I think it is likely that the high alcohol and/or acetone concentration inactivated the enzyme. Another possibility is that the enzyme is unstable in the storage conditions. How do you store the enzyme resin?

Actually I haven't stored it yet. Immediately after immobilization , I run the first batch then I wash by my buffer and run the second batch. Then the activity yield decreases by 50% .

So I will try to work with lower concentrations .

Are you sure that all the enzyme was covalently attached to the resin during the first experiment? Perhaps half of it was associated loosely with the resin, but this half was washed off prior to the 2nd run.

I don't think so , because I wash my resin by 25 ml , 2M NaCl before the first run and I get approximately 90% activity yield . In my opinion  a non-covalently bound protein can't afford the first wash and give 90 % activity yield. and even to get sure of that I extended my immobilization coupling time from optimum  the period ( 6 hours ) to 30 hours and there was no significant change.

Best Regards