Endoglucanase Sensitivity for Substituents in Methyl Cellulose Hydrolysis Studied Using MALDI-TOFMS for Oligosaccharide Analysis and Structural Analysis of Enzyme Active Sites

Abstract

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.

Liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose

“…T. reesei endoglucanases have been applied for hydrolysis of cellulose derivatives in several recent studies. [4][5][6][11][12][13][14] The selectivity of the endoglucanases on various substrates was studied and interesting conclusions, based on the detection of specific enzyme products or the extent of the hydrolysis, were drawn.…”

Biochemical Characterization and Structural Analysis of a Bifunctional Cellulase/Xylanase from Clostridium thermocellum

“…hydrogen bonds to C2-OH of the bound cellulodextrins (26 -28) or can better tolerate an equatorial or axial 2-OH because of the relatively flexible structures (6,29). In contrast, the Asn-208 side chain of CtCel5E cannot form a hydrogen bond with the axial 2-OH.…”

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

“…Cellulases are commonly employed for the characterization of derivatized celluloses with respect to their substitution pattern. Many of the enzymes used, such as Cel5A, Cel7B and Cel45 from Trichoderma reseei [ 15 , 30 ] , EGII from Trichoderma longibrachiatum [ 16 ], Cel5A from Bacillus agaradhaerens [ 24 , 30 , 38 ] or Cel5A from Humicola insolens [ 30 ] contain one or more carbohydrate-binding modules linked to either the C- or N-terminus of the catalytic module. The goal of this work was to investigate if CBMs on their own have the ability to make a distinction in the distribution pattern of cellulose derivatives based on their substitution pattern.…”

“…Enzyme-aided characterization of cellulose derivatives is an attractive approach as it is based on specific protein-carbohydrate interactions [ 30 ]. Other proteins that can specifically recognize natural cellulose are carbohydrate-binding modules (CBMs).…”