Abstract: In order to reduce the cost in synthesis of glutathione（GSH）,and concurrently to realize the enzyme storage and the commercialization,the glutathione synthetase was precipitated through appropriate precipitant,and immobilized crosslinked enzyme aggregates（CLEAs）were prepared by glutaraldehyde crosslinking. By optimizing the precipitation and crosslinking conditions,as well as the uses of protein concentration and additives,finally the optimal preparation condition was determined as：4℃,30 g/L protein concentration,adding PEI-600（additive W∶enzyme protein w = 1∶5）fully mixed with the enzyme solution,then 60% saturated ammonium sulfate added for precipitation for 4 h,and finally crosslinked by 0.5% glutaraldehyde. The final protein precipitation rate was 94.9% and the recovery of enzyme activity was 48.2%,enzyme activity per CLEAs was 4.9 U/g,respectively,compared to the control group 28.7% and 2.57 U/g,and increased by 19.5% and 2.33 U/g. The highest yield of GSH reached 11.26 g/L,GSH yield was 9.03 g/L in the 12th batches of conversion,and the relative enzyme activity was 78.4%;after 33 d storage at 4℃,the relative enzyme activity remained 92.12%. In conclusion,after optimizing the preparation conditions,the CLEAs of glutathione synthetases presented higher initial enzyme activity,which could be stably converted more than 10 batches,with favorable storage stability and solid industrialization prospects.

Key words: glutathione synthetases, immobilization, crosslinked enzyme aggregates, additives