【作者】 王静；

【导师】 辛志宏；

【作者基本信息】 南京农业大学 ， 食品科学， 2011， 硕士

【摘要】 大蒜中蒜氨酸酶(alliinase)是生成大蒜功能成分的关键酶,可催化S-烯丙基-L-半胱氨酸亚砜,即蒜氨酸(alliin)反应生成大蒜功能性物质。目前,有关温度、pH、E/S对蒜氨酸酶活性影响的研究较多,但有关金属离子对蒜氨酸酶活性作用机理的研究罕见报道。本研究以新鲜大蒜为原料,采用硫酸铵分级沉淀法、PEG6000沉淀法、葡聚糖G-200柱层析法提取分离蒜氨酸酶,用SDS-PAGE电泳鉴定其纯度,获得蒜氨酸酶提取分离纯化的适宜条件；利用正交试验、Box-behnken试验优化了金属离子对蒜氨酸酶活性影响的工艺参数；研究了金属离子对蒜氨酸酶反应动力学及紫外可见光谱的影响,初步探索了金属离子对蒜氨酸酶的激活与抑制机制。由于国内大部分热风干制蒜片产品品质较差,而高品质蒜片产品如冷冻干燥生产成本较高,本研究在单一干燥方式的基础上,采用真空微波-热风组合干燥技术研究开发高品质脱水蒜片。具体研究结果如下：1.蒜氨酸酶的提取分离对比实验表明,PEG6000沉淀法比硫酸铵沉淀法有更高的提取率,采用20%PEG6000纯化粗酶液,纯度是粗酶液的2.96倍,回收率达到72.75%；应用葡聚糖G-200柱层析纯化粗酶液,纯化后的酶液经SDS-PAGE电泳鉴定显示单一条带,凝胶成像系统分析其分子量约为54.7KD,这与文献报道值基本一致,说明本研究方法可有效纯化蒜氨酸酶。2.蒜氨酸酶性质研究在单因素试验的基础上进行正交及Box-behnken实验设计,优化金属离子激活蒜氨酸酶的参数,结果显示,E/S=0.20,反应温度36℃,pH为6.4,Mn2+浓度8.15mM时,蒜氨酸酶活性达到最大值,为0.156±0.0053umol/min。酶动力学试验结果显示,激活剂Mn2+使蒜氨酸酶的Km值、Vmax值变大,反应速度提高,这可能是Mn2+提供了适合蒜氨酸酶与底物反应的适宜环境,从而提高了反应速度；而抑制剂半胱氨酸使蒜氨酸酶Km、Vmax值减小,反应速度降低,这可能是因为半胱氨酸在这个反应体系中是一种反竞争性抑制剂,影响了酶与底物形成复合物向产物的转化,即反应过程中酶与底物结合形成中间复合物,复合物与反竞争性抑制剂半胱氨酸结合形成酶-底物-半胱氨酸复合物,而不能转化为产物,从而降低反应速度。紫外可见光谱试验结果显示,激活剂和抑制剂对特征吸收波长无影响,仅对吸收峰的大小有影响。添加激活剂Mn2+后,蒜氨酸酶在280nm、325nm以及430nm附近的吸收峰均变大；添加抑制剂半胱氨酸后,蒜氨酸酶在280nm、325nm附近吸收峰变大,在430nm处的吸收峰变小,这可能是因为激活剂和抑制剂影响了蒜氨酸酶辅助因子5’-磷酸吡哆醛的结构以及其与蒜氨酸酶主链的结合,从而影响了蒜氨酸酶的活性。初步探索了金属离子提高蒜氨酸酶活性的作用机制,探讨了蒜氨酸酶活性与大蒜素生成量的关系,结果表明蒜氨酸酶活性与大蒜素生成量呈正相关。3.无臭脱水蒜片的开发在护色与脱臭的基础上,采用真空微波-热风组合技术开发无臭脱水蒜片,以大蒜素含量、色值、复水率为指标,优化了组合干燥生产蒜片的最佳工艺参数：前期真空微波干燥20min(375w,真空度-90kpa),后期热风干燥60min(60℃),所得产品品质明显高于单一干燥方式的产品品质。更多还原

【Abstract】 Alliinase (alliin lyase, EC 4.4.1.4) form garlic is the key enzyme, which catalyzes the conversion of alliin to allicin, the main biologically active component in garlic. Currently, there were many studies on factors of the alliinase activity, such as temperature, pH, E/S, however, for metal ions on the mechanism of alliinase activity rare reports. In this study, alliinase was purified from fresh bulbs using various steps, including Ammonium sulfate method, PEG6000 method, Sephadex G-200 column chromatography, the purity of alliinase was identified by SDS-PAGE, then got the optimal method of the alliinase extraction and purification from fresh garlic.We optimized the parameters of metal ions on the alliinase activity by orthogonal test and box-behnken test, studied the metal ions on kinetics and UV-Visible spectrum of alliinase, preliminary explored the activation and inhibition mechanism of the metal ions on alliinase. The quality of hot-air drying garlic slices is poor, while high-quality products such as freeze-drying garlic slices are costly in the domestic. Based on the method of single drying, develop high-quality dehydrated garlic slices by the technology of combined microwave-vacuum drying (MVD) with air-drying. The main conclusions stated as follows:1. Extraction of alliinaseContrast tests showed that, PEG6000 method has a higher extraction rate and alliinase activity recovery, using 20% PEG6000 to purify the crude enzyme, the purity was 2.96 times as much as crude enzyme and the recovery can reach to 72.75%. Then the crude enzyme was passed the Sepadex G-200 column. SDS-PAGE showed that no unpurposed protein was found, and one most intensive band at Mr=54.7 was detected, which is consistent with literature reports.2. Properties of alliinaseBased on the single-factor test, Orthogonal Test and Box-behnken test were used to optimize the parameters that activation effect of metal ions on allinase. The results showed that the optimal condition was:E/S value of 0.20, enzyme reaction temperature of 36℃, pH value of 6.4, Mn2+concentration of 8.15 mM. The allinase activity under this condition was 0.156±0.0053μmol/min.The kinetics of enzymatic activity results showed that, Mn2+as an activator increase the Km, Vmax and reaction speed, it is possible that Mn2+provides a suitable environment for the reaction alliinase with substrate, then improve the reaction speed. The role of L-cys as an inhibitor was opposite, it is possible that L-cys is an anti-competitive inhibitor in the reaction system, which affected the transformation ES complex to product. During the reaction, enzyme and substrate combined to form intermediate compound, which combined L-cys to form E-S-L-cys complex, not to transform product, then decrease the reaction speed.UV-Vis spectroscopy of alliinase showed that addition of activator did not move the absorption peak, just affected the strength of absorption peak. Mn2+as an activator increased the value of the absorption at 280nm,325nm and 430nm. L-cysteine as an inhibitor had the same effect at 280nm,325nm, however, decreased the value of the absorption at 430nm. It is possible that the activator and inhibitor affectd the structure of PLP and the combination of PLP as the cofactor to the main chain of alliinase, which affects the activity of alliinase.Preliminary explored of the activation and inhibition mechanism of the metal ions on alliinase, discussed the relationship between the alliinase activity and allicin formation. We found a significant positive correlation between the alliinase activity and allicin formation.3. Develop of odorless dehydrated garlic slicesBased on the color protection and deodorization, the technology of combined microwave-vacuum drying (MVD) with air-drying (AD) were optimized by orthogonal experiment. The results showed that the best processing was using microwave-vacuum drying for 20min (vacuum-90kpa, microwave power 375w), then air-drying 60min (60℃), the process obviously got higher quality garlic products than single method.更多还原