蒜氨酸酶動(dòng)力學(xué)性質(zhì)研究

Agricultural Science& Technology, 2008, 9(1): 139-142Copynight C 2008, Information Institute of HAAS. All rights reserved.Plant Physiology and Bio-chemistryStudy on Kinetic Characteristics of AllinaseGE Yan-hui, ZHAO Jun-ying, MIN Di,FENG Xin1. College of Environmental Science and Safety Engineering, Tianiin University of Technology, Tianyin 300191; 2. College of Enuironment Science and Tech-nology, Tianjin University, Tianjin 300071e] The kinetic characteristics of allinaseto select the optimum reaction perfoperature was 29c and ph value was 6.m04y1mm如m.8圖ivity was activated when the k,MCd'eisted and alliinase activity was inhibsion] Results showed that the kinetic characteristics o alliinase supply the academic foundation for development andased to as natural foods and original medical plants in many Drawing standard curve of pyruvate emovedAllium sativum is a kind of Liliaceae, A. sativum was (8000 r/min), and the supematant wascountries throughout the world. A. sativum contains sulfur0.01 mol/L sodium pyruvate solution was prepared ascompound named as allicin which is produced by the enzy- follows, 110 g sodium pyruvate was dissolved, and final vol-matic reaction between alliinase and alliin. Allicin is also ume was 100 ml. 0. 2, 0.4,0.6,0.8, 1.0 ml sodium pyruvaterery important in medicinal application. The alliinase con- was added to volumetric flasks(100 ml), respectively, andent is low and sensitive to the environment that it is unsta. distilled water was added to 100 ml. 4 ml sodium pyruvateble. In addition, the current production conditions often lead solution from every volumetric flask was removed to tubesto the destruction of the allinase activity, and pharmacologi- respectively. 1 ml 0. 005 mol/L 2, 4-dinitrophenylhydrazineal action of alliinase is effected. Therefore, study on physi- solution was added to above tubes respectively, and reactedcochemical properties of alliinase is important to development for 5 min at 25C, then 5 ml 1.0 mol/L NaoH solution wasand application of garlic medical products.added and reacted for 5 min at 25 C. The absorbance valuewas determined at 420 nm, respectively. According to theMaterials and methodsabsorbance value and pyruvate content, standard curve wasMaterialdrew( Fig. 1). Fig. I showed that R was0.998 and the linFresh red-skin-garlic from Tianjiear relationship between absorbance value and pyruvate warExtraction of alliin and alliinasegood, so pyruvate content could be obtained by determiPreparation of alliin was referred to the reference [2] absorbance value.and improved slightly. The compositions of 50 ml solution(MCW)were methanol, chloroform and water( the ratiowas 12: 5: 3).10 g garlic was put in MCW and was hold at-20C for 48 h. Adding 22.5 ml chloroform and 27. 5 mlwater respectively, according to the principle of adding 4.5000ml chloroform and 5.5 ml water per 10 ml MCW. The samplestanded until there was a sharp demarcation line between thetwo layers after it was concussed by seperatory funnel. Upper0.43104750.522solution was removed to rotary evaporator and was evaporatedto 10 ml. The solution was diluted to 60 ml with waterPreparation of alliinase was referred to the referenceFg. 1 The standard curve of pyruvate[1, 3] and improved slightly. 100 g fresh garlic pre-cooled at Determination of allinase activity4C, ground for 15-20 min at 4 C with quartz sand andsodium phosphate buffer (pH=6.5-7.5). When the method. 1 ml enzyme solution and 1 ml substrate weregrinding solution presented thick paste, it was transferred to held at 25 C for 5 min, and the reaction was ended bybeaker with sodium phosphate buffer, and was dipped for adding 2 ml 10% trichloroacetic acid to sample for 2 min, 1more than 30 min at 4C. Centrifuged for 30 min at 4C ml 2, 4中國煤化工 ed and the reactionCNMHlution(1.0 mol/L)Received: March 28, 2008 Accepted April 10, 200810 min,, and then theSupported by the Natural Science Foundation Program of Tianjin Sci- absorbance was determined at 420 nm. When 1ence Committee(043611111)and the Science and Technology Development Foundation Program of Tianjin Colleges and Universities(20050901)vate per minute was catalyzed by alliinase at 25C, the activ-Corresponding author. E-mail: xeng2100@ tjut. edu.enity of alliinase was defined as 1 unit (Iu)gricultural Science Technology Vol 9, No 1, 2008Resuits and analysisEffects of temperature on alliinase activity29C and pH =5. 5-6.1, activity of alliinase was deter-Like other enzymes, allinase was a kind of biocatalyst mined, respectively. The result showed that the activity ofhich was produced by living cells and also a kind ofalliinase improved with pH value increased in the range ofteins. Activity of alliinase was influenced by temperature pH= 5.5-6. 1. When pH was 6.1, the activity of alliinaseThe activity of alliinase at 15-70C was detected. The re- was the highest. However, when, pH value was over 6. 1, thesult showed that the allinase activity was high at 20-50c activity of alliinase decreased with pH value increased. SoFig 2). The successive tests showed that the activity of al. the optimum pH value of alliinase was 6. 1 which is lowerliinase was lost when the temperature reached to 50C. So than 6. 24 and 6. 6 which was determined by QIAO Xu-alliinase could keep thermal stability and activity at 20-50 guang and GoU Ping, respectiveC. Fig 3 showed that the activity was high at 20-50Cfurther research was conducted in this temperature range.0.0Fig 4 showed that the optimum reaction temperature of allii-nase activity was 29C and lower than other optimum reac-0.060tion temperature which was determined by Rostand GOU0.040Ping, respectively. And this may be caused by substrateS-allyl-L-cysteine sulfoxide used by Krost and GOU Pingwhile natural substrate was used in this study5.65.75859606162636465Fig 5 Efects of pH value on allinase actlvityEffects of substrate concentration on alliinase activitynd determination of Michaelis constant( Km)Alliinase activity was determined in different substrateconcentration( the reaction temperature was 29C when theconcentrations of natural extract as substrate and was 1, 2,3, 4, 5,6, 7,8,9, 10 mmol/L, respectively ) RusultsFig-2 Thermal stability of alliinaseshowed that the vmax was 0. 439 IU/mg. and the Km was0.483 mmol/L. It was inconsistent with the results whichwas determined by GoU Ping, QIAO Xu-guang)and Kazaryan, respectively. And maybe because of the different是0302025303540455560sTamperature』℃Fig 3 Ehects of temperature on alliinase activityFig 6 Determination of Km of alliinaseEffects of metal ions on alliinase activityAllinase activity was determined while the alliinase0096tion system included the same concentration cations of K022222Mg, Na, Cd, Caand Cu, respectively(Fig. 7)In Fig. 7, the alliinase activity can be enhanced when the中國煤化工 existed respectively,Fig 4 Enects of temperature on allinase activityandent when the cation ofEffects of pi value on alliinase actiCNMHGcan be inhib-油可山咖如,GE Yan-hui et al. Study on Kinetic Characteristics of Alliinasested and alliinase activity was inhibited when Cu*existeda100[1] GOU P, L Y, WANG R. Purification and properties of allinase from garlicower[J ]. Plant Physiology Communications, 2004, 40(3): 355-357(in Chinese ).[2]QIN XC. Stries an the separation, purification ad propeties d alliin[D]a.02andong Agricultural University 2004.(in Chinese[3]LU J. Protein purification technology and its application[M ]. BeijingChemical Industry Press, 2005. (in Chinese).Mctal ins[4]Y00 KS, PIKE LM. Determination o background pyruvic acid comcen-ations in onion, Allium species, and other vegetable [J]. Scientia Hor-Fig 7 Efects of postive ions on alliinase activityultimate,201(8):249-256Conclusion[5] KROST I, KEUSGEN M. Quality o herbal remedies from Allium satiumResults showed that alliinase was an enzyme with therifferences between alliinase from gartic powder and fresh gartic[J].Medica,199(65):139-143.mal instability. Its optimum reaction temperature was 29C [6]QIAO XG, ZHANG ZH, QIN XC. Study on the purification and kineticnd pH was 6. 1. The Vmax was 0. 439 IU/mg and Kmaracteristics o alliinase[J]. Food and Fermentation Industrie, 2004483 mmol/l by using natural extract as substrate30(9):1-4.( in Chinese)[7]KAZARYAN RA. Allinase: purification and characterization[J]. Biactivity was activated when the K, Mg, Na'and Cd*ex-chemistry Engish Translation, 1978(43): 1502-1508[8]MAZELIS M, CREWS L Purification of allinase[ J]. analytical Biochem-isry,1968(72):248-254.蒜氨酸酶動(dòng)力學(xué)性質(zhì)研究葛艷輝12,趙俊英!,閔笛,馮炘(1.天津理工大學(xué)環(huán)境科學(xué)與安全工程學(xué)院天津300191;2.天津大學(xué)環(huán)境科學(xué)與工程學(xué)院,天津3001)大蒜中含有一類(lèi)被稱(chēng)之為大蒜素的含硫化合物,主要由蒜0.005mo/L2,4-二硝基苯肼溶液,25℃反應5min;再加入5ml氨酸酶與蒜氨酸發(fā)生酶解反應生成,但蒜氨酸酶含量低,對環(huán)境1.0mlL/ L NaOH溶液,25℃反應10min。最后在420mm處測較敏感,因而活性表現不穩定,加之目前的生產(chǎn)條件常導致該酶定各管吸光度值。依據各吸光度值及對應的丙酮酸含量繪制標活性被破壞使得蒜類(lèi)產(chǎn)品的藥理作用不顯著(zhù)。因此研究蒜氨酸準曲線(xiàn)(圖1)。由該標準曲線(xiàn)可知,R2為0.9982,說(shuō)明吸光度酶的理化性質(zhì)對于開(kāi)發(fā)應用蒜類(lèi)藥用產(chǎn)品具有重要的指導作用。值與丙酮酸線(xiàn)性關(guān)系良好,可以用測定吸光度值的方法獲得丙1材料與方法酮酸的含量11材料天津紅皮新鮮大蒜。12蒜氨酸和蒜氨酸酶的提取蒜氨酸的制備參考文獻[2]方法略有改進(jìn)。將甲醇、氯仿、水按12:5:3的比例配成50m的體系(MCW),混合均勻后取10g大蒜放入其中置于-20℃48目a8h,然后按每10mMCW加4.5m氯仿55m水的原則分別加入2.5ml氯仿和27.5ml水。在分液漏斗中震蕩混勻后靜置分層取上層水溶液于旋轉蒸發(fā)器中蒸發(fā)至10叫,然后加水稀釋至60ml。蒜氨酸酶的制備參照文獻[1,3]的方法略有改進(jìn)。稱(chēng)取經(jīng)0149a261035a4310.475a5224℃預冷的新鮮大蒜100g,加入適量的石英砂和磷酸鈉鹽緩沖吸光度值溶液(pH值為6.5~67),于4℃下研磨15-20min。當研磨液呈現厚糊狀時(shí)轉移至燒杯中,并加入上述緩沖溶液4℃浸提圖1丙酮酸標準曲線(xiàn)30min以上;4℃下8000r/min離心30min,取上清液1.3丙酮酸標準曲線(xiàn)的繪制準確稱(chēng)取110mg丙酮酸鈉溶14酶活性的測定用丙酮酸法測定酶活性。精確吸取1ml解后定容于100m容量瓶中,制成0.01mo/L丙酮酸鈉溶液。酶液和1ml底物于試管中,搖勻,25℃保溫5min,加人2ml取7只100m容量瓶分別加人0.4、0.8、1.2、1.6,2.0、2.4、2.80%三氯乙酸終止反應2min,再加入1m2,4-二硝基苯肼,反m丙酮酸鈉用蒸餾水定容至100m分別從各容量瓶中吸取應5mn后加入1.0m/ L NaOH溶液5m,反應10min后于4m丙酮酸鈉溶液加入7只試管中。然后各管分別加入1m420m處比色。以25℃條件下每分鐘產(chǎn)生1umd丙酮酸為1個(gè)活力2結果中國煤化工基金項目天津市科委自然科學(xué)基金項目(0436111);天津市高等2.1CNMH(Q我酶在20~50℃時(shí)活學(xué)?？萍及l(fā)展基金項目(20050901)。作者簡(jiǎn)介葛艷輝(197-),女,黑龍江雙城人,在讀博士,講師從事性較高(圖2)。由后續試驗可知當環(huán)境溫度達到50℃時(shí),酶生物化學(xué)和環(huán)境生物學(xué)研究?！ねㄓ嵶髡?。已開(kāi)始失活。圖3表明,在25-30℃范圍內蒜氨酸酶的活性最收稿日期2008403-28修回日期200804-10高故對該溫度范圍進(jìn)行進(jìn)一步試驗研究。結果表明(圖4),蒜氨酸酶的最適反應溫度為29℃。這比Kroe和茍萍測得的最適反應溫度都低,可能是由于試驗中采用的底物不同引起的,他們采用S烯丙基L半胱氨酸亞砜,而該試驗采用的是天然底物。0056575859606162636465圖5pH值對蒜氨酸酶活性的影響圖2蒜氨酸酶的熱穩定性0區度蒜氨酸酶Km值測定圖3溫度對蒜氨酸酶活性的影響2.4金屬離子對蒜氨酸酶活性的影響選用K、Mg、Na、Cd2·、Ca2和Cu2·6種陽(yáng)離子以相同的陰離子、相同濃度加入酶反應體系,分別測定其對蒜氨酸酶活性的影響,K、MgCa2+Na‘和Cd2能增加酶活性,其中Ca2的增強效果最強Cu2對酶活性有抑制作用(圖7)。與Mali測定的結果基本a60溫度℃圖4溫度對蒜氮酸酶活性的影響2.2pH值對蒜氨酸酶活性的影響在29℃下,pH為5.56.5范圍內,分別測定蒜氨酸酶的活力情況。結果表明(圖5)金屬高子在55-61范圍內隨著(zhù)pH值增大蒜氨酸酶的活性逐漸增加蒜氨酸酶的最適pH值為61,比喬旭光和茍萍所得的結果要低(他們得到的最適pH值分別為624和66)。圖7陽(yáng)離高子對蒜氨酸酶活性的影響23底物濃度對蒜氨酸酶活性的影響及米氏常數的測定以3結論自然抽提物為底物,底物濃度分別設為1、2、3、4、5、6、7、8、該研究中蒜氨酸酶動(dòng)力學(xué)試驗結果為:蒜氨酸酶為熱不穩9、10mmL,測定不同底物濃度下蒜氨酸酶的活性(反應的環(huán)定性酶,其最適反應溫度為c,最適PH值為61:以其天然境溫度為29℃)。依據結果(圖6)得到最大反應速度(Wmax)抽提物為底物,測得的最大反應速度及米氏常數分別為0.439為0.439Um,進(jìn)一步算得米氏常數(Km)為0.483molL。uag.0.483mol/L;K、Mg”Na‘和c對蒜氨酸酶活性有與茍萍喬旭光、Km等報道的結果不一致,可能是反應底定的激活作用,C對酶活性有抑制作用。物不同所致·?！簟?。0·60?！??！??！?00400·0·?！ぁ选??！?00·0000·00·00·⊙·?！?。90··?！?。0。0·0·00·00·?！?。?！?上接第116頁(yè))其重要的生理作用和藥用價(jià)值,對商城肥鯢在低溫時(shí)保持膜的肥鯢肌肉脂肪酸的組成有2個(gè)特點(diǎn):①不飽和脂肪酸含很高。流動(dòng)性和滲透性有重要意義。占總脂肪酸的80.02%,遠高于飽和脂肪酸,為飽和脂肪酸的44結論倍以上;②不飽和脂肪酸中高度不飽和脂肪酸的含量很高。平商中國煤化工類(lèi)型,是種可貴的野均含量高達39.50%。這可能與商城肥鯢的生活環(huán)境有關(guān)其生自然生活在山澗溪水中,水體平均溫度較低,高度不飽和脂肪酸的出來(lái)作CNMHG質(zhì)量,這對豐富人們存在有利于生理代謝的正常進(jìn)行。另外,商城肥鯢富含花生五的物質(zhì)生活增強人們的體質(zhì)促進(jìn)人們身體健康頗有益處。烯酸,其含量分別達到368%和4.37%。EPA和DHA具有極