1、酶工程酶的固定化與修飾課件酶工程酶的固定化與修飾課件第一節(jié) 概述第二節(jié) 固定化酶的性質(zhì)及其影響因素第三節(jié) 固定化酶的制備第四節(jié) 酶的修飾第五節(jié) 固定化新技術(shù)與發(fā)展趨勢(shì)第六節(jié) 固定化酶的應(yīng)用第一節(jié) 概述什么是固定化酶？固定化酶（Immobilized enzyme） ：固定在一定載體上，在一定空間范圍內(nèi)起催化作用的酶。什么是固定化酶？固定化酶（Immobilized enzym為什么要對(duì)酶進(jìn)行固定化？對(duì)酶而言：1、穩(wěn)定性差2、回收困難，一次使用3、產(chǎn)物的分離純化困難核心：成本問題！為什么要對(duì)酶進(jìn)行固定化？對(duì)酶而言：優(yōu) 點(diǎn)固定化酶可以重復(fù)，使用效率高，成本低極易將固定化酶與底物、產(chǎn)物分開；在大多數(shù)

2、情況下，能提高酶的穩(wěn)定性；具有一定的機(jī)械強(qiáng)度可以在較長(zhǎng)時(shí)間內(nèi)進(jìn)行反復(fù)分批反應(yīng)和裝柱連續(xù)反應(yīng)；酶反應(yīng)過程能夠加以控制；產(chǎn)物溶液中沒有酶的殘留，簡(jiǎn)化了提純工藝；較游離酶更適合多酶反應(yīng)；可以增加產(chǎn)物的收率，提高產(chǎn)物的質(zhì)量； 酶的使用效率提高，成本降低。固定化酶的優(yōu)缺點(diǎn)優(yōu) 點(diǎn)固定化酶的優(yōu)缺點(diǎn)缺 點(diǎn)固定化時(shí)，酶活力有損失；工廠初始投資大；只能用于可溶性底物；胞內(nèi)酶必須經(jīng)過酶的分離。缺 點(diǎn)第二節(jié) 固定化酶的性質(zhì)及其影響因素一 影響固定化酶性質(zhì)的因素二 固定化后酶性質(zhì)的變化三 評(píng)價(jià)固定化酶的指標(biāo)第二節(jié) 固定化酶的性質(zhì)及其影響因素一 影響固定化酶性質(zhì)的因一 影響固定化酶性質(zhì)的因素1 酶本身的變化，主要是由于活

3、性中心的氨基酸 殘基、高級(jí)結(jié)構(gòu)和電荷狀態(tài)等發(fā)生了變化。2 載體的影響（1） 分配效應(yīng)（2） 空間障礙效應(yīng)（3） 擴(kuò)散限制效應(yīng)3. 固定化方法的影響一 影響固定化酶性質(zhì)的因素1 酶本身的變化，主要是由于活二、固定化后酶性質(zhì)的變化1 固定化對(duì)酶活性的影響：酶活性下降，反應(yīng)速度下降。2 固定化對(duì)酶穩(wěn)定性的影響（1） 操作穩(wěn)定性提高（2） 貯存穩(wěn)定性比游離酶大多數(shù)提高。（3 ） 對(duì)熱穩(wěn)定性，大多數(shù)升高，有些反而降低。（4）對(duì)分解酶的穩(wěn)定性提高。（5） 對(duì)變性劑的耐受力升高二、固定化后酶性質(zhì)的變化1 固定化對(duì)酶活性的影響：酶活性下固定化后酶穩(wěn)定性提高的原因：a. 固定化后酶分子與載體多點(diǎn)連接。b. 酶活

4、力的釋放是緩慢的。c. 抑制自降解，提高了酶穩(wěn)定性。固定化后酶穩(wěn)定性提高的原因：a. 固定化后酶分子與載體多點(diǎn)連pH的變化pH對(duì)酶活性的影響：1） 改變酶的空間構(gòu)象2）影響酶的催化基團(tuán)的解離3）影響酶的結(jié)合基團(tuán)的解離4）改變底物的解離狀態(tài)，酶與底物不能結(jié)合或結(jié)合后不能生成產(chǎn)物。pH的變化pH對(duì)酶活性的影響：固定化酶pH的變化： 微環(huán)境是指在固定化酶附近的局部環(huán)境，而把主體溶液稱為宏觀環(huán)境。1） 載體帶負(fù)電荷，pH向堿性方向移動(dòng)。 載體帶正電荷，pH向酸性方向移動(dòng)。2）產(chǎn)物性質(zhì)對(duì)pH的影響 催化反應(yīng)的產(chǎn)物為酸性時(shí)，固定化酶的pH值比游離酶的pH值高；反之則低。固定化酶pH的變化： 微環(huán)境是指在固

5、定化酶附近最適溫度變化：一般與游離酶差不多，但有些會(huì)有較明顯的變化。底物特異性變化：作用于低分子底物的酶 特異性沒有明顯變化。既可作用于低分子底物又可作用于大分子底物的酶 特異性往往會(huì)變化。最適溫度變化：一般與游離酶差不多，但有些會(huì)有較明顯的變化。底米氏常數(shù)Km的變化，Km值隨載體性質(zhì)變化：由于分配效應(yīng)：=Si微環(huán)境/S宏觀環(huán)境 Km=Km/(表觀米氏常數(shù))（1） 載體與底物帶相同電荷，SiS, Km 固定化酶降低了酶的親和力。（2） 載體與底物電荷相反，靜電作用，SiS，則1,Km5校對(duì)功能的性質(zhì)，配合適當(dāng)條件，以很低的比率向目的基因中隨機(jī)引入突變，構(gòu)建突變庫(kù)，憑借定向的選擇方法，選出所需性

6、質(zhì)的優(yōu)化酶(或蛋白質(zhì))，從而排除其他突變體。定向進(jìn)化的基本規(guī)則是“獲取你所篩選的突變體”。定向進(jìn)化=隨機(jī)突變+選擇。前者是人為引發(fā)的，后者雖相當(dāng)于環(huán)境，但只作用于突變后的分子群，起著選擇某一方向的進(jìn)化而排除其他方向突變的作用，整個(gè)進(jìn)化過程完全是在人為控制下進(jìn)行的定向進(jìn)化的原理在待進(jìn)化酶基因的PCR擴(kuò)增反應(yīng)中，利用Taq 第五節(jié) 固定化新技術(shù)與發(fā)展趨勢(shì)仿生粘附材料納米材料（石墨烯）多酶組裝固定發(fā)展趨勢(shì)第五節(jié) 固定化新技術(shù)與發(fā)展趨勢(shì)仿生粘附材料BackgroundP. B. Messersmith, et al. (2007). ScienceDOPA and other catechol com

7、pounds perform well as binding agents for coating inorganic or organic surface.PTFE仿生粘附材料BackgroundP. B. Messersmith, ePreparation of Bio-adhesive magnetic nanoparticles Ren, et al. (2011). BMC Biotechnology Ren, et al. (2012). PLoS OneSynthesis of PD-MNPs Synthesis of CCS-MNPs 仿生粘附材料Preparation of

8、Bio-adhesive maCharacterization of PD-MNPsThe finally weight loss of the PD-MNPs is 48.8%, while the MNPs lost 12.1% weight. The nitrogen-to-carbon (N/C) ratio of the PD-MNPs is 0.119. Additionally, the O1s peak of iron oxide at 529.5 eV replaced by the organic oxygen peak (532 eV).XPS of MNPsXPS of

9、 PD-MNPsPD-MNPsMNPs仿生粘附材料Characterization of PD-MNPsTheCharacterization of CCS-MNPsThe finally weight loss of the MNPs (a), CS- MNPs (b) and CCS-MNPs (c) is 12%, 36% and 57%, respectively. The nitrogen-to-carbon (N/C) ratio of the nanoparticles is decreased from 0.105 (CS-MNPs) to 0.038 (CCS-MNPs).X

10、PS of CS-MNPsXPS of CCS-MNPs仿生粘附材料Characterization of CCS-MNPsThSEM &TEM of the nanoparticlesSEMTEMMNPsPD-MNPsCCS-MNPs仿生粘附材料SEM &TEM of the nanoparticlesSImmobilization of lipase using the PD-MNPsThe nitrogen peak (399.5 eV) significantly increases and the ratio of N/C is reach at 0.139 after lipase

11、 immobilized. NanoparticlesAmount of particles (mg)Amount of lipase (mg)Protein binding (mg)Immobilization yield (%)Activity yield (%)MNPs10103.1 0.531.2 5.0 18.8 1.71051.8 0.4 36.0 4.026.4 1.4PD-MNPs10107.5 0.6 75.5 6.048.2 1.91054.3 0.4 86.0 8.063.4 1.6 仿生粘附材料Immobilization of lipase usingStabilit

12、y of the immobilized lipase SolventsImmobilization enzyme (%)Free enzyme (%)Toluene993.5552.1Hexane962.9682.7Chloroform502.2351.5Acetone51.2553.0Acetonitrile20.5502.5Ethyl Acetate20.5401.9Methanol00.400.5Ethanol00.800.6DMF00.6141.4DMSO00.500.6仿生粘附材料Stability of the SolventsImmobMore than 70% of its

13、initial activity was still remained after 21 reuses.Reusability of the Immobilized lipase仿生粘附材料More than 70% of its initial aImmobilization of -transaminase using the CCS-MNPsCompositesBound enzyme (mg/g)Activity (U/mg carrier particle)Specific activity (U/mg)Activity retention (%)MNPs195.312.41.630

14、.058.350.4933.4CS-MNPs107.36.90.920.068.540.0795.72.4CCS-MNPs681.711.65.510.187.970.2589.50.6ABCDRen, et al. (2012). PLoS One仿生粘附材料Immobilization of -transaminaIn situ aggregation of Gluconobacter oxydans using PD-MNPsRen, et.al., (2012) Biotechnology & BioengineeringRen, et.al., (2013) Journal of B

15、iotechnologyPD-MNPsMNPsMNPs-Cells aggregatesPD-MNPs-Cells aggregatesIn situ aggregation of GluconoThe factors during the preparation of PD-MNPs effecting on G.oxydan aggregating efficiency5.0mg/ml0.5mg/ml120min45min仿生粘附材料The factors during the preparaMaterialsAmount of Nanoparticles (mg)Added Cells

16、(mg)Aggregated Cells (mg)Cell Recovery (%)Activity Recovery (%)MNPs2509.60.919.21.818.30.5PD-MNPs25042.61.285.22.482.71.0Free cells50100Effect of temperature and pH on G.oxydan aggregating efficiencyMNPsPD-MNPsMNPsPD-MNPsMNPsPD-MNPs仿生粘附材料MaterialsAmount of NanoparticlCells aggregated by the optimize

17、d protocol show better stability than free cells, leading to maintenance of high cell biocatalytic activity over more cycles.Magnetic Isolation and ReusePD-MNPsFree CellsMNPs仿生粘附材料Cells aggregated by the optimi 納米材料(納米石墨烯） Jingyan Zhang etc. Nanotechnol Rev. 2013; 2910:27-45 納米材料(納米石墨烯） Jingyan Zhan

18、g石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯石墨烯其它納米技術(shù)其它納米技術(shù)酶工程酶的固定化與修飾課件組裝固定方法Nature biotechnology 2009, 27(8):753-759.ChemBioChem 2010, 11, 1517 1520Angew. Chem. Int. Ed. 2012, 51, 8787 8790Nano Lett., Vol. 9, No. 12, 2009組裝固定方法Nature biotechnology 200發(fā)展趨勢(shì)理性設(shè)計(jì)酶的固定化技術(shù)In silico analysis to predict the orientat

19、ion of enzymesAn array of algorithms (based on the protein size and the textural properties of the support)for the rational design of immobilized derivativesOrdered mesoporous materialsTailor immobilized enzymes with high volumetric activity Fluorescence confocal microscopyUnderstand the diffusional

20、 restrictions and the distribution of biomolecules within the support發(fā)展趨勢(shì)理性設(shè)計(jì)酶的固定化技術(shù)IntroductionThe importance of enzyme immobilizationEasy separation and reuse of the biocatalyst, makes product recovery easier and very often enhances enzyme stabilityFor analytical, energetic and biomedical applicat

21、ions (e.g., biosensors, biofuel cells)Enzyme immobilization protocolsEnzyme binding to a prefabricated supportEnzyme entrapment or encapsulationCarrier-free cross-linking with bifunctional regentsDesign a robust immobilized biocatalystNon-catalytic requirements: separation, reuse, downstream process

22、ingCatalytic function: productivity, space-time yield, productivityIrrational vs rational (by predicting the location of amino acid residues or protein domains implicated in the binding with the support)IntroductionThe importance of In silico analysisHudson et al. J. Phys. Chem. B, Vol. 109, No. 41

23、cytochrome c and xylanasephysicochemical properties of mesoporoussilica surface potential of the biomoleculesWeber et al. J. Mol. Catal. B-Enzym, Vol 64They studied the adsorption of P450 enzymes on mesoporous MCM-41 and SBA-15modeling the 3D enzyme structureperforming electrostatic potential calcul

24、ationspredicted the pH-dependence of the P450 immobilization proposed the possible orientations of the protein on such mesoporous materialsIn silico analysisHudson et alSelection of a proper supportSelection of a proper supportComputational Methods Applied to Covalent Binding of Enzymes to SupportsC

25、omputational Methods Applied Reactivity of the amino acid residuesintrinsic chemical naturethe microenvironment and, state of ionizationLIGRe algorithm:it represents the proportion between active (e.g., deprotonated in the case of NH2 ) and inactive (e.g., protonated) groups at immobilization pHLIGR

26、e = 10(pHpKa)LIGRe 0.1 low0.1 LIGRe 1 half-reactiveLIGRe 1 reactiveThe priority of reactivity of amino acid residuesReactivity of the amino acid rExample for LIGRe algorithmMyceliophthora thermophila laccase (MtL)Polymethacrylate-based polymer (Sepabeads EC-EP3) activated with epoxy groupsanalyzed t

27、he reactivity of amino and phenolic residues on the solvent accessible areaExample for LIGRe algorithmMycN-term was almost opposite to the active site of the enzymeTwo other -NH2 (Lys128 and Lys339), located far away from the active siteAt pH 10.0 the increase on reactivity of other -NH2 on the prot

28、ein solvent accessible area (Lys205, Lys325 and Lys454) could affect the catalytic efficiency of the immobilized derivative as they were in close proximity to the active siteTyr286 and Tyr391, offered an optimal orientation for the immobilized derivativeHowever, (Tyr214) was located in the vicinity

29、of the active site and could exert an egative effectLIGRe prediction leads to select pH 9.0 as the optimal immobilization pH in terms of protein orientation with acceptable group reactivityN-term was almost opposite to In Silico Analysis of Adsorption of Enzymes to SupportsThe amount of adsorbed enz

30、yme depends on the size of the protein molecule, the specific surface area of the carrier, the pore size and volume, and the number of sites available for protein adsorptionRDID 1.0 software: optimization of immobilization processestMQ , theoretical maximum protein quantityIn Silico Analysis of Adso

31、rptiThe chemical nature of the surfaces of two enzymes, lipase B from Candida antarctica (CALB) and penicillin G acylase from Providence rettgeri (PGA)GRID computational method:establish either hydrophobic or hydrophilic interactions using a chemical probeOrientation of CALB can be modulated varying

32、 the hydrophilicity of the carrierThe chemical nature of the surMesoporous Silica as Protein BindersZeolites: small pore diameter (below 1.2 nm)Amorphous mesoporous silicas(uniform and higher pore diameters (240 nm)Ordered mesoporous silicasAdsorbed the lipase CALB in the cage-like material SBA-16On

33、e-step processes for the simultaneous synthesis of ordered porous silica networks and enzyme encapsulation within the poresMesoporous Silica as Protein BAnalysis of the Distribution of the Enzyme in the SupportA typical confocal image for sterol esterase labeled with fluorescein isothiocyanate (FTIC

34、) and covalently immobilized in epoxy activated Dilbeads TA, varying the observation depththe enzyme was not uniformly distributed in the beads, as most of the enzyme molecules were confined in an outer shell of approximately 10.5 m widthThe depth of the enzyme layer was very similar analyzing beads of different radiusAnalysis of the Distribution o（一）固定化酶在工業(yè)生產(chǎn)中的應(yīng)用1、固定化氨基?；?/p>