Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin

The presence of lignin has shown to play an important role in the enzymatic degradation of softwood. The adsorption of enzymes, and their constituent functional domains on the lignocellulosic material is of key importance to fundamental knowledge of enzymatic hydrolysis. In this study, we compared the adsorption of two purified cellulases from Trichoderma reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on steam pretreated softwood (SPS) and lignin using tritium labeled enzymes. Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its catalytic domain. Removal of cellulose binding domain decreased markedly the binding efficiency. Significant amounts of CBH I and EG II also bound to isolated lignin. Surprisingly, the catalytic domains of the two enzymes of T. reesei differed essentially in the adsorption to isolated lignin. The catalytic domain of EG II was able to adsorb to alkaline isolated lignin with a high affinity, whereas the catalytic domain of CBH I did not adsorb to any of the lignins tested. The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.     

Mechanism of melatonin-induced oscillations in the peroxidase-oxidase reaction

Melatonin induces oscillations in the peroxidase-oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.     

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.     

Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer

Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C(14)H(29)) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12 degrees C in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase.     

Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes

Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40 degrees C) and enzyme loading (0.16 micromol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 micromol/g at 4 degrees C and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40 degrees C when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4 degrees C, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain.     

Separation of amino acids and peptides by temperature induced phase partitioning. Theoretical model for partitioning and experimental data

There is a strong interest in use of ‘smart polymers’ in separation systems. These are polymers which can react on external influence, such as temperature or pH change. With such polymers it is possible from the outside to affect the properties of a separation system. Amphiphilic copolymers show drastic changes in solubility properties, such as self-association and phase separation, at e.g. temperature increase. The random copolymers of ethylene oxide and propylene oxide units (EOPO-polymers) can form aqueous two-phase systems above the copolymer cloud point temperature. Two phases are formed, one consisting of 40–60% polymer in water and the other of almost 100% water. Amino acids and peptides can be partitioned in the thermoseparating systems. The partitioning strongly depends on the solute hydrophobicity, where aromatic amino acids and peptides are partitioned to the polymer phase and hydrophilic to the water phase. Salt effects can be used to enhance the partitioning of charged molecules. The thermodynamic driving forces which govern the partitioning of molecules in a thermoseparated aqueous phase system is described with use of the Flory-Huggins theory for polymer solutions. Expressions are derived which show the entropic and enthalpic effects on solute partitioning. This revised version was published online in July 2006 with corrections to the Cover Date.     

Single cell studies and simulation of cell-cell interactions using oscillating glycolysis in yeast cells

The observation of oscillations in the concentrations of NADH and other intermediates in glycolysis in dense yeast cell suspensions is generally believed to be the result of synchronization of such oscillations between individual cells. The synchrony is believed to be a property of cell density and the question is: does metabolism in each individual yeast cell continue to oscillate, but out of phase, in the absence of synchronization? Here we have used high-sensitivity fluorescence microscopy to measure NADH in single isolated yeast cells under conditions where we observe oscillations of glycolysis in dense cell suspensions. However, we have not been able to detect intracellular oscillations in NADH in these isolated cells, which cannot synchronize their metabolism with other cells. However, addition of acetaldehyde to a single cell as pulses with a frequency similar to the oscillations in dense cell suspensions will induce oscillations in that cell. Ethanol, another product of glycolysis, which has been proposed as a synchronizing agent of glycolysis in cells, was not able to induce oscillations when added as pulses. The experiments support the notion that the intracellular oscillations are associated with the cell density of the yeast cell suspension and mediated by acetaldehyde and perhaps also other substances.     

Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites

The properties of modified cellulose polymers, such as methylcellulose, are significantly influenced by the distribution of substituents along the polymer backbone. This distribution is difficult to determine due to the lack of suitable analytical methods. One approach is to use cellulose-degrading enzymes to gain information from the capability of the enzymes to cleave the bonds between glucose units. Endoglucanases are cellulase enzymes that can break internal glycosidic linkages and degrade low substituted regions of modified cellulose where the substituents do not interfere with the enzyme active site. In this work methyl cellulose was degraded using five endoglucanases from glycosyl hydrolase families 5 and 7 from three different species. The products were analyzed with reducing end analysis, chromatography (SEC-MALS-RI), and MALDI-TOFMS. The results were correlated with available determined enzyme structures and using structural alignment for unknown enzyme structures. This was performed in order to elucidate the relationship between active site structures and sensitivity for substituents on derivatized cellulose. The evaluation of endoglucanase hydrolysis of methyl cellulose showed that differences in sensitivity could be related to differences in steric hindrance of substituents in the active site, which could explain differences within family 5 and 7 enzymes, as well as the generally higher substituent tolerance for family 5 enzymes. This information is important for use of endoglucanases as tools for characterization of substituent distribution. The results are also valuable since soluble cellulose derivatives are generally used as substrates during enzyme characterization and in endoglucanase activity assays.     

On-line monitoring of CO2 production in Lactococcus lactis during physiological pH decrease using membrane inlet mass spectrometry with dynamic pH calibration

Monitoring CO2 production in systems, where pH is changing with time is hampered by the chemical behavior and pH-dependent volatility of this compound. In this article, we present the first method where the concentration and production rate of dissolved CO2 can be monitored directly, continuously, and quantitatively under conditions where pH changes rapidly ( approximately 2 units in 15 min). The method corrects membrane inlet mass spectrometry (MIMS) measurements of CO2 for pH dependency using on-line pH analysis and an experimentally established calibration model. It is valid within the pH range of 3.5 to 7, despite pH-dependent calibration constants that vary in a non-linear fashion with more than a factor of 3 in this interval. The method made it possible to determine the carbon dioxide production during Lactococcus lactis fermentations, where pH drops up to 3 units during the fermentation. The accuracy was approximately 5%. We used the method to investigate the effect of initial extracellular pH on carbon dioxide production during anarobic glucose fermentation by non-growing Lactocoocus lactis and demonstrated that the carbon dioxide production rate increases considerably, when the initial pH was increased from 6 to 6.8.