Immobilization of alkaline protease on polyacrylamide and poly(Acrylamide-co-methacrylic acid) beads by inverse suspension polymerization

Abstract

The alkaline protease was entrapped during inverse suspension polymerization of acrylamide monomer. N,N ́-methylene-bis-acrylamide(MBA), Pluronic PE 8100, and paraffin wax were utilized as a crosslinker, surfactant, and continous phase, respectively. Ammonium persulfate (APS) and N,N, N ́, N ́-tetraethylmethylenediamine (TEMED) were used as the redox initiator. The effects of concentration of monomer, crosslinker, enzyme, initiator, accelerator, and surfactant on enzyme activity were investigated. The effects of stirring rate, polymerization time, and temperature on enzymatic activity of the product were also carried out. The effects of each parameter were established for the best polymerization conditions for entrapment of the alkaline protease for the optimum enzymatic activity. The enzymatic activity was determined using casein as a substrate. Conditions that showed the optimum enzymatic activity were: acrylamide (3.14 mM), MBA (15 mM), alkaline protease (1.5 mg/5 cm3), APS (6.5 mM), TEMED (47.75 mM), at stirring rate of 300 rpm, polymerization time 2 h, and temperature 30℃. The enzymatic activity was 178 units, with 42% immobilization and 92% conversion. The effect of acrylamide/methacrylic acid ratios (100/0, 97.5/2.5, 95/5, 90/10% W/W) on the enzymatic activity were investigated. The enzymatic activity was decreased with increasing the methacrylic acid concentration. The water absorption of polyacrylamide and poly(acrylamide-co-methacrylic acid) in deionized water and saline solutions was also carried out for comparison. The water absorption was increased with increasing methacrylic acid concentration while the absorption in saline solutions was less than that in deionized water. The effects of pH and temperature on enzymatic activity of free- and immobilized enzyme were compared. The maximum enzymatic activity of free- and immobilized enzymes was shown at pH 10 and 10.5 at the same temperature of 45℃, respectively. The free- and immobilized enzymes kept at temperature -20 to 4℃ for one month were stable and without loss of enzymatic activity. The enzymatic activity of the free enzyme was decreased to 51%, while the enzymatic activities of immobilized enzyme on polyacrylamide and poly(acrylamide-co-methacrylic acid) were decreased to 37% and 42%, respectively after an one month storage at 60℃. At higher temperatures, the immobilized enzyme was thermally stable for a longer shelf life than the free enzyme.