蒜氨酸酶动力学性质研究

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图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中国煤化工 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中国煤化工 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.蒜氨酸酶动力学性质研究葛艳辉12,赵俊英!,闵笛,冯炘(1.天津理工大学环境科学与安全工程学院天津300191;2.天津大学环境科学与工程学院,天津3001)大蒜中含有一类被称之为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℃反应5min;再加入5ml氨酸酶与蒜氨酸发生酶解反应生成,但蒜氨酸酶含量低,对环境1.0mlL/ L NaOH溶液,25℃反应10min。最后在420mm处测较敏感,因而活性表现不稳定,加之目前的生产条件常导致该酶定各管吸光度值。依据各吸光度值及对应的丙酮酸含量绘制标活性被破坏使得蒜类产品的药理作用不显著。因此研究蒜氨酸准曲线(图1)。由该标准曲线可知,R2为0.9982,说明吸光度酶的理化性质对于开发应用蒜类药用产品具有重要的指导作用。值与丙酮酸线性关系良好,可以用测定吸光度值的方法获得丙1材料与方法酮酸的含量11材料天津红皮新鲜大蒜。12蒜氨酸和蒜氨酸酶的提取蒜氨酸的制备参考文献[2]方法略有改进。将甲醇、氯仿、水按12:5:3的比例配成50m的体系(MCW),混合均匀后取10g大蒜放入其中置于-20℃48目a8h,然后按每10mMCW加4.5m氯仿55m水的原则分别加入2.5ml氯仿和27.5ml水。在分液漏斗中震荡混匀后静置分层取上层水溶液于旋转蒸发器中蒸发至10叫,然后加水稀释至60ml。蒜氨酸酶的制备参照文献[1,3]的方法略有改进。称取经0149a261035a4310.475a5224℃预冷的新鲜大蒜100g,加入适量的石英砂和磷酸钠盐缓冲吸光度值溶液(pH值为6.5~67),于4℃下研磨15-20min。当研磨液呈现厚糊状时转移至烧杯中,并加入上述缓冲溶液4℃浸提图1丙酮酸标准曲线30min以上;4℃下8000r/min离心30min,取上清液1.3丙酮酸标准曲线的绘制准确称取110mg丙酮酸钠溶14酶活性的测定用丙酮酸法测定酶活性。精确吸取1ml解后定容于100m容量瓶中,制成0.01mo/L丙酮酸钠溶液。酶液和1ml底物于试管中,摇匀,25℃保温5min,加人2ml取7只100m容量瓶分别加人0.4、0.8、1.2、1.6,2.0、2.4、2.80%三氯乙酸终止反应2min,再加入1m2,4-二硝基苯肼,反m丙酮酸钠用蒸馏水定容至100m分别从各容量瓶中吸取应5mn后加入1.0m/ L NaOH溶液5m,反应10min后于4m丙酮酸钠溶液加入7只试管中。然后各管分别加入1m420m处比色。以25℃条件下每分钟产生1umd丙酮酸为1个活力2结果中国煤化工基金项目天津市科委自然科学基金项目(0436111);天津市高等2.1CNMH(Q我酶在20~50℃时活学校科技发展基金项目(20050901)。作者简介葛艳辉(197-),女,黑龙江双城人,在读博士,讲师从事性较高(图2)。由后续试验可知当环境温度达到50℃时,酶生物化学和环境生物学研究。·通讯作者。已开始失活。图3表明,在25-30℃范围内蒜氨酸酶的活性最收稿日期2008403-28修回日期200804-10高故对该温度范围进行进一步试验研究。结果表明(图4),蒜氨酸酶的最适反应温度为29℃。这比Kroe和苟萍测得的最适反应温度都低,可能是由于试验中采用的底物不同引起的,他们采用S烯丙基L半胱氨酸亚砜,而该试验采用的是天然底物。0056575859606162636465图5pH值对蒜氨酸酶活性的影响图2蒜氨酸酶的热稳定性0區度蒜氨酸酶Km值测定图3温度对蒜氨酸酶活性的影响2.4金属离子对蒜氨酸酶活性的影响选用K、Mg、Na、Cd2·、Ca2和Cu2·6种阳离子以相同的阴离子、相同浓度加入酶反应体系,分别测定其对蒜氨酸酶活性的影响,K、MgCa2+Na‘和Cd2能增加酶活性,其中Ca2的增强效果最强Cu2对酶活性有抑制作用(图7)。与Mali测定的结果基本a60温度℃图4温度对蒜氮酸酶活性的影响2.2pH值对蒜氨酸酶活性的影响在29℃下,pH为5.56.5范围内,分别测定蒜氨酸酶的活力情况。结果表明(图5)金属高子在55-61范围内随着pH值增大蒜氨酸酶的活性逐渐增加蒜氨酸酶的最适pH值为61,比乔旭光和苟萍所得的结果要低(他们得到的最适pH值分别为624和66)。图7阳离高子对蒜氨酸酶活性的影响23底物浓度对蒜氨酸酶活性的影响及米氏常数的测定以3结论自然抽提物为底物,底物浓度分别设为1、2、3、4、5、6、7、8、该研究中蒜氨酸酶动力学试验结果为:蒜氨酸酶为热不稳9、10mmL,测定不同底物浓度下蒜氨酸酶的活性(反应的环定性酶,其最适反应温度为c,最适PH值为61:以其天然境温度为29℃)。依据结果(图6)得到最大反应速度(Wmax)抽提物为底物,测得的最大反应速度及米氏常数分别为0.439为0.439Um,进一步算得米氏常数(Km)为0.483molL。uag.0.483mol/L;K、Mg”Na‘和c对蒜氨酸酶活性有与苟萍乔旭光、Km等报道的结果不一致,可能是反应底定的激活作用,C对酶活性有抑制作用。物不同所致·。◆·。0·60。·。·。·000400·0·。·⊙·。·000·0000·00·00·⊙·。·。90··。·。0。0·0·00·00·。·。。·(上接第116页)其重要的生理作用和药用价值,对商城肥鲵在低温时保持膜的肥鲵肌肉脂肪酸的组成有2个特点:①不饱和脂肪酸含很高。流动性和渗透性有重要意义。占总脂肪酸的80.02%,远高于饱和脂肪酸,为饱和脂肪酸的44结论倍以上;②不饱和脂肪酸中高度不饱和脂肪酸的含量很高。平商中国煤化工类型,是种可贵的野均含量高达39.50%。这可能与商城肥鲵的生活环境有关其生自然生活在山涧溪水中,水体平均温度较低,高度不饱和脂肪酸的出来作CNMHG质量,这对丰富人们存在有利于生理代谢的正常进行。另外,商城肥鲵富含花生五的物质生活增强人们的体质促进人们身体健康颇有益处。烯酸,其含量分别达到368%和4.37%。EPA和DHA具有极