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RESEARCH PAPER

Immobilization of Horseradish Peroxidase on Modified Cellulose Carriers via Hydrophobic Interactions: Catalytic Properties and Stability Sasa Savic1

•

Sanja Petrovic1 • Sanela Savic2 • Zivomir Petronijevic1

Received: 7 May 2020 / Accepted: 3 November 2020 Ó Shiraz University 2020

Abstract Cellulose is one of the most common biopolymers on earth with well-known non-toxic, biocompatible, and environmentally friendly properties. Structural and chemical properties of cellulose fibers give additional opportunities for surface modification of cellulose and its use as a carrier for immobilization of various bioactive compounds, including enzymes. For the proposes of horseradish peroxidase (HRP) immobilization, five different cellulose-based carriers were synthesized by acylation of microcrystalline cellulose. After HRP immobilization, based on the saved catalytic activity, protein equilibrium, and immobilization parameters, the benzoyl- and cinnamoyl-cellulose carriers were selected for further characterization. Kinetic studies have shown that the apparent Michaelis constants for HRP immobilized on benzoylcellulose and cinnamoyl-cellulose carriers were 58.1 lM and 60.8 lM for hydrogen peroxide and 113.4 lM and 115.2 lM for ABTS, respectively. Experimental investigations of temperature and pH value influence on immobilized HRP on two selected cellulose carriers have shown that the optimal values of temperature and pH were 35 °C and 6.8, respectively. Thermal and storage stability tests showed that immobilized HRP could exhibit improved thermal and storage stability compared to the free enzyme. Operational stability tests showed that HRP immobilized on modified cellulose carriers can do up to 20 successive batch operations, without the decrease in initial activity during the first eight cycles. Keywords Immobilization  Horseradish peroxidase  Cellulose  Hydrophobic interaction

1 Introduction Cellulose is one of the most common biopolymers on earth that is non-toxic, biocompatible, and environmentally friendly. These facts are the main reason for its wide use as a carrier for the immobilization of many bioactive

& Sasa Savic [email protected] Sanja Petrovic [email protected] Sanela Savic [email protected] Zivomir Petronijevic [email protected] 1

Faculty of Technology, University of Nis, Bulevar Oslobodjenja 124, 16000 Leskovac, Serbia

2

DCP Hemigal, Tekstilna 97, 1600 Leskovac, Serbia

compounds (Di Risio and Yan 2009; Klemm et al. 2005). Structurally, cellulose represents a linear polysaccharide composed of D-glucose molecules connected by b(1–4) linkages with variable degrees of polymerization depending on the origin and processing of raw material. The presence of numerous inter- and intramolecular hydrogen bonds in cellulose chains leads to their organization into fibrils with high-organized, crystalline and low-organized, amorphous regions. Besides, cellulose has a system of pores with capillaries, voids, and interspace