Studies on New Synthetic Substrates for Cellulolytic Enzymes

Thomson, John W (1977) Studies on New Synthetic Substrates for Cellulolytic Enzymes. PhD thesis, University of Glasgow.

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Abstract

The introduction consists of a review of the occurrence of cellulose, of the organisms responsible for cellulose degradation and of the cellulolytic enzymes, A new method has been developed for the large scale fractionation of peracetylated cello-oligosaccharides using a large silica column and a solvent system consisting of carbon tetrachlaride/chloroform in the ratio 70 : 30 with 1% methanol added. The carbon-13 NMR spectra of the peracetylated cello-oligosaccharides were determined and are discussed in relation to the carbon-13 NMR spectrum of cellulose acetate, 3,4-Dinitrophenyl beta-D-glucopyranoside and the 3,4-dinitrophenyl beta-cello-oligosaccharides with D.P. from 2 to 4 were prepared. This involved coupling of the acetobromo-sugars with 3,4-dinitrophenol in the presence of anhydrous potassium carbonate and dry acetone. The resultant peracetylated aryl oligosaccharides were de-O-acetylated by the method of Zemplen. Some modified 3,4-dinitrophenyl beta-D-glucopyranosides were also prepared. An affinity column specific for beta-glucosidases was prepared by coupling 4-aminophenyl beta-D-thioglucopyranoside with Affigel 10, a commercial affinity column support matrix with a 10A spacer arm. Two enzymes from a commercial cellulase from Trichoderma viride were purified, A beta-1,4-glucan glueanohydrolase, designated cellulase El, was obtained by ion exchange chromatography on DEAE-Sephadex A-25, passage through Sephadex G-75 (twice) and finally passage through the affinity column, and a cellobiase, designated cellulase ELI, obtained by ion exchange chromatography on DEAE-Sephadex A-25, passage through Sephadex G-75 ard then Sephadex G-100. Both enzymes behaved as single proteins as judged by SDS-gel electrophoresis and Sephadex G-75 and G-100 chromatography. The molecular weights of cellulase EI and cellulase EII were estimated to be 12,000 and 74,400 respectively. The aryl cello-oligosaccharides were used as substrates for the cellulase enzymes. The values of kcat/Km for cellulase EI-catalysed hydrolysis of 3,4-dinitrophenyl beta-cellobioside, cellotrioside and cellotetraoside were determined to be 161, 334 and 423 l.M-1.s-1 respectively. These values are compared with the lysozyme-catalysed hydrolyses of 3,4-dinitrophenyl beta-chitobioside, chitotrioside and chitotetraoside under similar conditions. Induced hydrolyses of 3,4-dinitrophenyl beta-D-glucopyranoside and p-nitrophenyl beta-D-glucopyranoside were observed in the presence of cellotriose, cellotetraose or cellopentaose and cellulase EI. Modified 3,4-dinitrophenyl beta-D-glucopyranosides were incubated with cellopentaose and cellulase El and a rapid rate of induced hydrolysis was observed with the 6-deoxy-glucopyranoside and the xylopyraroside as well as the glueopyranoside. With 3,4-dinitrophenyl 6-O-raethyl and 6-chloro-6-deoxy beta-D-glucopyranosides a much reduced induced rate of hydrolysis was observed. With p-nitrophenyl 2-deoxy-beta-D-glucopyranoside there was no induced hydrolysis. These results are discussed and compared with previously reported results for induced hydrolyses catalysed by lysozyme. The reaction rates for cellulase Ell-catalysed hydrolysis of 3,4-dinitropheryl and p-nitrophenyl beta-D-glucopyranosides were compared with the reaction rates for the corresponding modified gluco-pyranosides. Any changes to the glucose moiety resulted in a considerable loss of activity compared to the parent glucopyranoside. With p-nitrophenyl 2-deoxy-beta-D-glucopyranoside no enzymic hydrolysis was observed. 'These results are discussed and compared with the hydrolyses of other modified glucopyranosides by various beta-glucosidases which have been previously reported. The Michaelis-Menten constants for catalysed hydrolysis of 3,4-dinitrophenyl B-D-glucopyranoside were determined for cellulase ElI. The value of Vmax was 1.44 x 10e-8 M/1/s and the value of Km was 7.09 x 10e-5M/1. The value of kcat/Km was 8830 1M-1s-1.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Organic chemistry
Date of Award: 1977
Depositing User: Enlighten Team
Unique ID: glathesis:1977-78773
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 14:55
Last Modified: 30 Jan 2020 14:55
URI: http://theses.gla.ac.uk/id/eprint/78773

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