Purification of R‐phycoerythrin from Porphyra haitanensis (Bangiales, Rhodophyta) using expanded‐bed absorption¹

Abstract

R-phycoerythrin (R-PE) was purified from leafy gametophyte of Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) by a simple, scaleable procedure. Initially, phycobiliproteins were extracted by repeated freeze-thaw cycles, resulting in release from the algal cells by osmotic shock. Next, R-PE was recovered by applying the crude extract with a high concentration of (NH 4 ) 2 SO 4 salt directly to the expanded-bed columns loaded with phenyl-sepharose. An expanded-bed volume twice the settled-bed volume was maintained; then low (NH 4 ) 2 SO 4 concentration was used to develop the column. After two rounds of hydrophobic interaction chromatography (HIC), R-PE was purified by anion-exchange column. The method was also successful with free-living conchocelis of P. haitanensis. The purified R-PE was identified with electrophoresis, and absorption and fluorescence emission spectroscopy. The results were in agreement with those previously reported. The yield with a spectroscopic purity (OD 565 ⁄ OD 280 ) higher than 3.2 (the ratio of A 565 ⁄ A 620 £ 0.02) was 1.4 mg AE g )1 of leafy gametophyte of P. haitanensis. For the free-living conchocelis of P. haitanensis extract, R-PE could be purified successfully with only one round of HIC. The yield with a spectroscopic purity (OD 565 ⁄ OD 280 ) higher than 3.2 (the ratio of A 565 ⁄ A 620 £ 0.02) was 5.0 mg AE g )1 of free-living conchocelis of P. haitanensis. The method described here is a scaleable technology that allows a large quantity of R-PE to be recovered from the unclarified P. haitanensis crude extract. It is also a high protein recovery technology, reducing both processing costs and times, which enhances the value of this endemic Porphyra of China.

Pilot Scale Recovery of Phycocyanin from Spirulina platensis Using Expanded Bed Adsorption Chromatography

“…In comparison with previous papers that describe similar methods [13][14][15][16][17][18][19][20][21] for phycobiliproteins purification using expanded bed chromatography, we find important differences. So, Niu et al [19] described a method based on EBAC, using a 25 mm ID column.…”

“…So, Niu et al [19] described a method based on EBAC, using a 25 mm ID column. This size column is relatively small in comparison with the columns used in this work (15,25,40, 60, 90 and finally 150 mm ID columns). In this sense, Niu et al do not study the scale-up process and only focused their attention in 25 mm column.…”

“…This technique can be used in general primary protein purification [12] and more specifically in phycobiliproteins purification. In this sense, there are different studies about phycoerythrins [13][14][15][16][17] and phycocyanins [18][19][20][21] purification using EBAC at small, medium and large scale obtaining diverse protein quantities, but always in the order of milligrams.…”

Large scale preparation of phycobiliproteins from <i>Porphyra yezoensis</i> using co-precipitation with ammonium sulfate

“…Many types of procedures and materials were used for large scale preparation of PBP [13][14][15][16] and the latest approach was the application of hydrophobic chromatography. For example, 28 g P. yezoensis was smashed and applied to phenyl-sepharose column to obtain 27 mg PE with a purity of 2.0 -2.5.…”

“…In these procedures, 33 g PC and 2.3 g APC with purity of 3.4 and 4.0 respectively were obtained from 550 g Microcystis aeruginosa using DEAE-sepharose chromatography column [13]. Using hydrophobic chromatography, 98 mg PE with a purity of 4.4 were obtained from 700 g Gracilaria verrucosa [14], 67 mg PE with a purity of 3.5 from 550 g Palmaria palmate [15], and 23 mg PE with a purity of 4.8 from 15 g Porphyra haitanensis [16]. Recently, novel techniques such as immuno-absorption and genetic recombination have been utilized for purification of PBP.…”