木瓜蛋白酶的化学修饰及固定化研究
摘要
酶的化学修饰能够改进酶的物理化学性能、提高其生物活性,已受到越来越多的重视。其主要方法包括交联技术,定点突变和化学修饰结合技术、小分子修饰、单功能聚合物化学修饰等。酶的固定化处理能够进一步提高其稳定性。
木瓜蛋白酶(EC 3.4.22.2)作为水解蛋白酶属于半胱氨酸蛋白酶家族,主要存在于番木瓜的乳汁中,其应用广泛,包括细胞分离、化妆品、洗涤剂、食品和医药等行业,由于受到环境和其他条件的影响,容易变性失活,降低催化效率,从而限制了其大规模的应用。
本论文首次以三种新型的酸酐修饰剂在底物保护和无底物保护下对木瓜蛋白酶进行化学修饰,以三硝基苯磺酸法测定修饰酶的平均氨基修饰率,对修饰前后的木瓜蛋白酶分别纯化并通过UV-vis和IR对其结构进行了表征。考察了温度、pH值、表面活性剂SDS和会属离子对化学修饰酶活力的影响,并与天然木瓜蛋白酶进行了比较,对天然酶和修饰酶进行了动力学和热力学研究。结果表明,化学修饰酶的最适反应温度为80℃;最适pH值为9.0;在SDS浓度为5 mg/mL时修饰酶酶活仍能保持在50%左右;在所有酶中,均苯四甲酸酐修饰酶的催化效率最高,为2.442×10~2,ΔH~*,ΔG~*和ΔS~*分别为131.8 kJ/mol,80.17 kJ/mol和151.9J/mol·K,显示了化学修饰提高了木瓜蛋白酶的稳定性。与天然酶相比,化学修饰酶的热稳定性、耐碱性和耐洗涤性得到了明显提高。
本论文通过合成方法制备了两种不同分子量的单甲氧基聚乙二醇(mPEG)衍生物,对木瓜蛋白酶进行了修饰,对修饰酶的热稳定性、耐酸碱性、在有机溶剂中的稳定性和动力学等相关参数进行了测定并对其结构进行了一系列表征。结果表明,木瓜蛋白酶经过不同分子量的修饰剂修饰后,其活性均得到了很好的保留;热稳定性测定显著提高,且热稳定性与修饰剂分子量有关;修饰酶K_m值的测定表明在活泼位点不被修饰、整体结构不发生较大改变的情况下可以保持较高的对底物的表观亲和力。随着修饰剂分子量的增大,修饰酶的SDS聚丙烯酰胺凝胶电泳速度减慢。
另外,本论文将化学修饰酶进行了固定化处理,先对棉布进行活化,以活化后的棉布为载体,戊二醛为交联剂,对修饰前后的木瓜蛋白酶分别进行固定化,考察了固定化酶性质,对固定化酶棉织物的贮存稳定性、回潮率和透气性能进行了测定,并首次探讨了木瓜蛋白酶对酪氨酸的抑制作用。结果表明,棉布经过活化以后,在温和条件下实现木瓜蛋白酶固定化,固定化酶的热稳定性和酸碱稳定性有了明显的提高。在不加任何保护剂的条件下,在室温条件下放置30 d后游离酶仅保持一半的酶活,固定化能保持70%以上的酶活力,固定化酶的回潮率达到公定标准并具有良好的透气性。分别以_L-酪氨酸和_L-多巴(_L-DOPA)为底物,木瓜蛋白酶对酪氨酸酶的单酚酶和二酚酶有一定的抑制作用。
As an effective technique to improve the physical and chemical characteristics,such as enzyme activity,enzyme chemical modification has received considerable attention.The methods includes enzyme crosslinking,site-directed mutagenesis,small molecular compounds, homo-functionality polymerization and so on.Immobilization of papain after chemical modification will further increase its stability.
Papain(EC 3.4.22.2) is a powerful proteolytic enzyme,belonging to the cysteine protease family.It is a minor constituent among the papaya endopeptidases.The enzyme has found many application in cell isolation,cosmetic,detergents,food and pharmaceutical industries.
In this paper,Under the substrate protecting and non-substrate protecting,papain was firstly modified by using three novel anhydrides.The average ratio of modified -NH_2 was tested by a trinitrobenzenesulfonic acid method.The native and modified papain samples were purified by dialysis and their structures were characterized by UV and IR.The factors related with the activity of the modified papain,such as temperature,pH value,kinetic constant,thermodynamics,metal ion and the concentration of SDS were studied and compare with those of the native papain.The results showed that the optimum reaction temperature and pH for modifying papain were 80℃and 9.0,respectively.When the concentration of SDS was 5 mg/mL,about 50%of the activity of modified papain were maintained.Among these modified enzymes,the enzyme modified with pyromellitic dianhydride was found to be best in catalytic efficiency.ΔH*,ΔG andΔS*were found as 131.8 kJ/mol,80.17 kJ/mol and 151.9 J/mol·K,respectively.Compared with the native papain, the thermal stability and the resistance of modified enzyme to alkali and washing detergent were improved considerably.
Several mPEG derivatives with different molecular weight were synthesized in this dissertation,by which papain was modified,and relative characteristics of the enzyme modified was determined.It has been discovered in this thesis that the enzyme activity is retained to a great degree through modification,and the thermal stability is improved greatly,too.And it is proved that the thermal stability is related directly with the molecular weight of modifiers.As the molecular weight of modifiers increased,the enzyme's rate of SDS-PAGE slowed down.
The modified papain was immobilized on the cotton fabric with the aid of glutaraldehyde. The properties of immobilized enzyme were studied,the enzyme stability,moisture regain and air permeability fabric were also measured.The inhibiton of tyrosine by papain was firstly investigate. The results showed that after treated with glutaraldehyde,papain was immobilized to the support. Compared with immobilized nature papain,the thermal stability and the resistance to alkali were improved obviously.Storaged,the free enzyme only kept half of the original activity but the immobilized enzyme still retained 70%of the activity.The regain of the cotton fabric with enzyme immobilized have been up to scratch and has good air permeability.Papain inhibited both monophenolase and diphenolase activities of mushroom tyrosinase when _(L)-tyrosine and _(L)-DOPA were assayed spectrophotometrically,respectively.
木瓜蛋白酶(EC 3.4.22.2)作为水解蛋白酶属于半胱氨酸蛋白酶家族,主要存在于番木瓜的乳汁中,其应用广泛,包括细胞分离、化妆品、洗涤剂、食品和医药等行业,由于受到环境和其他条件的影响,容易变性失活,降低催化效率,从而限制了其大规模的应用。
本论文首次以三种新型的酸酐修饰剂在底物保护和无底物保护下对木瓜蛋白酶进行化学修饰,以三硝基苯磺酸法测定修饰酶的平均氨基修饰率,对修饰前后的木瓜蛋白酶分别纯化并通过UV-vis和IR对其结构进行了表征。考察了温度、pH值、表面活性剂SDS和会属离子对化学修饰酶活力的影响,并与天然木瓜蛋白酶进行了比较,对天然酶和修饰酶进行了动力学和热力学研究。结果表明,化学修饰酶的最适反应温度为80℃;最适pH值为9.0;在SDS浓度为5 mg/mL时修饰酶酶活仍能保持在50%左右;在所有酶中,均苯四甲酸酐修饰酶的催化效率最高,为2.442×10~2,ΔH~*,ΔG~*和ΔS~*分别为131.8 kJ/mol,80.17 kJ/mol和151.9J/mol·K,显示了化学修饰提高了木瓜蛋白酶的稳定性。与天然酶相比,化学修饰酶的热稳定性、耐碱性和耐洗涤性得到了明显提高。
本论文通过合成方法制备了两种不同分子量的单甲氧基聚乙二醇(mPEG)衍生物,对木瓜蛋白酶进行了修饰,对修饰酶的热稳定性、耐酸碱性、在有机溶剂中的稳定性和动力学等相关参数进行了测定并对其结构进行了一系列表征。结果表明,木瓜蛋白酶经过不同分子量的修饰剂修饰后,其活性均得到了很好的保留;热稳定性测定显著提高,且热稳定性与修饰剂分子量有关;修饰酶K_m值的测定表明在活泼位点不被修饰、整体结构不发生较大改变的情况下可以保持较高的对底物的表观亲和力。随着修饰剂分子量的增大,修饰酶的SDS聚丙烯酰胺凝胶电泳速度减慢。
另外,本论文将化学修饰酶进行了固定化处理,先对棉布进行活化,以活化后的棉布为载体,戊二醛为交联剂,对修饰前后的木瓜蛋白酶分别进行固定化,考察了固定化酶性质,对固定化酶棉织物的贮存稳定性、回潮率和透气性能进行了测定,并首次探讨了木瓜蛋白酶对酪氨酸的抑制作用。结果表明,棉布经过活化以后,在温和条件下实现木瓜蛋白酶固定化,固定化酶的热稳定性和酸碱稳定性有了明显的提高。在不加任何保护剂的条件下,在室温条件下放置30 d后游离酶仅保持一半的酶活,固定化能保持70%以上的酶活力,固定化酶的回潮率达到公定标准并具有良好的透气性。分别以_L-酪氨酸和_L-多巴(_L-DOPA)为底物,木瓜蛋白酶对酪氨酸酶的单酚酶和二酚酶有一定的抑制作用。
As an effective technique to improve the physical and chemical characteristics,such as enzyme activity,enzyme chemical modification has received considerable attention.The methods includes enzyme crosslinking,site-directed mutagenesis,small molecular compounds, homo-functionality polymerization and so on.Immobilization of papain after chemical modification will further increase its stability.
Papain(EC 3.4.22.2) is a powerful proteolytic enzyme,belonging to the cysteine protease family.It is a minor constituent among the papaya endopeptidases.The enzyme has found many application in cell isolation,cosmetic,detergents,food and pharmaceutical industries.
In this paper,Under the substrate protecting and non-substrate protecting,papain was firstly modified by using three novel anhydrides.The average ratio of modified -NH_2 was tested by a trinitrobenzenesulfonic acid method.The native and modified papain samples were purified by dialysis and their structures were characterized by UV and IR.The factors related with the activity of the modified papain,such as temperature,pH value,kinetic constant,thermodynamics,metal ion and the concentration of SDS were studied and compare with those of the native papain.The results showed that the optimum reaction temperature and pH for modifying papain were 80℃and 9.0,respectively.When the concentration of SDS was 5 mg/mL,about 50%of the activity of modified papain were maintained.Among these modified enzymes,the enzyme modified with pyromellitic dianhydride was found to be best in catalytic efficiency.ΔH*,ΔG andΔS*were found as 131.8 kJ/mol,80.17 kJ/mol and 151.9 J/mol·K,respectively.Compared with the native papain, the thermal stability and the resistance of modified enzyme to alkali and washing detergent were improved considerably.
Several mPEG derivatives with different molecular weight were synthesized in this dissertation,by which papain was modified,and relative characteristics of the enzyme modified was determined.It has been discovered in this thesis that the enzyme activity is retained to a great degree through modification,and the thermal stability is improved greatly,too.And it is proved that the thermal stability is related directly with the molecular weight of modifiers.As the molecular weight of modifiers increased,the enzyme's rate of SDS-PAGE slowed down.
The modified papain was immobilized on the cotton fabric with the aid of glutaraldehyde. The properties of immobilized enzyme were studied,the enzyme stability,moisture regain and air permeability fabric were also measured.The inhibiton of tyrosine by papain was firstly investigate. The results showed that after treated with glutaraldehyde,papain was immobilized to the support. Compared with immobilized nature papain,the thermal stability and the resistance to alkali were improved obviously.Storaged,the free enzyme only kept half of the original activity but the immobilized enzyme still retained 70%of the activity.The regain of the cotton fabric with enzyme immobilized have been up to scratch and has good air permeability.Papain inhibited both monophenolase and diphenolase activities of mushroom tyrosinase when _(L)-tyrosine and _(L)-DOPA were assayed spectrophotometrically,respectively.
引文
[1]Montes T,Grazffl V,Manso I.Improved stabilization of genetically modified penicillin acylase in the presence of organic cosolvents by co-immobilization of the enzyme with polyethyleneimine[J].Advanced Synthesis and Catalysis.2007,349(3):459-464.
[2]Fernandez M,Fragoso A.Stabilization of α-chymotrypsin by chemical modification with monoamine cyclodextrin[J].Process Biochemistry,2005,40(6):2091-2094.
[3]Lopez Gallego F,Montes T.Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports[J].Journal of Biotechnology,2005,116(1):1-10.
[4]周海梦,王洪睿.蛋白质化学修饰[M].北京:清华大学出版社,1998:1-50.
[5]Desantis G,Jones J B.Chemical modification of enzymes for enhanced functionality[J].Current Opinion Biotechnology,1999,10(4):324-330.
[6]Garcia D,Ortega F,Marty J L.Kinetics of thermal inactivation of horseradish peroxidase:stabilizineffect of methoxypoly(ethylene glycol)[J].Biotechnology and Applied Biochemitry,1998,27:49.
[7]Beak W O,Wijayalakshmi M A.Efect of chemical glycosylation histidines accessibility in immobilized metal ion affinity electrophoresis(IMAGE) system [J].Biochemitry Biophysics Acta,1997,136(2):397-400.
[8]Khajeh K,Naderi Manesh H,Ranjbar B.Chemical modification of lysine residues in bacillus a-amylases:efect on activity and stability[J].Enzyme and Microbial Technology,2001,28(6):543-549.
[9]史凌洋,魏东芝.用活化的单甲氧基聚乙二醇修饰L-天冬酰胺酶[J].华东理工大学学报,2001.27(6):601-604.
[10]Veronese,Francesco M.Polyethylene glycolsuper oxide dismutase a conjugate in search of exploitation[J].Advanced DruDelivery Reviews,2002,54(4):587-606.
[11]St Clair Nancy L,Navia Manuel A.Cross linked enzyme crystals robust biocatalysts[J].Journal of the American Chemical Society,1992,144(18):7314.
[12]罗贵民,曹淑贵.酶工程[M].北京:化学工业出版社,2008:75-84.
[13]Khajeh K,Ranjbar B etal.Chemical modification of lysine residues in Bacillus alpha-amlases:effect on activity and stability[J].Enzyme and Microbial Technology,2001,28(6):543-549.
[14]Spreti N,Marte L.Activation and stabilization of alpha-chymotrypsin by cationic additives[J].European Journal of Biochemistry,2001,268(24):6491-6497.
[15]王金辉,李铣.中药现代化与天然药物化学的研究[J].中药研究与信息,2000,2(5):13.
[16]Fernandez M,Villalonga M L,Fragoso A etal.α-Chymotrypsin stabilization by chemical conjugation with O-carboxymethyl-poly-β-cyclodextrin[J].Process Biochemistry,2004,39:535.
[17]Roberts M J,Bentley M D,Harris J M.Chemistry for peptide and protein PEGylation[J].Advanced Drug Delivery Reviews,2002,54:459.
[18]王颖达,郭丽,钱世钧等.多聚唾液酸对L-天冬酰胺酶的修饰及修饰酶特性研究[J].生物工程学报,2000,16(5):13.
[19]吴梧桐,刘景晶等.右旋糖苷对大肠杆菌L-天冬酰胺酶的化学修饰[J].药物生物技术,1997,4(4):208.
[20]Polgar L,Bender M L.A new enzyme containing a synthetically formed active site[J].Journal of the American Chemical Society,1966,88:3152.
[21]Antonic P,Kirsch F.Reengeneering the catalytic lysine of aspartate aminotransferease by chemical elaboration of a genetically introduced cystein[J].Biochemistry,1991,30:8268.
[22]Tai D F,Chang Y F,Chiou T W.Thiolsubtilisin acts on acetyltransferase in organic solvent[J].FEBS Letters,2002,517:24.
[23]Zaborsky O.Immobilized Enzymes[M].Clevelund Ohio,CRC Press,1973.
[24]周晓云.酶学原理与酶工程[M].北京:中国轻工业出版社,2005.
[25]Mohapatra B R,Gould W D etal.Optimizaion of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species[J].Journal of Biotechnology,2006,124(3):523-531.
[26]Uragami T,Watanabe M etal.Preparation of urease-immobilized polymeric membranes and their function[J].Catalysis Today,2006,118(1-2):158-165.
[27]Gowoun L,Jungbae K,Jung-heon L.Development of magneticall separable polyaniline nanofibers for enzyme immobilization and recovery[J].Enzyme and Microbial Technology,2008,42(2):466-472.
[28]Galina A K,Larisa V P.Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin[J].Carbohydrate Research,2008,343:1202-1211.
[29]Ye P,Xu Z K,Che A F etal.Chitosan-tethered polyacylonitrile-co-maleic acid ultrafiltration hollow fiber membrane for lipase immobilization[J].Biomaterials,2005,26(32):6394-6403.
[30]Liu X Q,Guan Y P etal.Immobilization of lipase onto micron-size magnetic beads[J].Journal of Chromatography B,2005,822(1):91-97.
[31]肖海军,贺筱蓉.固定化酶及其应用研究进展[J].生物学通报,2001,36(7):9-10.
[32]蒋雯.固定化木瓜蛋白酶载体的研究[J].化工纵横,2003,17(6):15-18.
[33]ChanS W,Shaw J F,YanK H etal.Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(γ-glutamic acid) by RSM[J].Bioresource Techology,2008,99:2800-2805.
[34]Barrett A J,Rawlings N D.Families and clans of cysteine peptidases[J].Perspectives in DruDiscovery and Design,1996,6:1-11.
[35]Morikawa M,Izawa Y,Rashid N etal.Purification and characterization of a thermostalbe thiol protease from a newly isolated hyperthermophilic pyrococcus sp.[J].Applied and Environment Microbiology,1994,60:4559.
[36]Andrew C,Storer,Robert M.Catalytic mechanism in papain family of cysteine peptidases[J].Methods in Enzymology,1994,244:486-490.
[37]Roy J J,Sumi S,Sangeetha T,Abraham T E.Chemical modification and immobilization of papain[J].Journol of Chemical Technology and Biotechnology,2005,80:184-188.
[38]Martin J H,Neil A B,Ian H H.Catalytic mechanism of enzyme papain: predictions with a hybrid quantum mechanical/molecular mechanical potential[J].Journal of the American Chemical Society,1997,119:12285-12291.
[39]邓健,吴华昌,周健.木瓜蛋白酶研究进展[J].广西轻工业,2003,3:5-7.
[40]吴显荣.木瓜蛋白酶的开发与应用[J].中国农业大学学报,2005,10(6):11-15.
[41]Sangeetha K,Abraham T E.Chemical modification of papain for use in alkaline medium[J].Journal of Molecular Catalysis B:Enzymatic,2006,38:171-177.
[42]Khaparde S S,Singhal P S.Chemically modified papain for applications in detergent formulations[J].Bioresource Technology,2001,78:1-4.
[43]Drenth J,Kalk K H,Swen H R.Bindinof Chloromethyl Ketone Substrate Analogues to crystalline papain[J].Biochemistry,1976,15:3731.
[44]Kaiser E T,Lawrence D S.Chemical mutation of enzyme active sites[J].Science,1984,226:505-511.
[45]SucklinC J,Zhu L M.Carbon-carbon bond formation mediated by papain chemically modified by thiazolium salts[J].Bioorganic and Medicinal Chemistry Letter,1993,13:531-534.
[46]马晓建,吴勇等.化学修饰酶的研究进展[J].化学与生物工程,2004,6:7-9.
[47]Padma V L,Laxmi A.Enzyme stability and stabilization-aqueous and non-aqueous environment[J].Process Biochemistry,2008,43:1019-1032.
[48]Habeeb A F S A.Determination of free amino groups in proteins by trinitrobenzenesulfonic acid[J].Analytical Biochemistry,1966,14:179-212.
[49]Kasy A J,Wildi B S.Proteases of the genus Bacillus.I.Neutral proteases[J].Biotechnology and Bioengineering,1970,12:179-212.
[50]王镜岩,朱圣庚,徐长法.生物化学[M].北京:高等教育出版社,2002:359-361.
[51]Rosiele L,Florencia C etal.Kinetics and thermodynamics of thermal inactivation of the antimicrobial peptide cerein 8A[J].Journal of Food Engineering,2009,91:223-227.
[52]Canan T,Nergiz D,Nihan G.Biochemical and thermal characterization of crude exo-polygalacturonase produced by Aspergillus sojae[J].Food Chemistry,2008, 111:824-829.
[53]Benjamin D.Chemical modification of biocatalysts[J].Current Opinion in Biotechnology,2003,14:379-386.
[54]Witold K S,Henry H M,Dennis C.Determination of protein secondary structure by Fourier transform infrared spectroscopy:A critical assessment[J].Biochemistry,1993,32:389-394.
[55]Yue Y Y,Chen X etal.A study of the bindinof C.I.direct yellow 9 to human serum albumin usinoptical spectroscopy and molecular modelin[J].Dyes and Pigments,2008,79:176-182.
[56]黄惠华,梁汉华,郭乾初.超声波对大豆胰蛋白酶抑制剂活性及二级结构的影响[J].食品科学,2004,25(3):29-33.
[57]高荣强,范世福.现代近红外光谱分析技术的原理及应用[J].分析仪器,2002,8(3):9-13.
[58]Xue Y,Nie H L etal.Immobilization of modified papain with anhydride groups on activated cotton fabric[J].Applied Biochemistry and Biotechnology,2009,doi:10.1007/s 12010-009-8588-x.
[59]张为灿,戴旭慧等.十二烷基磺酸钠对大豆过氧化物酶活性和构想的影响[J].化学学报,2008,66(6):669-674.
[60]Selvaraj R,Paramasamy G.Purification and characterization of keratinase from recombinant Pichia and Bacillus strains[J].2009,64(1):24-31.
[61]阎隆飞,孙之荣.蛋白质分子结构[M].北京:清华大学出版社,1999:11-170.
[62]Bowden A C.Fundamentals of enzyme kinetic[M].London:Portland,1996,554-561.
[63]刘广林.蛋白质的PEG修饰[J].生命科学仪器,2008,6(2):7-10.
[64]党琳,李明.化学修饰酶在天然药物研究和生物技术中的应用[J].陕西中医学院学报,2003,26(5):60-62.
[65]施险峰,詹家荣,廖本仁.聚乙二醇修饰技术研究进展[J].上海化工,2007,32(12):30-32.
[66]刘志国.生物化学实验[M].湖北:华中科技大学出版社,2007:93-101.
[67]应国清,易喻等.单甲氧基聚乙二醇修饰蚓激酶的制备及性质研究[J].高校化学工程学院,2005,19(4):527-531.
[68]张霖琳,郑春杨等.不同分子量聚乙二醇单修饰重组人干扰素α-2a[J].过程工程学报,2006,6(4):619-622.
[69]黄河.PEG修饰蛋白等高分子[J].化学推进剂与高分子材料,2004,2(5):18-22
[70]Liu J Z.Improvement of activity and stability of chloroperoxidase by chemical modification[J].BMC Biotechnology,2007,doi:10.1186/1472-6750-23.
[71]Guerrero F Q,Duarte M A etal.Chemical modification of turnip peroxidase with methoxypolyethylene glycol enhances activity and stability for phenol removal usinthe immobilized enzyme[J].Journal of Agricultural and Food Chemistry,2008,56:8058-8065.
[72]皮肤美容及整容[J].中国医学文摘-皮肤科学,2009,26(4):263-269.
[73]Fenoll L G,Penalver J N etal.Tyrosinase kinetics:discrimination between two models to explain the oxidation mechanism of monophenol and diphenol substrates[J].The International Journal of Biochemistry and Biology,2004,36(2):235-246.
[74]王丽萍,李创等.棉纤维回潮率及含水率测试实验误差分析[J].北京纺织,2005.26(6):45-47.
[75]Chen Q X,SonK K etal.Inhibitory effects on mushroom tyrosinase by p-alkoxybenzoic acids[J].Food Chemistry,2005,91:269-274.
[76]章基凯,赵陈超.有机硅整理剂及其在纺织工业上的应用[J].纺织学报,1990,11(6):43-45.
[77]Adams J B.Review:enzyme inactivation durinheat processinof food-stuffs [J].International Journal of Food Science and Technology,1991,26:1-20.
[78]Pawan G,Tariq M,Saleemuddin M.Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support[J].Journal Molecular Catalysis B:Enzymatic,2007,45(3-4):78-83.
[2]Fernandez M,Fragoso A.Stabilization of α-chymotrypsin by chemical modification with monoamine cyclodextrin[J].Process Biochemistry,2005,40(6):2091-2094.
[3]Lopez Gallego F,Montes T.Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports[J].Journal of Biotechnology,2005,116(1):1-10.
[4]周海梦,王洪睿.蛋白质化学修饰[M].北京:清华大学出版社,1998:1-50.
[5]Desantis G,Jones J B.Chemical modification of enzymes for enhanced functionality[J].Current Opinion Biotechnology,1999,10(4):324-330.
[6]Garcia D,Ortega F,Marty J L.Kinetics of thermal inactivation of horseradish peroxidase:stabilizineffect of methoxypoly(ethylene glycol)[J].Biotechnology and Applied Biochemitry,1998,27:49.
[7]Beak W O,Wijayalakshmi M A.Efect of chemical glycosylation histidines accessibility in immobilized metal ion affinity electrophoresis(IMAGE) system [J].Biochemitry Biophysics Acta,1997,136(2):397-400.
[8]Khajeh K,Naderi Manesh H,Ranjbar B.Chemical modification of lysine residues in bacillus a-amylases:efect on activity and stability[J].Enzyme and Microbial Technology,2001,28(6):543-549.
[9]史凌洋,魏东芝.用活化的单甲氧基聚乙二醇修饰L-天冬酰胺酶[J].华东理工大学学报,2001.27(6):601-604.
[10]Veronese,Francesco M.Polyethylene glycolsuper oxide dismutase a conjugate in search of exploitation[J].Advanced DruDelivery Reviews,2002,54(4):587-606.
[11]St Clair Nancy L,Navia Manuel A.Cross linked enzyme crystals robust biocatalysts[J].Journal of the American Chemical Society,1992,144(18):7314.
[12]罗贵民,曹淑贵.酶工程[M].北京:化学工业出版社,2008:75-84.
[13]Khajeh K,Ranjbar B etal.Chemical modification of lysine residues in Bacillus alpha-amlases:effect on activity and stability[J].Enzyme and Microbial Technology,2001,28(6):543-549.
[14]Spreti N,Marte L.Activation and stabilization of alpha-chymotrypsin by cationic additives[J].European Journal of Biochemistry,2001,268(24):6491-6497.
[15]王金辉,李铣.中药现代化与天然药物化学的研究[J].中药研究与信息,2000,2(5):13.
[16]Fernandez M,Villalonga M L,Fragoso A etal.α-Chymotrypsin stabilization by chemical conjugation with O-carboxymethyl-poly-β-cyclodextrin[J].Process Biochemistry,2004,39:535.
[17]Roberts M J,Bentley M D,Harris J M.Chemistry for peptide and protein PEGylation[J].Advanced Drug Delivery Reviews,2002,54:459.
[18]王颖达,郭丽,钱世钧等.多聚唾液酸对L-天冬酰胺酶的修饰及修饰酶特性研究[J].生物工程学报,2000,16(5):13.
[19]吴梧桐,刘景晶等.右旋糖苷对大肠杆菌L-天冬酰胺酶的化学修饰[J].药物生物技术,1997,4(4):208.
[20]Polgar L,Bender M L.A new enzyme containing a synthetically formed active site[J].Journal of the American Chemical Society,1966,88:3152.
[21]Antonic P,Kirsch F.Reengeneering the catalytic lysine of aspartate aminotransferease by chemical elaboration of a genetically introduced cystein[J].Biochemistry,1991,30:8268.
[22]Tai D F,Chang Y F,Chiou T W.Thiolsubtilisin acts on acetyltransferase in organic solvent[J].FEBS Letters,2002,517:24.
[23]Zaborsky O.Immobilized Enzymes[M].Clevelund Ohio,CRC Press,1973.
[24]周晓云.酶学原理与酶工程[M].北京:中国轻工业出版社,2005.
[25]Mohapatra B R,Gould W D etal.Optimizaion of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species[J].Journal of Biotechnology,2006,124(3):523-531.
[26]Uragami T,Watanabe M etal.Preparation of urease-immobilized polymeric membranes and their function[J].Catalysis Today,2006,118(1-2):158-165.
[27]Gowoun L,Jungbae K,Jung-heon L.Development of magneticall separable polyaniline nanofibers for enzyme immobilization and recovery[J].Enzyme and Microbial Technology,2008,42(2):466-472.
[28]Galina A K,Larisa V P.Immobilization of glucoamylase by adsorption on carbon supports and its application for heterogeneous hydrolysis of dextrin[J].Carbohydrate Research,2008,343:1202-1211.
[29]Ye P,Xu Z K,Che A F etal.Chitosan-tethered polyacylonitrile-co-maleic acid ultrafiltration hollow fiber membrane for lipase immobilization[J].Biomaterials,2005,26(32):6394-6403.
[30]Liu X Q,Guan Y P etal.Immobilization of lipase onto micron-size magnetic beads[J].Journal of Chromatography B,2005,822(1):91-97.
[31]肖海军,贺筱蓉.固定化酶及其应用研究进展[J].生物学通报,2001,36(7):9-10.
[32]蒋雯.固定化木瓜蛋白酶载体的研究[J].化工纵横,2003,17(6):15-18.
[33]ChanS W,Shaw J F,YanK H etal.Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(γ-glutamic acid) by RSM[J].Bioresource Techology,2008,99:2800-2805.
[34]Barrett A J,Rawlings N D.Families and clans of cysteine peptidases[J].Perspectives in DruDiscovery and Design,1996,6:1-11.
[35]Morikawa M,Izawa Y,Rashid N etal.Purification and characterization of a thermostalbe thiol protease from a newly isolated hyperthermophilic pyrococcus sp.[J].Applied and Environment Microbiology,1994,60:4559.
[36]Andrew C,Storer,Robert M.Catalytic mechanism in papain family of cysteine peptidases[J].Methods in Enzymology,1994,244:486-490.
[37]Roy J J,Sumi S,Sangeetha T,Abraham T E.Chemical modification and immobilization of papain[J].Journol of Chemical Technology and Biotechnology,2005,80:184-188.
[38]Martin J H,Neil A B,Ian H H.Catalytic mechanism of enzyme papain: predictions with a hybrid quantum mechanical/molecular mechanical potential[J].Journal of the American Chemical Society,1997,119:12285-12291.
[39]邓健,吴华昌,周健.木瓜蛋白酶研究进展[J].广西轻工业,2003,3:5-7.
[40]吴显荣.木瓜蛋白酶的开发与应用[J].中国农业大学学报,2005,10(6):11-15.
[41]Sangeetha K,Abraham T E.Chemical modification of papain for use in alkaline medium[J].Journal of Molecular Catalysis B:Enzymatic,2006,38:171-177.
[42]Khaparde S S,Singhal P S.Chemically modified papain for applications in detergent formulations[J].Bioresource Technology,2001,78:1-4.
[43]Drenth J,Kalk K H,Swen H R.Bindinof Chloromethyl Ketone Substrate Analogues to crystalline papain[J].Biochemistry,1976,15:3731.
[44]Kaiser E T,Lawrence D S.Chemical mutation of enzyme active sites[J].Science,1984,226:505-511.
[45]SucklinC J,Zhu L M.Carbon-carbon bond formation mediated by papain chemically modified by thiazolium salts[J].Bioorganic and Medicinal Chemistry Letter,1993,13:531-534.
[46]马晓建,吴勇等.化学修饰酶的研究进展[J].化学与生物工程,2004,6:7-9.
[47]Padma V L,Laxmi A.Enzyme stability and stabilization-aqueous and non-aqueous environment[J].Process Biochemistry,2008,43:1019-1032.
[48]Habeeb A F S A.Determination of free amino groups in proteins by trinitrobenzenesulfonic acid[J].Analytical Biochemistry,1966,14:179-212.
[49]Kasy A J,Wildi B S.Proteases of the genus Bacillus.I.Neutral proteases[J].Biotechnology and Bioengineering,1970,12:179-212.
[50]王镜岩,朱圣庚,徐长法.生物化学[M].北京:高等教育出版社,2002:359-361.
[51]Rosiele L,Florencia C etal.Kinetics and thermodynamics of thermal inactivation of the antimicrobial peptide cerein 8A[J].Journal of Food Engineering,2009,91:223-227.
[52]Canan T,Nergiz D,Nihan G.Biochemical and thermal characterization of crude exo-polygalacturonase produced by Aspergillus sojae[J].Food Chemistry,2008, 111:824-829.
[53]Benjamin D.Chemical modification of biocatalysts[J].Current Opinion in Biotechnology,2003,14:379-386.
[54]Witold K S,Henry H M,Dennis C.Determination of protein secondary structure by Fourier transform infrared spectroscopy:A critical assessment[J].Biochemistry,1993,32:389-394.
[55]Yue Y Y,Chen X etal.A study of the bindinof C.I.direct yellow 9 to human serum albumin usinoptical spectroscopy and molecular modelin[J].Dyes and Pigments,2008,79:176-182.
[56]黄惠华,梁汉华,郭乾初.超声波对大豆胰蛋白酶抑制剂活性及二级结构的影响[J].食品科学,2004,25(3):29-33.
[57]高荣强,范世福.现代近红外光谱分析技术的原理及应用[J].分析仪器,2002,8(3):9-13.
[58]Xue Y,Nie H L etal.Immobilization of modified papain with anhydride groups on activated cotton fabric[J].Applied Biochemistry and Biotechnology,2009,doi:10.1007/s 12010-009-8588-x.
[59]张为灿,戴旭慧等.十二烷基磺酸钠对大豆过氧化物酶活性和构想的影响[J].化学学报,2008,66(6):669-674.
[60]Selvaraj R,Paramasamy G.Purification and characterization of keratinase from recombinant Pichia and Bacillus strains[J].2009,64(1):24-31.
[61]阎隆飞,孙之荣.蛋白质分子结构[M].北京:清华大学出版社,1999:11-170.
[62]Bowden A C.Fundamentals of enzyme kinetic[M].London:Portland,1996,554-561.
[63]刘广林.蛋白质的PEG修饰[J].生命科学仪器,2008,6(2):7-10.
[64]党琳,李明.化学修饰酶在天然药物研究和生物技术中的应用[J].陕西中医学院学报,2003,26(5):60-62.
[65]施险峰,詹家荣,廖本仁.聚乙二醇修饰技术研究进展[J].上海化工,2007,32(12):30-32.
[66]刘志国.生物化学实验[M].湖北:华中科技大学出版社,2007:93-101.
[67]应国清,易喻等.单甲氧基聚乙二醇修饰蚓激酶的制备及性质研究[J].高校化学工程学院,2005,19(4):527-531.
[68]张霖琳,郑春杨等.不同分子量聚乙二醇单修饰重组人干扰素α-2a[J].过程工程学报,2006,6(4):619-622.
[69]黄河.PEG修饰蛋白等高分子[J].化学推进剂与高分子材料,2004,2(5):18-22
[70]Liu J Z.Improvement of activity and stability of chloroperoxidase by chemical modification[J].BMC Biotechnology,2007,doi:10.1186/1472-6750-23.
[71]Guerrero F Q,Duarte M A etal.Chemical modification of turnip peroxidase with methoxypolyethylene glycol enhances activity and stability for phenol removal usinthe immobilized enzyme[J].Journal of Agricultural and Food Chemistry,2008,56:8058-8065.
[72]皮肤美容及整容[J].中国医学文摘-皮肤科学,2009,26(4):263-269.
[73]Fenoll L G,Penalver J N etal.Tyrosinase kinetics:discrimination between two models to explain the oxidation mechanism of monophenol and diphenol substrates[J].The International Journal of Biochemistry and Biology,2004,36(2):235-246.
[74]王丽萍,李创等.棉纤维回潮率及含水率测试实验误差分析[J].北京纺织,2005.26(6):45-47.
[75]Chen Q X,SonK K etal.Inhibitory effects on mushroom tyrosinase by p-alkoxybenzoic acids[J].Food Chemistry,2005,91:269-274.
[76]章基凯,赵陈超.有机硅整理剂及其在纺织工业上的应用[J].纺织学报,1990,11(6):43-45.
[77]Adams J B.Review:enzyme inactivation durinheat processinof food-stuffs [J].International Journal of Food Science and Technology,1991,26:1-20.
[78]Pawan G,Tariq M,Saleemuddin M.Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support[J].Journal Molecular Catalysis B:Enzymatic,2007,45(3-4):78-83.