N.G. Bakalova

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.

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.

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.

Isolation of Xylanase Preparations for Biotechnological Applications from Aspergillus Niger A3

ABSTRACTThe crude xylanase preparation (P-1) for biotechnological applications was obtained from the culture supernatant of Aspergillus niger A3 by precipitation with ethanol. The pH optimum (5.0) and temperature optimum (40 °C) were estimated. This crude xylanase preparation was stable at pH 5.0 (80 % retained initial activity for 24 h) and temperature between 20 - 45 °C (75 % retained initial activity for over 24 h). At the optimal experimental conditions for enzyme hydrolysis (2.5 % substrate concentration, 0.1 % concentration of the crude xylanase preparation, 50 °C—temperature of hydrolysis, pH 5.0 and 72 h time of hydrolysis) the xylans of two substrates (xylan ex larch sawdust and pretreated com stalks with 1 % NaOH) were hydrolyzed to 55 and 65%, respectively. Six forms of xylanases were detected by molecular-sieve column chromatography of the P-1. The maximal active xylanase peaks (Fr-II and Fr-IV) were purified further by anion-exchange column chromatography. The two xylanase preparations P-2 (F...

Kinetic Method for the Study of Xylan Hydrolysis by Xylan Hydrolases

SummaryThe Somogyi-Nelson colorimetric method was used in a new manner more suitable for evaluating the kinetics of the enzyme hydrolysis of xylan catalyzed by xylan hydrolases. The values of the Michaelis parameters (Km=5.56 g l−1 andV=2.94 · 10−5M s−1) were determined.ZusammenfassungDie kolorimetrische Methode nach Somogyi-Nelson wurde nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Xylan, katalysiert durch Xylan-Hydrolasen vonAspergillus oryzae, angewandt. Es wurden die Michaelis-Parameter (Km=5.56 g l−1 undV=2.94 · 10−5M s−1) bestimmt.

Properties of two endoglucanases from a mutant strain Trichoderma sp. M7 with potential application in the paper industry

Two endoglucanases were purified to electrophoretic homogeneity from the culture filtrate of a mutant strain Trichoderma sp. M7. EG-III and EG-IV had Mr of 49.7 and 47.5 kDa, and estimated pi values of 3.7 and 6.35, respectively. The optimal pH and temperature values were determined to be pH 5.0 and 60°C for the first cellulase, whereas pH 5.2 and 50 °C were optimal for the other. Endoglucanases exhibited typical Michaelis-Menten kinetics with Km and V values of 2.9 mg ml−1 and 60498.5 μmol min−1 mg−1 for EG-III and 3.8 mg ml−1 and 22650.9 μmol min−1 mg−1 for EG-IV, respectively. Mn2+, Cu2+ and Pd2+ strongly inhibited the enzymes. EC-IV catalyzed the hydrolysis of Na-CMC and hydroxyethyl cellulose (HEC) only, whereas EG-III displayed high activity towards xylans, also. Different preferences towards cellulosic substrates and their regions define a different role of the investigated enzymes in the degradation of plant biomass.

Purification and Biochemical Characteristics of β-Glucosidase from Aspergillus Awamori K-1

ABSTRACT An extracellular β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) produced by Aspergillus awamori K-1 was purified to electrophoretic homogeneity. The purified enzyme, which had a glycoprotein nature, was a monomer with a relative molecular mass of 116 kDa and pI= 5.8. The β-glucosidase was the most active against aryl-β-glucosides and cellobiose. In addition, the enzyme was able to catalyze the hydrolysis of β-1→2 and β-1 →6-linked diglucosides. The apparent Km and V values for p-nitrophenyl-β-D-gluco-pyranoside were calculated to be 0.8 mM and 5.29 μmol.min−1 respectively. Maximal β- glucosidase activity occurred at 60 °C and pH 5.0. This enzyme was competitively inhibited by β-D-glucose with K1 value of 13.15 mM.

Immunological Investigations of Purified Xylanase from Aspergillus Oryzae

ABSTRACTRabbit an tisera against xylanase fraction (F-III-II-I) from Aspergillus oryzae with Mr 40 kDa were prepared. In double diffusion experiments, antiserum to this fraction formed a precipitation band. Western blotting analysis using antibodies against the xylanase showed that antibodies obtained from us recognized the main xylanase fraction and showed a single immunopositive protein with Mr of 40 kDa. The inhibitory effects of the antibody on xylanase activity were assayed. Maximum inhibition occurred at 0.72 mg/ml concentration of antibodies. Three enzyme preparations were tested against the antibodies from Aspergillus oryzae.