生物工藝學(xué)教學(xué)課件：Chapter 09 Immobilization of Biocatalysts

1、Chapter 9Immobilization of BiocatalystsMatrix-Entrapped9.1. Introduction 9.2. Biocatalysts 9.2.1. Enzymes Specificity Catalytic Power 9.2.2. Ribozymes RNA 9.2.3. Abzymes9.2.4. Multienzyme complex PDC (the pyruvate dehydrogenase complex) Proteosome Cellulosome Multienzyme Complexes and Immobilization

2、 Technology9.2.5. Cells Animal Cells Plant Cells Microorganisms (Bacteria, Yeast and Filamentous Funji)9.2.6. Biocatalyst Selection Repeated use of biocatalyst in batch reactions Simple separation of biocatalyst from product after reaction Continuous use of biocatalyst in a continuous reactor system

3、9.3 Immobilization InorganicOrganicBiologicalAluminium OxidePolyethyleneCelluloseNickel oxidePolysty;reneDextranStainless steelPolyacrylateAgarosePorous glassNylonStarchPorous silicaPolyacrylamideAlginateActivated carbonPolymethacrylateCarrageenanDiatomaceous earthPolypyrroleChitinIron oxide Polyani

4、lineBoneTitanium oxidePolyphenolChitosanPumice stonePolyesterCollagen, gelatinZirconium oxidePolyvinyl alcoholLiposomeVanadium oxide-Cells (yeast, red blood)9.3.1 Choice of Support material9.3.2 Choice of Immobilization ProcedureAdvantages:Little or no damage to the biocatalystSimple, cheap and quic

5、k to obtain immobilizationNo chemical changes required for the support or biocatalystEasily reversed to allow regeneration with fresh biocatalystDisadvantages:Leakage of biocatalyst from the support/contamination of productNon-specific binding of other material to the supportOverloading of the suppo

6、rt with biocatalystSteric hindrance of the biocatalyst by the support(1) Adsorption(2) Covalent Binding酶分子之間共價(jià)交聯(lián)和與水不溶性載體共價(jià)偶聯(lián)(3) Entrapment(4) Encapsulation(5)Cross-linking 9.4.1. Stability9.4.2. Catalytic activity9.4 Properties of Immobilized Biocatalysts9.5 Applications Hydrolysis Esterication Alco

7、holysis Transfer- esterification9.5.1 Applications of lipase脂肪酶催化制備單一手性胺兩步酶法拆分獲得型和型脂肪酶拆分生產(chǎn)擬除蟲菊酯的工藝Scheme1. An enzymatic process for the preparation of chiral C-13 paclitaxel side-chain synthon. Anticancer(paclitaxel semisynthesis) Captopril and zofenopril (antihypertensive drugs) prevent the convers

8、ion of angiotensin I to angiotensin II by inhibition of the angiotensin-converting enzyme (ACE).Captopril zofenopril Angiotensin-converting enzyme (ACE) inhibitors S-(-)-3-benzoylthio-2- methylpropanoic acid is a key chiral intermediate for the synthesis of captopril or zofenopril; both are antihype

9、rtensive drugs.Scheme2 A process for the resolution of 3-benzoylthio-2- methylpropanoic acid by lipase Squalene synthase is the first pathway-specific enzyme in the biosynthesis of cholesterol. Farnesyl pyrophosphate (FPP) analogs are a major class of inhibitors of squalene synthase. BMS-188494 is a

10、 potent squalene synthase inhibitor effective as an anticholesterol drug. Anticholesterol drugs (S)1-(acetoxyl)-4-(3-phenyl)butylphosphonic acid, diethyl ester , is a key chiral intermediate required for the total chemical synthesis of BMS-188494.Scheme3 Resolution of 1-(hydroxy)-4-(3-phenyl)butyl p

11、hophonic acid, diethyl ester by lipaseD-Hydroxyl phenylglycine is used in the production of amoxicillin and cefadroxyl, and D-phenylglycine is required for the semisynthetic antibiotic amphicillin and cephalexin.amoxicillincefadroxylcephalexin Anti-infective drugs Crich et al. have developed a gener

12、al enzymatic asymmetric synthesis of -amino acids. Scheme4 Enzymatic synthesis of -amino acids by lipase Purine nucleoside analogs have been used as antiviral agents. lamivudine, zidovudine, and didanosine are effective antiviral agents. Lamivudine is a highly promising drug candidate for HIV 2 and

13、HIV 3 infection. Didanosine zidovudine Lamivudine Antiviral agents An enzymatic resolution process has been developed for the preparation of chiral intermediate for lamivudine synthesis.Scheme5 Enzymatic resolution of chiral intermediate of lamivudine by lipasePalm oil midfraction ethyl stearateDryi

14、ng with vacuumInteresterification (40,48-72h with hexane as the medium)Immobilised lipaseSeparation of lipase by filtration and removal of solvent by vacuumDistillation to remove ethyl estersSolvent fractionation and refiningCBE productFigure Process scheme for the production of cocoa butter equival

15、ents(CBE) via lipase-catalyzed interesterification in a stirred-tank reactorFigure Acyl migration and formation mechanisms of nonspecific by-products during enzyme production of specific structure lipidsFigure Reaction schemes of enzymatic randomization by alcoholysis between a triacylglycerol(LLL)

16、and glycerol(Gly)Figure The relationships between substrate molar ratio(n) and the contents of acylglycerol species at equilibrium during reactions catalyzed by nonspecific lipaseLLL,triacylglycerolDAG,diacylglycerolMAG,monoacylglycerolGly,glycerolLipaseSubstrateProductreferencesLipozyme IM and PSLS

17、ingle-cell oil and CyA23% incorporation with Lipozyme IMIwasaki,Y. et.alRSL, RML, and RDLOOO, LLL, cottonseed oil, and CyA45% CyOCy and 46% CyOO with acidolysis from OOO, 95% sn-1,3 incorporation with the two-step processSummanouM.M. et.alLipozyme IMCanola oil and CyA59.9% new triacylglycerols with

18、acidolysis and 82.8% with esterificationWillis, W.M. et.alLipozyme IMPeanut oil and CyA30% incorporationLee, K.T. et.alLipozyme IMRapseed oil and CANet incorporation 77% with less than 2% of DAGXu,X. et.alTable Lipase-catalyzed synthesis and production of specific structured lipids in batch reactors

19、LipaseSubstrateProductreferencesLipozyme IMBorage oil and CA, EPAIncorporation of 10.2% CA and 27.3% EPA was obtainedAkoh, C.C. et.alLipozyme IM, RDL, and RJLOOO, LLL, peanut oil and CyASn-1,3 incorporation 90.9%, CyA at sn-2 position below 0.1%Soumanou, M.M. et.alLipozyme IM, RDL, and RJLOOO, LLL,

20、cotton seed oil and CyASn-1,3 incorporation 94%, CyA at sn-2 position less than 1.9%Soumanou, M.M. et.alLipozyme IMLLL and CyA-EEDi-incorporation SSL 32.1%Lee,K.T. et.alLipozyme IMOOO and caproic acid, butyric acid49% di-incorporated and 38%mono-incorporated SSLFomuso,L.B. et.alLipozyme IMOOO and Cy

21、A-EE41.7%MLM was obtainedHuang,K.S. et.alLipozyme IMMCT and EPA-EE32-36% EPA was incorporated into MCTLee,K.T. et.alLipaseSubstrateProductreferencesRDLSaffower oil linseed oil and CyAIncorporation 45-50%. 0.7% CyA was found in sn-2 positionShimada, Y.A. et.alRDLTuna oil and CyASn-1,3 incorporation 65%, less than 1% CyA at sn-2 positionShimada, Y.A. et.alLipozyme IMTrisum 90 and CAC49 and C41 total yield 95-96%Shieh, C.J. et.alLipozyme IMMenhaden oil Products containing 40% CyA and 30% EPA and DHA with less than 3% acyl migrationXu,X. et.alLipozyme