Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens

A .beta.-glucosidase and an endocellulase were purified from the culture filtrates of a thermophilic cellulolytic fungus Humicola insolens. Both the preparations were homogeneous by PAGE, ultracentrifugation and gel filtration (Mr 45,000). Ouchterlony immunodiffusion showed complete cross reactivity between the antibodies and the two enzyme antigens, indicating the presence of a common epitope on the two enzyme proteins. The two enzymes, however, differ in their amino acid composition and their substrate specificity. .beta.-Glucosidase acts on p-nitrophenyl .beta.-D-glucopyranoside and hydrolyses cellulose to release mainly glucose and small amounts of cellobiose from the non-reducing end. On the other hand, endocellulase hydrolyses cellulose to release cellopentaose, cellotetraose, cellotriose along with cellobiose and glucose and also hydrolyses larch wood xylan.

Purification of goat serum retinol-binding protein and preparation of its antibodies

The method for the purification of goat serum retinol-binding protein consists of DEAE-cellulose chromatography of the serum followed by preparative polyacrylamide disc gel electrophoresis. After electrophoresis, the retinol-binding protein containing zone is identified by the specific fluorescence of retinol. For raising the antibodies, the portion of the gel containing retinol binding protein is homogenized and injected intradermally and intramuscularly to rabbits. The availability of this simple method for the isolation of retinol-binding protein and production of its antibodies enables the development of a radioimmunoassay for this protein.

Studies on LDPE-cyanoethylated lignocellulosics blends using epoxy functionalized LDPE as compatibilizer

Rubberwood flour and cellulose have been plasticized by cyanoethylation and then blended with low-density polyethylene (LDPE). A small quantity of epoxy functionalized polyethylene i.e., polyethylene-co-glycidyl methacrylate (PEGMA) has been added to further enhance the mechanical properties. The mechanical properties were measured according to the standard ASTM methods. SEM analysis was performed for both fractured and unfractured blend specimens. The mechanical properties were improved by the addition of PEGMA compatibilizer. LDPE blends with cyanoethylated wood flour (CYWF) showed higher tensile strength and modulus than cyanoethylated cellulose CYC-LDPE blends. However CYC-LDPE blends exhibited higher relative elongation at break values as compared with the former. The TGA analysis showed lowering of thermal stability as the filler content is increased and degradation temperature of LDPE is shifted slightly to lower temperature. DSC analysis showed loss of crystallinity for the LDPE phase as the filler content is increased for both types of blends. Dielectric properties of the blends were similar to LDPE, but were lowered on adding PEGMA. (c) 2006 Wiley Periodicals, Inc.

Nanoscale studies of materials using x-ray synchrotron radiation and mesoscopic modelling

X-ray synchrotron radiation was used to study the nanostructure of cellulose in Norway spruce stem wood and powders of cobalt nanoparticles in cellulose support. Furthermore, the growth of metallic clusters was modelled and simulated in the mesoscopic size scale. Norway spruce was characterized with x-ray microanalysis at beamline ID18F of the European Synchrotron Radiation Facility in Grenoble. The average dimensions and the orientation of cellulose crystallites was determined using x-ray microdiffraction. In addition, the nutrient element content was determined using x-ray fluorescence spectroscopy. Diffraction patterns and fluorescence spectra were simultaneously acquired. Cobalt nanoparticles in cellulose support were characterized with x-ray absorption spectroscopy at beamline X1 of the Deutsches Elektronen-Synchrotron in Hamburg, complemented by home lab experiments including x-ray diffraction, electron microscopy and measurement of magnetic properties with a vibrating sample magnetometer. Extended x-ray absorption fine structure spectroscopy (EXAFS) and x-ray diffraction were used to solve the atomic arrangement of the cobalt nanoparticles. Scanning- and transmission electron microscopy were used to image the surfaces of the cellulose fibrils, where the growth of nanoparticles takes place. The EXAFS experiment was complemented by computational coordination number calculations on ideal spherical nanocrystals. The growth process of metallic nanoclusters on cellulose matrix is assumed to be rather complicated, affected not only by the properties of the clusters themselves, but essentially depending on the cluster-fiber interfaces as well as the morphology of the fiber surfaces. The final favored average size for nanoclusters, if such exists, is most probably a consequence of these two competing tendencies towards size selection, one governed by pore sizes, the other by the cluster properties. In this thesis, a mesoscopic model for the growth of metallic nanoclusters on porous cellulose fiber (or inorganic) surfaces is developed. The first step in modelling was to evaluate the special case of how the growth proceeds on flat or wedged surfaces.

Litter quality and its response to water level drawdown in boreal peatlands at plant species and community level

Changes in the structure of plant communities may have much more impact on ecosystem carbon (C) cycling than any phenotypic responses to environmental changes. We studied these impacts via the response of plant litter quality, at the level of species and community, to persistent water-level (WL) drawdown in peatlands. We studied three sites with different nutrient regimes, and water-level manipulations at two time scales. The parameters used to characterize litter quality included extractable substances, cellulose, holocellulose, composition of hemicellulose (neutral sugars, uronic acids), Klason lignin, CuO oxidation phenolic products, and concentrations of C and several nutrients. The litters formed four chemically distinct groups: non-graminoid foliar litters, graminoids, mosses and woody litters. Direct effects of WL drawdown on litter quality at the species level were overruled by indirect effects via changes in litter type composition. The pristine conditions were characterized by Sphagnum moss and graminoid litters. Short-term (years) responses of the litter inputs to WL drawdown were small. In longterm (decades), total litter inputs increased, due to increased tree litter inputs. Simultaneously, the litter type composition and its chemical quality at the community level greatly changed. The changes that we documented will strongly affect soil properties and C cycle of peatlands.

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.

The effects of beta-mercaptoethanol and sodium dodecyl sulfate on the Humicola insolens beta-glucosidase

Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol. This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme. In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions. Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme. These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme.

Konjac- ja galaktoglukomannaanipohjaiset emulsiokalvot

Emulsiokalvolla tarkoitetaan kalvoa, joka on valmistettu haihduttamalla ylimääräinen vesi pois emulsiosta. Polysakkaridipohjainen emulsiokalvo koostuu kalvonmuodostuspolysakkaridista, rasvasta, emulgointiaineesta ja pehmittimestä. Kirjallisuusosassa selvitettiin, mitä raaka-aineita polysakkaridipohjaisissa emulsiokalvoissa käytetään ja mitkä tekijät vaikuttavat emulsiokalvojen vesihöyrynläpäisevyyteen ja mekaanisiin ominaisuuksiin. Tutkimuksen kokeellisen osan tavoitteena oli selvittää, miten konjac-glukomannaani (KGM) ja kuusen galaktoglukomannaani (GGM) soveltuvat emulsiokalvon raaka-aineiksi. Lisäksi selvitettiin, miten rasvan tyyppi ja rasvapitoisuus vaikuttavat GGM-KGM-pohjaisten emulsiokalvojen mekaanisiin ominaisuuksiin ja vesihöyrynläpäisevyyteen. Mehiläisvahasta, mäntyöljystä ja rypsiöljystä valmistettiin emulsiokalvot, joissa oli 30 %:n (paino-% GGM:sta) rasvapitoisuudet. Lisäksi mehiläisvahasta valmistettiin emulsiokalvot, joissa oli 10 ja 50 % mehiläisvahaa. Emulsiokalvoja verrattiin vertailukalvoon, jossa ei ollut rasvaa. Kalvoissa käytetty KGM:n ja GGM:n suhde oli 1:1. Kalvoista mitattiin vesihöyrynläpäisevyys ja -läpäisynopeus, vetolujuus, Youngin moduuli ja murtovenymä. Näiden lisäksi kalvojen poikkileikkaus kuvattiin pyyhkäisyelektronimikroskoopilla. GGM ja KGM soveltuvat emulsiokalvon raaka-aineiksi. Huoneenlämpötilassa kuivatuista kalvoista saatiin tasaisemman näköisiä kuin lämpökaapissa kuivatuista. Pyyhkäisyelektronimikroskooppikuvissa vahapisarat olivat öljypisaroita pienempiä, mikä mahdollisesti vaikutti siihen, että vahapisarat pysyivät paremmin kiinnittyneenä kalvomatriisissa. Öljypisaroiden koko oli kalvoissa noin 10 ?m ja vahapisaroiden 2–6 ?m. Vesihöyrynläpäisynopeus oli pienin 50 %:n mehiläisvahakalvolla (p < 0,05). Vesihöyrynläpäisevyys laski lineaarisesti mehiläisvahapitoisuuden suurentuessa. Öljykalvot ja 10 %:n mehiläisvahakalvo eivät eronneet tilastollisesti merkitsevästi vesihöyrynläpäisevyyden suhteen vertailukalvosta. Pienin vetolujuus ja Youngin moduuli oli 50 %:n mehiläisvahakalvolla. Vertailukalvo oli kestävin ja jäykin. Murtovenymän suhteen kalvot eivät eronneet toisistaan tilastollisesti merkitsevästi. Tutkimuksessa onnistuttiin valmistamaan GGM-KGM-pohjaisia emulsiokalvoja, jotka pidättivät vesihöyryä vertailukalvoa paremmin ja silti säilyttivät mekaaniset ominaisuutensa kohtuullisen hyvin.

Purification and functional characteristics of an endocellulase from Chaetomium thermophile var. coprophile

An endocellulase (1→4)-β-d-glucan 4-glucanohydrolase was isolated from the culture filtrates of Chaetomium thermophile. The enzyme was homogeneous by PAGE and SDS-PAGE. The molecular weight was 36 000 by SDS-PAGE and 38 000 by gel filtration. It was a glycoprotein. From the amino acid composition, it was found to be rich in glycine, threonine, and aspartic and glutamic acids, but contained only low proportions of histidine and sulfur-containing amino acids. It was optimally active at pH 6 and at 60°. The enzyme did not hydrolyze cellobiose and cellotriose, but hydrolyzed cello-tetraose, -pentaose, and -hexaose at comparable rates. It was specific for molecules containing β-(1→4) linkages. It showed high activity towards amorphous cellulose, and the reaction products contained cellobiose to cellopentaose, showing that it effects random cleavage of cellulose.

Purification and characterization of an extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity

Multiple forms of beta-glucosidase (EC 3.2.1.21) of Sporotrichum thermophile were produced when the fungus was grown in a cellulose medium. One beta-glucosidase was purified 16-fold from 6-d-old culture filtrates by ion-exchange and gel-filtration chromatography. The purified enzyme was free of cellulase activity. It hydrolysed aryl beta-D-glucosides and beta-D-linked diglucosides. It was optimally active at pH 5.4, at 65-degrees-C. The apparent K(m) values for p-nitrophenyl beta-D-glucoside (PNPG) and cellobiose were 0.29 and 0.83 mm, respectively. Glucose, fucose, nojirimycin and gluconolactone inhibited beta-glucosidase competitively. At high (> 1 mm) substrate concentration, beta-glucosidase catalysed a parallel transglycosylation reaction. The transglycosylation product formed from cellobiose appeared to be a beta-linked tetramer of glucose. Admixtures of beta-glucosidase and cellulase components showed that the concept of cellobiose inhibition of cellulases was not valid for all components of the cellulase system of S. thermophile. Beta-Glucosidase supplementation also stimulated cellulose hydrolysis by cellulases when there was no accumulation of cellobiose in reaction mixture.

Localization and release of ?-glucosidase in the thermophilic and cellulolytic fungus, Sporotrichum thermophile

In a medium containing cellulose as the carbon source, the rapid growth of Sporotrichum thermophile, the secretion of cellulases and the utilization of cellulose were well-correlated events. The production of beta-glucosidase in culture medium lagged behind cellulases, coinciding with the time of extensive autolysis of mycelia. By contrast, neither apparent autolysis nor secretion of beta-glucosidase occurred when S. thermophile was grown in medium containing cellobiose; the enzyme activity remained associated with mycelia. The release of beta-glucosidase in cellulose-grown cultures was correlated with the activity of the lytic enzyme in the cell wall. Immunocytochemical localization and biochemical characterization showed that a beta-glucosidase released in the cellulose medium was the same as that which remained associated with mycelia grown on cellobiose. The results indicated that the release of beta-glucosidase in the cellulose culture is incidental to the activity of the lytic enzymes which are strongly induced by cellulose. The observations minimize a functional role of the culture fluid beta-glucosidase in cellulolysis by the fungus. Rather, the available information suggests that the cellulases and beta-glucosidases associated with the hyphal cell wall may play a role in cellulolysis by the fungus. (C) 1994 Academic Press, Inc.

Localization and release of β-glucosidase in the thermophilic and cellulolytic fungus, Sporotrichum thermophile

n a medium containing cellulose as the carbon source, the rapid growth of Sporotrichum thermophile, the secretion of cellulases and the utilization of cellulose were well-correlated events. The production of beta-glucosidase in culture medium lagged behind cellulases, coinciding with the time of extensive autolysis of mycelia. By contrast, neither apparent autolysis nor secretion of beta-glucosidase occurred when S. thermophile was grown in medium containing cellobiose; the enzyme activity remained associated with mycelia. The release of beta-glucosidase in cellulose-grown cultures was correlated with the activity of the lytic enzyme in the cell wall. Immunocytochemical localization and biochemical characterization showed that a beta-glucosidase released in the cellulose medium was the same as that which remained associated with mycelia grown on cellobiose. The results indicated that the release of beta-glucosidase in the cellulose culture is incidental to the activity of the lytic enzymes which are strongly induced by cellulose. The observations minimize a functional role of the culture fluid beta-glucosidase in cellulolysis by the fungus. Rather, the available information suggests that the cellulases and beta-glucosidases associated with the hyphal cell wall may play a role in cellulolysis by the fungus. (C) 1994 Academic Press, Inc.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Role of length scales on microwave thawing dynamics in 2D cylinders

Microwave (MW) thawing of 2D frozen cylinders exposed to uniform plane waves from one face, is modeled using the effective heat capacity formulation with the MW power obtained from the electric field equations. Computations are illustrated for tylose (23% methyl cellulose gel) which melts over a range of temperatures giving rise to a mushy zone. Within the mushy region the dielectric properties are functions of the liquid volume fraction. The resulting coupled, time dependent non-linear equations are solved using the Galerkin finite element method with a fixed mesh. Our method efficiently captures the multiple connected thawed domains that arise due to the penetration of MWs in the sample. For a cylinder of diameter D, the two length scales that control the thawing dynamics are D/D-p and D/lambda(m), where D-p and lambda(m) are the penetration depth and wavelength of radiation in the sample respectively. For D/D-p, D/lambda(m) much less than 1 power absorption is uniform and thawing occurs almost simultaneously across the sample (Regime I). For D/D-p much greater than 1 thawing is seen to occur from the incident face, since the power decays exponentially into the sample (Regime III). At intermediate values, 0.2 < D/D-p, D/lambda(m) < 2.0 (Regime II) thawing occurs from the unexposed face at smaller diameters, from both faces at intermediate diameters and from the exposed and central regions at larger diameters. Average power absorption during thawing indicates a monotonic rise in Regime I and a monotonic decrease in Regime III. Local maxima in the average power observed for samples in Regime II are due to internal resonances within the sample. Thawing time increases monotonically with sample diameter and temperature gradients in the sample generally increase from Regime I to Regime III. (C) 2002 Elsevier Science Ltd. All rights reserved.

Grafting of m-isopropenyl-alpha, alpha-dimethylbenzyl-isocyanate (m-TMI) onto isotactic polypropylene: Synthesis and characterization

A novel vinyl monomer with an isocyanate functional group, m-isopropenyl-alpha,alpha-dimethylbenzyl-isocyanate (m-TMI), was grafted onto isotactic polypropylene (i-PP) using dicumyl peroxide (DCP) as the initiator. This would open up the possibility of using the grafted polymer with the reactive isocyanate group as compatibilizer for blending carbohydrates such as cellulose with. polypropylene. The grafting was carried out in a Brabender Plasticoder at 180degreesC. The effects of monomer and initiator concentrations on the yield of grafting were investigated by performing statistical analysis. While the grafting yield increased with the concentration of DCP at any given concentration of m-TMI, the variation of the grafting yield with m-TMI concentration, for a given concentration of DCP, went through a maximum, the optimum yield of 7.8% (w/w) being obtained at 10 wt.% concentration of both DCP and m-TMI. The grafting reaction is. accompanied by considerable chain scission of I-PP, resulting in a decrease in the molecular weight of the grafted polymer. While the molecular weight drops sharply even at a low concentration of DCP, there occurs no further significant change in the molecular weight even at much higher concentrations of the initiator.