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

Abstract

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.

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

“…Of these, endo-␤-1,4-glucanase plays a key role in cellulose degradation, as it can randomly break down the cellulose backbone internally to release free chain ends. According to the classification of carbohydrate enzymes (http://www.cazy.org/), endo-␤-1,4-glucanases are often found in GH families 6,7,8,9,12,44,45,48,51,74, and 126 (5). Among them, GH12 is a unique group possessing both endoglucanase and xyloglucanase activities (6), and GH12 endoglucanases usually attract attention due to their higher endo-␤-1,3-1,4-glucanase activity.…”

Purification and characterization of an endoglucanase from Aspergillus terreus highly active against barley β-glucan and xyloglucan

“…EG having xyloglucan-degrading activity may be particularly useful for degradation of cell wall material having xyloglucan content, for instance in the wine and fruit industry, for pectin-extraction and for removal of hemicelluloses from textile fibers (Dalboege et al 2001;Hasper et al 2002). Previously two of seven cellulases from Chrysosporium lucknowense (Cel12A and Cel45A) have been reported to possess notable activity towards xyloglucan, together with activities towards CMC and barley b-glucan (Bukhtojarov et al 2004). …”

Identification and Characterization of a Xyloglucan-Specific Family 74 Glycosyl Hydrolase from Streptomyces coelicolor A3(2)

“…Although many researchers have studied xyloglucan-degrading enzymes, little is known about them. Some cellulases (endo-1,4-␤-glucanases) have been reported to have hydrolytic activity against xyloglucan as well as major activities toward carboxymethyl cellulose (CMC) and barley ␤-glucan (2,22). A few specific xyloglucanases that either completely lack activity or display low levels of activity toward CMC and ␤-glucan have been discovered in recent years (7,9,10,15,19,23,25).…”