Abstract:

In this paper, stepwise salting-out combined with aqueous two-phase extraction was used to purify phycocyanin
from Nostoc spharoids Kützing. The extraction process was optimized by using a three-factor quadratic regression
orthogonal rotation combination design. Using the Design-Expert 7.0 software, a mathematical regression model was
established indicating the effect of various extraction conditions on phycocyanin purity. The optimal extraction process was
determined as follows: after discarding of the precipitate salted out by 20% (NH4)2SO4, phycobiliproteins were salted out
from the supernatant by 40% and 50% (NH4)2SO4 before being subjected to aqueous two-phase extraction with 10% PEG
6 000 and 18% (NH4)2SO4. The purity (the absorbance ratio at 615 nm and 280 nm) and recovery of the purified
product under the optimized conditions were 8.6 and 71.40%, respectively. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis of the product revealed two clear bands having molecular weights of about 35 and
15 ku, respectively. Mass spectrometry (MS) analysis confirmed that the molecular weight of band 2 was about 15 ku . Its
isoelectric point was 5.35 and its partial amino acid sequences were TPLTEAVAAADSQGR and DIGYYLR, respectively.
Through simulation of protein structure according to the SWISS-MODEL website, we speculated that band 2 might be the
α-subunit of phycocyanin from Nostoc spharoids Kützing.

Key words: Nostoc spharoids Kützing, phycocyanin, stepwise salting-out, aqueous two-phase extraction, SDS-PAGE electrophoresis, mass spectrometry