[生物学]酵母蔗糖酶的提取工艺.doc

酵母蔗糖酶的提取工艺摘要蔗糖酶是一种水解酶, 广泛存在于动物、植物、微生物等各种生物体内。它可以不可逆的催化蔗糖水解为D-葡萄糖和D-果糖，为微生物的生长提供碳源和能源。采用甲苯自溶法、冻融法、SDS抽提法3种方法从酵母中提取蔗糖酶1，冻融法和SDS抽提法的提取效率远高于传统的甲苯自溶法。其中冻融法的效率最高（纯化倍数比活力与总活力），加之其操作简便，更适合于酵母蔗糖酶大规模的制备提取。比较了乙醇分级沉淀、硫酸铵分级沉淀对于冻融法得到的粗提物的沉淀效果，结果表明：50%(w/w)乙醇分级沉淀效果较好（比活力与总活力），乙醇分级沉淀所得蔗糖酶经DEAE-Sepharose 离子交换层析纯化后，制得高纯度的酵母蔗糖酶（比活力与总活力）。纯化倍数为16.14倍，比活性为947.805U/mg，回收率为51.6%。蔗糖酶的酶促动力学性质表明，蔗糖酶的最适PH值为4.5，最适温度为50，酶的特征米氏常数Km值为13.8mmol/L，最大反应速度Vmax为5.98ug/min。关键词：酵母；蔗糖酶；提取；纯化Study on Purification of Invertase from YeastAbstractSucrase is widespread in prokaryotes and eukaryotes .Sucrase catalyzes the irreversible hydrolysis of sucrose into glucose and fructose.the mainfroms of carbon and energy supplies in microorganism growth and development.This paper used three methods to extract invertase from yeast,which included in this manuscript, three different extraction method breaking cells by adding methylbenzene,frost grinding,and adding SDS for extracting invertase from yeast were investigated.Then the purified invertase was obtained by precipitatation with 50% ethyl alcohol、sequential ammonium sulpate precipitation and DEAE-Sepharose lon-exchange chromatography.The purified sucrase was characterized by SDS-PAGE.The results showed all three methods had both advantages and disadvantages.The invertase extracted by adding SDS and frost grinding had much more total activity than that of extracted by adding methylbenzene.A highest total invertase activity was found in the forst grinding,and it was a convent and economical method for commercial production of invertase from yeast.The results of our study were followed:1、 Purification of invertase from yeastThe specific activity was 947.805U/mg,purification fold was 32.28.The activity recovery of sucrase was 51.6%.2、 Properties of sucrase The kinetic characters of the enzyme have been studied.The optimum PH and optimum temperature for the enzyme are PH4.5 and 50.Km is 21mmol/Land Vmax is 6.57ug/min.Key words : yeast;invertase;extraction;purification第一部分 文献综述蔗糖酶（Sucrase,EC 6）又称转化酶（Invertase），是将蔗糖水解成D-葡萄糖和D-果糖的-D-果糖苷酶的一种。除广泛分布于微生物、植物外，类似的活性也在动物的消化液中发现。传统的酵母蔗糖酶的提取方法是甲苯自溶法，尽管此方法所用试剂简单、价格低廉，但由于其消耗时间长、重复性差、酶活性较低等缺陷，现已很少采用。目前也有采用乙醇沉淀法和硫酸铵分级沉淀法制得的蔗糖酶，再用柱层析的方法提纯，由于柱层析工艺复杂，处于实验室探索研究阶段。经查新，已见采用冻融法和SDS抽提法的文献报道。文献说明，相对于甲苯自溶法，这两种方法提取的酶的活性要高出很多。SDS抽提法对酵母蔗糖酶的提取效率比较高，是冻融法的2倍，但其中总蛋白量提取也较高，是冻融法的3倍，因此如果采用SDS法，对酵母蔗糖酶的纯化需要去除较多的杂蛋白本实验结合传统方法和新兴方法，寻找从经济和效率上最优的酵母蔗糖酶的提取工艺3.3 图33.3.2 图43.3.3 3.6.2 最适温度的测定在2170范围内， 设置一系列不同的温度梯度，分别测定各个温度梯度下蔗糖酶的活性，活性最高的温度即为蔗糖酶的最适温度。表六管数01234567891mol/L蔗糖（ml）0.1水（ml）0.55缓冲液（ml）0.25酶液200ul处理温度（1h）室温（21）253035404550556070DNS（ml）1.5沸水浴10min，取出迅速用自来水冷却水（ml）10A54000.1790.2690.5550.7470.8100.8270.7960.6610.33400.1810.2670.5550.7420.8110.8260.7950.6580.32900.1820.2700.5540.7460.8270.7960.6580.333A平均00.1810.2690.5550.7450.8100.8270.7960.6590.3323.6.3 蔗糖酶Km及Vmax的测定以蔗糖为底物，在最适条件下，分别设置不同的底物梯度，测定蔗糖酶催化蔗糖的反应速度，以1/S为横坐标，1/V为纵坐标，按双倒数法作图，可以得到Lineweaver Burk双倒数直线方程，在1/V纵轴的截距是1/Vmax,在1/S横轴上的截距是-1/Km，求出Km和Vmax，双倒数方程如下：表七管数123456781mol/L蔗糖（ml）00.020.030.060.00水（ml）0.60.580.570.540.520.500.450.40缓冲液（PH4.5）（ml）0.25酶液（ml）1恒温55，水浴保温1hDNS（ml）1.5沸水浴10min，取出迅速用自来水冷却水（ml）10A54000.1000.1280.3470.4300.4540.8160.99900.0980.1300.3430.4340.4550.8171.003A平均00.0990.1290.3450.4320.4550.8161.001S016.9518.14222.72833.24783.154580.230980.3021/S0.0590.0550.0440.0300.0120.0020.001V1.1831.2211.3971.8902.6385.7478.8501/V0.8450.8190.7160.5290.3790.1740.1133.7 实验结果与分析3.7.1 离子交换层析图酵母经过冻融法粗提蔗糖酶、硫酸铵分级沉淀后进行DEAE-Sephrose离子交换层析。其洗脱曲线如下图，蔗糖酶集中在稳定后一个小时至第二个小时之间。图5酵母经过冻融法粗提蔗糖酶、乙醇分级沉淀后进行DEAE-Sephrose离子交换层析。其洗脱曲线如下图，蔗糖酶集中在稳定后20min至第二个小时结束之间。图63.7.2蔗糖酶的分纯化计算各步的总蛋白、总活性、比活、回收率和纯化倍数，蔗糖酶比活力为 947.805U/mg，最后纯化倍数为16.14倍，回收率为51.6%。表八步骤方法总蛋白（mg）总活性（U）比活性（U/mg）回收率（%）纯化倍数粗提甲苯自溶法 862.7794475028.6861001冻融法434.1044950058.7421001SDS法511.8444775056.2621001一级纯化乙醇分级沉淀法冻融法24.81434375385.30769.46.56SDS法49.42836375335.91976.25.97硫酸铵分级沉淀法冻融法3.98514750371.38029.86.32SDS法7.76215625313.01232.75.56二级纯化乙醇沉淀+冻融法2.68525667947.80551.616.14乙醇沉淀+SDS法4.47720600733.08043.112.48从上表可以看出，选用乙醇沉淀+冻融法和乙醇沉淀+SDS法是最优组合，但SDS法得到的酶液 ，容易随时间的改变而变化，见下表；表九总活力比活力两周前4775056.262两周后2675038.471这极有可能是因为，SDS为强的蛋白质变性剂，随着时间的变化，其对蛋白质的活性有很大的影响，这在酶的生产过程中对周期的要求就大大增加了，而冻融法却没有这种生产限制：表十总活力比活力两周前4950058.742两周后4892657.6743.7.3 蔗糖酶纯度鉴定 图71冻融法+硫酸铵分级沉淀法得到的酶液2Marker（由Amersham公司提供）3冻融法+乙醇分级沉淀法得到的酶液4冻融法+乙醇分级沉淀法+DEAE-Sephrose离子交换层析得到的酶液5冻融法+硫酸铵分级沉淀法得到的酶液6冻融法+硫酸铵分级沉淀法+DEAE-Sephrose离子交换层析得到的酶液7冻融法+乙醇分级沉淀法得到的酶液8冻融法+乙醇分级沉淀法+DEAE-Sephrose离子交换层析得到的酶液9Marker（由TaKaRa公司提供）10冻融法+硫酸铵分级沉淀法+DEAE-Sephrose离子交换层析得到的酶液从上图可以看出，除了2号泳道和9好泳道有Marker条带，其余没有条带跑出。初步认为，是由于上样量太少（10uml），但由于存样量不够继续加样实验。3.7.4 酶促反应动力学研究 最适PH值 用不同PH值的缓冲液体系代替原来的缓冲液体系，然后按常规测定条件和方法测定酶活。结果表明，酵母蔗糖酶在PH4.5的条件下活性最大（如下图所示）。图 蔗糖酶最适温度 在一系列不同温度下测定蔗糖酶的活性，结果表明，蔗糖酶的最适温度在50左右。（如下图所示）图 蔗糖酶Km及Vmax 在蔗糖酶的最适温度和PH下，测定不同底物浓度时的反应速度，按测定结果计算1/S和1/V，并按双倒数法作图，得到线性回归方程（如下图），求得蔗糖酶的Km为13.8mmol/L，最大反应速度Vmax为 5.98ug/min。图10第四部分 讨论4.1 蔗糖酶的分离纯化 在进行实验之前，根据已有的文献报道和实验室现有的条件设计了一条常规的纯化路线，即粗提、分级沉淀、DEAE-Sepharose离子交换层析。在本实验中经过DEAE-Sepharose离子交换层析后，得到了纯的蔗糖酶。酵母蔗糖酶的提取较常使用甲苯自溶法的，冻融法和SDS抽提法报道较少。本实验中比较了上述三种酵母蔗糖酶的提取方法（见表八），可以看出SDS抽提法和冻融法的酶活性远高于甲苯自溶法的。尽管甲苯自溶法所用试剂简单、价格低廉，但由于其耗时长、重复性差、酶活性低等缺陷，提取效率远不如其它两种方法。通过对不同放置时间相同SDS浓度提取酵母蔗糖酶效果的比较，可以看出（见表九），随着放置时间的增加，酶的总活力和比活力都大大下降了，而冻融法却不然（见表十），随着放置时间的增加，酶的总活力和比活力几乎没变，因此，SDS抽提法在提取酵母蔗糖酶的过程中对操作周期的要求要比冻融法要严格的多！综合各个资料中对几种物种蔗糖酶的分离纯化实验，一般都是通过乙醇分级沉淀。乙醇分级沉淀法是指在混合组分的溶液中加人与该溶液能互溶的溶剂，通过改变溶剂的极性而改变混合组分溶液中某些成分的溶解度，使其从溶液中析出。如在含有糖类或蛋白质的水溶液中，分次加入乙醇,使含醇量逐步增高，逐级沉淀出分子量段由大到小的蛋白质、多糖、多肽在含皂苷的乙醇溶液中分次加入乙醚或丙酮可使极性有差异的皂苷逐段沉淀出来等。与硫酸铵分级沉淀相比，该方法更简便易行，时间周期要求更短，且对操作温度没有限制，还可以有杀菌的作用。离子交换层析是分离蛋白质应用最广泛的技术，蛋白质的两性特征意味着他们可以依PH值不同而呈阳离子或阴离子状态存在，与离子交换剂之间进行结合。因此，蛋白质的洗脱可用离子强度递增的梯度方式进行，也可以用PH梯度方式进行。由于PH值的变化可能导致酶的失活，故一般用离子强度递增的方法来纯化酶。蔗糖酶分为酸性蔗糖酶、中性蔗糖酶和碱性蔗糖酶。本文利用DEAE-Sephardse柱层析后得到的纯蔗糖酶，其最适PH为4.5，应属酸性蔗糖酶，和资料18中的实验结果大体上是一致的。4.2 蔗糖酶的酶学性质实验结果表明，蔗糖酶的最适反应条件为PH4.5，55C。反应条件较容易满足，不会给生产厂家带来控制反应条件的压力。酵母蔗糖酶的Km和Vmax与微生物来源的蔗糖酶进行比较，与其他微生物的相近，和文献中的相近。但和植物中的Km有一定差别，植物中的Km一般都比微生物中的小，提示植物叶片组织比其他物种具有更强的糖代谢水平，可能是由于在植物的一生都需要蔗糖酶的参与来利用蔗糖等，为植物的生长发育、渗透调节来发挥功能19。参考文献页：101 李楠,庄苏星,丁益. 酵母蔗糖酶的提取方法J食品与生物技术学报, 2007,(04). 页：102刘晓雯.小肠蔗糖酶的分离纯化及部分性质:硕士学位论文.成都:四川大学,2002.41-49页：103Isla M,Salerno,Ponitis H,et al.Purification and Properties of the acid invertase from oryza sativa.Phytochemistry.1995,38:321-3254冯希平，刘正.测定葡萄糖水平观察儿童唾液蔗糖酶活性.上海第二医科大学学报.1996,16(4);269-2715Karialainen S.Sucrase activity in relation to other salivary factors and DMFS values of dental students .Acta Odontal Scand.1990,48:1836翁梅倩,吴圣嵋.肠发育于糖消化、吸收的分子学研究进展.国外医学儿科学分册.1996,26(3):155-1577Zweivaum A,Triadou N,Kedinger M,et al.Sucrase isomaltase:a marker of fetal and malignant epithelial cells of the human colon ,Int J,Cancer.1989,44:2388陈未顺,于皆平,沈志祥,等.蔗糖酶作为大肠腺瘤恶变倾向标志的研究.中国肿瘤临床.1996,23(8):542-5449NS Jensen,TB Stanton.Production of an inducible sucrade activity by hyodysenieriae.Appl Environ.Microbiol.1994,60(9):3429-343210陈庆森.固定化活性干酵母细胞生产转化糖的研究.天津商学院学报.1994,3:11-1511陈庆森.王育,张莉,等.利用产黄青霉制备高活力蔗糖转化酶,食品科学.1997,18(12):18-2212Dan V.Selection of come active strains of Aaspergillus niger as -fructofuranosidase producers.Industrial Microbioligy.1984,4:355-36713周世萍,段吕群,韩青,等. 毒死蜱对土壤蔗糖酶活性的影响。生态环境.2005,14(5):672-67414王聪颖,和文祥,何敏超,等.乙醇对土壤转化酶活性的影响.中国环境科学.2006,34(9):125-129页：1015E.Sahmetlioglu et al./Reactive&Functional Polymers 66(2006)365371页：1016 Mohammadia.Aminea.Rhazi.et al.Use the amperometric determination of phenylmercury based on invertase enzyme inhibition.Talanta.2004.62:951-958.页：1017 Laemmli U K.Cleavage lf structural proteins during the assembly of the head of bacretiophage T41J.Nature,1970,227:680-6851.18余冰宾.生物化学实验知道.北京:清华大学出版社,200419张振清,夏叔芳.大豆叶片蔗糖酶的分离纯化及其特征.植物生理学报.1984,10(1);19-26致谢时光飞逝，随着即将到来的论文答辩日子的临近，四年的大学生活即将结束，就要离开这个曾经生活、奋斗了四年的地方，每想及此，心中就有许多的牵挂和舍不得，更有许多的感谢！首先感谢化工学院给我这次学习的机会，在这几年的学习生活中得到了化工学院的各位老师和领导的大力支持和帮助。感谢所有教育帮助我的各位老师，是你们给予我的专业知识，为我完成论文打下基础。本论文是在导师崔志芳教授的悉心指导和严格要求下完成的。从论文的选题论证，实验方案的确定与实施，实验的进行，论文的构思与撰写，直至定稿，无不倾注导师的大量心血和汗水。回首大学生活，崔老师务实创新的科研精神、严谨治学的科学态度、高尚的道德情操、兢兢业业的工作作风、平易近人的学者风范，潜移默化的影响着我的人生观、价值观，使我提高了对科学研究的认识，使我在思想上学习上不断前进，恩师在学习上的关怀使我感激至深，在此，谨向崔志芳教授致以最崇高的敬意和最真挚的感谢。在此感谢师姐季爱云和同学刘娜、尹传志、景新秀、郭兴华、张旭、刘荣明、王雷、宁停波，有了你们的帮助，实验才顺利进行；有了你们的支持，实验过程多了更多的欢声笑语，实验生活不再枯燥。也在此感谢室友吴少婷以及孙洪林、刘彩平、王云先和宋新妥在学习和生活中给予的关心帮助。最后还要感谢我的父母和亲人多年来对我学习的关心与鼓励，感谢我的好朋友长期以来给予我的帮助和支持。 任娜 2008-6-5Immobilization of invertase and glucose oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol)with pyrroleErtugrul Sahmetlioglua,Huseyin Yuruka,Levent Toppareb,*,Ioan Ciangac,1,Yusuf YagcicaDepartment of Chemistry,Nigde University,51100 Nigde,TurkeybDepartment of Chemistry,Middle East Technical University,06531 Ankara,TurkeycDepartment of Chemistry,Istanbul Technical University,80626 Maslak,Istanbul,TurkeyReceived 22 November 2004;received in revised form 10 August 2005;accepted 10 August 2005Available online 21 September 2005AbstractIn this study,immobilizations of invertase and glucose oxidase were achieved in conducting thiophene functionalizedcopolymers of vinyl alcohol with thiophene side groups and pyrrole(PVATh/PPy)via electrochemical polymerization.Thekinetic parameters,Vmax(maximum reaction rate)and Km(substrate a?nity),of both free and immobilized enzymes weredetermined.The e?ect of supporting electrolytes,p-toluene sulfonic acid and sodium dodecyl sulfate,on the enzyme activ-ity and film morphologies was examined.The optimum temperature,operational and storage stabilities of immobilizedenzymes were determined.PVATh/PPy copolymer was found to exhibit significantly enhanced properties compared topristine polypyrrole.2005 Elsevier B.V.All rights reserved.Keywords:Immobilization;Invertase;Glucose oxidase;Poly(vinyl alcohol);Electropolymerization1.IntroductionIn development of biosensors composed of conductive polymers and enzymes1,2,glucose oxidase or invertase entrapments are usually made by electrochemical polymerization of pyrrole in aqueous solution containing glucose oxidase or invertase.This preparation is much attractive for microelectronic fabrication technique3.The advantages of electropolymerization can be easily summarized as achievement of new properties by using various supporting electrolytes or monomers and the control of the film thickness by regulating the amount of charge passed48.Electrochemical immobilization;covalent attachment3and chemical crosslinking911are additional examples of techniques used in glucose oxidase or invertase immobilization .1381-5148/$-see front matter 2005 Elsevier B.V.All rights reserved.doi:10.1016/j.reactfunctpolym.2005.08.009*Corresponding author.Tel.:+90 3122103251;fax:+903122101280.E-mail address:.tr(L.Toppare).1On leave fromPetru PoniInstitute of MacromolecularChemistry,Iasi,Romania.Biosensors containing enzymes have been widely applied in chemistry and biology due to their high sensitivity and potential selectivity,in addition to low cost and possibility of miniaturization or automation.Glucose oxidase(E.C.)catalyzes the oxidation of b-D-glucose to D-glucono1,5-lactone and hydrogen peroxide,using molecular oxygen as the electron acceptor.Glucose oxidase is widely used for the determination of glucose in body fluids and removing residual glucose and oxygen from beverages and foodstu?s46.Invertase,known as b-froctofuranosidase(E.C.6),catalyzes the hydrolytic breakdown of sucrose to glucose and fructose.The mixture of theseproducts has a lower crystallinity than sucrose at high concentrations and does not crystallize out like sucrose.The usage of invertase confectionary thus ensures that the products remain fresh and soft even when kept for a long time.Therefore,it is widely used in the production of artificial honey and to a small extent in the industrial production of liquid sugar4,6.Poly(vinyl alcohol)is a polymer that is frequently used as a matrice for the immobilization of various enzymes and cells because of its availability,low price,hydrophilic character and hydroxyl groups on the surface that is capable of chemical reactions 12,13.In this study,immobilization of invertase within poly(vinyl alcohol)with thiophene side groups and pyrrole copolymer matrices were investigated.The synthesis and characterization of conducting copolymer of PVATh/PPy were reported in an earlier study14.Kinetic parameters(reaction rate Vmax and Michaelis Menten constant Km)of free enzyme and enzyme electrodes were determined.Temperature optima,operational and storage stabilities of immobilized invertase or glucose oxidase were examined.2.Experimental2.1.Material and technique Invertase(EC 6)Type V,glucose oxidase (EC )Type II-S,peroxidase(EC ) Type II,o-dianisidine were purchased from Sigma and used as received without further purification.Poly(vinyl alcohol)with thiophene side chains (PVATh)were prepared as described previously (Scheme 1)14.Pyrrole(Merck)was distilled before use and stored at 4 C.Sodium dodecyl sulfate (SDS)and p-toluene sulfonic acid(PTSA),sulfuric acid,hydrogen peroxide and sodium hydroxide were supplied from Merck. Potentioscan Wenking POS-73 and ST-88 potentiostats,Shimadzu UV-160-A model spectrophotometer and Leon 440 model scanning electron microscope(SEM)were used.2.2.Immobilization of invertase and glucose oxidase in PVATh/PPy matricesImmobilization of invertase and glucose oxidase was performed in a typical three electrode cell containing platinum foil(1 cm1 cm)working and counter electrodes and an Ag/Ag + (0.01 M)reference electrode by constant potential electrolysis at room temperature.Electrolysis solution consists of invertase(1 mg/mL)or glucose oxidase(2 mg/ mL);SDS(0.6 mg/mL)or PTSA(0.6 mg/mL)as the supporting electrolytes,pyrrole(0.02 M)and acetate bu?er(pH 5).Polymerization reactions were carried out by applying 1.0 V potential di?erence for 1 h between the reference and working electrodes.Immobilization of enzymes was carried out on both bare and PVATh coated electrodes.After electrolysis,enzyme electrodes(30 lm)were washed with distilled water in order to remove both the excess supporting electrolyte and un-bound enzyme,and kept in acetate bu?er at 4 C when not in use.2.3.Determination of invertase activity Determination of immobilized and free invertase activities was performed by using Nelson?smethod15.Di?erent concentrations of sucrose solution were preincubated for 10 min at 25 C.Then,enzyme electrode was placed in sucrose solutions for specific reaction times(2,4 and 6 min).After removing the electrode,1 mL aliquots were drawn and added to 1 mL Nelson?s reagent to terminate the reaction.The tubes were then placed in boiling water bath for 20 min,then they were cooled down and 1 mL arsenomolybdate reagent was added.Finally,7 mL of distilled water was added to each test tube and mixed byvortexing.After mixing,absorbances for the blank and the substrate solutions were determined at 540 nm with a double beam spectrophotometer.One unit of invertase activity was defined as the amount of enzyme required to release 1 lmol glucose from sucrose per minute at pH 5 and 25 C.2.4.Determination of glucose oxidase activityThe activity determination was performed by using a modified version of Sigma Bulletin16.For free glucose oxidase activity determination,glucose solutions were placed in test tubes and incubated at 25 C.After addition of 0.1 mL enzyme solution,enzyme and substrate were allowed to react for specific times(2,4 and 6 min).Then,0.1 mL POD(60 U/mL)to catalyze the reaction of hydrogen peroxide and 2.4 mL o-dianisidine (0.21 mM)as the coloring agent were added.The reaction was terminated with the addition of 0.5 mL(2.5 M)sulfuric acid,and spectrophotometric measurements were performed at 530 nm.For the determination of the activity of immobilized enzyme,the reaction was started by placing enzyme electrode in glucose solution and after specific reaction times(2,4 and 6 min),0.5 mL of aliquots were drawn,the rest of the procedure was the same given for free enzyme activity.H2O2 standard calibration curve was used in order to define enzyme activity.One unit of glucose oxidase activity(EU)was defined as the amount of enzyme required to produce 1 lmol of D-gluconic acid and H2O2 per minute at pH 5 and 25 C.2.5.Determination of kinetic parametersIn order to determine maximum velocity of the reaction(Vmax)and the MichaelisMenten constant (Km)for each electrode,activity assay was applied for di?erent concentrations of sucrose and glucose.2.6.Determination of optimum temperature Optimum temperatures for immobilized invertase and glucose oxidase were determined by changing incubation temperature between 1570 C and 1560 C,respectively,while keeping substrate concentration constant.2.7.Morphologies of the films The morphologies of polymer films both with and without enzyme were investigated.After peeling o?the films from electrode and washing with bu?er solution for several times to remove unbound enzyme and excess supporting electrolyte from the surface of film,SEM analyses were perfo