【作者】 薛勇；

【导师】 朱利民；

【作者基本信息】 东华大学 ， 生物化工， 2010， 硕士

【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。更多还原

【Abstract】 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 -NH₂ 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.更多还原