Isolation and properties of xyloglucanases of <Emphasis Type="Italic">Penicillium</Emphasis> sp.

Using chromatographic technique, xyloglucanase (XG) A (25 kDa, pI 3.5, 12th glycosyl hydrolase family) was isolated from the enzyme complex secreted by the mycelial fungus Penicillium canescens, and xyloglucanases XG 25 (25 kDa, pI 4.1, 12th glycosyl hydrolase family) and XG 70 (70 kDa, pI 3.5, 74th glycosyl hydrolase family) were isolated from the enzyme complex of Penicillium verruculosum. Properties of the isolated enzymes (substrate specificity, optimal ranges of pH and temperature for enzyme activity and stability, effect of metal ions on catalytic activity) were compared with the properties of xyloglucanases XG 32 of Aspergillus japonicus, XG 78 of Chrysosporium lucknowense, and XG of Trichoderma reesei. The gene xegA encoding XG A of P. canescens was isolated, and the amino acid sequence of the corresponding protein was determined.     

Specific xyloglucanases as a new class of polysaccharide-degrading enzymes

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 beta-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-beta-glucanases.     

Recombinant Endo-β-1,4-xylanase from Penicillium canescens

Recombinant endo-beta-1,4-xylanase (Xyl-31rec, 31 kD, pI 8.2-9.3, the tenth family of glycosyl hydrolases) was isolated from the culture liquid of Penicillium canescens (strain with the amplified homologous xylanase gene) by chromatofocusing on Mono P and hydrophobic chromatography on phenyl-Superose. It is shown that the biochemical and kinetic parameters, substrate specificity, stability, and other properties of the recombinant and native enzymes are almost the same. It was found that Xyl-31rec can be used for biobleaching of cellulose, the recombinant P. canescens strains providing a high yield of extracellular Xyl-31rec (up to 800-900 U/ml of culture liquid) and not secreting cellulases.     

Isolation and properties of pectinases from the fungus Aspergillus japonicus

Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI5.6 and 3.3), pectin lyase (50 kD, pI3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI(3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50°C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30°C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.     

Change in composition of hydrolyzed lignin during composting

Microbial assemblages were composed for composting hydrolysed lignin. Data on bioconversion of aromatic compounds with various types of substitution in the ring were used for this purpose. Composting of hydrolysed lignin reduced the contents of lignin, low-hydrolyzable polysaccharides, resins, and low-molecular-weight phenols and resulted in accumulation of humic acids. The resulting compost showed no phytotoxicity.     

Change in composition of structural components of leaf cellulose sulfate during fermentation bleaching by xylanases

The possibility of the use of xylanase preparations for hydrolysing hemicelluloses in a non-bleached kraft pulp in order to facilitate its bleaching was studied. The effects of enzymatic preparations of the fungal and bacterial origins were examined, and the optimal conditions for xylanase activity were determined. UV spectroscopy demonstrated that the treatment of kraft pulp with the enzymatic preparations containing xylanase facilitated the subsequent removal of lignin and increased the brightness by 5%. The effect of enzymatic treatment was retained in the case of peroxide bleaching. The enzymatic preparations studied are promising for the development of chlorine-free pulp bleaching technologies.     

Studies of hydrolytic activity of enzyme preparations of Penicillium and Trychoderma fungi

Enzyme preparations were isolated from the culture liquid of five mutant strains of the cellulase producer Penicillium verruculosum. The hydrolytic activities of these preparations against unbleached eucalypt cellulose was compared to that of commercial preparations of Trichoderma reesei (T. longibrachiatum). In the majority of cases, P. verruculosum enzymes provided higher yields of reducing sugars (RSs) and glucose. A correlation was found between the yield of RSs and the avicelase activity of the preparations in the reaction mixture.     

Localization of motor and speech cortex in the brain with functional magnetic resonance tomography

Functional magnetic resonance imaging (MRI) can be used for non-invasive mapping of cerebral cortex. The purpose of the study was evaluation of applicability of functional MRI for studies of neurosurgical patients. 32 volunteers (mean age 37.8 +/- 20.9 years) and 16 patients with brain tumors (mean age 36.2 +/- 24.2 years) were included in the study. Statistical analysis of the data obtained was performed. Activation maps were superimposed on anatomical images and discussed with neurosurgeons. Functional MRI studies were successful in localising the motor cortex and Broca's area in 89% of cases. In 69% of cases, results of the functional MRI influenced the patients' treatment.     

Viscometric method for assaying of total endodepolymerase activity of pectinases

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40°C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.     

Isolation and Properties of Cellobiase from Penicillium verruculosum

Cellobiase (-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i. e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose--lactone competitively inhibited cellobiase (K _i0.19 mM and 17 M, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.