Influence of cultivation conditions on the production of cellulolytic enzymes withTrichoderma reesei Rutgers C30 in aqueous two-phase systems

Cellulolytic enzyme production in aqueous two-phase systems withTrichoderma reesei Rutgers C30 has been investigated. The influ ence of different phase systems, as well as addition of media compo nents and substrate on enzyme production have been studied. Extractive enzyme production in fed-batch cultivations was per formed in a phase system composed of PEG 8000 5%-Dextran T500 7% with 1% Solka-Floc BW 200 as substrate. The cellulolytic enzyme system was intermittently withdrawn with the top phase. Addition of media components every 24 h and cellulose every 72 h gave an aver age enzyme activity in the withdrawn top phase of 2.2 FPU/mL dur ing 170 h cultivation. The corresponding productivity was 18 FPU/lh. The productivity was increased to 24 FPU/l.h when media compo nents and cellulose were added every 72 h. The average enzyme con centration was then 1.6 FPU/mL. The results are discussed in relation to methods for cellulolytic enzyme production involving immobiliza tion and cell recycling.     

Isotherms for adsorption of cellobiohydrolase I and II fromtrichoderma reesei on microcrystalline cellulose

Adsorption to microcrystalline cellulose (Avicel) of pure cellobiohydrolase I and II (CBH I and CBH II) fromTrichoderma reesei has been studied. Adsorption isotherms of the enzymes were measured at 4‡C using CBH I and CBH II alone and in reconstituted equimolar mixtures. Several models (Langmuir, Freundlich, Temkin, Jovanovic) were tested to describe the experimental adsorption isotherms. The isotherms did not follow the basic (one site) Langmuir equation that has often been used to describe adsorption isotherms of cellulases; correlation coefficients (R²) were only 0.926 and 0.947, for CBH I and II, respectively. The experimental isotherms were best described by a model of Langmuir type with two adsorption sites and by a combined Langmuir-Freundlich model (analogous to the Hill equation); using these models the correlation coefficients were in most cases higher than 0.995. Apparent binding parameters derived from the two sites Langmuir model indicated stronger binding of CBH II compared to CBH I; the distribution coefficients were 20.7 and 3.7 L/g for the two enzymes, respectively. The binding capacity, on the other hand, was higher for CBH I, 1.0 Μmol (67 mg) per gram Avicel, compared to 0.57 Μmol/g (30 mg/g) for CBH II. The isotherms when analyzed with the combined Langmuir-Freundlich model indicated presence of unequal binding sites on cellulose and/or negative cooperativity in the binding of the enzyme molecules.     

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.     

Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.     

Design of a multi-channel dropping segmenter for liquid-liquid extraction continuous flow-injection analysis

A simple dual-channel dropping segmenter was used for the simultaneous introduction of aqueous solutions of a sample and an organic analytical reagent directly into a continuous flow of an immiscible organic solvent. Droplets of precisely defined volume of the homogeneous reaction mixture are formed at the junction of the inlet capillary system. Reaction and extraction take place simultaneously in the individual segments of the aqueous phase during the transport through the reaction/extraction coil. An analytical signal is measured by an “on-tube” fast-reading spectrophotometric detector and processed mathematically. The applicability of the method is demonstrated with the determination of Cu(II) ions with tetramethylene-dithiocarbamate in neutral medium in 0.1 M ammonium acetate buffer.     

On the relation between two Minkowski functions

The definition of Minkowski's “Fragefunktion”? (x) is recapitulated. This function is compared to a function L(x) introduced by the author in 1926. It is shown that the inverse function P(w) = x to the function L(x) is related to the Fragefunktion through ?(w) = P(w)?1.