TRYPSIN IMMOBILIZATION ON SHRIMP CHITIN WITH FORMALDEHYDE AND ITS APPLICATION TO CONTINUOUS HYDROLYSIS OF CASEIN

A new method of trypsin immobilization on chitin was developed to impr ove the specific activity, activity recovery and stability of the immo bilized trypsin for practical applications. Sliced shrimp-shell chitin , pretreated with 40% (w/v) sodium hydroxide at 60 degrees C for 8 h, was activated by 4% (v/v) formaldehyde at 4 degrees C and pH 8.0 for 1 2 h. After removal of the residual formaldehyde, the immobilized tryps in was prepared by covalent coupling of the enzyme to the chitin at 4 degrees C in a 0.1 M phosphate buffer, pH 7.5. The unbound trypsin was recovered and proved to be reusable for immobilization. Operational s tability of the immobilized trypsin was further improved by cross-link ing with 0.2% glutaraldehyde at 4 degrees C for 4 h. The method was ch aracterized by the high specific activity (2114 units/g), recovery of activity (66.90%) and storage stability (73.80%) of the immobilized en zyme, which was much better than the previous method for immobilizing the enzyme on chitin with glutaraldehyde. The potential application of the enzyme was investigated by the continuous hydrolysis of casein in a pilot-scale column reactor. The enzyme activity survival ratio was 80% after 45 days of operation at 50 degrees C.