The coil-globule transition in rigid-chain amphiphilic macromolecules was studied by means of computer simulation, and the phase diagrams for such molecules in the solvent quality-persistence length coordinates were constructed. It was shown that the type of phase diagram depends to a substantial extent on the degree of polymerization of a macromolecule. Relatively short amphiphilic macromolecules in the poor-solvent region always form a spherical globule, with the transition to this globule involving one or two intermediate conformations. These are the disk globule if the Kuhn segment is relatively large and the string of spherical micelles or the disk globule in the case of relative flexible chains. The phase diagram of a long rodlike amphiphilic chain turned out to be even more complex. Namely, three characteristic regions were distinguished in the region of a poor solvent, depending on the chain rigidity: the region of a cylindrical globule without certain order in the main chain, the region of the cylindrical globule with blobs having the collagen ordering of the chain, and the region of coexistence of collagen-like and toroidal globules. In the intermediate transitional region, not only conformations of strings of spherical micelle beads but also the necklace conformations in which the polymer chain in each bead has collagen ordering can occur in this case.

Conformational properties of amphiphilic stiff-chain macromolecules in concentrated solutions in poor solvent have been studied via computer modeling. We have found that the conformational state of macromolecules in such systems depends on the macromolecular stiffness and on the way the solution has been prepared. Thus, if the concentration of globules increased from a very diluted solution, the globules remain stable, independent of the macromolecular stiffness, and do not aggregate even in concentrated solutions. On the other hand, if the solvent quality is gradually decreased in a solution with a concentration much larger than that of a semidilute solution, then relatively flexible chains form separate globules, whereas semirigid macromolecules tend to aggregate and form braid-like conformations. The results obtained agree with the published experimental data and can be used for directed synthesis of macromolecules modeling the behavior of biopolymers.

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained earlier based on the strain 1 Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pI 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, β-xylosidase (β-XYL; 80 kDa, pI 4.5) and α-L-arabinofuranosidase I (α-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of preparations Celloviridin G20x and Xybeten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (α-L-AF I or β-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with α-L-AF I.

Commercial and pilot enzyme preparations from fungi of the genera Penicillium and Trichoderma have been compared with regard to their action on conifer wood pretreated with acidified aqueous ethanol (organosolve). In most experiments, enzymes from the genus Penicillium allowed higher yields of reducing sugars and glucose than those from Trichoderma . High β-glucosidase activity is essential for deep pulp hydrolysis.

Seven cellulase preparations from Penicillium and Trichoderma spp. were evaluated for their ability to hydrolyze the cellulose fraction of hardwoods (yellow poplar and red maple) pretreated by organosolv extraction, as well as model cellulosic substrates such as filter paper. There was no significant correlation among hydrolytic performance on pretreated hardwood, based on glucose release, and filter paper activity. However, performance on pretreated hardwood showed significant correlations to the levels of endogenous β-glucosidase and xylanase activities in the cellulase preparation. Accordingly, differences in performance were reduced or eliminated following supplementation with a crude β-glucosidase preparation containing both activities. These results complement a previous investigation using softwoods pretreated by either organosolv extraction or steam explosion. Cellulase preparations that performed best on hardwood also showed superior performance on the softwood substrates.

BACKGROUND:DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA.RESULTS:Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species.CONCLUSION:In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations.

Seven fungal cellulase preparations from Trichoderma and Penicillium spp. were evaluated for their ability to hydrolyze the cellulose component of lignocellulose samples prepared from softwoods by steam explosion or organosolv pretreatment. Hydrolysis was quantified using two indices: mean specific rate and specific conversion. The activities of the cellulase preparations on model cellulosic substrates (filter paper, carboxymethylcellulose and Avicel) were also determined, together with their β-glucosidase, xylanase, pectinase, galactomannanase and β-glucanase activities. The ability of a cellulase preparation to hydrolyze pretreated softwood showed little correlation with its activity on filter paper, carboxymethylcellulose and Avicel; however, there was a significant correlation with its level of endogenous β-glucosidase and xylanase activity. Differences in the performance of the various cellulase preparations were substantially reduced following supplementation with a commercial β-glucosidase preparation from Aspergillus niger. In addition to β-glucosidase, this preparation was shown to contain significant levels of endogenous xylanase activity. It appears that the levels of endogenous β-glucosidase and xylanase are important factors in determining the ability of a cellulase preparation to hydrolyze pretreated softwood and that deficiencies in the levels of both enzymes can be compensated by supplementation with corresponding activities present in the β-glucosidase preparation. Presumably, β-glucosidase improves cellulose hydrolysis by reducing end-product inhibition by cellobiose, while xylanase activity increases the accessibility of cellulose to cellulases.

Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75–105 kDa, pI 4.1–4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley β-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-β-glucanases.

Two forms of cellobiohydrolase I (CBH I, Cel7A) were purified from the culture ultrafiltrate of a mutant strain of the fungus Chrysosporium lucknowense, an industrial producer of cellulases and hemicellulases. The enzymes had different molecular masses (52 and 65kDa, SDS-PAGE data) but the same pI (4.5). Peptide sequencing showed that a single gene encodes both proteins. Both enzymes displayed maximum activity at pH 5.0–5.5; they had similar specific activities against soluble substrates. However, the 65kDa CBH I was much more efficient in hydrolysis of Avicel and cotton cellulose, and its adsorption ability on Avicel was notably higher in comparison to the 52kDa enzyme. Using the in-gel tryptic digestion followed by MALDI-TOF mass-spectrometry, it was shown that the 52kDa enzyme represents the core catalytic module of the intact 65kDa CBH I without a cellulose-binding module and major part of glycosylated linker. Both enzymes were stable at 50°C for 24h. At higher temperature, the 65kDa enzyme showed better thermostability: it retained >90% of activity after 7h at 60°C and 50% of activity after 3h at 65°C. The intact CBH I is also notably more thermostable than the Trichoderma reesei CBH I (by 6°C, the data of differential scanning microcalorimetry study). The cbh1 gene was cloned and then the amino acid sequence of Cel7A was deduced from the gene sequence. The enzyme had high degree of similarity (up to 74%) to family 7 cellobiohydrolases and lower degree of similarity (up to 41%) to family 7 endoglucanases.

Adsorption of several crude and purified cellulases (from Trichoderma reesei, Penicillium verruculosum and Chrysosporium lucknowense) on indigo particles and Avicel cellulose was studied. Much higher amounts of protein were bound to indigo than to cellulose under similar conditions. For different purified enzymes, the quantity of bound protein per mg of adsorbent (indigo or cellulose) varied in the range of 57–111 and 0–62 μg mg−1, respectively. However, in general, the enzyme adsorption on indigo was less specific than the adsorption on cellulose. Three endoglucanases, having the highest indigo-binding ability, demonstrated the best washing performance in the process of enzymatic denim treatment. These data confirmed our previous findings that certain cellulases, which have indigo-binding sites (clusters of closely located aromatic and other non-polar residues) on the surface of their molecules, may remove indigo from the denim fabric better than cellulases with lower content of hydrophobic residues exposed to solvent.

Study of cellulase adsorption on indigo particles and insoluble cellulose, as well as experiments on indigo interaction with immobilized amino acids together with theoretical analysis of three-dimensional structures of enzyme molecules, provided an evidence that certain cellulases, which have hydrophobic domains (clusters of closely located aromatic and non-polar residues) on their surface, may bind signicant amounts of indigo and thus act as emulsiers helping the dye to oat out of cellulose bers to the bulk solution in the process of enzymatic denim treatment. Only those cellulases, which had such indigo-binding domains, could eciently remove indigo from the denim fabric providing high abrasive eects on the surface of the material.

A method for determination of endo-1,4-beta -D-glucanase activity of cellulase samples based on the indirect measurement of decrease in viscosity of a carboxymethylcellulose solution in an electrochemical cell in the presence of an electron carrier was developed. A rotating disk electrode is used as the working electrode. When two reactions (enzymatic and electrochemical) proceeded in the cell simultaneously, the limiting diffusion current at a constant applied potential increases as the viscosity of the solution decreases. Conditions where the initial rate of change of diffusion current (dI/dt) is proportional to the enzyme concentration were found. A good correlation between the new method and a previously known viscometric method for determination of endoglucanase activity was observed.

A model microassay system was developed to measure indigo back-staining on cotton fabrics in the presence of enzymes on a small laboratory scale. Backstaining indexes for 11 cellulase samples were measured, and the enzymes were ranked from lower to higher backstaining. Two multienzyme cellulase preparations were separated into fractions using chromatofocusing on a Mono P column. Adsorption ability and backstaining properties of purified enzyme fractions were studied. Evidence was obtained that protein adsorption on cotton fabrics is a crucial parameter causing backstaining (both for crude cellulase samples and purified enzyme components).