Boris B. Ustinov

Cellulase Complex of the Fungus Chrysosporium lucknowense: Isolation and Characterization of Endoglucanases and Cellobiohydrolases

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), β-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-β-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against β-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified β-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.

Properties of Hemicellulases of the Enzyme Complex from Trichoderma longibrachiatum

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained earlier based on the strain 1 Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pI 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, β-xylosidase (β-XYL; 80 kDa, pI 4.5) and α-L-arabinofuranosidase I (α-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of preparations Celloviridin G20x and Xybeten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (α-L-AF I or β-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with α-L-AF I.

ansa-Ytterbocenes(+3) with a short bridge and bulky substituents: synthesis and crystal structure of meso-(CH3)2Si[3-(CH3)3SiC5H3]2YbCl(THF), rac-(CH3)2C[3-tBuC5H3]2Yb(μ2-Cl)2Li(OEt2)2, and [meso-(CH3)2C[3-tBuC5H3]2Yb(μ2-OCH3)]2

Abstract (CH 3 ) 2 E(3-(CH 3 ) 3 EC 5 H 3 ) 2 Li 2 (E=C, Si) compounds react with YbCl 3 to yield the ate -complex rac -(CH 3 ) 2 C(3- t BuC 5 H 3 ) 2 Yb( μ 2 -Cl) 2 Li(OEt 2 ) 2 ( 1 ) and the monosolvate meso -(CH 3 ) 2 Si(3-(CH 3 ) 3 SiC 5 H 3 ) 2 YbCl(THF) ( 2 ), respectively. The reaction between (CH 3 ) 2 C(3- t BuC 5 H 3 ) 2 Li 2 and YbI 2 in 1,2-dimethoxyethane (DME) proceeds with solvent decomposition, and the complex [ meso -(CH 3 ) 2 C(3- t BuC 5 H 3 ) 2 Yb( μ 2 -OCH 3 )] 2 ( 3 ) crystallises from a DME–hexane mixture. The structures of the compounds obtained were determined by X-ray structural analysis.