2酶的發(fā)酵工程

1、Chapter 2 Production of Enzyme 1The Methods of Enzyme Productionextractionbiosynthesischemical synthesisorgan/tissue/cellmicroorganism fermentationplant cell cultureanimal cell culturefew example2Unit 1 Biosynthesis and Regulation of Enzymes 1. Biosynthesis Process of Enzyme - Central dogma - Transc

2、ription - Translation - Regulation 3Reverse transcriptionCentral DogmaQ: The enzyme used to creat DNA from RNA is reverse transcriptase.4Transcription Making RNA from a DNA template to transfer information from the genome IngredientsrNTPs（ribonucleotides）DNA templateRNA polymerase 5 Stages Recogniti

3、on of initiation site Initiation Elongation Termination Processing or modification67 The synthesis of protein from mRNA template. What is necessary?Template - mRNARibosomes tRNAs (Linked to amino acids)Many accessory proteinsSome energy Translation mRNAproteintranslation template product89Five stage

4、s Preinitiation Initiation Elongation Termination Post-translational modification1011 2. Metabolic Regulation-Genetic Level Regulation (enzyme concentration)InductionFeedback repressionCatabolite repression (glucose effect)-Cellular Level Regulation (enzyme activity)Allosteric regulationZymogen acti

5、vationsReversible covalent modification 14-412Genetic Level RegulationSome genes are regulated.（ The level of gene expression can vary under different conditions.）Others are not (constitutive).They have essentially constant levels of expression. Frequently, constitutive genes encode proteins that ar

6、e necessary for the survival of the organism. 13operonstructural gene, Scontrol regionoperator gene, Opromoter gene, P Genetic Regulatory Theory Jacob and Monod-operon theory 14 regulatory gene promoter gene operator gene structural gene DNA transcription （-） RNA polymerase （+） transcription transla

7、tion mRNA translation repressor protein inducercontrol region information regionoperonOperon presence15Structure of a typical operon 16 Regulatory gene Produces a repressor (regulatory protein) that inhibits an operator gene. A repressor have two binding sitesOne for a small effector（inducer, co-rep

8、ressor）moleculeThe other for DNACo-repressor - is a small molecule that triggers repression of transcription by binding to a regulatory protein. 17 Promoter geneA DNA molecule to which RNA polymerase binds, initiating the transcription of mRNA.18Operator gene - the site on DNA at which a repressor p

9、rotein binds to prevent transcription.Structural gene -codes for any RNA or protein product.19 Types of Genetic Level Regulation 1) Induction -constitutive enzymes -inducible enzymes 2) Repression -catabolite repression -feedback repression20 Constitutive Enzyme Enzymes that are always present and a

10、ctive in cell regardless of the amount of substrate; Inducible Enzyme Enzymes whose synthesis is stimulated in the presence of a chemical (substrate) or physical stimulus (heat, light); 21(1) Induction refers to the ability of bacteria (or yeast) to synthesize certain enzymes only when their substra

11、tes are present; Inducer -a small molecule that triggers gene transcription by binding to a regulatory protein.The Regulatory Mechanism22Induction of the Lac Operon Gene transcription can be switched on and off by gene regulation proteins. The lac operon in E.coli is an example of that dual control.

12、 Glucose and lactose levels control the initiation of transcription of the lac operon.23In an E. Coli cell growing in the absence of lactose, a repressor protein binds to the operator, preventing RNA polymerase from transcribing the lac operons genes. The operon is OFF. When the inducer, lactose, is

13、 added, it binds to the repressor and changes the repressors shape so as to eliminate binding to the operator. The operon is ON.24Regulator geneOperator geneStructural genesPGLacZLacYLacamRNArepressor（active）InactiveNo transcriptionNo translationPromoter geneOGRGPGLacZLacYLacaOGRGmRNAZmRNAYmRNAa Rep

14、ressor inducer complexmRNAA、No inducerB、Inducer(lactose)lactoseRepressor joins and turns off operator geneturned offStructural genesturned onRNA polymerasetranscriptiontranslation2526(2)Feedback Repression (end-product repression ) Enzymes whose synthesis can be decreased by adding the end products

15、( co-repressor).27Expression is regulated by the Trp repressor protein which is encoded by the trpR gene. TrpR protein is unable to bind to the operator by itself. Once the level of tryptophan builds up, the Trp repressor will block further transcription of the operon and the synthesis of those enzy

16、mes will decline.The Tryptophan Operon2829m-RNARNA polymeraserepressorPromoterOperatorGene 1Gene 2Gene 3Normal TranscriptionRNA polymeraserepressorPromoterOperatorGene 1Gene 2Gene 3Co-repressorTranscription BlockedDNA templateinactiveactiveDNA template30methodsrepressoradditivebind with operator gen

17、eeffectsexamplesinductionactivetranscription off and translation off.inducertranscription and translation .Lac operon repressioninactiveCo-repressortranscription off and translation off.Trp operonInducible Operon and Repressible Operon31InductionRepression1. It turns the operon on.1. It turns the op

18、eron off.2. It starts transcription and translation.2. It stops transcription and translation.3.It is caused by a new substance, which needs enzymes to get used.3. It is caused by an excess of existing metabolite.4. It operates in a catabolic pathway.4. It operates in an anabolic pathway.5. Represso

19、r is prevented by the inducer from joining the operator gene.5. Repressor is enabled by a co-repressor to join the operator gene.32(3)Catabolite Repression （Glucose Effect）When exposed to both lactose and glucose E. coli uses glucose first, and catabolite repression prevents the use of lactoseWhen g

20、lucose is depleted, catabolite repression is alleviated, and the lac operon is expressedglucose ？33The small effector molecule in catabolite repression is not glucose.This form of genetic regulation involves a small molecule, cyclic AMP (cAMP) It is produced from ATP via the adenylate cyclasecAMP bi

21、nds an activator protein known as the Catabolite Activator Protein (CAP) Also termed ： the cyclic AMP acceptor protein (CAP)34Catabolite Repression RLacZLacYLacamRNAmRNAZmRNAYmRNAaCAPgeneStructural geneTCAPOCAP binding site RNApolymeraseTcAMP -CAPPglucosecatabolitecAMPasePhosphodiesteraseATPcAMP5-AM

22、PinhibitactivateGlucose catabolite and cAMPcAMPLower cAMPInactivate CAP35 The sequential use of two sugars by a bacterium is termed diauxic growth36Genes for transport and catabolism of secondary carbon source ( )TimeCell density= Usually glucose37Genes for transport and catabolism of secondary carb

23、on source ( )TimeCell density= Usually glucose38Genes for transport and catabolism of secondary carbon source ( )TimeCell density= Usually glucose39Genes for transport and catabolism of secondary carbon source ( )TimeCell density= Usually glucose40Genes for transport and catabolism of secondary carb

24、on source ( )TimeCell density= Usually glucose41Genes for transport and catabolism of secondary carbon source ( )TimeCell density= Usually glucose42Genes for transport and catabolism of secondary carbon source ( )TimeCell density= Usually glucose43Genes for transport and catabolism of secondary carb

25、on source ( )TimeCell density= Usually glucose44TWO questions:How does cAMP control the enzyme levels (or gene expression)?How does glucose (the favored carbon source) control the levels of cAMP?Catabolite Repression45Cellular Level Regulation -Allosteric regulation46Whats the difference between All

26、osteric regulation47The effects of substrates on allosteric enzymes are referred to as _ effects.homotropic heterotropicEnzymes that do not obey Michaelis-Menten kinetics are_.isoenzyme allosteric enzymes 48What are the differences between feedback repression and feedback inhibition?49Feedback repre

27、ssionFeedback inhibitionRegulation levelgenetic: RNA transcriptioncellular: activity of enzymeComplex formedend product +repressorend product + enzymeEffectoperator on DNA template occupied by the complexreduced enzyme activityConsequenceblocked transcription The respective reaction is inhibited.50U

28、nit 2 Method for Improving Productivity of Enzyme51 Genetic Control 1. gene mutation Inducible Constitutive Repressed De-repressed522. Gene Recombination (1) Gene recombination in vivo -Conjugation（接合）： genetic material between two bacterial cell via direct cell-to-cell contact -Transduction（轉(zhuǎn)導(dǎo)）：a v

29、irus -Transformation（轉(zhuǎn)化）： donor DNA free in the environment -Protoplast fusion（原生質(zhì)體融合）: cytoplasmic hybrid from two different species5354protoplast fusion1.Two different cells with their cell walls removed by enzymes.2. The cells get together and fuse after exposing them to Polyethylene glycol.3. A

30、single cell is produced, often with 2 nuclei and organelles of the 2 cells.55(2) Gene recombination in vitro （recombinant DNA technique or gene engineering） Donor Vector Receptor56 Condition ControlAdding inducer -substrate -product -substrate analog 2. Controlling repressor concentration -Control e

31、nd-product concentration -Using not-easily-used carbon source (e.g. starch) -Add a certain amount of cAMP573. Adding surface active agent -ionic harmful to cell -non-ionic increase cell permeability (Tween80, TritonX100)4. Adding enzyme accelerator -determined by experiments58Unit 3 Fermentation Kin

32、etics of Enzyme Production1. Cell growth kinetics2. Biosynthetic mode of enzyme3. Enzyme producing kinetics591. Cell growth kinetics 4 stages of the cell growth cycle: Lag stage, exponential stage, stationery stage and death stage60Monod equation: specific growth ratemax： maximum growth rateS： conce

33、ntration of limiting nutrientKs： Monod coefficient6162Synchronization biosynthetic mode Mid-term biosynthetic modeContinue biosynthetic modeLag biosynthetic mode 2. Patterns of enzyme synthesis63 Enzyme synthesis is synchronous with cells growth. It would be stopped after the inducer was removed or

34、cells growth entered into the equilibrium phase. CharacteristicsCan be reduced, but not be repressed by catabolite repression and feedback repression.mRNA is unstable. 1) Synchronization biosynthetic mode 64 2) Mid-term biosynthetic mode Enzyme synthesis started after a period of cells growth and st

35、opped after cells growth entered into the equilibrium phase. CharacteristicsEnzyme synthesis is repressed by catabolite repression and feedback repression.mRNA is unstable. 65 3) Continue biosynthetic mode Enzyme synthesis followed along with the cells growth and lasted to the end of the equilibrium

36、 phase. CharacteristicsCan be reduced, but not repressed by catabolite repression and feedback repression.mRNA is stable. 66 4) Lag biosynthetic mode Enzyme synthesis started only after cells growth entered into the equilibrium phase. Characteristics Repressed by catabolite repression. mRNA is stabl

37、e. 67 Summary The major factors which affect the mode of enzyme biosynthesis are: 1）mRNA stability (When cell growth stopping) high：enzyme synthesis continues low：enzyme synthesis stops immediately 2） repressor Be repressed ：lag behind Not be repressed ：go along with68To select ： The most desirable

38、mold Continue biosynthetic mode To transform ： Non-perfect mode Synchronization biosynthetic mode -increasing the stability of mRNA. How? Lag biosynthetic mode -de-repressing (catabolite repression) Mid-term biosynthetic mode -start from two aspects693. Kinetics of Enzyme ProductionXcell concentrati

39、oncell specific growth rategrowth coupling specific enzyme production coefficientnon-growth coupling specific enzyme production rateSynchronization biosynthetic mode Mid-term biosynthetic modeContinue biosynthetic modeLag biosynthetic mode dE/dt=XdE/dt= X+XdE/dt= XdE/dt=X70 Unit 4 Producing Enzyme b

40、y Microorganism An industrial microorganism -high yield -cheap media -grow rapidly -stable ( not easily mutated or degenerated ) -non-pathogenic 71 Common microorganisms in enzyme productionEscherichia coli Pseudomonas Bacillus subtilis Micrococcus Streptococcus StreptomycesAspergillus nigerAspergil

41、lus oryzaeMonascusPenicilliumTrichoderma Rhizopus Mucor Absidia Saccharomyces cerevisiae Candida72 Isolation and Screening Sampling Enrichment Pure culture Primary screening：quantity Secondary screening：quality 73 Screening of microorganisms for the production of -amylase74Screening of microorganism

42、s for the production of protease75 The Biotechnical Process7677Fermentor78High Cell Density FermentationOptimization and cost reduction79High cell-density culture 10 x bio wet mass, 10 x product cost reductionMethodsMedia optimization (media formulation)Change supply mode OxygenationRemoval of inhib

43、itory components80Unit 5 Enzyme Production by Plant and Animal Cell Cultures 81animal cellplant cellThe main difference is that- plant cells have cell wall and animal cells dont.82 A comparison of three types of cell kinds of cellplant cell microorganism cell animal cell size /um20030011010100double

44、 times/h120.3-615nutritionsimplesimplecomplexilluminationyes no no shearing forcesensitivenon-sensitive（mostly) very sensitivemain productspigment、medicine(s)、fragrance、enzyme et al.alcohol、organic acid、amino acid、antibiotics、nucleotide、enzyme et al.vaccine、hormone、monoclonal antibody、enzyme et al.831. Enzyme production by plant cel