Cross-linked enzyme aggregation (CLEA) is a novel carrier-free immobilization technique. In this study, trypsin-CLEA has been prepared and its preparation routine, application condition, stability and structure have been studied systematically. (1) Every step for CLEA preparation was investigated to evaluate the effect on the enzyme-activity retention with special aims at the precipitant concentration and type. The experimental results indicated that pure alcohol was an ideal precipitant. (2) The best temperature and pH for CLEA catalysis were determined respectively as 70 ℃ and 9.0, and a detailed explanation was given to clarify the phenomena about shift of the temperature-activity and pH-activity curves. (3) The enzyme stability versus temperature and solvent was tested and the enzyme leakage was examined. This investigation result confirms that the CLEA technique can fundamentally improve the stability of trypsin. (4) The structure of CLEA was characterized by scan electronic microscopy, optical microscopy and laser particle size analysis, respectively. The relationship between the structure and property of CLEA was also discussed. The obtained result of the present research may benefit the further application of CLEA technique, and the observed rules can be applied to other enzyme-type CLEA.

Key words: trypsin, cross-linked enzyme aggregation (CLEA), immobilization, enzyme stability