Cr(Ⅲ)对Alcalase碱性蛋白酶催化动力学影响及机理  

Effect of Cr(Ⅲ)on the Catalytic Kinetics of Alcalase Alkaline Protease and their Action Mechanism

作　　者：杨柳 由耀辉[1,2] 熊林颖 王丹 欧洁[1] 陈虹 YANG Liu;YOU Yaohui;XIONG Linying;WANG Dan;OU Jie;CHEN Hong(College of Chemistry and Chemical Engineering,Neijiang Normal University,Neijiang 641100,China;Key Laboratory of Fruit Waste Treatment and Resource Recycling of the Provincial Higher Learning Institutes,Neijiang 641100,China)

机构地区：[1]内江师范学院化学化工学院,四川内江641100 [2]四川省高等学校果类废弃物资源化重点实验室,四川内江641100

出　　处：《皮革科学与工程》2020年第6期6-12,共7页Leather Science and Engineering

基　　金：国家自然科学基金(201506103);四川省科技厅应用基础项目(2019YJ0399);大学生创新创业项目(X2018066)。

摘　　要：Alcalase碱性蛋白酶(AAP)水解含铬废皮渣是当前皮革废弃物资源化处理的重要方法之一,但Cr(Ⅲ)对AAP活性的影响及作用机理却鲜有报道。研究Cr(Ⅲ)对AAP活性影响及二者间的作用机理,为科学化实施AAP水解含铬废皮渣提供理论依据。结果表明,在AAP最适pH条件下,Cr(Ⅲ)的主要存在形态是Cr(OH)3,对AAP活性有显著的抑制作用,动力学研究证实该抑制类型为非竞争性抑制。紫外光谱和圆二色谱证实Cr(Ⅲ)导致AAP的二级结构发生改变,肽链结构更加舒展,该结果得到三维荧光光谱印证。荧光光谱表明Cr(Ⅲ)对AAP内源荧光(酪氨酸和色氨酸残基)有猝灭作用,且猝灭类型为碰撞引起的动态猝灭。同步荧光光谱和三维荧光光谱证实Cr(Ⅲ)导致AAP酪氨酸残基和色氨酸残基的微环境趋于亲水,且对色氨酸残基影响更为显著。Hydrolyzation of chrome-containing leather waste by using Alcalase alkaline protease(AAP)is one of the important methods for recycling leather waste.However,the effect of Cr(Ⅲ)on the activity of AAP and their action mechanisms have rarely been reported.Hence,the effect of Cr(Ⅲ)on the catalytic kinetics of Alcalase alkaline protease(AAP)and their action mechanism are investigated in order to provide the theory basis for the hydrolysis of chrome-containing leather waste using AAP.Under the optimal pH condition of APP,the main distribution of Cr(Ⅲ)speciation was Cr(OH)3,which significantly inhibited the AAP activity,and the inhibition was non-competitive type inhibition.The results of ultraviolet spectrum and circular dichroism spectrum showed that the secondary structure of AAP was obviously changed and its peptide chain was more loose after interaction with Cr(Ⅲ),these results are also verified by three-dimensional fluorescence spectra.The analyses of fluorescence spectra showed that Cr(Ⅲ)can quench the intrinsic fluorescence of AAP(tyrosine and tryptophan residues)and the quenching mechanism is a dynamic quenching caused by collision.In addition,based on the analysis of synchronous fluorescence and three-dimensional fluorescence spectra,it was found that Cr(Ⅲ)changes the micro-environment of tyrosine and tryptophan residues to be more hydrophilic,and the changes were more obvious for tryptophan residues.

关 键 词：酶 三价铬 动力学 抑制类型 作用机理 

分 类 号：TS59[轻工技术与工程—皮革化学与工程]