180
M. R. Nikolic et al.
Keywords Phycobiliproteins · Phycocyanin · Allophycocyanin · Phycoerythrin ·
Phycocyanobilin · Chromopeptides
8.1 Introduction
Phycobiliproteins (PBPs) are a family of water-soluble, highly fluorescent bioactive
molecules composed of apoproteins and phycobilin open-chain tetrapyrrole chromophores covalently bound via cysteine amino acid. There are three PBP classes in
cyanobacteria and red algae: phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), containing phycocyanobilin (PCB) and phycoerythrobilin (PEB) as
pigments that differ in their spectral properties. Given its increasing application in
the various fields, the aim of this chapter is to review and discuss methodology and
protocols in PBPs research focusing on recent and the most relevant literature data.
8.2 PBPs Isolation and Purification
PBPs are produced by the photoautotrophic, mixotrophic, or heterotrophic cultivation
of cyanobacteria and red algae.
Isolation of PBPs in high yield requires en efficient extraction process. There are
several effective approaches used for their extraction: freezing and thawing, sonication, microwave assisted extraction, pulsed electric field extraction, high-pressure
homogenization, osmotic shock, acid treatment, enzymatic treatment, organic solvent
extraction, etc. (reviewed in Bleakley and Hayes 2017). Extraction of algal proteins
could be very challenging due to the presence of mechanically robust, multilayered cell wall, and application of different polysaccharides-hydrolyzing enzymes
(xylanase, cellulase, etc.) significantly increases PBPs extraction (Dumay et al.
2013).
Procedures for PBPs purification use single or combination of several chromatographic steps (ion-exchange chromatography, hydrophobic chromatography,
gel filtration, hydroxyapatite chromatography, and expanded bed adsorption chromatography), as well as preparative electrophoresis or two-phase aqueous extraction
(Sonani et al. 2016). One of the main challenges during the purification of PBPs is the
separation of individual PBP from PBPs mixture because of their similar properties
(pI, molecular mass, chromophore spectral properties). Recently, ultrasound assisted
three-phase partitioning was employed for efficient extraction and purification of PC
from Arthrospira platensis (Zhang et al. 2017b).
PBPs purity is evaluated using the ratio between absorbance in the visible region
(652 nm, 620 nm, 565 nm, or 540 nm for APC, PC, R-PE, and B-PE, respectively)
and the absorbance at 280 nm. PC preparations with A 620 /A 280 > 0.7 are considered as food grade, while those with A 620 /A 280 > 4.0 have an analytical grade of
purity (Vernès et al. 2015). Absorbance ratio A 652 /A 280 > 4.0 means analytical purity
M. R. Nikolic et al.
Keywords Phycobiliproteins · Phycocyanin · Allophycocyanin · Phycoerythrin ·
Phycocyanobilin · Chromopeptides
8.1 Introduction
Phycobiliproteins (PBPs) are a family of water-soluble, highly fluorescent bioactive
molecules composed of apoproteins and phycobilin open-chain tetrapyrrole chromophores covalently bound via cysteine amino acid. There are three PBP classes in
cyanobacteria and red algae: phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), containing phycocyanobilin (PCB) and phycoerythrobilin (PEB) as
pigments that differ in their spectral properties. Given its increasing application in
the various fields, the aim of this chapter is to review and discuss methodology and
protocols in PBPs research focusing on recent and the most relevant literature data.
8.2 PBPs Isolation and Purification
PBPs are produced by the photoautotrophic, mixotrophic, or heterotrophic cultivation
of cyanobacteria and red algae.
Isolation of PBPs in high yield requires en efficient extraction process. There are
several effective approaches used for their extraction: freezing and thawing, sonication, microwave assisted extraction, pulsed electric field extraction, high-pressure
homogenization, osmotic shock, acid treatment, enzymatic treatment, organic solvent
extraction, etc. (reviewed in Bleakley and Hayes 2017). Extraction of algal proteins
could be very challenging due to the presence of mechanically robust, multilayered cell wall, and application of different polysaccharides-hydrolyzing enzymes
(xylanase, cellulase, etc.) significantly increases PBPs extraction (Dumay et al.
2013).
Procedures for PBPs purification use single or combination of several chromatographic steps (ion-exchange chromatography, hydrophobic chromatography,
gel filtration, hydroxyapatite chromatography, and expanded bed adsorption chromatography), as well as preparative electrophoresis or two-phase aqueous extraction
(Sonani et al. 2016). One of the main challenges during the purification of PBPs is the
separation of individual PBP from PBPs mixture because of their similar properties
(pI, molecular mass, chromophore spectral properties). Recently, ultrasound assisted
three-phase partitioning was employed for efficient extraction and purification of PC
from Arthrospira platensis (Zhang et al. 2017b).
PBPs purity is evaluated using the ratio between absorbance in the visible region
(652 nm, 620 nm, 565 nm, or 540 nm for APC, PC, R-PE, and B-PE, respectively)
and the absorbance at 280 nm. PC preparations with A 620 /A 280 > 0.7 are considered as food grade, while those with A 620 /A 280 > 4.0 have an analytical grade of
purity (Vernès et al. 2015). Absorbance ratio A 652 /A 280 > 4.0 means analytical purity