Bioconjugation of enzyme with silica microparticles: a promising platform for α-amylase partitioning

Abstract

Here, we report the implementation of α-amylase conjugated silica microparticles for improvement of α-amylase partitioning in a PEG–organic salt-based aqueous two phase system. A direct reduction method was employed for the synthesis of silica microparticles with simultaneous introduction of α-amylase. In this context, we synthesized three different silica α-amylase conjugated microparticles with variation of tetraethyl orthosilicate concentration, and thus the effect of final particle size and enzyme loading on partitioning was also studied. The partition coefficient ratio of α-amylase to Si:α-amylase of 2.186 : 21.701 validated an almost tenfold increase in separation. The microscopic structure of the system was thoroughly investigated in order to understand the extraction mechanism and any possible denaturation. Improved partition coefficients can be interpreted by the formation of α-amylase–silica–PEG carriers. Furthermore, circular dichroism (CD) spectra validated partial unfolding of the enzyme.