Antifreeze proteins as non-toxic cryoprotective agents have extensive prospect in fermentation strain preservation.To test the cryoprotective effect of antifreeze protein MpAFP698 from the desert beetle Microdera punctipennis on S.cerevisiae,antifreeze protein MpAFP698 was prokaryotic expressed and purified.Fusion protein MBPMpAFP698 with high activity was obtained.1,10,30 μg/mL of MBP-MpAFP698 were added to S.cerevisiae and then treated at-7 and-20 ℃ for different time lengths;in other experiments yeast cells were treated at 4,0,-50 ℃ for different time lengths with 10 μg/mL MpAFP698 as well as repeated freeze-thaw at-20 ℃.The morphology and survival rate of the yeast cells after these treatments were observed or calculated.The results showed that the survival rate of S.cerevisiae with the protection of MBP-MpAFP698 were significantly better than the control group without MBP-MpAFP698.Even the concentration of 1 μg/mL showed protective effect,and 10 μg/mL showed the best protective effect,indicating that insect antifreeze protein can function as cyroprotective agent at very low concentrations.This result lay a foundation for the development of efficient protective agent at low temperatures.%昆虫抗冻蛋白作为天然无毒副作用的低温和冷冻保护剂,在发酵菌种的保藏方面具有很好应用前景.为了验证荒漠昆虫小胸鳖甲(Microdera punctipennis)抗冻蛋白MpAFP698对酵母菌的抗冻和抗低温保护效果,对MpAFP698抗冻蛋白进行原核表达和纯化,获得了具有活性的融合抗冻蛋白MBP-MpAFP698.将质量浓度为1、10、30 μg/mL的MBP-MpAFP698添加到酿酒酵母(Saccharomyces cerevisiae)中,在-7和-20℃冷冻处理酵母菌液不同时间;以10 μg/mL的抗冻蛋白在4、0、-50℃处理酵母菌液,在-20℃反复冻融处理,观察菌落形态并统计菌种存活率.结果表明,添加抗冻蛋白组的酵母存活率显著优于未加抗冻蛋白的对照组,1 μg/mL就有保护效果,10 μg/mL保护效果最好,说明昆虫抗冻蛋白能以极低的浓度发挥低温保护作用.
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