ABSTRACT
Gene egl2 of secreted endo-(1-4)-beta-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding beta-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60 degrees C, respectively, exhibited specific activity of 33 IU, and had K(m) and V(max) in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 micromol/sec per mg, respectively.
Subject(s)
Cellulase/genetics , Cellulase/metabolism , Fungal Proteins/metabolism , Genes, Fungal , Penicillium/enzymology , Amino Acid Sequence , Base Sequence , Calorimetry, Differential Scanning , Carboxymethylcellulose Sodium/metabolism , Cellulase/biosynthesis , Cellulase/isolation & purification , Enzyme Stability , Escherichia coli/genetics , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Galactosidases/genetics , Galactosidases/metabolism , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Nitrate Reductase/genetics , Nitrate Reductase/metabolism , Protein Sorting Signals , Recombinant Proteins/metabolism , Sequence Alignment , Transformation, BacterialABSTRACT
The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.
Subject(s)
Cellulase/biosynthesis , Cellulase/chemistry , Fungal Proteins/biosynthesis , Fungal Proteins/chemistry , Penicillium/enzymology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Cellulase/genetics , Cellulase/isolation & purification , Cloning, Molecular/methods , Fungal Proteins/isolation & purification , Gene Expression , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Penicillium/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (Eap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The Eap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (Ep/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one- and two-electron oxidation are proposed.
Subject(s)
Antioxidants/chemistry , Flavonoids/chemistry , Hydroxybenzoates/chemistry , Phenols/chemistry , Plants/chemistry , Potentiometry/methods , Hydrogen-Ion Concentration , Kinetics , Molecular StructureABSTRACT
The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)2]PF6 and Ru(bpy)2CO3 under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)2]PF6 and 3 for Ru(bpy)2CO3. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.