Monday, May 5, 2008
9-35

Liquid-liquid extraction using Triton X-114 for nisin and green fluorescent protein (GFPuv) extraction with electrolytes

Priscila G. Mazzola, Angela F. Jozala, Larissa M. Sato, Adalberto Pessoa Jr., and Thereza C. V. Penna. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutics Sciences, Avda. Professor Lineu Prestes, 580, Bloco 16, São Paulo, 05508-900, Brazil

Green fluorescent protein (GFP), expressed by E.coli DH5-α, is widely applied with as biossensor and can be detected by spectrofluorometry or using hand UV lamp, it has became a versatile tool for a variety of biotechnological uses and as a potential biological indicator, for preservation of manufactured and processed products. Nisin, antimicrobial peptide, produced by L.lactis ATCC 11454, used as natural agent of biopreservation; and it has been accepted as a safe and natural preservative in different areas of food and pharmaceutical industry. This study aimed to evaluate the aqueous two-phase system (ATPS) composed by Triton X-114 (TX), in presence or absence of electrolytes, to separete nisin and GFP. The system was composed by 2%TX with and without MgSO4 or NH2SO4. Nisin activity was determined by agar diffusion with L.sakei as bioindicator. GFP concentration was determined by fluorimetry. The phase diagram of the TX in buffer was obtained by the cloud-point method. Each solution was placed in a thermo-regulated device set at a temperature of 36.2ºC for 2h to attain partitioning equilibrium. The coexisting phases formed were withdrawn and the biomolecules concentration was determined in each phase, in TX system without electrolytes. Results indicated that nisin partitions preferentially to the micelle rich-phase, with significant antimicrobial activity increase (up to 10-fold). GFP partitioned evenly between the phases in TX system without electrolytes. After partitioning in the presence of salts, nisin was more extremely driven to the micelle-rich phase, while GFP was driven to the micelle-poor phase.

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