Covalent bonding immobilization of a Bacillus licheniformis protease on chitosan and its application in protein hydrolysis

Abstract

Enzyme immobilization makes its structure more rigid and insoluble, increasing its stability. This can be done by forming a covalent bond between the enzyme and an inert, insoluble, and preferably low-cost support. Proteases are widely applied in protein hydrolysis; however, most studies focus on the use of proteases in free form. In this work, for the first time, a commercial alkaline protease from Bacillus licheniformis (Protezyn APP 3000) was immobilized by covalent bonding on chitosan modified by glutaraldehyde and ethylenediamine; the immobilized enzyme was applied in the hydrolysis of different proteins sources. The best immobilization conditions observed were as follows: 5% (m/v) chitosan support modified by 5% (v/v) glutaraldehyde diluted in water, immobilization solution with the enzyme diluted in water, and 2 h as immobilization time. Under these conditions, the amount of immobilized enzyme was 69.9%, the efficiency was 33.6% and effectiveness was 48.3%. The biochemical characterization showed that the immobilized enzyme exhibited maximum catalytic activity at the same pH and temperature as the free enzyme (pH 9 and 60 °C); however, the immobilization process extended the optimal temperature range of enzyme activity (50–70 °C). When reused, the immobilized enzyme retained 47.08% of its initial activity after three cycles. The immobilized enzyme showed promise in hydrolyzing casein, hemoglobin, and soy proteins and was more efficient on gelatin than the free enzyme. Therefore, the immobilization performed showed promise in stabilizing a commercial protease that is being studied for the first time and can be optimized to improve the process parameters.