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1、张露娟：鸭肉磷脂水解酶的分离纯化及其酶学性质研究三摘要f螋必荆南京板鸭是中国老式的腌腊肉制品之一，具有悠久的历史和独特的风味。众多学者的研究都证明腌腊肉制品特有的风味与脂类物质特别是磷脂的降解有关，而磷脂的变化与磷 脂水解酶系的作用密不可分。曾有学者从宏观角度在腌腊肉制品加工过程中跟踪过磷脂水 解酶系的活力变化，这就更进一步肯定了磷脂水解酶系在腌腊肉制品风味形成中的地位， 因此研究磷脂水解酶系在肉制品加工过程中的活性变化与作用机制，对于进一步理解腌腊肉 制品生产过程中脂类物质的变化机理，揭示脂类物质变化与风味形成之间的关系具有重要 的意义。目前只有少部分研究者检测过南京板鸭生产过程中总磷脂酶的
2、活力，但从原材料 中将磷脂水解酶系提取出来并进一步到分子水平去研究腌腊肉制品中磷脂水解酶系的作用 机制的有关资料却很少。本课题初次通过结合Sourcel 5Q离子互换层析法和Superdex200凝胶层析法从新鲜鸭 肉中分离纯化得到一种具有磷脂酶A2活性的磷脂水解酶，并初步研究了这种酶的部分酶学 性质，为南京板鸭风味形成的机理研究、加工过程的调控以及老式工艺的改善提供理论依据。具体研究内容和成果如下： (1)鸭肉磷脂粗酶液的提取及酶活分析。以鸭股二头肌为原料，优化了鸭肉肌内磷脂水解酶的提取过程并检测了多种鸭肉肌内磷脂水解酶的酶活(酸性脂肪酶、中性脂肪酶、 总磷脂酶、磷脂酶A2、磷脂酶C和磷脂酶
3、D)。成果显示，鸭股二头肌磷脂酶A2活性远高 于胸脯肉；股二头肌中磷脂酶A2的活性远高于其她磷脂水解酶的活性；肌内磷脂水解酶提 取缓冲液01molL1嘶sHCl的最佳pH为80，最佳盐析条件为70饱和度的硫酸铵溶液。(2)分离纯化鸭肉磷脂酶A2。以上述获得的粗酶液为原料，本实验一方面尝试使用Sourcel5Q(15x55cm)离子互换层析进行纯化，并对离子互换层析的条件进行优化，最后确 定为pH80，50mmolL。1TrisHCl为A相，含1molLdNaCl的pH80，50retoolL1TrisHCl 为B相，平衡流速为10mlmin1，洗脱流速为15mlmin，35min达到100B，
4、上样量为500p1。然后将得到的活性峰再次上样于Superdex200(10x30cm)凝胶层析，最后经 SDSPAGE证明，获得了一种电泳的具有磷脂酶A2活性的磷脂水解酶，并通过薄层析法和 液相色谱法证明了这种酶的活性。(3)部分酶学性质的初步研究。SDSPAGE电泳测得该具有磷脂酶A2活性的磷脂水解 酶的表观分子量约为823KDa：通过研究该种酶的基本酶学性质发现，NaF对其活性酶液 没有克制作用，因此这肯定不是脂肪酶，这种磷脂酶A2的最适反映温度为25，最适反 应pH为75，Ca2+对该酶的活性基本没有影响，纯化的酶液经超滤浓缩后在4C下可以保扬州大学研究生学位论文2一存最佳时间约为3d
5、，若要长期保存，则需要将浓缩后的酶液冷冻干燥后再保存。 核心词：鸭肉磷脂水解酶系提取纯化酶学性质张露娟：鸭肉磷脂水解酶的分离纯化及其酶学性质研究3一Ab stractNanjing cureddry duck is one of the China traditional cureddry products，which has long history and special flavorMany researchers have proved that the special flavor is related witIl the degradation of lipids especial
6、ly phospholipidand the change of phospholipid is related witll phospholipid hydrolysis enzymeSome scholars have follow the track of the activityS change of phospholipid hydrolysis enzyme from macro perspective during the processing of cured-dry meat products，which confirmed the function of phospholi
7、pid hydrolysis enzyme in the flavorSform of cured-dry meat products furthen Studyhag the activityS change and mechanism of actionis thus significant meaningful for better understanding the mechanism of the change of lipids in the processing of cured-dry meat products and revealing the relationship b
8、etween the change of lipids、历m flavorThere were only a little researchers who have detected the total activity of phospholipases，however，the references about the extraction and mechanism of action of the phospholipid hydrolysis enzymes in cureddry meat products from molecular level were littleThis e
9、xperimem of the subject purified a phospholipid hydrolysis enzyme having the phospholipase A2 activity firstly from fresh duck by combining the Sourcel 5Q ionexchange and Superdex200 gel-filtration chromatography,and studied the properties of this enzymepreliminarily,which provided the theoretical b
10、asis for the studying of the mechanism of flavorS forming in Nanjing cureddry ducL controlling of the processing and improvement of tradition craftThe concrete studying contents and results were嬲follows：(1)The extraction of crude phospholipid hydrolysis enzyme from duck and analyzing ofactivity of v
11、arious phospholipid hydrolysis enzymeThe extraction processing Was optimized and the activities of various phospholipid hydrolysis enzymes(acid lipase，neutral lipase，total phospholipase，phospholipase A2，phospholipase C and phospholipase D)were detectedTheresults showed that the activity of phospholi
12、pase A2 in biceps femoris WaS much higher than phospholipase A2 in chest of duck,the activity of phospholipase A2 Was much higher than other phospholipid hydrolysis enzyme in biceps femoris，the most suitable pH for intramuscular phospholipid hydrolysis enzyme was 80 and 70saturation of ammonuium sul
13、fate Was the most suitable precipitation condition(2)Separation and purification of phospholipase A2 in duckThe experiment use the crudeenzyme solution above嬲material and firstly tried to USe Source 1 5Q ionexchange扬州大学研究生学位论文4一chromatography(15x55cm)，moreover,the separation condition was optimized：
14、mobile phase A Was pH80，50mmolL1sHCl and mobile phase B Was phase A containing lmolL1 NaCl， equilibrium velocity Was 10mlminl，elution flowing rate was 15mLmin1 and the elution gradient Was O1 00B 35min and sample size Was 5009LThen the activity peak Was separated again by Superdex 200 gel-filtration
15、 chromatography and a electrophoretic homogeneityphospholipid hydrolysis enzyme with the activity of phospholipase A2was abtained finally SDS-PAGE confirmed that the enzyme abtained Was electrophoresis pureMoreover,the activity of this enzyme was demonstrated by thin layer chromatography and high performance liquid chromatography(3)Preliminary investigation on some enzymatic propertiesSDS-PAGE measured theapparent molecular weight of the PLA2 is about 823KDaNaF Was found that it didnt inhibit the activity of this enzyme，SO it Was certainly not a lipase and its optim
