实验十 溶菌酶提取、分离、纯化及生物学性质、理化性质

1、实验七 溶菌酶的制备及其性质【实验目的】同羧肽酶实验【实验要求】同羧肽酶实验【实验内容】1.溶菌酶Y活性检测 酶活力测定方法 酶活力单位定义 比活力定义 蛋白含量测定方法 2. 蛋清溶菌酶的提取 每组45个新鲜鸡蛋 利用等电点沉淀及树脂层析等方法进行溶菌酶提取3. 溶菌酶的分离、纯化 利用各种方法，从上述提取液分离 每步纯化前蛋白纯度检测 分离纯化过程中跟踪检测活性组份的蛋白质含量和酶活性4. 溶菌酶理化性质 溶菌酶的分子量测定 溶菌酶的等电点测定5. 溶菌酶的酶学性质 在活力单位定义确定后，求出溶菌酶的Vmax和Km 初步确定溶菌酶的最适pH 初步确定溶菌酶的最适温度 提出最少两种溶菌酶的可

2、逆抑制剂并验证抑制类型 提出溶菌酶的激活性并验证 (6) 溶菌酶的热稳定性【实验原理】溶菌酶（lysozyme）是由弗莱明在1922年发现的，它是一种有效的抗菌剂，全称为1，4-N-溶菌酶，又称作粘肽N-乙酰基胞壁酰水解酶或胞壁质酶。活性中心为天冬氨酸52和谷氨酸35，是一种糖苷水解酶，能催化水解粘多糖的N-乙酰氨基葡萄糖（NAG）与N-乙酰胞壁酸（NAM）间的-1，4糖苷键，相对分子质量14700Da，由129氨基酸残基构成，由于其中含有较多碱性氨基酸残基，所以其等电点高达10.8左右，最适温度为50OC，最适PH为67左右。在280nm的消光系数为13.0。该酶活性可被一些金属离子Cu2+

3、、Fe2+、Zn2+（10-510-3M）以及N-乙酰葡萄糖胺所抑制，能被Mg2+、Ca2+（10-510-3M）、NaCl所激活。溶菌酶广泛存在于动植物及微生物体内，鸡蛋(含量约为2%4%)和哺乳动物的乳汁是溶菌酶的主要来源，目前，溶菌酶仍属于紧销的生化物质，广泛应用于医学临床，具有抗感染、消炎、消肿、增强体内免疫反应等多种药理作用。溶菌酶常温下在中性盐溶液中具有较高天然活性 ，在中性条件下溶菌酶带正电荷，因此在分离制备时，先后采用等电点法，D152型树脂柱层析法除杂蛋白，再经Sephadex G-50层析柱进一步纯化。最后用SDS-PAGE鉴定为一条带。采用福林酚法测蛋白含量，分光光度法测

4、定酶活性。【实验材料】1.实验器材循环水式真空泵 HSB-I; 蛋白紫外检测仪; 记录仪; 紫外分光光度计; 梯度混合器(500mL); 721型光分光光度计; 冰冻离心机；冰箱；透析袋；酸度计；部分收集器；恒流泵；圆盘电泳装置；恒温水浴锅；层析柱(2.6×50cm)(1.6×30cm)；布氏漏斗(500mL)；吸滤瓶(1000mL)；G-3砂芯漏斗(500mL)2实验试剂 鸡蛋清（鲜鸡蛋） 底物微球菌粉 D152大孔弱酸性阳离子交换树脂 固体氯化钠（NaCl）；固体硫酸铵(NH4)2SO4；固体磷酸氢二钠（Na2HPO4·12H2O）；固体磷酸二氢钠（NaH2P

5、O4·2H2O）；固体磷酸钠（Na3PO4） 乙醇；蒸馏水；甲醇；考马斯亮蓝；三氯醋酸；丙酮 (6) 溶菌酶标准品；Sephadex G50 N-乙酰葡萄糖胺；硫酸铜；硫酸亚铁；硫酸锌；氯化镁；氯化钙；氢氧化钠；盐酸 SDS聚丙烯酰胺凝胶电泳试剂（见实验六）；蛋白含量测定（福林法）试剂（见实验二） 聚乙二醇-20000、两性电解质【实验操作】1.蛋清的制备将45个新鲜的鸡蛋两端各敲一个小洞，使蛋清流出（鸡蛋清pH值不得小于8），轻轻搅拌5分钟，使鸡蛋清的稠度均匀，用两层纱布过滤除去脐带块，量体积约为100ml.2.鸡蛋清粗分离按过滤好的蛋清量边缓慢搅拌边加入等体积的去离子水，均匀后在

6、不断搅拌下用1mol/L HCl调pH值至7左右，用脱脂棉过滤收滤液。3.D152大孔弱酸性阳离子交换树脂层析 D152树脂处理：将D152树脂先用蒸馏水洗去杂物，滤出，用1mol/L NaOH搅拌浸泡并搅拌48小时，抽滤干NaOH, 用蒸馏水洗至近pH7.5, 抽滤干, 再用1mol/L HCl按上述方法处理树脂，直到全部转变成氢型，抽滤干HCl , 用蒸馏水洗致近pH5.5，保持过夜，如果pH之不低于5.0，抽滤干HCl,用2mol/LNaOH处理树脂使之转变为钠型，pH值不小于6.5。吸干溶液，加pH6.5 0.02mol/L的磷酸盐缓冲液平衡树脂。 装柱：取直径1.6cm，长度为30c

7、m的层析柱，自顶部注入经处理的上述树脂悬浮液，关闭层柱出口，待树脂沉降后，放出过量的溶液，再加入一些树脂，至树脂沉积至1520cm高度即可。于柱子顶部继续加入pH6.5, 0.02mol/L磷酸盐缓冲液平衡树脂，使流出液pH为6.5为止，关闭柱子出口，保持液面高出树脂表面1cm左右。 上柱吸附：将上述蛋清溶液仔细直接加到树脂顶部，打开出口使其缓慢流入柱内，流速为1ml/min。 洗脱：用柱平衡液洗脱杂蛋白，在收集洗脱液的过程中，逐管用紫外分光光度计检验杂蛋白的洗脱情况，当基线开始走平后，改用含1.0mol/L NaCl的pH值6.5，浓度为0.02 mol/L磷酸钠缓冲液洗脱，收集洗脱液。 聚

8、乙二醇浓缩：将上述洗脱液合并装入透析袋内，置容器中，外面覆以聚乙二醇，容器加盖，酶液中的水份很快就透析膜外的聚乙二醇所吸收。当浓缩到5mL左右时，用蒸馏水洗去透析膜外的聚乙二醇，小心取出浓缩液。(6) 透析除盐：蒸馏水透析除盐24小时。4.Sephadex G50分子筛柱层析 装柱：先将用20%乙醇保存的Sephadex G50抽滤除去乙醇，用6g/LNaCl溶液搅拌Sephadex G50数分钟，再抽滤，反复多次直至无醇味为此。(如果Sephadex G50是新的，则按实验五中的方法处理凝胶)。加入胶体积1/4的6g/L NaCl溶液，充分搅拌，超声除去气泡，装入玻璃层析柱（1.6×

9、;50cm），柱床45cm。 上样：与实验五中的方法相同。 洗脱:样品流完后，先分次加入少量6g/L NaCl洗脱液洗下柱壁上的样品，连接恒流泵，使流速为0.5mL/min，用部分收集器收集，每10分钟一管。 聚乙二醇浓缩：合并活性峰溶液，用聚乙二醇浓缩到5mL左右时，用蒸馏水洗去透析膜外的聚乙二醇，小心取出浓缩液。 透析除盐:蒸馏水透析除盐24小时。收集透析液，量取体积。5. 溶菌酶活力测定 酶液配制：准确称取溶菌酶样品5mg, 用0.1mol/L,pH6.2磷酸缓冲液配成1mg/ml的酶液，再将酶液稀释成50µg/ml. 底物配制：取干菌粉5mg加上述缓冲液少许，在乳钵中（或匀浆

10、器中）研磨2分钟，倾出，稀释到1525ml，此时在光电比色上的吸光度最好在0.50.7范围内。5.3 活力测定：先将酶和底物分别放入25 OC恒温水浴预热10分钟，吸取底物悬浮液4mL放入比色杯中，在450nm波长读出吸光度，此为零时读数。然后吸取样品液0.2mL（相当于10µg酶），每隔30s读1次吸光度，到90s时共计下四个读数。活力单位的定义是：在25，pH6.2，波长为450nm时，每分引起吸光度下降0.001为1个活力单位。 酶的活力单位数=A450nm/t×0.001比活力=酶的活力单位数/mg蛋白质6. 蛋白质含量的测定采用Folin-酚试剂法进行测定。（参见

11、实验二）7. 纯度检测采用SDS-聚丙烯酰胺凝胶电泳方法。（参见实验六）8. 理化和酶学性质的测定学生可根据酶纯化和活力测定的结果，运用已掌握的生化知识和实验技能自行设计方案进行探索研究。【实验结果】步骤项目体积(ml)总蛋白量(mg)总活力单 位比活力(单位/mg)回收率(%)1.制备蛋清2.溶菌酶分离3.D152树脂柱层析4. Sephadex G50层析【思考题】1请说出其它提取和纯化溶菌酶的方法吗？请写出相关的方法及原理。2根据自身的实验体会，写出优化本实验的措施。Experiment 19 Preparation of Lysozyme and Its Properties【Purp

12、ose】The same as carboxypeptidase Y experiment【Requirement】The same as carboxypeptidase Y experiment【Contents】 1. Assay of lysozyme activity Method of enzyme activity assay. The definition of enzyme activity unit The definition of specific activity Lowry method to quantitate protein2. Extraction of l

13、ysozyme form the egg white Give every group 45 eggs Extract by some methods such as isoelectric point and resin chromatography3. Separation and purification of lysozyme(1) Separation lysozyme from above-mentioned solution by various methods.(2) Assay of protein purification before every step to puri

14、fy.(3) Assay of protein content and enzyme activity in every step.4. The physical and chemical properties of lysozyme(1) Assay the molecular weight of lysozyme.(2) Assay the isoelectric point of lysozyme5. The enzymatic properties of lysozyme(1) Calculate Vmax and Km of lysozyme(2) First make sure t

15、he optimum pH of lysozyme(3) First make sure the optimum temperature(4) Propose at least two inhibitors of lysozyme and prove the inhibition style(5) Propose the activators of lysozyme and then prove them (6) Prove the stability of lysozyme【Principle】Lysozyme, discovered by the Scottish bacteriologi

16、st Alexander Fleming in 1922, is an effective antimicrobial reagent and hydrolytic enzyme, whose full name is 1,4-N-lysozyme, also called muramidase. The active site of this enzyme is Asp62 and Glu36. It hydrolyzes the -1, 4 glucosidic linkages between N-acetylmuramic acid and N-acetylglucosamine wh

17、ich occur in the mucopeptide cell wall structure of certain microorganisms, such as Micrococcus lysodeikticus. It can hydrolyze the polysaccharide of the cell walls of gram-negative bacteria and some gram-positive bacteria, which cause the lysis of cell wall. The molecular weight of lysozyme is abou

18、t 14700Da, and is composed of 129 amino acid residues. Because it contains many basic amino acids, the isoelectric point is about 10.8, where the optimum temperature is about 50 . At 280nm wavelength, the extinction coefficient is 13.0. The inhibitor of lysozyme is Cu2+, Fe2+, Zn2+（10-510-3M） and N-

19、acetylglucosamine, while the enzyme can be activated by Mg2+, Ca2+（10-510-3M）, NaCl. Lysozyme extensively exists in the biosphere, including the animals, plants and microbes. The content of lysozyme in avian ovum (about 2%4%) and latex of mammalian is especially rich. At present, lysozyme, widely us

20、ed in clinical, is still a biochemical drug in shortage. It has many pharmacological effects, including anti-infection, diminish inflammation, detumescence, improving the immunity and so on. Because lysozyme has comparatively high activity in neutral salt solution at room temperature, and has positi

21、ve charge under this condition, it can be absorbed by cation-exchange-resin in the approximately neutral environment. So in this experiment, the use of isoelectric point method can eliminate some of the unpurified proteins. D152 resin is used to remove most unpurified protein. In order to further pu

22、rify lysozyme, we use sephadex G50 to sieve out those unpurified protein. Examine by SDS-PAGE whether most unpurified protein is excluded from the final product. Protein content and enzyme activity are measured with Lowry method and spectrophotometric method respectively. 【Materials】1. Apparatus Vac

23、uum pump of recycle water HSB-I; Ultraviolet detector; Recorder; UV-Visible Spectrophotometer; Gradient mixer (500ml); 721-Mode spectrophotometer (Uv-9100); pH meter; Centrifuge; Refrigerator; Constant temperature water boiler; G-3 Core sand funnel (500mL); Dialytic bag; Automatic collector; Constan

24、t speed pump; Disk electrophoresis apparatus; Chromatography column (2.6×50cm) (1.6×30cm)2. Reagents(1) Albumen (egg)(2) Substrate: dried germ powder (3) D152 acidic cation-exchange resin (4) Solid NaCl; (NH)2SO; Na2HPO4·12H2O; NaH2PO4·2H2O; Na3PO4 (5) Ethanol; Distilled water; M

25、ethanol; Coomassie Brilliant Blue G250; Trichloroacetic acid ;Acetone (6) Standard sample of lysozyme; Sephadex G50 (7) N-Acetylglucosamine; CuSO4; FeSO4; Zn SO4; MgCl2; CaCl2(8) NaOH; HCl; Polyethylene glycol-20000 (PEG-20000); Ampholyte(9) Folin-phenol reagent for determination of protein content

26、(the same as experiment 2); Reagents for SDS-PAGE (the same as experiment 6)【Procedures】1. Preparation of albumenCollection of egg white: choose 45 eggs and knock holes on each side to make the egg white flow out (the pH of egg white is no lower than 8), stir gently for 5 min to get suitable viscosi

27、ty, and then sieve with gauze, the total volume is about 100 ml. 2. Crude separation of lysozyme Stir the egg white gently while adding the same volume of deionized water, adjust the pH to 7.0 with 1 mol/L HCl, and then filter with pledget.3. Chromatography on D152 porous weak acidic cation-exchange

28、-resin Dispose of Resin: Wash D152-mode resin with clear water to eliminate the slight impurity, add 1 mol/L sodium hydroxide solution, and lay it aside for 48 hours. Stir it in intervals, then suction the basic liquid, wash it to pH7.5 with distilled water. Soak the resin with 1mol/L hydrogen chlor

29、ide solution as above. Add hydrogen chloride excessively with stirring to assure that the resin changes to hydrogen mode totally. Then suction the acid liquid, and wash it to pH5.5 with distilled water, and keep it balance over night. If the pH is not below 5.0, suction it, change the resin to sodiu

30、m mode with 2mol/L sodium hydroxide solution, but control the pH to not be over 6.5. Suction it, soak the resin with 0.02mol/L phosphoric acid buffer of pH6.5 over night. Packing: Take a chromatography column with the diameter of 1.6cm and the length of 30cm; Pour the above prepared resin from the t

31、op. Close the exit of chromatography column .When the resin sediments, let out the excessive solution ,add more resin until the height of the resin sediment is about 1520cm .Add 0.02mol/L phosphoric acid buffer of pH6.5 from the top to wash it until the flowing liquor reaches pH6.5. Close the exit o

32、f the column, and keep the level of the liquor surface about 1cm higher than that of the resin. Loading: Add above albumen solution to the top of resin directly and carefully, and open the exit to make it flow into the column slowly. The speed is about 1ml/min. Elution: Elute the unpurified protein

33、by balanced solution; Collect the eluent and check the protein in the eluent with ultraviolet detector tube by tube. When base line recoveries, elute with 0.02 mol/L phosphate sodium buffer containing 1.0 mol/L NaCl, pH6.5. The eluent is collected. PEG condensation: Combine and move eluent into dial

34、ytic bag; cover dialytic bag with PEG; Water in the enzyme solution will soon be absorbed by PEG outside the dialytic bag. When the final volume is about 5 ml, wash away the PEG outside the dialytic bag with distilled water; take out the condensed solution carefully. Desalt by dialysis: In order to

35、eliminate the salt, dialysis above solution against distilled water for about 24 hours.4. Chromatography on Sephadex G50(1) Stuffing: Suck the ethanol from the sephadex G50 storing solution which contains 20% ethanol, add 6 g/L NaCl and stir several minutes; Suction; Repeat till there is no ethanol

36、in solution. (If the sephadex G50 is new, dispose the gel as described in experiment 5. Add 6 g/L NaCl about 1/4 of gel volume, ultrasonic to eliminate the bubble, stuff the gel into the glass chromatography column (2.6×50), the column bed height is 45 cm.(2) Loading sample: the same as experim

37、ent 5. (3) Elution: After all of the sample enter into the gel, Add 6 g/L NaCl eluent respectively to wash the sample on the inner wall of glass chromatography column. Connect with constant speed pump; the flowing speed is 0.5ml/min. Collect the eluent about one test tube every 10 minutes with autom

38、atic collector. (4) Condense by PEG: Collect and combine the elutent; Condensed to 5 ml with PEG; Wash away the PEG outside dialytic bag with distilled water; Take out condensed liquid carefully.(5) Desalt by dialysis: Dialysis for 24 hours to desalt against distilled water. Calculate the volume. 5.

39、 Assay of activity Preparation of enzyme solution: Accurately weigh 5mg of lysozyme, dissolve it with 0.1mol/L pH6.2 phosphoric acid buffer to be 1mg/ml enzyme solution, and then dilute it to 50µg/ml. Preparation of substrate: Take 5mg of dried germ powder, add a little above buffer, and tritur

40、ate it in the mortar (or homogenizer) for 2 minutes. Pour it out and dilute it to 1525ml. It is the best that the optical density assayed by the photoelectric colorimeter is between 0.50.7. Activity determination: Preheat the enzyme and substrate respectively in aqueous thermostat of 25 for ten minutes. Suck 3ml of substrate suspension, and put it in cuvette. Then record the optical density at 450nm, and this is value at zero. Them draw 0.2ml of sample solution (equal to 10µg of enzyme), read the optical density every 30 seconds. Write down four