Immobilized protease on magnetic particles for enzymatic protein hydrolysis of poultry by-products

Wilhelm R Glomm,Sileshi G Wubshet,Diana Lindberg,Katinka R Dankel,Nils K Afseth,Per M Stenstad,Heidi Johnsen

doi:10.1016/j.lwt.2021.112327

Abstract

Immobilization of enzymes onto magnetic particles can potentially allow for enzyme reuse, which can significantly reduce the cost associated with e.g. enzymatic protein hydrolysis (EPH) of fish and meat by-products. Here, we report glutaraldehyde-mediated immobilization of a food-grade protease (Subtilisin A) onto magnetic silica particles using three different amine ligands; a short, brush-like linker (aminopropyl trimethoxysilane), a long, flexible linker (Jeffamine), and a gel-like coating (chitosan). The three coupling strategies were evaluated and compared with respect to the amount of immobilized protease, enzyme activity and catalytic performance in the hydrolysis of chicken meat and turkey tendons, respectively. The particle systems showed high reusability (≤85% activity remaining after six consecutive cycles) and storage stability (≤93% activity remaining after 25 months storage). Particle-immobilized enzyme systems were able to catalyze degradation and extraction of protein from chicken meat and turkey tendons.

Highlights

Immobilization of enzymes – both on solid supports and on sus pended particles - is an acknowledged method in many fields of bio catalysis for stabilizing enzymes in harsh production environments encompassing e.g. high temperature, pressure, and organic solvents (Ansari & Husain, 2012; Garcia-Galan, Berenguer-Murcia, Fernande z-Lafuente, & Rodrigues, 2011)
Aminopropyl trimethoxysilane (ATMS), 3-glycidyloxypropyl trime thoxysilane, glutaraldehyde, phosphate buffer, acetic acid, trichloroacetic acid, Bradford reagent B6916, ninhydrin, dimethyl sulfoxide (DMSO), Span80, toluene, methanol, nitric acid, lithium hy droxide (LiOH), tin (II) chloride (SnCl2), chitosan, protease from Bacillus licheniformis P5380 (Subtilisin A) and azo-casein were all purchased from Sigma Aldrich
Epoxy silica particles were coated with chitosan resulting in a hydrophilic gel-like coating with amino groups, which may be suitable for the protease activity of the immobilized enzymes

Summary

Introduction

Immobilization of enzymes – both on solid supports and on sus pended particles - is an acknowledged method in many fields of bio catalysis for stabilizing enzymes in harsh production environments encompassing e.g. high temperature, pressure, and organic solvents (Ansari & Husain, 2012; Garcia-Galan, Berenguer-Murcia, Fernande z-Lafuente, & Rodrigues, 2011). In the special case of proteases, a possible additional benefit is the prevention of autolysis (Garcia-Galan et al, 2011). As a detailed description of enzyme immobilization and the associated advantages and drawbacks is beyond the scope of the present paper, we recommend several excellent reviews (Barbosa et al, 2015; Garcia-Galan et al, 2011; Rodrigues et al, 2013) on the subject. The cost of industrial proteases for EPH varies greatly. A comparison performed in the fall of 2019 showed that the cost of 1 kg commercial food grade protease from 10 different vendors varied be tween £11 and £125 (Solstad et al, 2020). The possibility to immobilize proteases for enzyme reuse with a concomitant cost reduc tion becomes attractive. Immobilization is interesting from the perspective of reducing such undesired process-limiting properties

Methods

Results

Conclusion