Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose I and II cotton nanocrystalline preparations

Abstract

Lysozyme was attached through an amide linkage between some of the protein's aspartate and glutamate residues to amino-glycine-cellulose, which was prepared by esterification of glycine to preparations of cotton nanocrystals. The nanocrystalline preparations were produced through acid hydrolysis and mechanical breakage of the cotton fibers from a scoured and bleached cotton fabric and a scoured and bleached, mercerized fabric, which was shown to produce cellulose I (NCI) and cellulose II (NCII) crystals respectively. A carbodiimide-activation coupling reaction was used to create the lysozyme-aminoglycine-cellulose conjugates using both NCI and NCII in a polar solvent and gave yields of covalently linked lysozyme at 604 mg/gram of cotton nanocrystal. The incorporation of lysozyme conjugated to the NCI and NCII preparations gave very high activity (1,500 U/mg cotton) when assessed using a fluorescence tag assay to measure antimicrobial activity against Micrococcus lysodeikticus. Scanning electron micrographs demonstrated an aggregation of nanoparticles corresponding to lysozyme bound on the surface of larger cotton nanocrystalline sheets. The approach of producing high enzyme activity on cotton nanocrystals is discussed in the context of selectively presenting robust hydrolase activity on nanocrystalline surfaces.

Surface modification of cellulose nanocrystals

“…108 The nal DS was reported as 0.029, indicating 90% complexation of the surface graed terpyridine with ruthenium. 108 Esterication was used as a precursor to further modica-tion in the attachment of lysozyme to cellulose nanocrystals by Edwards et al 86 Cellulose nanocrystals were rst modied with Fmoc-glycine using DIC as a coupling agent, before deprotection and attachment of lysozyme by amidation. 86 The level of modication was reported as DS ¼ 0.09 for the esterication, but lateral dimensions for the nanocrystals are not given in the paper making conversion of this value into DS problematic.…”

“…The lysozyme attachment is then analysed by use of a "nitrogen to protein conversion factor" rather than direct measurement of the nitrogen concentration in the lysozyme starting material leading to gures that cannot be directly related to DS. 86 Amidation was used as a precursor to the popular azidealkyne cycloaddition reaction. Filpponen et al used EDC to attach both azide and alkyne functionalities to oxidized cellulose nanocrystals.…”

Nanocellulose-Based Biosensors: Design, Preparation, and Activity of Peptide-Linked Cotton Cellulose Nanocrystals Having Fluorimetric and Colorimetric Elastase Detection Sensitivity

“…Enzymes can be formulated with nanocellulose as covalent linkages, salt bridges, and with physical inclusion complexes [13][14][15][16][17][18][19][20][21]. The hydrophilic surface properties provide a biocompatible matrix for selective activity to occur.…”

“…Edwards et al reported very high lysozyme activity when the hydrolase was attached to cotton cellulose nanocrystals [18], which suggests the activity of anti-microbial enzymes can be bolstered when immobilized on cotton cellulose nanocrystals, and this approach may have further implications for use to detect as well as inhibit formation of microbial biofilms [19]. Finally it is noteworthy that cellulases have been used in an approach that demonstrates how enzymatic digestion of cellulose in combination with soft lithography can be used to obtain patterned surfaces [20].…”

Papain@Magnetic Nanocrystalline Cellulose Nanobiocatalyst: A Highly Efficient Biocatalyst for Dipeptide Biosynthesis in Deep Eutectic Solvents

“…NCC is generally prepared by acid-catalyzed hydrolysis of cellulose 5 , and has drawn increasing attention due to its excellent physicochemical properties (including high surface-to volume ratios, high aspect ratios, high stiffness, 3 good strength) and sustainability 6 . Glucose oxidase 7 , peroxidase 8 , and lysozyme 9 , as well as papain 10 were successfully immobilized onto NCC with enhanced activity, stability and catalytic efficiency. This indicated that the novel NCC-based enzyme carriers show great potential and are worthy of further study.…”