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

F. E. Bukhtojarov¹, B. B. Ustinov¹, T. N. Salanovich¹, A. I. Antonov¹, A. V. Gusakov^1*, O. N. Okunev², and A. P. Sinitsyn¹

¹Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia; fax: (7-095) 939-0997; E-mail: avgusakov@enzyme.chem.msu.ru

²Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142292, Moscow Region, Russia; fax: (7-095) 923-3602

^* To whom correspondence should be addressed.

Received September 23, 2003

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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 pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-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 beta-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 beta-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.

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KEY WORDS: cellulase, endoglucanase, cellobiohydrolase, chromatography, adsorption, saccharification

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