Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.
Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.
Karlsson
Johan
J
Department of Biochemistry, Lund University, P. O. Box 124, S-221 00 Lund, Sweden.
Momcilovic
Dane
D
Wittgren
Bengt
B
Schülein
Martin
M
Tjerneld
Folke
F
Brinkmalm
Gunnar
G
D001203:Ascomycota / Q000201:enzymology*; D001673:Biodegradation, Environmental; D001704:Biopolymers / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002850:Chromatography, Gel; D005656:Fungal Proteins / Q000378:metabolism; D006026:Glycoside Hydrolases; D006868:Hydrolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
D001704:Biopolymers; C435103:CEL45A protein, Piromyces equi; D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C407024:endoglucanase Cel5A; D002480:Cellulase; C019826:carboxymethylcellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D002266:Carboxymethylcellulose Sodium