[生物学]酵母蔗糖酶的提取工艺

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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 Yeast Abstract

Sucrase is widespread in prokaryotes and eukaryotes .Sucrase catalyzes

the irreversible hydrolysis of sucrose into glucose and fructose.the mainfroms 实用文档

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 yeast

The specific activity was 947.805U/mg,purification fold was 32.28.The activity recovery of sucrase was 51.6%.

2、 Properties of sucrase

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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 3.2.1.26）又称转化酶（Invertase），是将蔗糖水解成D-葡萄糖和D-果糖的ß-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

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3.3

葡萄糖浓度标准曲线0.60.50.40.30.20.1000.10.20.3葡萄糖浓度（mg/mL）0.40.5

y = 1.625x - 0.11412R = 0.9988OD值图3

3.3.2

标准蛋白曲线0.60.50.4y = 0.0538x - 0.00882R = 0.9958OD值0.30.20.10-0.1024681012

标准蛋白浓度（mg/mL）

图4

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3.3.3

3.6.2 最适温度的测定

在21℃～70℃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 1mol/L蔗糖（ml）水（ml）缓冲液（ml）酶液 0 1 2 3 4 0.1 5 6 7 8 9 0.55 0.25 200ul 温25℃ 30℃ 35℃ 40℃ 45℃ 50℃ 55℃ 60℃ 70℃ 处理温室度（1h）（21℃） DNS（ml）沸水浴10min，取出迅速用自来水冷却水（ml） A540 0 0 10 0.179 0.269 0.555 0.747 0.810 0.827 0.796 0.661 0.334 0.181 0.267 0.555 0.742 0.811 0.826 0.795 0.658 0.329 1.5 实用文档

0 0 0.182 0.270 0.554 0.746 —— 0.827 0.796 0.658 0.333 A平均

0.181 0.269 0.555 0.745 0.810 0.827 0.796 0.659 0.332 3.6.3 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：

1Km[S]Km11 VVmax[S]Vmax[S]Vmax表七

管数 1mol/L蔗糖（ml）水（ml） 1 0 0.6 2 3 4 0.06 0.54 5 0.08 0.52 6 0.10 0.50 7 0.15 0.45 8 0.20 0.40 0.02 0.03 0.58 0.57 缓冲液（PH4.5）（ml） 0.25 酶液（ml） 1 恒温55℃，水浴保温1h DNS（ml） 1.5 沸水浴10min，取出迅速用自来水冷却水（ml）实用文档

10 A540 0 0.100 0.128 0.347 0.430 0.454 0.130 0.343 0.434 0.455 0.129 0.345 0.432 0.455 18.142 22.728 33.247 83.154 0.816 0.999 0 0.098 0.817 1.003 A平均 0 0.099 0.816 1.001 [S] 0 16.95 580.230 0.002 980.302 0.001 1/[S] 0.059 0.055 0.044 0.030 0.012 1.221 1.397 1.0 2.638 0.819 0.716 0.529 0.379 V 1.183 5.747 8.850 1/V 0.845 0.174 0.113 3.7 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。

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示数6050示数 4030201000离子交换层析图离子交换层析图50100时间离子交换层析图150200 605060 4050306040图5 205030104020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。03010050100150200-1020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。0时间10050100150200-100时间50100150200 -100示数示数示数时间

图6

3.7.2蔗糖酶的分纯化

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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

步骤方法总蛋白总活性（U）比（mg）活性回收率纯化倍（%） 100 100 100 69.4 76.2 数 1 1 1 6.56 5.97 （U/mg） 28.686 58.742 56.262 385.307 335.919 粗提甲苯自溶法冻融法 SDS法 862.779 44750 434.104 49500 511.844 47750 24.814 49.428 34375 36375 一级纯乙醇分冻融法化级沉淀SDS法法硫酸铵冻融法分级沉SDS法淀法二级纯乙醇沉淀+冻融法化乙醇沉淀+SDS法

3.985 7.762 14750 15625 371.380 313.012 29.8 32.7 6.32 5.56 2.685 4.477 25667 20600 947.805 733.080 51.6 43.1 16.14 12.48 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；实用文档

表九

总活力 47750 26750 比活力 56.262 38.471 两周前两周后

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：

表十

总活力 49500 426 比活力 58.742 57.674 两周前两周后

3.7.3 蔗糖酶纯度鉴定

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图7

1——冻融法+硫酸铵分级沉淀法得到的酶液 2——Marker（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 9——Marker（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

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3.7.4.1 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）。

1相对吸光度0.80.60.40.20024蔗糖酶最适PH值6PH8

图8

3.7.4.2 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）

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0.90.80.70.60.50.40.30.20.1020304050607080温度（℃）相对吸光度蔗糖酶最适温度图9

3.7.4.3 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。

10.80.60.40.200y = 12.115x + 0.1671R2 = 0.97480.020.040.060.08

蔗糖酶Km及Vmax测定

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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

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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，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PH4.5，55°C。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]。

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参考文献页：16

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[3]Isla M,Salerno,Ponitis H,et al.Purification and Properties of the acid invertase from oryza sativa.Phytochemistry.1995,38:321-325

[4]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);269-271

[5]Karialainen S.Sucrase activity in relation to other salivary factors and DMFS values of dental students .Acta Odontal Scand.1990,48:183

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[7]Zweivaum A,Triadou N,Kedinger M,et al.Sucrase isomaltase:a marker of fetal and malignant epithelial cells of the human colon ,Int J,Cancer.19,44:238 [8]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

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[9]NS Jensen,TB Stanton.Production of an inducible sucrade activity by hyodysenieriae.Appl Environ.Microbiol.1994,60(9):3429-3432

[10]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.1994,3:11-15 [11]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):18-22

[12]Dan V.Selection of come active strains of Aaspergillus niger as ß-fructofuranosidase producers.Industrial Microbioligy.1984,4:355-367 [13]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):672-674

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[17] Laemmli U K.Cleavage lf structural proteins during the assembly of the head of bacretiophage T41[J].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,2004

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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。实用文档

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 2008-6-5

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Immobilization of invertase and glucose oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol)with pyrrole

Ertugrul

Sahmetlioglua,H¨useyin

Y¨ur¨uka,Levent

Toppareb,*,Ioan

Ciangac,1,Yusuf Yagcic

aDepartment of Chemistry,Nigde University,51100 Nigde,Turkey

bDepartment of Chemistry,Middle East Technical University,06531 Ankara,Turkey

cDepartment

Chemistry,Istanbul

Technical

University,80626

Maslak,Istanbul,Turkey

Received 22 November 2004;received in revised form 10 August 2005;accepted 10 August 2005

Available online 21 September 2005

Abstract

In this study,immobilizations of invertase and glucose oxidase were achieved in conducting thiophene functionalized

copolymers of vinyl alcohol with thiophene side groups and pyrrole(PVATh/PPy)via electrochemical polymerization.The 实用文档

kinetic parameters,Vmax(maximum reaction rate)and Km(substrate a?nity),of both free and immobilized enzymes were

determined.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 immobilized

enzymes were determined.PVATh/PPy copolymer was found to exhibit significantly enhanced properties compared to

pristine polypyrrole.

1.Introduction

In development of biosensors composed of conductive polymers and enzymes[1,2],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

technique[3].The

advantages

oxidase;Poly(vinyl

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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 passed[4–8].Electrochemical

immobilization;covalent

attachment[3]and

chemical crosslinking[9–11]are additional examples of techniques used in glucose oxidase or invertase immobilization .

*Corresponding author.Tel.:+90 3122103251;fax:+90 3122101280.

E-mail address:toppare@metu.edu.tr(L.Toppare).

1On leave from‘‘Petru Poni’’Institute of Macromolecular Chemistry,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.1.1.3.4)catalyzes the oxidation of b-D-glucose to D-glucono1,5-lactone and hydrogen peroxide,using molecular oxygen as the 实用文档

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?s[4–6].

Invertase,known

b-froctofuranosidase(E.C.3.2.1.26),catalyzes

the

hydrolytic breakdown of sucrose to glucose and fructose.The mixture of these

products 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 sugar[4,6].

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 study[14].Kinetic parameters(reaction rate Vmax and

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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.Experimental

2.1.Material and technique Invertase(EC 3.2.1.26)Type V,glucose oxidase (EC 1.1.3.4)Type II-S,peroxidase(EC 1.11.1.7) 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 matrices Immobilization of invertase and glucose oxidase was performed in a typical three electrode cell containing platinum foil(1 cm·1 cm)working and counter electrodes and an Ag/Ag + (0.01 M)reference electrode by constant potential 实用文档

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?smethod[15].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 实用文档

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 by

vortexing.After mixing,absorbances for the blank and the substrate solutions

were

determined

540

with

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 activity

The activity determination was performed by using a modified version of Sigma Bulletin[16].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

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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 parameters

In order to determine maximum velocity of the reaction(Vmax)and the Michaelis–Menten 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 15–70 C and 15–60 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 performed.2.8.Operational stabilities Operational

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stabilities of immobilized enzymes in polymer films were tested(at optimum activity assay conditions)by performing 30 activity assays during one day.3.Results and discussion 3.1.E?ect of supporting electrolytes on immobilization

Di?erent

supporting

electrolytes,p-toluene

sulfonic

acid(PTSA)and sodium dodecyl sulfate (SDS)were used for the immobilization of invertase and glucose oxidase[4–7].Apparently,invertase immobilized matrices in the presence of SDS,exhibited the highest activity.On the other hand,for glucose oxidase immobilized matrices no activity was detected when PTSA was used as the electrolyte.The electrolysis times for immobilizations of invertase and glucose oxidase were also di?er.Sixty minutes of electrolysis was su?cient with SDS whereas 120 min were required when PTSA was used.Therefore,SDS was used as the supporting electrolyte for both enzymes due to minimum time requirement for the immobilization and high activity.

3.2.E?ect of temperature on the activity of enzymes

The e?ect of temperature on the enzyme electrodes was investigated and given in Fig.1(a)and (b).The maximum activity was found at 50 C for the enzyme electrode.The activity of the free invertase is strongly dependent on temperature,with the optimum temperature being observed between 40 and

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60 C.As the temperature is further increased above the optimum temperature,the structure of the enzyme becomes altered and consequently,its catalytic properties are reduced,and eventually destroyed.The optimum temperatures for free invertase and enzyme entrapment in pristine PPy were determined previously as 50 and 60 C,respectively [4].We have also investigated the enzyme activity for glucose oxidase with respect to temperature for PVATh/PPy and PPy matrices(Fig.2(a)and(b),respectively).The maximum activity was observed at 30 C for both matrices.For free glucose oxidase,optimum temperature of about 30 C was reported [5].PVATh/PPy enzyme electrode exhibits higher stability against temperature change when compared to PPy electrode between 40 and 60 C.

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3.3.Kinetic parameters of immobilized enzymes

The maximum reaction rate,Vmax,and Michealis–Menten constant,Km were obtained from Lineweaver–Burk plots[17](Tables 1 and 2).The kinetic behavior for invertase was significantly changed via immobilization.Kinetic constant,Km values for the immobilized invertase were found to be higher than that of the free invertase.The increase in Km results from the change in the a?nity of the enzyme to its substrate and is probably caused by the structural change of the 实用文档

enzyme upon entrapment in the matrice consisting polymer

chains.Moreover,the di?erence in the Km values of the di?erent polymer matrices may be due to the porosity di?erences of the matrices.The decrease in the reaction rate,Vmax,may be due to the restricted di?usion of substrate to the enzyme.Km values for immobilized glucose oxidase are slightly higher than that of the free counterpart.The increase of the Km values upon immobilization is due to a lower a?nity of the immobilized enzyme for the substrate compare to that of the free enzyme.Increased Km and decreased Vmax values for the immobilized enzymes have been reported in previous immobilization studies[4–6,18]

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3.4.Operational stability and shelf life of the enzyme electrodes

In order to determine the operational stabilities of immobilized enzymes,activity assays were performed during the same day.For both invertase (Fig.3(a))and glucose oxidase(Fig.3(b)),immobilized electrodes activities were 实用文档

determined.This study was carried out for both PVATh/PPy and PPy matrices for 30 successive measurements at 25 C.There were small fluctuations for the operational stabilities of both immobilized enzyme electrode activities.For both enzymes;PVATh/Ppy copolymer exhibited higher stability compared to PPy matrice.Invertase immobilized in PVATh/PPy has reasonable storage stability(Fig.4).In 30 days 80%of the invertase activity is lost.Immobilization glucose oxidase has a storage stability (Fig.5)of only 15 days.It should be pointed out that in general these matrices exhibit no storage stability[6].In our case the best operational stabilities were recorded.

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3.5.Morphology of immobilized enzymes

Scanning electron microscopy(SEM)was used to observe the surface changes of the films when the enzyme was immobilized.The films were washed in order to remove unbound enzymes.The surface morphologies of these films were completely di?erent compared to the films produced in the absence of 实用文档

invertase and glucose oxidase.On the solution side of films,the cauliflower like structure was significantly damaged when invertase(Fig.6(a)and(b)),and glucose oxidase (Fig.7(a)and(b))were entrapped in each polymer matrice.

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4.Conclusions

Immobilization of invertase and glucose oxidase were successfully achieved for PVATh/PPy and PPy matrices.SDS was found to be the best supporting electrolyte for the entrapments.The electrolysis conditions and their influence on several parameters such as enzyme activity,temperature and Km and Vmax values were investigated.Km values for the immobilized invertase were found to be significantly higher than that of the free invertase.The corresponding value for immobilized glucose oxidase was only slightly higher.

Acknowledgement

This work is partially supported by Research Foundation of Nigde University(FEB 2002-01),Nigde,Turkey.

References

[1] S. Cosnier, Biosens. Bioelectron. 14 (1999) 443. [2] W. Schuhmann, Biosens. Bioelectron. 10 (1995) 181. [3] K. Kojima, T. Unuma, T. Yamauchi, M. Shimomura, S. Miyauchi, Synth. Met. 85 (1997) 1417.

[4] A. Gu¨rsel, S. Alkan, L. Toppare, Y. Yagci, Reac. Func. Polym. 57 (2003) 57.

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[5] S. Tirkes?, L. Toppare, S. Alkan, U. Bakir, A. Onen, Y. Yagci, Int. J. Bio. Macr. 30 (2002) 81.

[6] S. Is??k, S. Alkan, L. Toppare, I. Cianga, Y. Yagci, Eur. Polym. J. 39 (2003) 2375.

[7] A. Cirpan, S. Alkan, L. Toppare, Y. Hepuzer, Y. Yagci, Bioelectrochemistry 59 (2003) 29.

[8] R. Erginer, L. Toppare, S. Alkan, U. Bakir, Reac. Func. Poly. 45 (2000) 227.

[9] S.F. D?Souza, S.S. Godbole, J. Biochem. Biophys. Meth. 52 (2002) 59.

[10] D. Pan, J. Chen, S. Yao, L. Nie, J. Xia, W. Tao, Sensors Actuators B, in press.

[11] S. Brahim, D. Narinesingh, A. Guiseppi-Elie, J. Mol. Catal. B 18 (2002) 69.

[12] S. Akgo¨l, Y. Kac?ar, A. Denizli, M.Y. Arica, Food Chem. 74 (2001) 281.

[13] T. Uhlich, M. Ulbricht, G. Tomaschewski, Enzy. Micr. Tech. 19 (1999) 124.

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[14] E. Sahmetlioglu, H. Yuruk, L. Toppare, I. Cianga, Y. Yagci, Polym. Int. 53 (2004) 2138. [15] N. Nelson, J. Biol. Chem. 153 (1944) 375.

[16] Sigma Techinal Bulletin No. 510. The enzymatic colorimetric determination of glucose, Sigma Chemical Co., St Louis, MO, USA, 1983.

[17] H. Lineweaver, D. Burk, J. Am. Chem. Soc. 56 (1934) 658.

[18] S. Alkan, L. Toppare, Y. Yagci, Y. Hepuzer, J. Biomater.: Sci. Poly. Ed. 10 (1999) 1223.

用固定化蔗糖和葡萄糖氧化酶生产带有噻吩功能聚吡咯共聚物

摘要

在这项研究中，固定化蔗糖和葡萄糖氧化酶应用在通过电化学聚合生产带噻吩功能

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聚吡咯共聚物。动力学参数Vmax （最大反应速率）和Km（底物亲和系数）是由自由的和固定化酶共同决定的。要检查支持电解质对甲苯磺酸和十二烷基硫酸钠，对酶的活性的影响。已经测定出最适温度，操作和稳定储存固定化酶的状态。 PVATH/PPy被发现比传统的聚吡咯显示出更强的性能。

关键词：固定化;蔗糖;葡萄糖氧化酶;聚乙烯醇;电化学聚合 1 .简介

在由生产生物传感器中的聚合物和酶[ 1,2 ]发展过程中，葡萄糖或蔗糖氧化酶通常在含有葡萄糖氧化酶或蔗糖水溶液中由电化学聚合吡咯制得。这个准备技术需要微电子制造技术的支持[ 3 ] 。电化学优势可以很容易地概括为利用各种支持电解质或单体和通过调节通过的分子量控制薄膜厚度 [ 4-8 ] 获得新物质。还有其他像电化学固定化;共价结合[ 3 ]和化学交联[ 9-11]技术被应用在葡萄糖或蔗糖氧化酶固定化上。

含有酶的生物传感器除了成本低，可以小型化，或自动化外还由于其高灵敏性和潜在的选择性，已被广泛适用于化学和生物学。

葡萄糖氧化酶（ E.C. 1.1.3.4 ）用分子氧作为电子受体催化（？-D-glucose,5 ）生成（-内酯过氧化氢）的氧化反应。葡萄糖氧化酶被广泛应用于在体液和从饮料食品消除残余的葡萄糖和氧气后葡萄糖 [ 4-6 ]含量的测定。

众所周知蔗糖氧化酶也就是呋喃果糖酶（ E.C. 3.2.1.26 ），催化水解蔗糖生成葡萄糖和果糖。在高浓度下这些产物比蔗糖具有较低的结晶度并不像蔗糖发生结晶。使

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用蔗糖氧化酶糖果可确保产品在相当长的时间保持新鲜和柔软。因此，它被广泛用在人工蜂蜜生产中和在工业生产的液体糖[ 4,6 ] 也有一定的应用。

聚乙烯醇是一种聚合物是经常做为固定各种酶和细胞的一个胶联剂，因为它的这种能力，廉价，亲水性的性质和表面上有能进行化学反应的羟基 [ 12,13 ] 。

在这项研究中，对蔗糖氧化酶固定化中使用的聚乙烯醇与带有噻吩方官能团的吡咯共聚物胶联剂进行了研究。在早先的研究中已有有关生产PVATH/PPy 及其性质的报道[ 14 ] 。对自由酶和酶电极的动力学参数（反应速度,Vmax 和米氏常数Km）进行了测定。已经研究过固定化蔗糖或葡萄糖氧化酶的最适温度，条件和储存稳定性。 2 .实验

2.1.材料和方法

蔗糖氧化酶（E.C.3.2.1.26 ） V型，葡萄糖氧化酶（E.C.1.1.3.4 ）II- S，过氧化物酶（E.C.1.11.1.7 ） II型， O- dianisidine购自西格玛没有进一步的净化直接用。带有噻吩侧链聚乙烯醇（ PVATH ）以前面所述准备（计划1 ） [ 14 ] 。吡咯（默克公司）是蒸馏前使用和存放于4 0c 。十二烷基硫酸钠（ SDS ）和对甲苯磺酸（PTSA），硫酸，过氧化氢和氢氧化钠购自默克公司。

POS- 73和ST - 88，岛津紫外- 160 -模型分光光度计和里昂440模型扫描电子显微镜（ SEM ）。

2.2 . 在PVATH/PPy 的固定化蔗糖和葡萄糖氧化酶

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固定化蔗糖和葡萄糖氧化酶表现在铂箔（ 1厘米× 1厘米）一个典型的三极细胞的工作和反电极和一个银/银离子（ 0.01米）是指在室温电极恒电位电解法。电解解决方案：蔗糖（ 1毫克/毫升）或葡萄糖氧化酶（ 2毫克/ 毫升） ;的SDS （ 0.6毫克/毫升）或PTSA （ 0.6毫克/毫升）作为支持电解质，吡咯（ 0.02米）和醋酸缓冲液（ pH 5 ）。进行聚合反应了应用在1小时内潜在的差异为1.0 v的范围和工作电极。固定化酶进行列于都暴露了和PVATH涂层电极。电解后，（ 30体育馆）酶电极用蒸馏水水洗，以消除无论是过剩的支持电解质交联

酶的约束，并在不使用的时候保存在4 0c醋酸盐缓冲液中 2.3 .蔗糖酶活力测定

使用纳尔逊的方法[ 15 ]测定固定化和自由的蔗糖酶活力。不同浓度的蔗糖溶液在25 0c水浴10分钟。然后，将酶电极在蔗糖溶液中放置特定的反应时间（ 2 ， 4和6分钟）。在移出电极后，制备1毫升的aliquots并添加到1毫升纳尔逊试剂来终止反应。然后试管在沸水浴中放置20分钟，待试剂冷却下来，加入 1毫升arsenomolybdate试剂。最后， 7毫升蒸馏水被添加到每个试管中并混合均匀。混合实用文档

后，用双光束分光光度计在540 Nm测黑体和对照吸光度。一个单位的蔗糖酶活力定义为在pH 5和25 0c下每分钟，分解蔗糖释放1umol 葡萄糖所需酶的量。

2.4 .，葡萄糖氧化酶活力测定

活力测定的方法是根据修改后的版本西格玛公告来确定的[ 16 ] 。对于测定自由的葡萄糖氧化酶的活力，葡萄糖溶液被放在试管中在25 0c水浴。然后加入0.1毫升酶溶液，酶和底物在特定的时间（ 2 ， 4和6分钟）里反应。然后,加入0.1毫升过氧化物酶（ 60 U / ml的）和2.4毫升O型dianisidine （ 0.21毫米）作为着色剂催化过氧化氢反应。再加上0.5毫升（ 2.5M）硫酸终止反应，，在530 nm测量吸光度。为了测定固定化酶的活力，反应是从把酶电极在葡萄糖溶液后开始，反应特定的时间（ 2 ， 4和6分钟）， 0.5毫升的aliquots人得出的，其余的程序与测定自由酶活性相同。 H2O2标准校准曲线用以确定酶活性。一个单位的葡萄糖氧化酶的活力（欧盟）是在pH值5和25 0c每分钟生成1 u mol的葡萄糖酸和H2O2所需的酶量。

2.5 。测定动力学参数

为了确定每个电极的最大速度的反应速率（Vmax）和有关米氏常数（Km），应用不同浓度的蔗糖和葡萄糖进行活性测定。

2.6 .测定最适温度

固定化蔗糖和葡萄糖氧化酶的最适温度是分别在不同的水浴温度之间的15-70 0c和15-60 0c，同时保持底物浓度不变的情况进行测定。

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2.7 .膜的形态

对有和没有酶的聚合物薄膜进行了研究。从电极表面剥去膜和用缓冲液多次洗涤，以消除结合不牢固的酶和过剩的支持电解质后，用扫描电镜分析观察。

2.8 .操作的稳定性

固定化酶在聚合物薄膜操作的稳定性进行了研究（在最佳活性测定条件）在24h内检测30个酶活力单位的衰减情况。 3 .结果与讨论

3.1 .固定化中支持电解质的影响

不同的支持电解质，p-toluene sulfonic（ PTAS）和十二烷基硫酸钠

（ SDS ）经常用于固定蔗糖酶和葡萄糖氧化酶[ 4-7 ] 。显然，固定化矩阵用SDS固定化的蔗糖酶具有很高的酶活。在另一方面，当PTAS被用来作为支持电解质时固定化的葡萄糖氧化酶检测不到活性。电解时间对固定化的蔗糖和葡萄糖氧化酶也有不同的影响。在SDS需要反应60分钟而在需要120分钟才能达到相同的效果。因此，由于电解时间要求短和高活性，SDS经常用做这两种酶的交联剂。

3.2 .温度对酶活性的影响

温度对酶电极影响的研究， Fig 1（ a ）和（b）中给出。酶电极在50 C被发现活力最高。最适温度在40-60之间的自由蔗糖酶的活力强烈依赖于温度。随着温度的进一步在上述的最适温度上增加，酶的结构成改变，因此它的催化性能降低，并最终

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遭受破坏。之前人们确定包埋在聚吡咯中自由蔗糖酶和酶的最佳温度，分别是50和60 c [ 4 ] 。

我们也研究了固定化在PVATH /PPy和PPy的葡萄糖氧化酶酶活的最适温度（Fig 2 （ a ）及（ b ））。在两种介质中30 C可以观察到最大酶活。据报道自由葡萄糖氧化酶，最适温度约30 C [ 5 ] 。 PVATH /PPy酶电极与以PPy电极之间的40和60摄氏度对的温度变化展示出更高的稳定性。

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3.3 .固定化酶的动力学参数

从Lineweaver -伯克的图[ 17 ] （Table1和Table 2 ）, 获得了最大反应速率，Vmax ， Michealis - Menten常数，Km。蔗糖酶的动力学由于固定化发生了显著的变化。动力学常数，Km值固定化蔗糖酶高于该游离蔗糖酶。由于酶与底物的亲和力的改变，使Km值增加，也可能是酶包埋在交联剂中使其结构改的原因，此外，由于交联剂的不同使Km值改变。反应速率Km值的降低可能是由于底物与酶接触受到。固定化的葡萄糖氧化酶的Km值比游离的酶稍高，固定化酶Km值是由于固定化酶比游离实用文档

酶比对底物的亲和性降低。固定化酶的Km值升高和Vmax 的降低。在以前的固定化研究中已被报道[ 4-6,18 ] 。

3.4 操作稳定性和酶电极的寿命

为了测定固定化酶活性操作稳定性分别在同一天检测酶活。转化酶（Fig3 （ a ））和葡萄糖氧化酶（Fig4（ b ）），固定化酶电极的活性就被确定下来。这实用文档

个研究，在25℃下为PVATH /PPy和PPy连续测量30组数据。这两种固定化电极酶活力的操作稳定性有小幅波动。这两种酶在PVATH /PPy相比PPy显示更高的稳定。固定在PVATH /PPy的蔗糖酶有足够的稳定性（图4 ）。在30 天有80 ％的转化酶活性丧失。固定化化葡萄糖氧化酶只有15天贮存稳定性（图五）。应当指出，一般来说，这些交连介质显示没有足够的稳定性[ 6 ] 。在我们的实验中，记录了最佳操作稳定性。

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3.5 .固定化酶的形态

当酶固定化用扫描电子显微镜（ SEM ）观察膜表面的变化。清洗膜，以消除游离酶。这些膜表面形态与缺乏蔗糖酶和葡萄糖氧化酶相比完全不同在溶液一侧的膜表面，当蔗糖（Fig6 （ a ）和（ b ）），葡萄糖氧化酶（Fig7（ a ）和（ b ））被包埋在交联剂中，花椰菜结构显着损坏。

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4 .结论

成功地实现了用PVATH /PPy和PPy固定化蔗糖和葡萄糖氧化酶。SDS被发现是最好的包埋电解质。对该电解条件和他们的几个参数，如酶的活性，温度和Km和Vmax进行了研究。Km对固定化蔗糖酶的影响显著高于自由蔗糖酶。相应的影响在固定化葡萄糖氧化酶中只略高。