Extraction and purification of C-phycocyanin and genome analysis of an indigenous hypersaline cyanobacterium

Abstract

Cyanobacteria are photosynthetic microorganisms that inhabit diverse ecological habitats and are capable of producing wide range of natural products and bioactive metabolities including peptides, vitamins, enzymes and pigments such as phycobiliproteins. Amongst the group of phycobiliproteins, C-Phycocyanin (C-PC) is a light-harvesting accessory pigment known to possess excellent biotechnological applications due to their intense colour, fluorescent properties and health benefits. This study has focused on the characterisation and full genome analysis of a unique indigenous halophilic cyanobacterium capable of overproducing the pigment phycocyanin (C-PC). Further, development of a cost-effective extraction method for high purity C-PC and characterisation of the purified C-PC was accomplished. The strain was isolated from a hypersaline environment in KwaZulu-Natal, South Africa and was found to possess several unique traits such as its ability to accumulate high amount of phycocyanin, tolerance to high salinity (up to 180 g/L), ability to grow under varying growth conditions and high growth rate. The taxonomic identity of the isolate was revealed using a polyphasic approach including cell morphology, growth conditions, pigment composition, 16S rRNA analysis. The cells were oval to rod-shaped, 14-18 μm in size, and contained majority of C-PC, as well as some allophycocyanin and chlorophyll. The strain was moderately thermotolerant (35°C), alkalitolerant (pH 8.5) and was halophilic with an optimum NaCl of 120 g/L. Based on the 16S rRNA gene sequence phylogeny, the strain was found to be related to members of the ‘Euhalothece’ subcluster (99%). Further, the whole genome sequence was also determined, and the annotated genes have shown sequence similarity (90%) to the gas- vacuolate, spindle-shaped Dactylococcopsis salina PCC 8305. Based on the above results, the strain is considered to represent a novel species of Euhalothece. The size of the genome was determined to be 5,113,178 bp and contained 4332 protein-coding genes and 69 RNA genes with a GC content of 46.7%. The full genome sequence analysis also provided important information about the strain which facilitated the identification of key genes and proteins necessary for C-PC synthesis and salt acclimation. Genes encoding osmoregulation, oxidative stress, heat shock, persister cells, and UV-absorbing secondary metabolites, among others, were also identified. Further, single factor experiments were performed to optimise the factors (extraction buffers, freezing time, biomass:buffer ratio and lysozyme concentration) essential for C-PC extraction from cyanobacteria. A range of buffers viz., acetate, potassium phosphate (PPB), sodium phosphate (SPB), phosphate buffered saline (PPBS), Tris-chloride and double distilled water (control) with different pH and concentrations were investigated. Cell lysis was carried out by freezing the cells at different temperatures viz., at -196, and -80, and -20°C, and by thawing at 4 and 25°C. The freezing and thawing time varied from 0.5-24 h. Based on the results obtained, thawing temperature, enzyme concentration and biomass-buffer ratio were further selected for optimisation for maximum C-PC yield and purity using response surface methodology (RSM). Under optimised conditions, the yield of crude C-PC was increased to 78 mg/g (>90% percentage increase) with a purity index of 2.5 compared to extraction prior to optimisation. The crude C-PC was further purified using 6% w/v of activated charcoal combined with a two-step ammonium sulphate (NH4SO4) precipitation and ultrafiltration which resulted in high yield analytical grade C-PC with a purity index of 5. The purified C-PC showed a single absorption peak at 620 nm and emission at 640 nm. Based on the amino acid analysis the calculated molecular weight of α- and β-subunits were found to be 17.7 and 18.4 kDa respectively, which corresponded to the two bands seen on the SDS- PAGE. Additionally, the primary, secondary and tertiary structures of the C-PC was also evaluated based on the amino acid sequence obtained from the genome sequence. The C-PC physiochemical properties such as the molecular weight, isoelectric point, extinction coefficient, half-life, aliphatic index, amino acid property, instability index and Grand Average of Hydropathicity was predicted based on the in-silico analysis of the amino acid sequences. The physicochemical properties revealed that these proteins are non-polar and stable. Multiple sequence alignment analyses of the α- and β-subunits displayed significant differences amongst the amino acid residues of hypersaline/marine and freshwater cyanobacteria. These amino acids play a vital role in the stability of the C-PC. The secondary structure prediction of the α- and β -subunits consisted of > 50% of amino acid residues in α-helices, with 9-13% of amino acid residues in the extended strand. The stability of the purified C-PC under different conditions were investigated. The optimum pH range for purified C-PC was found to be 5.0–7.0 and was found to be stable up to 45oC. However, the relative concentration C-PC (CR%) and thermostability of the purified C-PC was observed to be pH dependent, a lower pH improved the stability at higher temperatures and vice-versa. An IC50 value of 0.540 ± 0.02 mg/mL was also observed using the DPPH assay indicating a higher antioxidant potential of the C-PC. C-phycocyanin exhibited a maximum absorbance of 1.37 ± 0.05 by ferric ion reducing assay. The presence of a high level of non-polar and aromatic residues such as Ala, Gly Glu, Leu, Arg, Ser, and Val could be regarded as an indication of higher antioxidant activity levels of the C-PC. Addition of preservatives sodium azide and sodium citrate (at 4°C) proved to be suitable for preservation of C-PC for up to 42 weeks. This research contributed to our understanding of molecular, cellular and biochemical mechanisms of the C-PC biosynthesis as well as newly identified metabolites in cyanobacteria. The study has also demonstrated an efficient extraction method for analytical grade C-PC from cyanobacterial strains for potential applications in biotechnological biomedical industries.