| Antifreeze proteins (AFPs) are macromolecular antifreezers first isolated from blood serum of Antarctic Cod.Various AFPs have been found from insects, plants, bacteria and fungi et cetera.Some of their gene sequences and several crystal structures have been determined.Recent studies are focused on their antifreeze mechanisms in attempting to better understand how exactly AFPs work. Antifreeze proteins inhibit the growth of ice crystals and decrease the freezing point of water by a non-colligative mechanism without depressing the melting point, therefore producing a difference between the freezing and melting points, which is termed as thermal hysteresis. The thermal hysteresis is one of the important characteristics of AFP. AFPs have three main founctional characteristics in that①having thermal hysteresis activity;②changing the shape of the ice crystal;③suppressing the recrystallization of ice crystals. AFPs have widely potential applications.They can aid not only in the food storage,but also in the preservation of cells, tissues and organs at low temperatures in improving their antifreeze ability. The thermal hysteresis of insect AFPs is 10～100 times higher than that of fish AFPs and much higher than that of plant AFPs.The research is focused on determining whether AFPs from desert insect could be used as cryoprotectant in the protection of Saccharomyces cerevisiae at temperatures achieved in common refrigerator, so that to make the protection of food yeast more easy and less cost..First, the MpAFP698 gene from desert insect Microdera punctipennis dzhungarica was expressed in prokaryotic system with pMAL-p2X prokaryotic expression vector. The expressed fusion protein MBP-MpAFP698 was purified by Amylose Resin. The quantity of MBP-MpAFP698 was measured by Bradford method and the concentration was700μg/mL～3000μg/mL. To check the activity of the expressed AFP, the shape of the ice crystals in MBP-MpAFP698 solution was observed with Nanolitre Osmometer. It looked as needle- shape, showing the high activity of MBP-MpAFP698. When MBP-MpAFP698 was incubated in boiling water 10 min, it showed high heat-stability. The successful expression and purification of MBP-MpAFP698 with high activity provided the raw materials for the next experiment procedure on the preservation of Saccharomyces cerevisiae.Next, the purified MBP-MpAFP698 protein of 50μg/mL was added to edible strain Saccharomyces cerevisiae and then placed at freezing temperature of -20℃for a period of times. The result showed that the MBP-MpAFP698 had significant freeze-resistance effect compared to the control. The preliminary results demonstrated that the MBP-MpAFP698 was activated, and could be used to protect yeast at freezing temperatures.Different concentrations of MBP-MpAFP698 (1μg/mL,10μg/mL,30μg/mL,50μg/mL,70μg/mL)all showed protective effect. The minimum effective concentration was as low as 1μg/mL, showing the high activity of MBP-MpAFP698. The most effective concentration was 10μg/mL. To determine whether this concentration works at different temperatures,,10μg/mL MBP-MpAFP698 was add to yeast cultures and then placed at -50℃, 4℃and 0℃. The results showed that the protective effect of the AFP group was apparent in comparing with the control group at -50℃, and there are not any significant differences between the two group at 4℃and 0℃. Furtherly, the maximum duration of the protection was investigated. The result showed that the effect of AFPs was lasted more than 50 days. These results laid the foundation for the application of insect AFP in preserving yeast at low temperatures.The influence of MpAFP698 on the growth of yeast and mouse were investigated. The results showed that the MBP-MpAFP698 had no obvious influence on the growth of strains and mice, which indicated the possibility of desert insect AFPs in the application of edible yeast strains cryoprotection. These finding provided the foundation of safety assessment of desert insect AFPs.
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