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This work studied the immobilization of Cercospora kikuchii lipases on chitosan microparticles by chemical attachment on chitosan acetate microparticles activated by glutaraldehyde (CAM) added before or after the enzyme and physical adsorption on highly deacetylated chitosan hydrochloride microparticles (CHM). Lipases covalently immobilized on pre-activated CAM showed better performance retaining 88.4% of the enzymatic activity, with 68.2% of immobilization efficiency (IE). The immobilized enzyme retained an activity of about 53.5 % after five reuses, using p-NPP as substrate. Physical adsorption of lipase onto highly deacetylated CHM showed 46.2 % of enzymatic activity and 28.6% of IE. This immobilized derivative did not lose activity up to 80 days of storage at 4°C, while lipases immobilized on pre-activated CAM maintained its activity up to 180 days at same conditions. Taken together the results indicate that chitosan microparticles provide an optimal microenvironment for the immobilized enzyme to maintain good activity and stability.

This work studied the immobilization of Cercospora kikuchii lipases on chitosan microparticles by chemical attachment on chitosan acetate microparticles activated by glutaraldehyde (CAM) added before or after the enzyme and physical adsorption on highly deacetylated chitosan hydrochloride microparticles (CHM). Lipases covalently immobilized on pre-activated CAM showed better performance retaining 88.4% of the enzymatic activity, with 68.2% of immobilization efficiency (IE

Xyloglucan (XG) is an abundant polysaccharide in plant cell walls and some seeds and requires at least four different enzymes for complete depolymerization. Recombinant xyloglucanase from Aspergillus niveus (XegA), α‐xylosidase from Escherichia coli (YicI), β‐galactosidase from Hypocrea jecorina (Bga1) and β‐glucosidase from H. jecorina (Bgl1) were covalently immobilized individually and in combination on chitosan‐coated ferromagnetic iron oxide nanoparticles functionalized with glutaraldehyde. All immobilized enzymes presented reduced specific catalytic activity, where immobilized YicI, XegA, Bga1 and Bgl1 retained 14.9, 76.9, 29.5 and 6.6% activity as compared with the free enzymes, respectively. Immobilized and free enzymes presented similar optimum catalytic pH and optimum catalytic temperatures differed by ±10°C between the free and immobilized enzymes. Bga1 and Bgl1 presented decreased maximum catalytic temperatures (Topt), while YicI presented an increased Topt. The Topt of XegA remained unaltered. Mass spectrometry confirmed that nanoparticles carrying all four co‐immobilized enzymes degraded XG to glucose, galactose and xylose, and higher proportions of co‐immobilized Bgl1 and XegA resulted in higher XG saccharification. Although levels of Bgl1 activity were limiting, five re‐use cycles of the co‐immobilized enzymes were demonstrated, providing proof‐of‐principle for the use of a four‐component multienzyme nanoparticle in the breakdown of a complex polysaccharide. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd.