Chitosan-tethered microspheres for lactase immobilization

Abstract

Abstract Surface interactions between an enzyme and carrier can reduce activity retention after immobilization. Using compatible polymers to tether the enzyme provides a means of maintaining bulk properties of the carrier while limiting disadvantageous associations that reduce activity retention of the enzyme. In this work, chitosan was attached to P(S/V-COOH) microspheres by carbodiimide chemistry. Lactase (Aspergillus oryzae) was then covalently conjugated to the microspheres having the chitosan tether as well as directly to microspheres without the chitosan tether. Relative to the soluble enzyme, activity retention of lactase attached directly to the microspheres without the chitosan tether resulted in 11% activity retention after immobilization. Comparatively, attachment of lactase to microspheres having the chitosan tether yielded a retained activity that was not significantly different from that of the soluble enzyme, under optimum conditions for the immobilized conjugate. Chitosan-tethered microspheres also enabled an increase in protein loading—achieving 18 mg/g of support compared to 5 mg/g of support on microspheres without the chitosan-tether. However, beyond a protein loading of 12 mg/g of support, the immobilization efficiency on chitosan-tethered microspheres decreased. Lactase conjugation to chitosan-tethered microspheres resulted in shift in optimum pH from pH 4.5 to pH 4.0, but no significant change in temperature stability relative to the soluble enzyme. The results suggest that altering the material surface interface can lead to greater enzyme activity retention after immobilization.