Cellulase cross-linked enzyme aggregates (CLEA) activities can be modulated and enhanced by precipitant selection

BACKGROUND: Crosslinked enzyme aggregates (CLEA) technology is a rapid and versatile method to produce immobilized enzymes via precipitation and cross-linking. A direct relationship between CLEA final activity and process parameters is however yet to be clarified. To address the issue, we have used a factorial design to test the formation and optimization of CLEA made from technical grade cellulase (EC 3.2.1.4). Three types of precipitant (ammonium sulfate, polyethylene glycol, tert-butyl alcohol) were used at varied levels of cross-linker concentration, cross-linking time, and temperature. RESULTS: It was found that the CLEAs produced with tert-butyl alcohol were inactive, whereas the polyethylene glycol- and ammonium sulfate-CLEA, recovered 29 and 17% of the free enzyme activity, respectively. Testing for re-usability, we observed that the polyethylene glycol-CLEA maintained 40% of the initial activity after four cycles, in contrast the ammonium sulfate-CLEA only maintained 10% of its activity after one cycle. CONCLUSION: Our study highlights the importance of evaluating the effect of the precipitant on final CLEA activity rather than re-solubilized enzyme activity. It was demonstrated that polyethylene glycol, despite not being able to precipitate the enzymes as readily as ammonium sulfate, resulted in better performing CLEA