| Antifreeze protein (AFP) can decrease the freezing point nonequilibriumly, referred to as thermal-hysteresis activity (THA), and retard recrystallization strongly. Even in frozen condition, AFPs inhibit the Ostwald ripening, particularly when ice approaches the melting point. Up to now, AFPs have been applied to food processing, cryopreservation of organs or cells, cryosurgery and aquaculture. In food processing, AFPs have been used to enhance the quality of the ice cream, frozen dough or meat, proving that the application of AFPs in food processing is feasible in the future. AFPs are widely distributed among organisms including prokaryotes, fungi, insects, plants, and fish. Therefore, it is an important subject for the food engineer to study. Winter-wheat (Triticum aestivum L.) bran antifreeze protein (TaAFP) was screened out and purified in this paper. The physicochemical and structure of TaAFP were also studied. Moreover, the ice-binding domain and the antifreeze mechanism of TaAFP were discussed.The measurement of THA was established at first. The differential scanning calorimetry (DSC) method was chosen for its advantages of the microsample, accurately controlling temperature, and precisely detecting ice crystal content. The effect of the temperature raising speed, ice crystal content, and protein content of the sample on the THA was studied. The stability, repetition, and accuracy of the DSC measurement were also evaluated. The results showed the stability, repetition, and accuracy of the DSC measurement were high enough for the following studies, with the RSD lower than 0.4%, relatively standard deviation of 3.84%. The process of the DSC measurement was also fixed: the sample was dissolved in 10 mmol/L PBS (pH 8.0), with the final protein content of 1.0 mg/mL. An aliquot of 10μL of the sample was sealed in the aluminum pan followed by the balancing on the control desk for 15 min. The temperature of the control desk was raised or decreased to Th at speed of 1.0 oC/min, with ice crystal content ranged from 10 % to 90 % (w/w).TaAFP was screened out from kinds of the corns, such as: spring-wheat bran, winter-wheat bran, rice, rice germ, barely, buckwheat, oat, etc.. TaAFP was purified about 300-fold to electrophoretic homogeneity with an overall yield at about 1.50 % from winter-wheat-bran protein, by the traditional purification process, specific binding purification process, and gel-sliced purification process, respectively. The traditional purification process can be used for kinds of samples, but needs long time and boring steps. The specific binding purification process can be used for the enlarged batch purification of TaAFP or other kinds of AFPs. The gel-sliced purification process can be used for structure determination, but the yield of the process is tiny.The physicochemical properties of TaAFP were also investigated. The molecular mass of TaAFP was 13860 Da by SDS-PAGE analysis. The Schiff-reagent dye showed TaAFP was not an antifreeze glycoprotein (AFGP). Amino acid analysis showed TaAFP consisted of 155 amino acid residues with molecular mass of 13085.2 Da, similar to the results from SDS-PAGE analysis. TaAFP was a glycine-rich protein (GRP) with glycine residues of 52.26 mol %, related to the cold-stressed protein. DSC analysis showed the denature temperature of TaAFP was 61.47oC, losing its THA after denature. The effect of pH and cations on THA of TaAFP was also investigated. TaAFP showed the stronger THA in pH7.0-9.0 than in other condition, consistent with the physiology condition of the plants. The THA of TaAFP was improved at the presence of Ca2+ instead of other cations. TaAFP was a Ca2+-dependend AFP, and its THA was improved at the presence of Ca2+. The hypothetical binding model of calcium to AFP was also discussed. The typical hydrophilicity and ice-binding capacity of AFP were also found in TaAFP by the TGA and specific binding analysis.The molecular weight of TaAFP was 13637.711 Da determined by MALDI-TOF-MS analysis. The primary structure and second structure of TaAFP was determined. The primary structure of TaAFP was determined by the combination of N-terminal sequencing and mass fingerprint overlapping analysis. The primary structure of TaAFP was MARKVIALAFLLLLTISLSKSNAARV KYNGGESGGGGGGGGGGGGGGNGSGSGSGYGYNYGKGGGQSGGGQGGGGGGGGGGGSNGSGSGSGYGYGYGQGNGGAQGQGSGGGGGGGGGGGGGGSGQGSGSGYGYGYGKGGGGGGGGGGDGGGGGGGGSAYVGRHE, with overlap rate of 100 %. The secondary structure of TaAFP was studied by the circular dichroistic spectra, Raman spectra, FI-IR spectra, and bioinformatics prediction. The results were summarized asα-helix of 10%-15%,β-sheet of 10%-20%, and random coil of 40%-60%.Finally, the antifreeze mechanism of TaAFP studied by the theory of the molecular mechanics and the quantum mechanics. The surface of TaAFP was divided into eight parts, denoted as surface 1 to surface 8, respectively. From the calculation, surface 1 (Met1, Lys4, Gly47, Asn48, Ser50, Gly51, Gly138, Gly139, Gly141, Gly142, Gly143, Gly161, Arg162, His163, Glu164) had the strongest binding capacity with ice surface (112 1). The Coulomb force and van de Wall force were the important factor between the TaAFP-ice binding domain instead of the hydrogen bond, consistent with the reported results. Meanwhile, surface 1 had 15 amino acid residues and the middle surface area among the tested surfaces, proving that the surface with the biggest area was usually not the best binding surface. The binding surface was usually flat and was suited for the ice crystal surface, just like surface 1. Furthermore, the semi-experiential mechanics including AM1 & PM3 was used to give a deeper insight of the interaction of TaAFP-ice, which could not be obtained by the molecular mechanics. The weak orbit interaction of surface 1 to ice was the strongest among the tested. Thus surface 1 had the more overlapped orbits to the ice than that of the other surfaces. Moreover, the results of the charge transfer also proved that surface 1 was the best surface binding to ice. The calculation of the charge transfer showed the charge of TaAFP moved to the ice and the ice-binding domain was improved. The bond level of surface 1 to ice was also the strongest in the surfaces. From the mentioned evidences, surface 1 was the optimal ice-binding domain. |
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