Investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques

Abstract

The benefits of applying cellulose selective enzymes as analytical tools for chemical structure characterization of cellulose derivatives have been frequently addressed over the years. In a recent study the high selectivity of cellulase Cel45A from Trichoderma reesei (Tr Cel45A) was utilized for relating the chemical structure to the flow properties of carboxymethyl cellulose (CMC). However, in order to take full advantage of the enzymatic hydrolysis the enzyme selectivity on the cellulose substrate must be further investigated. Therefore, the selectivity of Tr Cel45A on CMC was studied by chemical sample preparation of the enzyme products followed by mass spectrometric chemical structure characterization. The results strongly suggest that, in accordance with recent studies, also this highly selective endoglucanase is able to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (AGUs). Furthermore, the results also indicate that substituents on the nearby AGUs will affect the hydrolysis.

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

“…The filtered enzymatic hydrolysates were subjected to reduction and per-O-methylation as has been described earlier, 7 with the exception that NaBD 4 was used as reducing agent. The deuterium atom, which is introduced on the anomeric (C1) carbon atom on the reducing end, facilitates the fragment identification in MS/ MS.…”

“…Furthermore, peaks corresponding to 3,5 A-fragments, formed from crossring cleavages, were also detected. 7 From these fragments it is possible to deduce whether the substituent is located at the O-6 position or the O-2 or O-3 position. However, O-2 and O-3 substitution cannot be differentiated from the 3,5 A-fragments.…”

“…The sample preparation of the CM-oligomers obtained from enzymatic hydrolysis was performed as described by us earlier, 7 with the exceptions that NaBD 4 was used in the reduction instead of NaBH 4 , and that the reduced samples were evaporated under a stream of nitrogen instead of freeze-drying prior to per-O-methylation. In short, the enzymatic hydrolysates were filtered prior to reduction by NaBD 4 and per-O-methylated according to Ciucanu and Kerek 34 by using powderized NaOH in DMSO and iodomethane as reagents.…”

“…One attractive approach to study the substituent distribution in CMC is to perform enzymatic hydrolysis followed by chemical structure analysis of the oligomeric products by, for example, liquid chromatography (LC), 1-3 mass spectrometry (MS), 4,5 LC-MS, 6 or by tandem mass spectrometry (MS/MS). 7 Many of the commercially available cellulase preparations that are used for this purpose are obtained from the fungus Trichoderma reesei. [8][9][10] This fungus produces a compliment of cellulolytic enzymes, including the four cellulases (EC 3.2.1.4) Cel7B, Cel5A, Cel45A, and Cel74A.…”

Characterization of the endoglucanase and glucomannanase activities of a glycoside hydrolase family 45 protein from Penicillium decumbens 114-2

“…As no reducing sugars were released from locust bean gum, we concluded that the enzyme could not hydrolyze the β-1,4-linkage between two D-mannosyl residues. Thus the reason for the higher glucomannanase activity might be: (1) the enzyme hydrolyzed the bond between a glucosyl residue and a mannosyl residue faster than β-1,4-glycosidic bonds on konjac glucomannan, as discussed in Karlsson et al (2002); (2) as there was a lack of an absolutely amorphous but non-substituted cellulose substrate, the hydrolytic activities on CMCNa and PASC could not stand for the real endoglucanase activity of Cel45A (Enebro et al, 2009). The β-1,4-glycosidic bonds of KGM or lichenan (Eberhardt et al, 2000;Shibuya and Kikuchi, 2008) with very few substituents seem to be more suitable targets for GH family 45 enzymes.…”