The Discovery of Novel Carbohydrate Active Enzymes for Improved Detergent Sustainability

Abstract

Xyloglucan is an abundant hemicellulosic polysaccharide found within the primary cell walls of all land plants and is widely utilised throughout an array of industries. The polysaccharide consists of 43-45% glucose; 35-38% xylose, 15-17% galactose and small amounts of other monosaccharides which vary depending on the source of the plant, such as fucose. Xyloglucan may be responsible for a variety of consumer laundry problems due to its intimate association with cellulose and the fact that the polysaccharide is not completely removed during textile processing, suggesting protruding pills which cause fabrics to appear dull may consist of both cellulose and xyloglucan. Degradation of xyloglucan serves many important biotechnological applications and is achieved by specific endo-glucanases which target undecorated D-glucose residues constituting the β-1,4 linked glucose backbone and exo-glycosidases which trim the various side chain substitutions of the polysaccharide. To investigate which combination of glycoside hydrolase enzymes were most effective at degrading tamarind xyloglucan into its component mono- and oligo- saccharides, a search of the Carbohydrate Active Enzyme Database (CAZy) (http://www.cazy.org/) was initiated to identify characterised amino acid sequences within glycoside hydrolase families with activities of interest, including endo-β-1,4-glucanases from families 5 and 12; β-galactosidases from families 2 and 35; α-xylosidases from family 31 and αarabinofuranosidases from family 43, subfamily 12. These characterised sequences were selected and utilised to search the Prozomix metagenomic DNA database (ProzOMIGO!) (http://prozomigo.com/) with the top 10 sequences according to sequences identity selected and utilised to carry out primer design, PCR amplification, cloning into appropriately restricted pET vectors before expression in Escherichia coli and purification to near homogeneity.
The activity of the purified recombinant enzymes was assessed using a variety of methods including spectrophotometric assays utilising para-nitrophenol bound substrates and chromatographic methods such as thin layer chromatography and high-performance anion exchange chromatography ion chromatography (HPAEC-IC) following incubation with homogenous substrate, tamarind xyloglucan. Purified recombinant enzymes which exhibited initial activity toward para-nitrophenol bound substrates and homogenous substates were selected for incorporation into combination assays, in which enzymes with distinct activities were incubated simultaneously with tamarind xyloglucan, with the reaction products analysed using the HPAEC-IC following comparison to monosaccharide and oligosaccharide standards of known retention times.
A reaction incorporating a glycoside hydrolase family 5 endo-β-1,4-glucanase, termed GH5_B, and a glycoside hydrolase family 31 α-xylosidase, termed GH31_BO, was shown to be the most effective combination for the degradation of tamarind xyloglucan, apparent by the production of a main saccharide peak with a retention time of ten minutes with the greatest area and height, indicative of the greatest amount of component monosaccharides released (glucose, galactose and xylose), which may be due to the ‘trimming’ of the many xylose side chain substituents by the α-xylosidase allowing access of the endoglucanase to the β-glucan backbone. Data obtained clearly suggests that endo-glucanases require the synergistic action of various exo-glycosidases to effectively assimilate tamarind xyloglucan, supported by application testing in which a glycoside hydrolase family 5 endo-glucanase utilised without the concerted action of an exo-glycosidase lacked the ability to confer whiteness benefits to greying fabrics, a benefit which was apparent when the two enzyme classes worked in synergy.
During the identification of glycoside hydrolase enzymes with activities of interest, a GH1 β-fucosidase was selected, cloned and expressed in E. coli BL21 (DE3) and purified to near homogeneity before being utilised during spectrophotometric assays with para-nitrophenol-α-L-fucopyranoside. Theβfucosidase surprisingly exhibited strict substrate specificity towards the paranitrophenol bound substrate with a Km value of 0.227 mM, and a Kcat value of 8.150 μM s-1, comparable to that of literature derived α-fucosidases from GH families 29 and 95. The enzyme hydrolysed para-nitrophenol-α-Lfucopyranoside with an optimum pH of 7.8 using 50 mM sodium phosphate buffer, maintaining thermostability up to 60°C with maximum activity observed at 35°C. The recombinant glycoside hydrolase from family 1 is also able to hydrolyse α-1,2 linkages of 2’fucosyllactose, as well as α-1,3 and α-1,4 linkages in fucoidan from Macrocystis pyrifera, with this activity not previously documented within literature for a fucosidase from GH1.
During the study, two variants of carboxymethyl cellulose were utilised – standard detergent grade carboxymethyl cellulose and ‘blocky’ carboxymethyl cellulose. The homogenous carboxymethyl cellulose derivatives were incubated with purified recombinant endo-β-1,4-glucanases from glycoside hydrolase family 5 and 12, as well as various commercial detergent cellulases from families 5, 7 and 44 for various time points, with the reaction products analysed using the HPAEC-IC following comparison to monosaccharide and oligosaccharide standards of known retention times. The results suggest that the purified recombinant endo-β-1,4-glucanases are highly specific for tamarind xyloglucan, evident by the lack of released mono- and oligo- saccharide products released following the reaction as shown by HPAEC-IC. The commercial cellulases utilised released a variety of cellulosic oligosaccharides, with those from family 44 releasing cellobiose, cellotriose and cellotetraose, that from family 7 releasing glucose and cellobiose and that from family 5 releasing glucose, cellobiose and cellotriose. There is little difference in the released products between the two carboxymethyl cellulose derivatives, with the main distinguishing feature being the increased height of product peaks following incubation with standard detergent grade carboxymethyl cellulose as opposed to blocky carboxymethyl cellulose which may be attributable to the clustering of carboxymethyl substituents of blocky CMC which may influence enzyme activity, but explains the use of the polysaccharide in laundry detergents due to its ability to adsorb to the fabric surface and prevent soil redeposition and dye transfer between fabrics more effectively than standard carboxymethylcellulose.

Date of Award	30 Sept 2025
Original language	English
Awarding Institution	Northumbria University
Supervisor	Gary Black (Supervisor), Jose Munoz (Supervisor), Nicola Brown (Supervisor) & Hamish C.L. Yau (Supervisor)