Dimiter N. Kolev

An effective method for bioconversion of delignified waste-cellulose fibers from the paper industry with a cellulase complex.

Abstract An effective method for production of glucose was developed using enzymatic hydrolysis of waste-cellulose fibers by the cellulase complex from Trichoderma reesei . However, these cellulosic materials are strongly resistant to direct enzymatic hydrolysis (the degree of degradation after 48 h of enzyme treatment did not exceed 14%) apparently due to the presence of various chemical substances used in the process of paper production. This adhesive “envelope” around the cellulose fibers was effectively pretreated with 0.25% H 3 PO 4 and after that the free cellulose mass was extensively hydrolyzed by the cellulase complex (degree of degradation more than 80%). The HPLC analyses of the enzyme hydrolysates revealed glucose as the main component as well as some cellobiose and xylose.

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (Km and Vmax values of 3.7 mg ml−1 and 670 μmol min−1 xylose compared to the kinetic values of the purified wild type xylanase −5.1 mg ml−1 and 385 μmol min−1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.

A kinetic method for the direct determination of cellobiose hydrolysis by β-glucosidases

TheSomogyi—Nelson colorimetric method is applied in a new manner which is more suitable for following the kinetics of cellobiose hydrolysis catalyzed by β-glucosidase (EC 3.2.1.21). TheSomogyi—Nelson colour reagent, which is a mixture of the solutions of the reagent ofSomogyi and that ofNelson in a volume ratio of 1:1, is added to the enzyme-substrate solution at the very start of the reaction. The colour reagent reacts with the product (D-glucose). Under the reaction conditions (0.1M acetate buffer,pH = 5.0 and temperature 37°C) the colour reagent does not affect the enzyme activity. The method excludes any inhibition of the product, owing to the continuous removal of the latter by the colour reagent. The method suggested has been applied to monitor cellobiose hydrolysis with β-glucosidase, contained in four cellulase enzyme preparations from various fungal sources. The values of theMichaelis parameters (Km, V) were determined.ZusammenfassungDie kolorimetrische Methode nachSomogyi undNelson wird nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Cellobiose, katalysiert durch β-Glucosidase (EC 3.2.1.21), angewandt. Das Farbreagenz nachSomogyi undNelson (Mischung der Reagenzien vonSomogyi undNelson im Volumenverhältnis 1:1) wird der Enzym-Substrat-Lösung zu Beginn der Reaktion hinzugefügt. Das Farbreagenz tritt mit derD-Glukose in Reaktion, wobei unter den gegebenen Reaktionsbedingungen (0,1M Azetatpuffer,pH = 5,0 und 37°C) die Enzymaktivität nicht beeinflußt wird. Die entwickelte Methode wurde zur Verfolgung der Hydrolyse von Cellobiose durch ß-Glucosidasen, die in vier Enzympräparaten aus verschiedenen Pilzstämmen enthalten waren, angewandt. Es wurden dieMichaelis-Parameter (Km, V) bestimmt.

Catalytically important amino acid residues in endoglucanases from a mutant strain Trichoderma sp. M7.

Two endoglucanases, EG-III (49.7 kD) and EG-IV (47.5 kD), from a mutant strain Trichoderma sp. M7 were modified with several specific reagents. Water-soluble carbodiimide completely inactivated only one of the purified endoglucanases and kinetic analysis indicated that at least two molecules of carbodiimide bind to EG-IV for inactivation. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 3.57·10−5 mM−1·min−1. Both endoglucanases were inhibited by iodoacetamide, but the absence of substrate protection excluded direct involvement of cysteine residues in the catalysis N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate, since the presence of substrates or analogs prior to NBS modification protected the enzymes against inactivation.

Properties of two endoglucanases from a mutant strain Trichoderma sp. M7

Two endoglucanases (1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4) were purified to electrophoretic homogeneity from the culture filtrate of a mutant strain Trichoderma sp. M7. The purified endoglucanases had Mr of 57.4 and 55 kDa, and estimated pI values of 4.1 and 3.95/3.75, respectively. Optimal activity for the first cellulase was at pH 4.5 and 50 °C, and at pH 5.5 and 60 °C for the other. Carbodiimide inactivated the one of the purified endoglucanases, while the other was inhibited by iodoacetamide, indicating the involvement of carboxylic or thiol groups in the catalysis. N-Bromsuccinimide strongly inhibited both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate.

Kinetics and mechanism of the hydrolysis of sodium carboxymethylcellulose (Na-CMC) by a cellulase complex

TheSomogyi-Nelson colorimetric method is applied in a new manner more suitable for evaluating the kinetics of the enzyme hydrolysis of sodium carboxymethylcellulose (Na-CMC) catalyzed by the cellulase complex. By means of selective inhibition of a chosen enzyme from the cellulase complex it became possible to trace the effect of the other enzymes included in its composition.ZusammenfassungDie kolorimetrische Methode nachSomogyi undNelson wird nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Natriumcarboxymethylcellulose (Na-CMC), katalysiert durch den Cellulase-Komplex, angewandt. Durch selektive Inhibierung eines bestimmten Enzyms des Cellulase-Komplexes kann man die Wirkung der anderen zu seiner gesamten Zusammensetzung gehörenden Enzyme verfolgen.

Purification and Characterization of Two Endo-1,4-β-Xylanases from Aspergillus Awamori K-1

ABSTRACT Two xylanases (1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8) were isolated from the culture broth of Aspergillus awamori K-1. The enzymes were purified 9.81 and 18. 71-fold by ammonium sulfate precipitation, ultrafiltration, gel and anion-exchange chromatographic methods. The homogeneity of the purified endo-1,4-β-xylanases was assessed by SDS/PAGE and molecular-sieve chromatography on Superose 12 column. The enzymes have relative molecular masses (Mr) of 23 kDa and 27 kDa as detected by SDS/PAGE and isoelectric points (pIs of 3.45 and 3.75, respectively. The optimum pH and temperature values were 4.7 and 45 °C for xylanase II (Xyl II) and 3.0 and 50 °C for xylanase III (Xyl III). The products of the enzymatic hydrolysis of different xylans were analyzed by HPLC. The hydrolysis pattern showed that the xylanases purified in the present study were endo-acting enzymes. The Km and V values with three xylans as substrates were determined.

Kinetics of enzymatic hydrolysis of sodium carboxymethylcellulose with different degrees of polymerization by cellulase

AbstractThe reaction cellulase (EC 3.2.1.4)—sodium carboxymethylcellulose (Na-CMC) with different degrees of polymerization (n=140, 640 and 900) was investigated by the use of a modifiedMichaelis-Menten equation, valid for enzymatic hydrolysis of linear homopolymers. TheMichaelis-Menten constant [Km′ (M)=6.31·10−2mol/dm3] and the reaction rate constant (k′+2=4.07·10−6s−1), which correspond to the enzymatic hydrolysis of a single bond in the homopolymer substrates are determined. The free energy (Δ

Kinetic Method for the Study of Xylan Hydrolysis by Xylan Hydrolases

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