PROCESS TO PURIFY THE COLORED PROTEIN B-PHYCOERYTHRIN PRODUCED BY PORPHYRIDIUM CRUENTUM
Abstract
A simplified process for B-phycoerythrin (BPE) purification from Porphyridium cruentum exploiting aqueous twophase systems (ATPS) and isoelectric precipitation was developed. The effect of two different methods of cell disruption upon the release of the intracellular proteins produced by P. cruentum was evaluated. Cell disruption by sonication proved to be superior over manual maceration since a five time increase in the concentration of BPE released was achieved. The evaluation of partition behaviour of BPE in poly-ethylene-glycol (PEG)-sulphate, PEGdextran and PEG-phosphate ATPS was carried out to determine under which conditions the BPE and contaminants concentrated to opposite phases. ATPS formed with PEG of molecular weight of 1000 g/mol and phosphate proved to be suitable, after isoelectric precipitation at pH 4.0 for the recovery of highly purified BPE (defined as the absorbance ratio of A545nm to A280nm greater than 4.0) with a potential commercial value of $50 USD/mg. An ATPS extraction stage comprising PEG1000 29.5% w/w, phosphate 9.0% w/w, volume ratio (Vr) equal to 1.0, system pH of 7.0, loaded with BPE extract generated by precipitation (equivalent to the 40% w/w of the total extraction system), allowed BPE recovery with a purity of 4.1+0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9 fold after isoelectric precipitation and ATPS. The results reported herein demonstrated the benefits of the practical application of isoelectric precipitation together with ATPS for the
development of a process to purify BPE produced by Porphyridium cruentum.
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