PROTEASE ENCAPSULATION: EFFECT OF PARTICLE COMPOSITION AND SIZE ON ENZYMATIC REUSE

Enzymatic immobilization is used as a way to reduce costs by enabling enzyme reuse and continuously carried out processes. The objective of this work was to immobilize a microbial protease (Corolase, ABEnzymes) in particles with different composition (alginate, alginate-chitosan and alginate-chitosan-glutaraldehyde) and sizes (1.41 ± 0.06 and 3.26 ± 0.25 mm diameter) and to evaluate immobilization efficiency and enzymatic reuse. Alginate immobilization was performed by mixing the enzyme solution with a 2% sodium alginate solution (1:1); the protease-alginate mixture was then added dropwise in calcium chloride (0.2 M) to form the particles. Alginate-chitosan immobilization was performed as described above, with chitosan (0.5%) being added in the calcium chloride solution. Alginate-chitosan-glutaraldehyde immobilization was performed by immersing the alginate-chitosan particles in glutaraldehyde solution (0.5%) for 20 minutes. To obtain the different sized particles, a Pasteur pipette and syringe were used to make the drops. After immobilization, all particles of each treatment were weighed and used to evaluate proteolytic activity and calculate immobilization efficiency. Enzymatic reaction was carried out for 20, 40 and 60 min. Immobilization experiments were repeated 2 times and analyses were carried out in triplicate. Results were evaluated by ANOVA and means were compared by Tukey test (p<0.05). Results showed that particles made up of only alginate and of the smaller diameter yielded better recovery of enzymatic activity (60% immobilization efficiency). Enzymatic reactions performed for 60 min yielded significantly higher activities for both sizes and all compositions, suggesting that a longer time is needed to overcome diffusional restrictions and to provide more efficient enzyme-substrate interaction. Enzymatic reuse caused a progressive reduction of proteolytic activity with each new cycle independent of particle size and composition. Alginate particles exhibited significantly higher activity values and maintained more than 50% of activity after the fourth use.