重组别藻蓝蛋白(holo-apcB)的抗氧化活性研究

Antioxidant Activity of Recombinant Allophycocyanin β-Subunit(Holo-apcB)

本研究利用代谢工程技术原理在重组大肠杆菌表达合成了嗜热聚球藻(Thermosynechococcus elongatus BP-1)别藻蓝蛋白β亚基(holo-apcB),并利用亲和层析的方法对holo-apcB进行了分离纯化。SDS-PAGE电泳胶图显示,holo-apcB分子量与18.4 ku的蛋白条带相近,与理论分子量吻合。光谱学分析结果显示,其最大吸收峰为615 nm,最大荧光发射峰为640 nm,与天然别藻蓝蛋白β亚基具有相同的分子量和光谱学性质。在55℃条件下,holo-apcB的半衰期高达31.9 h。以嗜热聚球藻天然别藻蓝蛋白(APC)及脱辅基别藻蓝蛋白β亚基(apo-apcB)为参照,分析了holo-apcB的抗氧化活性。结果显示,holo-apcB具有最强的抗氧化能力,其清除羟自由基和氢过氧自由基半数抑制浓度(IC50)分别为224.9μg/mL和20.6μg/mL,显著低于apo-apcB和天然APC的IC50,这表明holo-apcB中的藻蓝胆素也参与了抗氧化过程。

In this study, a recombinant allophycocyanin β-subunit （holo-apcB） [Thermosynechococcus elongatus BP-1] was biosynthesized in recombinant Escherichia coli by using metabolic engineering technology. Holo-apcB was then isolated and purified using affinity chromatography. SDS-PAGE analysis showed that there was a band with a molecular weight close to 18.4 ku, which matched the theoretical molecular weight of holo-apcB. Spectrum analysis showed the maximum absorbance and maximum fluorescence emission of holo-apcB were at 615 nm and 640 urn, respectively. Thus, this subunit had the same molecular weight and spectroscopic properties as those of holo-apcB. At 55 ℃, the half-life of holo-apcB reached up to 31.9 h. Comparison of the antioxidant activity of holo-apcB with native allophycocyanin （APC） and apo-apcB showed that holo-apcB had the highest antioxidant activity. Moreover, the holo-apcB IC50 values for hydroxyl and peroxyl radical-scavenging activities were 224.9μg/mL and 20.6 μg/mL, respectively, which were significantly lower than those for apo-apcB and native APC. Therefore, it was suggested that phycocyanobilin also has antioxidant functions.