1、實驗七 溶菌酶的制備及其性質【實驗目的】同羧肽酶實驗【實驗要求】同羧肽酶實驗【實驗內容】1.溶菌酶Y活性檢測 酶活力測定方法 酶活力單位定義 比活力定義 蛋白含量測定方法 2. 蛋清溶菌酶的提取 每組45個新鮮雞蛋 利用等電點沉淀及樹脂層析等方法進行溶菌酶提取3. 溶菌酶的分離、純化 利用各種方法，從上述提取液分離 每步純化前蛋白純度檢測 分離純化過程中跟蹤檢測活性組份的蛋白質含量和酶活性4. 溶菌酶理化性質 溶菌酶的分子量測定 溶菌酶的等電點測定5. 溶菌酶的酶學性質 在活力單位定義確定后，求出溶菌酶的Vmax和Km 初步確定溶菌酶的最適pH 初步確定溶菌酶的最適溫度 提出最少兩種溶菌酶的可

2、逆抑制劑并驗證抑制類型 提出溶菌酶的激活性并驗證 (6) 溶菌酶的熱穩定性【實驗原理】溶菌酶（lysozyme）是由弗萊明在1922年發現的，它是一種有效的抗菌劑，全稱為1，4-N-溶菌酶，又稱作粘肽N-乙?；邗Ｋ饷富虬谫|酶?；钚灾行臑樘於彼?2和谷氨酸35，是一種糖苷水解酶，能催化水解粘多糖的N-乙酰氨基葡萄糖（NAG）與N-乙酰胞壁酸（NAM）間的-1，4糖苷鍵，相對分子質量14700Da，由129氨基酸殘基構成，由于其中含有較多堿性氨基酸殘基，所以其等電點高達10.8左右，最適溫度為50OC，最適PH為67左右。在280nm的消光系數為13.0。該酶活性可被一些金屬離子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。溶菌酶廣泛存在于動植物及微生物體內，雞蛋(含量約為2%4%)和哺乳動物的乳汁是溶菌酶的主要來源，目前，溶菌酶仍屬于緊銷的生化物質，廣泛應用于醫學臨床，具有抗感染、消炎、消腫、增強體內免疫反應等多種藥理作用。溶菌酶常溫下在中性鹽溶液中具有較高天然活性 ，在中性條件下溶菌酶帶正電荷，因此在分離制備時，先后采用等電點法，D152型樹脂柱層析法除雜蛋白，再經Sephadex G-50層析柱進一步純化。最后用SDS-PAGE鑒定為一條帶。采用福林酚法測蛋白含量，分光光度法測

4、定酶活性?！緦嶒灢牧稀?.實驗器材循環水式真空泵 HSB-I; 蛋白紫外檢測儀; 記錄儀; 紫外分光光度計; 梯度混合器(500mL); 721型光分光光度計; 冰凍離心機；冰箱；透析袋；酸度計；部分收集器；恒流泵；圓盤電泳裝置；恒溫水浴鍋；層析柱(2.650cm)(1.630cm)；布氏漏斗(500mL)；吸濾瓶(1000mL)；G-3砂芯漏斗(500mL)2實驗試劑 雞蛋清（鮮雞蛋） 底物微球菌粉 D152大孔弱酸性陽離子交換樹脂 固體氯化鈉（NaCl）；固體硫酸銨(NH4)2SO4；固體磷酸氫二鈉（Na2HPO412H2O）；固體磷酸二氫鈉（NaH2PO42H2O）；固體磷酸鈉（Na3P

5、O4） 乙醇；蒸餾水；甲醇；考馬斯亮藍；三氯醋酸；丙酮 (6) 溶菌酶標準品；Sephadex G50 N-乙酰葡萄糖胺；硫酸銅；硫酸亞鐵；硫酸鋅；氯化鎂；氯化鈣；氫氧化鈉；鹽酸 SDS聚丙烯酰胺凝膠電泳試劑（見實驗六）；蛋白含量測定（福林法）試劑（見實驗二） 聚乙二醇-20000、兩性電解質【實驗操作】1.蛋清的制備將45個新鮮的雞蛋兩端各敲一個小洞，使蛋清流出（雞蛋清pH值不得小于8），輕輕攪拌5分鐘，使雞蛋清的稠度均勻，用兩層紗布過濾除去臍帶塊，量體積約為100ml.2.雞蛋清粗分離按過濾好的蛋清量邊緩慢攪拌邊加入等體積的去離子水，均勻后在不斷攪拌下用1mol/L HCl調pH值至7左右

6、，用脫脂棉過濾收濾液。3.D152大孔弱酸性陽離子交換樹脂層析 D152樹脂處理：將D152樹脂先用蒸餾水洗去雜物，濾出，用1mol/L NaOH攪拌浸泡并攪拌48小時，抽濾干NaOH, 用蒸餾水洗至近pH7.5, 抽濾干, 再用1mol/L HCl按上述方法處理樹脂，直到全部轉變成氫型，抽濾干HCl , 用蒸餾水洗致近pH5.5，保持過夜，如果pH之不低于5.0，抽濾干HCl,用2mol/LNaOH處理樹脂使之轉變為鈉型，pH值不小于6.5。吸干溶液，加pH6.5 0.02mol/L的磷酸鹽緩沖液平衡樹脂。 裝柱：取直徑1.6cm，長度為30cm的層析柱，自頂部注入經處理的上述樹脂懸浮液，關

7、閉層柱出口，待樹脂沉降后，放出過量的溶液，再加入一些樹脂，至樹脂沉積至1520cm高度即可。于柱子頂部繼續加入pH6.5, 0.02mol/L磷酸鹽緩沖液平衡樹脂，使流出液pH為6.5為止，關閉柱子出口，保持液面高出樹脂表面1cm左右。 上柱吸附：將上述蛋清溶液仔細直接加到樹脂頂部，打開出口使其緩慢流入柱內，流速為1ml/min。 洗脫：用柱平衡液洗脫雜蛋白，在收集洗脫液的過程中，逐管用紫外分光光度計檢驗雜蛋白的洗脫情況，當基線開始走平后，改用含1.0mol/L NaCl的pH值6.5，濃度為0.02 mol/L磷酸鈉緩沖液洗脫，收集洗脫液。 聚乙二醇濃縮：將上述洗脫液合并裝入透析袋內，置容器

8、中，外面覆以聚乙二醇，容器加蓋，酶液中的水份很快就透析膜外的聚乙二醇所吸收。當濃縮到5mL左右時，用蒸餾水洗去透析膜外的聚乙二醇，小心取出濃縮液。(6) 透析除鹽：蒸餾水透析除鹽24小時。4.Sephadex G50分子篩柱層析 裝柱：先將用20%乙醇保存的Sephadex G50抽濾除去乙醇，用6g/LNaCl溶液攪拌Sephadex G50數分鐘，再抽濾，反復多次直至無醇味為此。(如果Sephadex G50是新的，則按實驗五中的方法處理凝膠)。加入膠體積1/4的6g/L NaCl溶液，充分攪拌，超聲除去氣泡，裝入玻璃層析柱（1.650cm），柱床45cm。 上樣：與實驗五中的方法相同。

9、洗脫:樣品流完后，先分次加入少量6g/L NaCl洗脫液洗下柱壁上的樣品，連接恒流泵，使流速為0.5mL/min，用部分收集器收集，每10分鐘一管。 聚乙二醇濃縮：合并活性峰溶液，用聚乙二醇濃縮到5mL左右時，用蒸餾水洗去透析膜外的聚乙二醇，小心取出濃縮液。 透析除鹽:蒸餾水透析除鹽24小時。收集透析液，量取體積。5. 溶菌酶活力測定 酶液配制：準確稱取溶菌酶樣品5mg, 用0.1mol/L,pH6.2磷酸緩沖液配成1mg/ml的酶液，再將酶液稀釋成50g/ml. 底物配制：取干菌粉5mg加上述緩沖液少許，在乳缽中（或勻漿器中）研磨2分鐘，傾出，稀釋到1525ml，此時在光電比色上的吸光度最好

10、在0.50.7范圍內。5.3 活力測定：先將酶和底物分別放入25 OC恒溫水浴預熱10分鐘，吸取底物懸浮液4mL放入比色杯中，在450nm波長讀出吸光度，此為零時讀數。然后吸取樣品液0.2mL（相當于10g酶），每隔30s讀1次吸光度，到90s時共計下四個讀數?；盍挝坏亩x是：在25，pH6.2，波長為450nm時，每分引起吸光度下降0.001為1個活力單位。 酶的活力單位數=A450nm/t0.001比活力=酶的活力單位數/mg蛋白質6. 蛋白質含量的測定采用Folin-酚試劑法進行測定。（參見實驗二）7. 純度檢測采用SDS-聚丙烯酰胺凝膠電泳方法。（參見實驗六）8. 理化和酶學性質的測

11、定學生可根據酶純化和活力測定的結果，運用已掌握的生化知識和實驗技能自行設計方案進行探索研究?！緦嶒灲Y果】步驟項目體積(ml)總蛋白量(mg)總活力單 位比活力(單位/mg)回收率(%)1.制備蛋清2.溶菌酶分離3.D152樹脂柱層析4. Sephadex G50層析【思考題】1請說出其它提取和純化溶菌酶的方法嗎？請寫出相關的方法及原理。2根據自身的實驗體會，寫出優化本實驗的措施。Experiment 19 Preparation of Lysozyme and Its Properties【Purpose】The same as carboxypeptidase Y experiment【Re

12、quirement】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 lysozyme form the egg white Give every group 45 e

13、ggs 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 purify.(3) Assay of protein content and enzyme activ

14、ity 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 the optimum pH of lysozyme(3) First make sure the

15、 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 bacteriologist Alexander Fleming in 1922, is an effective an

16、timicrobial 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 which occur in the mucopeptide cell wall structure

17、 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 about 14700Da, and is composed of 129 amino acid res

18、idues. 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-acetylglucosamine, while the enzyme can be activ

19、ated 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 used in clinical, is still a biochemical drug in s

20、hortage. 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 positive charge under this condition, it can be absorb

21、ed 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 purify lysozyme, we use sephadex G50 to sieve out

22、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 Vacuum pump of recycle water HSB-I; Ultraviolet det

23、ector; 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; Constant speed pump; Disk electrophoresis apparatus; Ch

24、romatography column (2.650cm) (1.630cm)2. Reagents(1) Albumen (egg)(2) Substrate: dried germ powder (3) D152 acidic cation-exchange resin (4) Solid NaCl; (NH)2SO; Na2HPO412H2O; NaH2PO42H2O; Na3PO4 (5) Ethanol; Distilled water; Methanol; Coomassie Brilliant Blue G250; Trichloroacetic acid ;Acetone (6

25、) 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 (the same as experiment 2); Reagents for SDS-PAGE (the same as experimen

26、t 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 viscosity, and then sieve with gauze, the total volume is about 100 ml. 2. Crud

27、e 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-resin Dispose of Resin: Wash D152-mode resin with clear water to elimin

28、ate 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 chloride solution as above. Add hydrogen chloride excessively with stirring t

29、o 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 sodium mode with 2mol/L sodium hydroxide solution, but control the pH to not

30、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 top. Close the exit of chromatography column .When the resin sediments, l

31、et 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 of the column, and keep the level of the liquor surface about 1cm higher

32、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 by balanced solution; Collect the eluent and check the protein in the el

33、uent 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 dialytic bag; cover dialytic bag with PEG; Water in the enzyme solution will

34、 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 eliminate the salt, dialysis above solution against distilled water for

35、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 in solution. (If the sephadex G50 is new, dispose the gel as described i

36、n 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 (2.650), the column bed height is 45 cm.(2) Loading sample: the same as experiment 5. (3) Elution: After all of the sample enter into the gel, Add 6 g/L NaCl

37、 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 automatic collector. (4) Condense by PEG: Collect and combine the elutent; Condense

38、d 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. Assay of activity Preparation of enzyme solution: Accurately weigh 5mg of lys

39、ozyme, dissolve it with 0.1mol/L pH6.2 phosphoric acid buffer to be 1mg/ml enzyme solution, and then dilute it to 50g/ml. Preparation of substrate: Take 5mg of dried germ powder, add a little above buffer, and triturate it in the mortar (or homogenizer) for 2 minutes. Pour it out and dilute it to 15

40、25ml. 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 10g of enzyme), read the optical density every 30 seconds. Write do