Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli

Abstract

A direct comparison of a chromatography and an aqueous two-phase system (ATPS) processes for the partial purification of penicillin acylase (PA) produced by a recombinant strain of E. coli, was performed. An established chromatography process for the recovery of PA was selected as a model system and characterised for comparison with a developed ATPS prototype process. PEG-phosphate systems were selected for the recovery of PA over PEG-citrate systems, since higher enzyme recovery and increased purity was obtained. ATPS proved to be suitable to process highly concentrated disrupted extract (35%, w/w) and maintain a high top phase enzyme recovery. In the direct comparison of the processes, the superiority of the ATPS approach was highlighted since a reduction of the number of unit operations from 7 to 4 was achieved. An outline economic analysis based on the cost of separation agent of the processes favour the ATPS process, in which a gross cost reduction of 37% (from 0.47 to 0.30 US dollars) was achieved. Such result was obtained considering a potential re-use of up to 100 times of the resin used in the chromatography process. Additionally, by assuming that all the unit operations are equivalent in investment and operating cost, further reduction of approximately 43% of the respectively involved cost can be obtained when the ATPS process is used. Overall, the proposed ATPS process comprising of PEG1450-phosphate, tie-line length (TLL) of 48.5% (w/w), volume ratio (Vr) of 1.0, pH of 7.0 and 35% (w/w) PA crude extract loaded into the system proved to be more efficient, recovering 97% of PA at the top phase (PEG rich phase) with a purity factor of 3.5. It is clear that the results reported herein raise the consideration for the potential substitution of the chromatography process for PA recovery from E. coli as a first step for the development of an optimised and economic process with evident commercial application.