Preparation and properties of trypsin chemically attached to EEDQ activated styrene–methacrylic acid copolymers

Abstract

Styrene-methacrylic acid copolymers of varying combinations crosslinked with p-DVB (1-2%) and porous structure were synthesized to be used as carriers in trypsin immobilization. The styrene-methacrylic acid copolymers containing free carboxy groups were activated by conversion into the mixed carbonic anhydride with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) at pH 4.0. The degree of activation of copolymers were determined from the amount of p-aminobenzoic acid each could bind. The activated copolymers were incubated with trypsin in phosphate buffer (pH 8.0) at 4 degrees C for 24 h. The optimum conditions for enzymatic activity measurements determined and the activity tests were carried out in 1.5 x 10(-2)M CaCl(2) solution (pH 8.0) at 0.05 ionic strength with a pH-stat instrument. The dependence of the activity of styrene-methacrylic acid (SMA)/trypsin derivatives to pH was investigated and it was observed that the optimum pH of the immobilized trypsin derivatives moved to the basic region compared to the native trypsin. It was found that as the ionic strength increased, the shift in the optimum pH decreased and the activity increased. The Michaelis constants for the SMA-trypsin derivatives were determined with aid of Lineweaver-Burk diagrams. The thermal, storage, and operational stabilities of SMA-trypsin derivatives were assessed. It was found that the above stabilities for all the immobilized trypsin derivatives were better than that for the native trypsin.