不同疏水参数有机溶剂中木瓜蛋白酶及其固定化酶活性的变化.pdf

第37卷 第3期2009年3月西北农林科技大学学报(自然科学版)Journal of Northwest A&F University(Nat.Sci.Ed.)Vol.37No.3Mar.2009不同疏水参数有机溶剂中木瓜蛋白酶及其固定化酶活性的变化3何 平,黄家乐,史清翠,巫光宏,初志战,黄卓烈(华南农业大学 生命科学学院,广东 广州510642)摘 要【目的】探寻疏水参数(logP)不同的有机溶剂对木瓜蛋白酶及其固定化酶活性的影响。【方法】制备固定化木瓜蛋白酶,在疏水参数不同(logP=-1.30,-1.10,-0.76,-0.33,-0.24,0.28,0.49,0.68,0.80,1.31,1.80,2.0,2.5,3.5和5.0)的15种有机溶剂及其不同体积分数(=5%,10%,15%,20%,25%,30%,35%,40%,50%,60%和70%)的溶液中,测定木瓜蛋白酶及其固定化酶的活性,以不含相应有机溶剂的0.1 mol/L p H 7.2磷酸缓冲液或Tris2盐酸缓冲液为对照,分析不同有机溶剂及其体积分数对酶活性的影响。【结果】在低疏水参数(logP-0.24)的有机溶剂中,有机溶剂二甲基亚砜、1,42二氧六环和乙腈具有在低体积分数(10%)下,激活木瓜蛋白酶及其固定化酶活性的特性,有机溶剂甲醇、乙醇虽然在低体积分数(10%)下不能激活木瓜蛋白酶及其固定化酶活性,但其活性与对照(=0)相当;低疏水参数有机溶剂在高体积分数(30%)下抑制酶活。当有机溶剂的疏水参数为0.281.31时,其对木瓜蛋白酶及其固定化酶活性有显著抑制作用。在有机溶剂疏水参数较高(logP1.80)条件下,低体积分数(10%)时酶活性下降较慢,超过一定体积分数(30%)后,酶活性开始升高。【结论】低logP(logP-0.24)有机溶剂在低体积分数(10%)时可激活或稳定木瓜蛋白酶及其固定化酶活性,而在高体积分数(30%)时对酶活有抑制作用;中logP(0.28logP1.31)有机溶剂对木瓜蛋白酶及其固定化酶活性具有显著抑制作用;高logP(logP1.80)有机溶剂在低体积分数(10%)时抑制木瓜蛋白酶及其固定化酶活性,但在高体积分数(30%)时酶活性升高。关键词 木瓜蛋白酶;固定化酶;有机溶剂;疏水参数;酶活性中图分类号Q556+.9文献标识码A文章编号167129387(2009)0320223207Enzyme activity changes of papain and immobilized papainin organic solvents with different log P valuesHE Ping,HUANGJia2le,SHI Qing2cui,WU Guang2hong,CHU Zhi2zhan,HUANG Zhuo2lie(College of Lif e Science,South China A gric.Univ.,Guangzhou,Guangdong510642,China)Abstract:【Objective】The aim of this paper was to study the effect of organic solvents with differentlogPvalues on enzyme activity papain and immobilized papain.【Method】Immobilization of papain on sodi2um alginate2chitosan and nylon cloth were prepared.Enzyme activity of papain and immobilized papainwere determined in 15 kinds of organic solvents with different logPvalues(logP=-1.30,-1.10,-0.76,-0.33,-0.24,0.28,0.49,0.68,0.80,1.31,1.80,2.0,2.5,3.5 and 5.0)and volume fractions(5%,10%,15%,20%,25%,30%,35%,40%,50%,60%and 70%),the effect of enzyme activity in different organicsolvents and volume fractions were analyzed by taking p H 7.2 Tris2hydrochloric acid buffer or p H 7.23收稿日期2008205216基金项目 国家自然科学基金项目(10074016);广东省自然科学基金项目(7006655);广东省科技计划项目(2008B011000006)作者简介 何 平(1967-),男,安徽枞阳人,副教授,博士,主要从事酶学研究。通信作者 黄卓烈(1950-),男,广东廉江人,教授,博士生导师,主要从事酶学研究。phosphate buffer without corresponding organic solvents as comparison.【Result】The results showed thatin lower logPvalues(logP-0.24)and lower volume fractions(10%)of the added organic solvents,enzyme activity of papain and immobilized papain was activated by some organic solvents(e.g.dimethylsulfoxide,1,42dioxane and acetonitrile),and it was observed in higher volume fractions(30%)of or2ganic solvents(logP-0.24)that the enzyme retained their activity in some organic solvents(e.g.metha2nol and ethanol),but decreased in their activity.In organic solvents with logPvalues between 0.28 and1.31,a dramatic reduction in their activity was observed.In organic solvents with higher logPvalues(logP1.80),it was observed at higher volume fractions(30%)enzyme activity decreased slowly inlower volume fractions(10%),but increased in the activity of the enzyme.【Conclusion】Enzyme activityof papain and immobilized papain were activated or stabilized in lower logPvalues(logP-0.24)and low2er volume fractions(10%)of the added organic solvents,but a reduction in their activity was observedin higher volume fractions(30%)of organic solvents.In organic solvents with middle logPvalues(0.28logP1.31)enzyme activity decreased remarkably.In organic solvents with higher logPvalues(logP1.80),it was observed at higher volume fractions(30%)enzyme activity decreased in lowervolume fractions(10%),but increased in the activity of the enzyme.Key words:papain;immobilized enzyme;organic solvent;logP;enzyme activity 近年来,针对非水酶学的研究十分活跃,显示了有机溶剂中酶的巨大应用潜力125。大量研究结果表明,酶在有机介质中的催化反应有很多优点,为许多重大理论的研究提供了新的方法和手段628。如在低水环境中,可用于稳定具有未知催化性质的构象异构体及在水中寿命极短的酶反应中间体,这有利于弄清酶反应机理;利用非水体系可以精确地控制蛋白质分子表面的含水量,以研究水分子与蛋白质结构和功能的关系等。但目前尚没有很好的理论预测反应介质的选择,仅粗略地参考有机溶剂的疏水参数1ogP(P为一种溶剂在正辛醇和水两相体系中的分配系数)来选择反应介质9210。有关学者较为详细地研究了有机溶剂对酶活的抑制作用及低体积分数有机溶剂对酶的激活作用等11213,但尚未见关于不溶性有机溶剂中酶活性变化的报道,也没有对疏水参数不同的有机溶剂及其不同体积分数下酶及其固定化酶活性的变化、及这些因素相互关系的系统研究。为此,本研究就不同体积分数、不同疏水参数的有机溶剂在单相共溶剂体系(水/水溶性溶剂)或水2有机溶剂两相体系中,对木瓜蛋白酶(papain,PA)及其固定化木瓜蛋白酶(Immobilized papain,IPA)活性的影响进行了详细研究,以期为有机溶剂中酶的应用提供理论依据。1 材料与方法1.1 试验材料部分纯化的木瓜蛋白酶粗酶由广西热带作物研究所提供;尼龙布(0.147 mm,100目)和壳聚糖(脱乙酰度 90%)购于试剂公司;酪蛋白,美国Sigma公司产品。本试验所用的15种有机溶剂 二甲基亚砜、1,42二氧六环、甲醇、乙腈、乙醇、丙醇、四氢呋喃、乙酸乙酯、丁醇、戊醇、己醇、苯、甲苯、环己烷和十二醇的logP依次为-1.30,-1.10,-0.76,-0.33,-0.24,0.28,0.49,0.68,0.80,1.31,1.80,2.0,2.5,3.5和5.014,均为国产分析纯试剂。1.2 研究方法1.2.1 有机溶剂中木瓜蛋白酶(PA)活性的测定 取0.2 mL(1 g/L)PA溶液,加入0.25 mL激活剂(0.1 mol/L p H 7.2磷酸缓冲液,内含36 mmol/L半胱氨酸和0.6 mmol/L EDTA)和2.3 mL有机溶液,用0.1 mol/L p H 7.2磷酸缓冲液配制,使反应体系中有机溶剂的体积分数分别达到5%,10%,15%,20%,25%,30%,35%,40%,50%,60%和70%,于37 下预热10 min,加入37 预热的10g/L酪蛋白溶液0.5 mL,准确反应10 min(可溶性有机溶剂如二甲基亚砜、1,42二氧六环、甲醇、乙腈、乙醇、丙醇、四氢呋喃等,于恒温水浴振荡反应,160次/min;不溶性有机溶剂如乙酸乙酯、丁醇、戊醇、己醇、苯、甲苯、环己烷、十二醇等,水浴搅拌器搅拌反应,250 r/min)后,加入1.0 mL 200 g/L三氯乙酸溶液终止酶反应。过滤,取其上清液于波长275nm处测定消光值。以不含相应有机溶剂的0.1mol/L p H 7.2磷酸缓冲液为对照(CK)。每项试验设3个重复,测定2次取平均值,下同。以37 下,422西北农林科技大学学报(自然科学版)第37卷PA水解酪蛋白时每min产生1g酪氨酸的酶量定义为1个酶活性单位(U),下同。1.2.2 海藻酸钠2壳聚糖固定化木瓜蛋白酶的制备及其在有机溶剂中酶活性的测定 海藻酸钠2壳聚糖固定化木瓜蛋白酶(Immobilized papain on sodi2um alginate2chitosan,IPSAC)的制备参考郑婉玲等15的方法并有所改进。称取2 g海藻酸钠溶于100 mL 0.1 mol/L p H 7.2的Tris2盐酸缓冲液中,再加入1 g壳聚糖粉末,微波炉加热至80 左右并搅拌均匀,冷却至40 左右,加入PA粉末,搅拌均匀。将此混合液缓慢滴入0.5 mol/L氯化钙溶液中形成小球,置于4 冰箱下30 min。使用前用蒸馏水洗涤3次,用粗滤纸吸干。取1 g IPSAC,加入0.25 mL激活剂(0.1mol/L p H 7.2 Tris2盐酸缓冲液,内含36 mmol/L半胱氨酸和0.6 mmol/L EDTA)和2.5 mL有机溶液,用0.1 mol/L p H 7.2 Tris2盐酸缓冲液进行配制,使反应体系的有机溶剂体积分数分别达到5%,10%,15%,20%,25%,30%,35%,40%,50%,60%和70%,于37 下预热10 min,加入37 预热的10 g/L酪蛋白溶液0.5 mL,准确反应10 min(对可溶性有机溶剂,如二甲基亚砜、1,42二氧六环、甲醇、乙腈、乙醇、丙醇、四氢呋喃等,恒温水浴160次/min振荡反应;不溶性有机溶剂,如乙酸乙酯、丁醇、戊醇、己醇、苯、甲苯、环己烷、十二醇等,水浴搅拌器搅拌反应,250 r/min),然后加入1.0 mL 200 g/L三氯乙酸溶液终止酶反应。过滤,取其上清液于波长275 nm处测定消光值。以不含相应的有机溶剂的Tris2盐酸缓冲液为对照(CK)。1.2.3 尼龙布固定化木瓜蛋白酶的制备及其在有机溶剂中酶活性的测定 尼龙布固定化木瓜蛋白酶(Immobilized papain on nylon cloth,IPN)制备参考徐凤彩等16的方法进行。取尼龙布(3 cm3 cm)洗净,晾干;用含186 g/L CaCl2和186 mL/L水的甲醇溶液在50 下保温10 s左右,并轻轻搅拌至尼龙布发粘,取出用水冲去污物,用吸水纸吸干;尼龙布用3.5 mo1/L HCl于室温水解45 min,用蒸馏水洗至p H中性,再用体积分数5%戊二醛在室温下浸泡偶联20 min;取出尼龙布,用0.1 mol/L p H7.2磷酸缓冲液反复冲洗去除多余的戊二醛,吸干,立即用酶液(酶液用量为每块布1 mL)在4 下固定3.5 h;从酶液中取出尼龙布,用0.5 mo1/L NaCl(用0.1 mol/L p H7.2磷酸缓冲液配制)洗去多余的酶蛋白,即得IPN。取一块固定化木瓜蛋白酶的尼龙布,加入0.25mL激活剂(0.1 mol/L p H 7.2磷酸缓冲液,内含36 mmol/L半胱氨酸和0.6 mmol/L EDTA)和2.5mL有机溶剂,用0.1 mol/L p H 7.2磷酸缓冲液配制,使反应体系中有机溶剂的体积分数分别达到5%,10%,15%,20%,25%,30%,35%,40%,50%,60%和70%,于37 下预热10 min,加入37 预热的10 g/L酪蛋白溶液0.5 mL,准确反应10 min(可溶性有机溶剂,如二甲基亚砜、1,42二氧六环、甲醇、乙腈、乙醇、丙醇、四氢呋喃等,恒温水浴振荡反应,160次/min;不溶性有机溶剂,如乙酸乙酯、丁醇、戊醇、己醇、苯、甲苯、环己烷、十二醇等,水浴搅拌器搅拌反应,250 r/min),然后加入1.0 mL 200g/L三氯乙酸溶液终止酶反应。过滤,取其上清液于波长275 nm处测定消光值。以不含相应有机溶剂的0.1 mol/L p H 7.2磷酸缓冲液为对照(CK)。1.2.4 数据统计与分析 用SAS(statistic analy2sis system)软件分析酶活性差异显著性。2 结果与分析2.1 低疏水参数(logP-0.24)有机溶剂对PA及其IPA活性的影响从表1可以看出,二甲基亚砜、1,42二氧六环、乙腈在低体积分数(10%)时,对PA及其IPA活性有激活作用,酶活性较对照(CK)有所升高。随着二甲基亚砜、1,42二氧六环、乙腈体积分数的进一步升高,PA及IPA的活性呈下降趋势。方差分析发现,二甲基亚砜体积分数为10%时,IPSAC和IPN的活性与对照(CK)相比差异显著;1,42二氧六环的体积分数为5%,10%,15%及乙腈的体积分数为5%,10%时,PA、IPSAC、IPN活性与对照差异显著。对甲醇、乙醇而言,在低体积分数(10%)时,PA及其IPA活性与对照基本相当;随着甲醇、乙醇体积分数的进一步提高,PA及其IPA活性逐渐降低。由以上分析可以看出,在logP较低(logP-0.24)的有机溶剂中,有机溶剂二甲基亚砜、1,42二氧六环和乙腈具有在低体积分数下激活PA及IPA活性的特性,有些有机溶剂如甲醇、乙醇虽然在低体积分数下不激活PA及其IPA活性,但其活性与对照相当。2.2 中疏水参数(0.28logP1.31)有机溶剂对PA及其IPA活性的影响从表2可以看出,随着丙醇、四氢呋喃体积分数522第3期何 平等:不同疏水参数有机溶剂中木瓜蛋白酶及其固定化酶活性的变化的逐渐升高,PA及其IPA的活性逐渐降低,且降低幅度加快。对乙酸乙酯而言,在低体积分数时,PA及其IPA活性下降较快,但当其体积分数超过30%时,酶活性虽开始升高,但仍低于对照(CK)中的酶活性。随着丁醇、戊醇体积分数的逐渐升高,PA及其IPA活性迅速下降。由此可见,当有机溶剂疏水参数为0.281.31时,其对PA及其IPA活性有显著抑制作用。表1 不同体积分数低logP有机溶剂对木瓜蛋白酶及其固定化酶活性的影响Table 1Effect of different volume fractions of organic solvents with lower logPvalues on activities of PA and IPA有机溶剂Organicsolvent酶Enzyme体积分数/%Volume fraction0(CK)510152025二甲基亚砜DimethylsulfoxidePA34.120.62 ab35.750.66 a36.960.36 a36.530.23 a35.770.72 a31.560.65 bIPSAC27.290.44 bc27.670.19 ab28.560.11 a26.480.18 c25.460.80 d23.590.71 eIPN21.581.09 b22.471.13 ab23.350.16 a22.500.11 ab18.510.61 c17.410.32 c1,42二氧六环1,42dioxanePA34.391.05 c38.781.35 b42.561.17 a38.341.49 b33.361.25 c26.870.21 dIPSAC25.760.79 c28.580.84 b32.030.68 a28.980.79 b25.920.89 c20.761.19 dIPN22.990.77 d25.370.43 c27.930.31 a26.481.08 b25.770.52 bc25.040.51 c甲醇MethanolPA34.190.96 a35.071.30 a34.450.77 a34.250.78 a32.760.48 ab30.662.59 bIPSAC25.640.72 a25.940.84 a25.110.57 a25.210.54 a24.220.43 ab23.441.62 abIPN22.910.64a22.950.52 a21.950.32 a20.541.73 a20.451.07 ab16.770.96 ab乙腈AcetonitrilePA31.810.36 b35.400.73 a34.250.39 a31.670.03 b27.151.27 c22.031.53 dIPSAC24.160.16 dc25.991.03 ab26.020.06 a24.360.09 bc22.690.75 d18.962.61 eIPN21.930.25 c25.411.96 a23.920.38 b23.470.07 b23.070.42 bc15.281.04 d乙醇EthanolPA33.321.11 a33.980.86 a34.150.30 a33.800.60 a33.640.38 a32.571.37 abIPSAC27.320.33 ab27.150.39 ab27.310.46 ab27.310.24 ab27.610.45 a26.241.04 bcIPN22.320.74 a22.280.32 a21.800.38 a21.950.26 a21.340.24 a18.250.92 b有机溶剂Organicsolvent酶Enzyme体积分数/%Volume fraction303540506070二甲基亚砜DimethylsulfoxidePA26.351.17 c26.182.61 c22.591.89 d20.770.35 d16.510.32 e13.050.43 fIPSAC22.120.62 f20.830.24 g18.440.88 h17.500.16 i14.710.43 j11.810.89 kIPN17.312.32 c14.370.41 d13.620.23 d11.910.39 e6.660.91 f6.090.71 f1,42二氧六环1,42dioxanePA26.220.28 de25.170.69 de24.630.69 eIPSAC19.560.36 d19.280.43 e18.450.51 eIPN22.070.53 d18.540.14 e18.090.19 e甲醇MethanolPA30.531.60 b25.031.43 c23.942.64 c23.902.56 c17.262.97 d13.373.08 eIPSAC22.371.35 b18.461.00 c17.311.92 c16.940.91 c14.003.43 d9.622.31 eIPN16.041.77 b16.011.72 c11.561.99 c9.102.06 c8.960.97 d6.720.43 e乙腈AcetonitrilePA20.450.60 e11.720.57 f9.200.64 g1.800.55 h00IPSAC15.310.68 f9.050.59 g7.020.36 h1.410.26 i00IPN14.200.41 d8.270.39 e6.560.43 f1.520.38 g00乙醇EthanolPA31.100.72 b28.740.16 c25.010.42 d24.571.08 d18.983.57 e16.481.14 fIPSAC25.370.24 c23.180.31 d20.490.32 e19.380.31 e15.101.62 f13.220.68 gIPN17.270.48 b15.680.11 c13.180.28 d11.590.79 e9.152.39 f7.470.76 g 注:同列数据后标不同小写字母者表示差异显著(P 0.05)。无数据的空格是因为滤液混浊而无法测定。下表同。Note:Data with different letters in the same column indicate remarkable differences(P 0.05).The blanks in the table above show that thedata were not determined by unclear filtrated solutions.The followings are the same.2.3 高疏水参数(logP1.80)有机溶剂对PA及其IPA活性的影响由表3可以看出,对有机溶剂己醇而言,在低体积分数时,PA及其IPA活性下降较快,但体积分数超过20%后,酶活性开始升高,但仍低于对照(CK)的酶活性。有机溶剂苯、甲苯、环己烷、十二醇中的情况与己醇的相似,不同的是超过一定体积分数(30%)后,酶活性开始升高,并逐渐高于对照中的酶活性。由表13可以看出,随体积分数上升,供试的15种有机溶剂对PA和IPA活性的作用趋势不同,但就每一种有机溶剂而言,其对PA和IPA活性的作用趋势表现一致。方差分析结果表明,15种有机溶剂在体积分数不同时,均对PA及其IPA的影响差异极显著。622西北农林科技大学学报(自然科学版)第37卷表2 不同体积分数中logP有机溶剂对木瓜蛋白酶及其固定化酶活性的影响Table 2Effect of different volume fractions of organic solvents with middle logPvalues on activities of PA and IPA有机溶剂Organicsolvent酶Enzyme体积分数/%Volume fraction0510152025丙醇PropanolPA34.750.32 a34.910.34 a28.301.19 b23.620.52 c21.880.50 d17.510.66 eIPSAC26.320.12 a26.150.09 a20.940.97 b14.110.55 c11.330.93 d9.011.22 eIPN20.940.97 a20.851.54 a18.570.72 b18.311.02 b14.911.23 c11.010.51 d四氢呋喃TetrahydrofuranPA33.321.11 a32.960.45 a30.580.37 b26.180.46 c18.250.39 d10.870.93 eIPSAC25.740.42 a24.450.52 a21.132.36 b16.580.48 c14.860.77 c8.401.95 dIPN22.010.45 a20.820.71 a17.710.58 b15.410.45 c13.280.30 d7.890.24 e乙酸乙酯Ethyl acetatePA34.490.42 a32.820.91 ab31.730.51 b28.001.92 c18.940.96 e15.711.43 fIPSAC26.811.10 a25.250.48 b23.271.46 c21.950.54 d19.260.69 ef16.920.37 hiIPN23.110.28 a21.990.61 ba21.260.34 b18.761.29 c13.451.06 e10.520.96 fg丁醇ButanolPA34.570.51 a29.602.08 b26.751.05 c19.380.89 d15.430.77 e9.680.40 fIPSAC26.650.17 a23.031.19 b19.380.89 c15.430.77 d12.490.27 e7.850.30 fIPN22.770.45 a21.640.28 a16.540.66 b13.920.79 c9.640.50 d7.280.35 e戊醇PentanolPA32.590.85 a24.640.46 b18.110.87 c14.951.12 d11.050.64 e9.061.32 fIPSAC25.060.64 a19.020.35 b14.060.66 c11.660.85 d8.700.48 e7.191.01 fIPN20.010.92 a12.830.79 b10.850.51 c9.770.59 d8.550.34 e7.620.39 f有机溶剂Organicsolvent酶Enzyme体积分数/%Volume fraction303540506070丙醇PropanolPA14.911.23 f6.470.20 g3.390.43 h000IPSAC5.710.26 f4.910.16 f2.580.33 g000IPN8.590.28 e6.350.53 f2.160.32 g000四氢呋喃TetrahydrofuranPAIPSACIPN乙酸乙酯Ethyl acetatePA13.701.39 f14.411.29 f14.140.86 f15.231.15 f21.022.18 e23.782.08 dIPSAC15.870.43 i17.800.67 gh17.940.63 gh18.830.61 fg19.631.08 ef20.310.51 eIPN9.180.93 g10.910.28 f11.640.99 f11.620.36 f13.701.39 e15.711.43 d丁醇ButanolPA8.070.59 g7.260.37 g5.350.33 h4.170.80 hi2.740.42 ij2.100.25 jIPSAC6.650.44 g6.040.28 g4.180.25 h3.250.63 h2.130.32 i1.640.19 iIPN6.130.31 fe5.510.36 f4.050.12 g3.450.33 g2.980.27 hg1.760.28 h戊醇PentanolPA5.770.78 g5.750.51 g4.860.74 gh5.001.07 gh4.320.42 hi3.050.40 iIPSAC4.660.59 g4.730.29 g3.990.56 gh3.830.67 gh3.580.32 hi2.620.30 iIPN4.880.39 g4.210.34 gh3.530.45 h3.390.12 h2.480.30 i2.160.18 i表3 不同体积分数高logP有机溶剂对木瓜蛋白酶及其固定化酶活性的影响Table 3Effect of different volume fractions of organic solvents with higher logPvalues on activities of PA and IPA有机溶剂Organicsolvent酶Enzyme体积分数/%Volume fraction0(CK)510152025己醇HexanolPA32.901.42 a27.531.28 de27.410.57 de26.791.28 de24.831.65 f25.660.95 feIPSAC23.580.87 a22.120.69 b20.030.23 cd18.230.30 ef15.750.61 g16.240.52 gIPN20.150.74 a20.130.23 a18.330.30 b15.630.79 d13.490.52 ef12.740.17 f苯BenzenePA33.480.50 gh32.670.70 h32.470.62 h31.180.53 i33.340.42 gh34.231.03 gIPSAC26.161.21 d25.530.94 de22.080.54 f21.640.96 f22.550.93 f22.790.51 fIPN22.430.33 e21.890.47 e22.080.54 e20.890.35 f22.340.28 e22.790.51 e甲苯ToluenePA33.840.25 ef30.560.46 h31.830.37 gh33.060.14 fg34.690.23 e34.831.05 eIPSAC26.600.36 fg24.580.62 h25.940.51 gh25.960.70 gh27.170.41 fg27.600.46 efIPN22.230.21 g21.040.27 h22.620.19 g23.710.30 f23.950.23 f25.140.12 e环己烷CyclohexanePA34.891.68 ef33.481.07 ef32.811.11 f33.840.19 ef34.470.51 ef36.100.64 deIPSAC26.852.04 de26.131.85 de25.270.88 e26.112.31 de26.231.24 de26.400.67 deIPN24.461.09 fg23.131.52 gh22.230.79 h23.620.51 fgh25.530.37 ef26.740.48 de十二醇DodecanolPA34.250.82 h33.270.55 h33.810.78 h36.940.71 gh39.350.28 fg40.880.37 fIPSAC26.610.44 h22.480.49 i20.010.84 j21.030.44 j25.820.56 h30.560.38 gIPN22.190.37 e18.740.41 f16.680.70 g17.540.37 fg19.230.65 f21.530.47 e722第3期何 平等:不同疏水参数有机溶剂中木瓜蛋白酶及其固定化酶活性的变化续表3Continue table 3有机溶剂Organicsolvent酶Enzyme体积分数/%Volume fraction303540506070己醇HexanolPA27.290.59 de27.791.30 d28.620.31 cbd28.281.18 cd29.950.60 cb30.321.73 bIPSAC16.030.99 g17.570.44 f19.260.97 de19.380.79 de20.250.71 cd20.630.96 cIPN13.860.66 e15.180.57 d15.720.15 d17.220.10 c17.350.62 c17.670.87 bc苯BenzenePA37.860.87 f41.850.51 e44.880.42 d47.940.56 c50.240.64 b52.620.68 aIPSAC24.510.83 e26.320.64 d28.300.75 c31.200.70 b33.270.85 a34.130.60 aIPN26.320.64 d28.040.34 c28.300.75 c29.330.66 b31.280.80 a32.080.57 a甲苯ToluenePA37.480.87 d39.030.69 c39.890.89 c42.300.92 b43.590.29 ab44.660.31 aIPSAC28.830.32 de29.660.37 d31.830.23 c32.970.45 bc33.971.11 b38.040.98 aIPN25.420.19 e27.460.27 d28.470.18 c28.110.11 cd29.150.13 b30.650.26 a环己烷CyclohexanePA38.421.62 cd40.060.97 bc41.041.31 abc40.483.58 bc42.662.40 ab43.610.55 aIPSAC26.950.19 de28.622.53 cd30.582.53 bc32.750.83 b37.351.02 a37.640.54 aIPN28.451.20 cd29.680.72 bc30.400.97 abc29.992.65 bc31.601.78 ab32.310.40 a十二醇DodecanolPA42.500.99 f48.190.75 e54.662.33 d58.727.08 c65.841.02 b74.331.40 aIPSAC33.890.73 f35.420.10 e36.940.71 d40.880.37 c48.190.75 b65.841.02 aIPN22.850.49 de24.152.32 d28.120.88 c28.850.76 c41.981.78 b5