Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites.
The properties of modified cellulose polymers, such as methylcellulose, are significantly influenced by the distribution of substituents along the polymer backbone. This distribution is difficult to determine due to the lack of suitable analytical methods. One approach is to use cellulose-degrading enzymes to gain information from the capability of the enzymes to cleave the bonds between glucose units. Endoglucanases are cellulase enzymes that can break internal glycosidic linkages and degrade low substituted regions of modified cellulose where the substituents do not interfere with the enzyme active site. In this work methyl cellulose was degraded using five endoglucanases from glycosyl hydrolase families 5 and 7 from three different species. The products were analyzed with reducing end analysis, chromatography (SEC-MALS-RI), and MALDI-TOFMS. The results were correlated with available determined enzyme structures and using structural alignment for unknown enzyme structures. This was performed in order to elucidate the relationship between active site structures and sensitivity for substituents on derivatized cellulose. The evaluation of endoglucanase hydrolysis of methyl cellulose showed that differences in sensitivity could be related to differences in steric hindrance of substituents in the active site, which could explain differences within family 5 and 7 enzymes, as well as the generally higher substituent tolerance for family 5 enzymes. This information is important for use of endoglucanases as tools for characterization of substituent distribution. The results are also valuable since soluble cellulose derivatives are generally used as substrates during enzyme characterization and in endoglucanase activity assays.
Schagerlöf
U
U
Departments of Molecular Biophysics, Biochemistry, and Technical Analytical Chemistry, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden. Herje.Schagerlof@biochemistry.lu.se
Schagerlöf
H
H
Momcilovic
D
D
Brinkmalm
G
G
Tjerneld
F
F
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry*; D006868:Hydrolysis; D008747:Methylcellulose / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014242:Trichoderma / Q000201:enzymology
D009844:Oligosaccharides; D008747:Methylcellulose; D002480:Cellulase