 1.1,20.8) was a significant risk factor for sensitization to wheat flour or enzymes in atopic workers only, corrected for current level and duration of exposure. This effect was not seen in non-atopic workers (OR 1.9, 0.2,17.9). Evidence of sensitization to less commonly encountered allergens was also seen to Aspergillus niger derived cellulase, hemicellulase and xylanase mix, in addition to glucose oxidase and amyloglucosidase mix. Conclusions The combination of health surveillance and exposure control in this population has been insufficient to prevent clinically significant workplace sensitization. Smoking may pose an additional risk factor for sensitization in atopic workers. Am. J. Ind. Med. 52:133,140, 2009. © 2008 Crown copyright. [source] Enzyme degradability of benzylated sisal and its self-reinforced compositesPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 10 2003Xun Lu Abstract To produce natural polymer based composite materials, sisal fibers were slightly benzylated and then molded into sheets. Because the modified skin portions of the fibers acquired certain thermoplasticity and the unmodified core parts remain constant, the resultant composites fall into the category of self-reinforced ones. The present article is devoted to the evaluation of the materials biodegradability with the help of cellulase. It was found that the inherent biodegradability of plant fibers is still associated with the benzylated sisal and the molded composites, as characterized by structural variation, weight loss and deterioration of mechanical performance of the materials. Reaction temperature and time, pH value of the enzyme solution, and dosage of the enzyme had significant influences on the decomposition behavior of the materials. In principle, the enzymolysis of sisal and its self-reinforced composites is a diffusion-controlled process. Due to the insusceptibility of lignin to cellulase and the hindrance of it to the cellulase solution, the degradation rates of the materials are gradually slowed down with an increase in time. Copyright © 2003 John Wiley & Sons, Ltd. [source] The cell wall and secretory proteome of a tobacco cell line synthesising secondary wallPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2009David J. Millar Abstract The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available. [source] Isolation and characterization of the "zooxanthellae" from soritid foraminifera and the giant clam Tridacna maximaTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005JOHN J. LEE Various methodological approaches are being used to characterize Symbiodinium spp isolated from Marginopora vertebralis and Amphisorus hemprichii and Amphisorus sp. from the Indo-pacific. While most of the cells in culture are non-motile vegetative cells, dinospores occur in batch cultures. We find consistant variance among the isolates in the duration and time of appearance of motile forms. Nitrate (0.146 mM) supports higher populations of the isolates than NH4 (0.146 mM). Higher concentrations of NH4 inhibit growth. Although Fensome et al. (1993) characterized the Order Suessiales (and Family Symbiodiniaceae) as gymnnodiniphycideans in which the amphiesmal vesicals are arranged in 7,10 longitudinal series we were unable to detect any plates on the surfaces of our isolates in the SEM. We were unsuccessful in revealing plates on non-motile forms by freeze fracture but they were present after treatment with various chemical agents (e.g. H2O2, HOCl,) and enzymes (cellulase, chitinase). They are not polygonal or arranged in the patterns anticipated for the Symbiodiniaceae. Polyclonal antibody studies of the Symbiodinium -like isolates are in agreement with molecular systematic studies in the sense that there is a common relation. The foram endosymbiotic dinoflagellates share some common surface antigens with each other and with the few invertebrate isolates we have tested. Studies of the foraminiferal symbionts' plastids revealed considerable morphological diversity. We isolated a diversity of endosymbionts from the mantle of Tridacna maxima from the Red Sea and the Marshall Islands. These included Symbiodinium, Amphidinium, Tetraselmis, and an unidentified chlorophyte. [source] Involvement of hydrogen peroxide in leaf abscission signaling, revealed by analysis with an in vitro abscission system in Capsicum plantsTHE PLANT JOURNAL, Issue 1 2008Masaru Sakamoto Summary Although auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion- and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H2O2) is involved in abscission signaling. Microscopic analyses revealed continuous H2O2 production by AZ cells. H2O2 scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H2O2 promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H2O2 production, whereas the expression of ethylene-responsive genes was unaffected by both H2O2 and an H2O2 inhibitor. These results indicated that H2O2 acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H2O2 production at the AZ, which preceded leaf abscission. These results indicate that H2O2 has roles in leaf abscission associated with ethylene both in vitro and in planta. [source] Relationships between the addition rates of cellulase or glucose and silage fermentation at different temperaturesANIMAL SCIENCE JOURNAL, Issue 3 2010Jian-Guo ZHANG ABSTRACT The influence of the application rates of cellulase preparation and glucose on silage fermentation at different temperatures was studied with the straw of naked barley (Hordeum vulgare L. emand Lam) and guineagrass (Panicum maximum Jacq.). Addition rate of cellulase and glucose, temperature and their interaction had significant effects on pH value, lactic acid content, butyric acid content and propionic acid content of naked barley straw silage and significant effects on all the parameters of guineagrass silage (P < 0.01). Temperature and interaction had significant effect on acetic acid content (P < 0.05) and no significant effect on NH3 -N content of naked barley straw silage (P > 0.05). Under all the temperatures, the pH values of barley straw and guineagrass silages were reduced by cellulase and glucose addition even at the lowest rate (P < 0.05), compared with their corresponding control. Lactic acid contents of silages were the highest within the same temperature and same additive when glucose and cellulase were added at the highest rates, whereas the effect of cellulase and glucose addition on butyric acid production varied with their application rates and silage storage temperature. The addition rate of restricting butyric acid fermentation was lower at 20°C than that at 30°C, and it was the lowest at 40°C where cellulase and glucose addition restricted butyric acid fermentation even at 0.1 g/kg and 10 g/kg, respectively, when compared to the control. While the addition rate was lower than the above level, cellulase and glucose addition also promoted butyric acid fermentation. [source] Effect of fortifying concentrate supplement with fibrolytic enzymes on nutrient utilization, milk yield and composition in lactating goatsANIMAL SCIENCE JOURNAL, Issue 3 2009Prokasananda BALA ABSTRACT Thirty lactating Beetle-sannen crossbred goats were randomly divided into 3 groups based on first fortnight milk yield, and then adjusted to body weights to study the effect of fibrolytic enzyme supplementation on digestibility and milk production. Goats in the control group (C) were fed 500 g of concentrate supplement (CS) without exogenous fibrolytic enzymes (control; CG) or CS fortified with cellulase and xylanases @ of 4000 and 12500 (EG1) or 8000 and 18 750 IU/kg (EG2), respectively. CS was offered in two equal proportions just after milking at 7 and 17 h. After feeding CS, goats were fed 1200 g of wheat straw as roughage in two equal parts at 08.00 and 18.00 hours. Milk yield was studied for 3˝ months. Milking was done by hand. Changes in body weight were also recorded. After a preliminary period of 60 days of feeding, a 7-day digestibility trial was conducted in two intervals with 5 animals from each group at a time. The increase in either wheat straw or NDF intake due to the enzyme mixture was only 3%. There was significant improvement (P < 0.05) in the diet digestibility of DM, OM, CP, NDF, ADF and total carbohydrates (TC) between the control and EG1 and EG2 goats. Improvements were greater (P < 0.05) in the latter group. Microbial protein (MBP) was estimated based on purine derivatives and creatinine excreted in spot urine samples. MBP increased about 10 and 15% in EG1 and EG2, but these values were statistically invalid. Phenomenally milk yield, fat and SNF were significantly (P < 0.001) changed with progression of lactation. On a temporal basis, fortnightly changes in FCM yield of this study could be grouped as 1 month each of first and last and 1˝ of middle phase of lactation. Repeated measure analysis showed 180 to 260 mL/day of higher FCM and 16 to 20 g/day of higher SNF yield (P < 0.001) in EG2 than control or even EG1 during 5 to 7th fortnight. However, no significant impact (P > 0.05) was noticed on TF. Feed intake, g/kg FCM yield was also 7% less in EG2 than either CG or EG1. The study concluded that apart from increased digestibility, fortification of CS with cellulase and xylanases enzymes improved FCM yield in the last quarter of lactation in goats, and improved body weights by 5% (P > 0.05). While the digestibility of the diet was higher in EG1 and EG2, the impact on FCM and SNF yield was noticed only on EG2. Our study indicated that CS of lactating goats may be fortified with 8000 and 18 500 IU/kg CS in the last quarter of lactation. [source] Thermogravimetric evaluation of perennial ryegrass (Lolium perenne) for the prediction of in vitro dry matter digestibilityANNALS OF APPLIED BIOLOGY, Issue 3 2008H.S.S. Sharma Abstract Thermogravimetry (TG) can be used for assessing the compositional differences in grasses that relate to dry matter digestibility (DMD) determined by pepsin,cellulase assay. This investigation developed regression models for predicting DMD of herbage grass during one growing season using TG results. The calibration samples were obtained from a field trial of eight cultivars and two breeding lines. The harvested materials from five cuts were analysed by TG to identify differences in the combustion patterns within the range of 30,600°C. The discrete results including weight loss, peak height, area, temperature, widths and residue of three decomposition peaks were regressed against the measured DMD values of the calibration samples. Similarly, continuous weight loss results of the same samples were also utilised to generate DMD models. The r2 for validation of the discrete and the best continuous models were 0.90 and 0.95, respectively, and the two calibrations were validated using independent samples from 24 plots from a trial carried out in 2004. The standard error for prediction of the 24 samples by the discrete model (4.14%) was higher than that by the continuous model (2.98%). This study has shown that DMD of grass could be predicted from the TG results. The benefit of thermal analysis is the ability to detect and show changes in composition of cell wall fractions of grasses during different cuts in a year. [source] | |||||||||||||||||||||||||||||||||||||