【作者】 张超；

【导师】 张晖； 陈正行；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2008， 博士

【摘要】 抗冻蛋白(AFP,antifreeze protein)是一种可以非依数性降低冰点、抑制体系发生重结晶特性的蛋白质,广泛地分布在鱼类、动物、昆虫、植物以及微生物中,是生命体抵御外界寒冷环境应激性反应所产生的一种蛋白。据报道,AFP具有提高产品质量,提高农作物抗寒能力,提高冷冻体细胞存活率等作用,广泛地应用于食品、生物、临床医学和化工等领域。本文以谷物原料进行筛选,发现冬小麦麸皮中存在AFP,并进一步对冬小麦麸皮抗冻蛋白(TaAFP)进行分离纯化,研究其理化特性、一级结构和二级结构,并以一级结构为基础,探讨TaAFP与冰晶的结合方式,揭示其抗冻机理。论文首先研究了AFP的检测方法。文中比较了目前常用AFP的检测方法,发现差热分析仪法(DSC)具有样品用量小、可以精确控制温度、可以获得准确结果等优点,因此确定DSC法为本文AFP抗冻活性(THA)的检测方法。还进一步研究了影响测定结果的各个因素,考察使用DSC法测定THA的稳定性、重复性和精密度,结果显示DSC法测定样品THA具有较高的稳定性、重复性和精密度,其RSD在0.4 %以下,相对标准偏差为3.84 %。具体的测定程序如下:样品溶解于10 mmol/L磷酸盐缓冲液(pH 8.0)达到最终蛋白浓度1.0 mg/mL,将10μL样品在坩埚中平衡15min后,按照1.0oC/min的速度进行升降温,在保留温度Th时,保持体系的冰晶质量含量占体系的10 % - 90 %(质量分数)。从多种谷物中筛选AFP,并对其进行分离纯化。筛选结果发现在冬小麦麸皮中存在AFP,进一步分别采用传统的分离法、特异性亲和法和条带切割法对冬小麦麸皮抗冻蛋白(TaAFP)纯化,均获得电泳纯的样品。比较三种纯化方法的优缺点:传统的分离纯化方法在AFP或其它活性物质的筛选中具有普遍适用性;特异性亲和分离纯化法将来可能会适用于AFP大规模的粗分离;电泳条带切割法适用于高精度和高纯度样品的纯化和分析。研究了TaAFP的物理与化学性质,结果发现TaAFP中无糖基。氨基酸分析结果推测TaAFP由155个氨基酸残基组成,相对分子质量13085.2。其中,Glycine残基占52.26%(摩尔百分含量),属于富甘氨酸蛋白(GRP)。使用ExPASy数据库检索与TaAFP相关的GRP发现,TaAFP是一种与冷诱导或其它诱导相关的蛋白。TaAFP的变性温度为61.47oC,变性后,THA丧失,属于热不稳定型AFP。TaAFP在微碱性的环境中(pH 7.0-9.0)显示较高的THA,该环境恰好与植物的生理状态环境一致。TaAFP属于Ca2+-dependend AFP,在Ca2+存在的情况下,THA得到增强。TaAFP还具有AFP所应有的亲水性和较高的分配系数(质量分数大于90%)。MALDI-TOF-MS分析结果显示TaAFP的相对分子质量为13637.711,与上述SDS-PAGE和氨基酸分析推测获得的结果基本吻合。进一步采用了结合N-末端测定和肽指纹图谱的分析方法测定TaAFP的一级结构,结果表明其一级结构为:MARKVIALAFLLLLTISLSKSNAAR VKYNGGESGGGGGGGGGGGGGGNGSGSGSGYGYNYGKGGGQSGGGQGGGGGGGGGGGSNGSGSGSGYGYGYGQGNGGAQGQGSGGGGGGGGGGGGGGSGQGSGSGYGYGYGKGGGGGGGGGGDGGGGGGGGSAYVGRHE,测定覆盖度达到100 %。TaAFP的二级结构分别采用圆二色性光谱、拉曼光谱、红外光谱以及生物信息学的预测方法测定。TaAFP中α-helix的含量为10%-15%;β-sheet的含量为10%-20%,random coil的含量为40%-60%。采用分子力学和量子力学的方法研究了TaAFP和(1121)冰晶面的结合情况。计算结果显示蛋白面M1(Met1、Lys4、Gly47、Asn48、Ser50、Gly51、Gly138、Gly139、Gly141、Gly142、Gly143、Gly161、Arg162、His163和Glu164)与冰晶面具有最强的结合能力。其中,静电作用和范德华作用大大超过了氢键的作用。另一方面,运用半经验量子力学的方法AM1和PM3进一步获得了TaAFP与冰晶面的微观相互作用。首先,当TaAFP与冰晶面作用时,蛋白面M1和冰晶之间发生弱轨道相互作用,AM1和PM3的计算结果均显示面M1与冰晶面之间的弱轨道相互作用是最强的。第二,半经验量子力学计算证实TaAFP与冰晶面之间存在电荷迁移,对整个体系而言,电子是从TaAFP迁移到冰晶上。因此,TaAFP与冰晶相互作用增强。第三,键级能的计算结果显示各个蛋白面与冰晶面的键级能基本上相当,其中,M1属于相互作用较强的面。因此,确定蛋白面M1是TaAFP与冰晶的最佳结合面。TaAFP的抗冻机理就在于M1通过库仑作用、范德华作用、轨道重合和电荷迁移等相互作用与冰晶面紧密的结合。当M1与冰晶面结合后TaAFP锚定于冰晶表面,冰晶如果继续生长,则冰晶的表面积需要增加,而冰晶表面积的增加则需要外界进一步提供能量。提供能量后,系统的熵降低,体系温度下降。在宏观上则表现为冰晶需要更低的温度才可以进一步生长,体系的冰点被降低,即产生热滞现象。更多还原

【Abstract】 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.更多还原