ENZYME PURIFICATION Enzymology and Enzyme Technology

PEMURNIAN ENZIMMata Kuliah Biokimia - MKK4213STF MUHAMMADIYAHWhy isolate enzymes??To understand enzyme properties:Enzymes work in a complex system (inside cell), == we must try first in a simple system ==

Used in food / industrial applications- Pectinase : Juice production - Chymosin : cheese production - Glucose isomerase : HFS productionWhy isolate enzymes??The isolated enzyme can be characterized:optimum activity in various conditions (pH, temperatur, ionic strengh etc)specificity kinetic parametermechanism of catalysisregulationStructure

Understand the role of enzymes in more complex systems

How to Separate These Objects

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64857458wood stone cotton wood wood cotton stone wood stone cotton stone cottoncottonwoodstoneShapeSizeDensityShapeDensitySizeSieving different sizesDifferent sedimentationDifferent rolling speed46785134678910111225Juang RH (2004) BCbasicsObjectives and StrategyObjectives:to isolate a particular enzyme from all other proteins (enzyme) and cell components with- maximum possible yield- maximum catalytic activity- maximum possible purity Strategy:Objectives and StrategyAffinity Separation MaterialWhole extractionCrude extractHomogenisationLarge scale separationPure enzymeSeries of small scale separationStrategy Protein are diverse in composition structure behaviour, you should know about their origin. As your purification strategy depends on it.Where is this enzyme or protein present in the cell? (intracellular, extracellular, membranous).How you can purify this protein in as few steps as possible without the loss of activity. Keeping in consideration of temperature and time.

Sources of EnzymesMicroorganism, animal, plant (tissue and cell)Rennin from Mucor mieheiRennin from stomach of calf

Heterologous expressionGenetically engineered bacteria, yeast to produce enzyme - Rennin / chymosin from g.e. yeast- Amyloglucosidase from g.e. Bacillus licheniformis

Industrial enzymes may be extracted from any living organism: fungi (over a half) bacteria (over 1 / 3) animal (8%) plant (4%)Sources of EnzymesSources of EnzymesMicrobes are preferred to plants and animals as sources of enzymes because:

They are generally cheaper to produceTheir enzyme contents are more predictable and controllablePlant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants)Enzyme EC

SourcesApplicationa-Amylase3.2.1.1AspergillusEBakingCatalase1.11.1.6AspergillusIFoodCellulase3.2.1.4TrichodermaEWasteDextranase3.2.1.11PenicilliumEFoodGlucose oxidase1.1.3.4AspergillusIFoodLactase3.2.1.23AspergillusEDairyLipase3.1.1.3RhizopusEFoodRennet3.4.23.6Mucor mieheiECheesePectinase3.2.1.15AspergillusEDrinksProtease3.4.23.6AspergillusEBakingE: extracellular enzyme; I: intracellular enzymeFungal Enzymes11Catalase:catalyzes the decomposition of hydrogen peroxide to water and oxygen.Enzyme

SourcesApplicationa-Amylase3.2.1.1BacillusEStarchb-Amylase3.2.1.2BacillusEStarchAsparaginase3.5.1.1Escherichia coliIHealthGlucose isomerase5.3.1.5BacillusIFructose syrupPenicillin amidase3.5.1.11BacillusIPharmaceuticalProtease3.4.21.14BacillusEDetergentBacterial Enzymes12Asparaginase:(EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid.Penicillin amidase: Sakaguchi and Murao1 reported on the presence of an enzyme in the mycelium of Penicillium chrysogenum and Aspergillus oryzae which would split penicillin G (I) into phenylacetic acid (II) and 'penicin' (III) : Basic Principles of Protein (Enzyme) PurificationCellOrganelleHomogenizationMacromoleculeNucleic acidCarbohydrate(Lipid)SizeChargeSolubilityAffinitySmall moleculeCell DebrisProteinAmino acid, Sugar,Nucleotides, etc Gel filtration,SDS-PAGE,UltrafiltrationIon exchange,Isoelectric focusingSalting-out (Ammonium sulfate )AffinitychromatographyGeneral protocol for enzyme purificationTaking the intact cell tissue.Homogenisation Separation of cell debris and insoluble stuffPrecipitation of protein with the saltGetting rid of salt by dialysisFurther purification by column and ion exchange chromatography ,Each above step is followed by enzyme assay activity(in case you lost your enzyme )Finding out the exact molecular weight by column chromatography and by SDS-Gel-electrophoresis

Typical Enzyme Purification Scheme16Develope Enzyme Assay How do we recognize the enzyme that we are looking for?

Develop analytical assayusually based on the reaction that the enzyme catalyzes in the cell

ONPGGalactoseONP+b galactosidaseDevelope Enzyme Assay Monitoring Progress of Purification ProtocolStepTotal protein (mg)Total activity (units)Specific activity (units/mg)Yield (%)Purification level (Purity factor)Initial extract15,000150,000101001(NH4)2SO4 precipitation4,600138,00030923Ion-exchange1,278115,50090779Size exclusion68.675,0001,10050110Affinity column1.7552,50030,000353,000(Berg, Tymoczko, & Stryer. (2002) Biochemistry, 5th ed. W.H. Freeman & Co., New York, NY, p. 86)PURIFICATION METHODSHomogenisation of sampleHomogenisation : the process of breaking open cells

Depend on the source- Mammalian tissue- Plant, fungal, bacterial material- Extraction of membrane-bound enzymesHomogenisation MethodsPhysicalFrench pressure cellSonicationGlass beads

ChemicalDetergents (SDS)Enzymes (lyzozyme)Hypotonic buffer

Cell Fractionation / Separation Separation of cell debris and insoluble stuff can be fractionated by centrifugation

In centrifugation, denser material will collect at the bottom of the tube in a pellet whereas material with lower density will remain in the soluble fraction called the supernatant

Only the fraction that contains the protein of interest will be further purified

Cell Fractionation CentrifugationSupernatantPellet

Filtration (other methods)

Stabilize SampleControl pHUse appropriate buffer

Control temperatureKeep samples on ice or work in cold room (0 4oC)Prechill instruments

Prevent frothing/foamingHandle gently.

Maintain concentrated sampleStabilize SampleProtease inhibitors

Phenylmethylsulfonyl fluoride (PMSF)

PMSFProtein SolubilitySalting inAt a low salt concentration, ions protect charges and allow proteins to fold.

Salting outIons compete with water to interact with side groups.

When [salt] is high enough, salt wins causing protein to precipitate. [salt]solubilitysalting insalting outSalting in / salting out

Salting-in and Salting-outDifferent proteins precipitate at different salt concentrations, so the salt concentration may be adjusted to precipitate the desired protein

Ammonium sulfate is the most commonly used reagent for salting out enzymes (proteins)

DialysisA form of size exclusion chromatography.

Used to desalt and concentrate protein samples.

Dialysis tubing has set molecular weight cut off (MWCO). Only molecules that smaller than MWCO will move out of the dialysis bag.

Dialysis

Column Chromatography

Most common method for separating enzymes and other proteins

Separating ProteinsChromatographyMobile phasePhase that carries sample throughout procedure.LiquidGasStationary phaseMatrix that retards the movement of sample being carried by the mobile phase.Enzymes are separated on the basis of size.

Large molecules exit first.

Mobile phaseLiquid

Stationary phaseInsoluble, porous carbohydrate beads

Gel-filtration ChromatographyAlso called size exclusion chromatography

Gel-filtration Chromatography

Gel-filtration ChromatographySample is applied to the top of a column containing porous beads / matrix

Small molecules will pass through the pores of the beads (while larger ones cannot)Large molecules flow more rapidly through the column and elute firstIntermediate size molecules will elute at an intermediate position (occasionally enter the beads) The small molecules will elute last, because take longer path

Ion Exchange ChromatographySeparates molecules based on charge.

Mobile phaseGenerally liquid

Stationary phaseElectrostatically charged ions bound to insoluble, chemically inert matrix.

Elution of protein Add salt to compete with binding of sample to stationary phase.Change pH (alters charge of protein).

cellulose

cellulose(Anion exchange)(Cation exchange)Ion exchange resins contain charged groups.

If these groups are acidic in nature they interact with positively charged proteins are called cation exchangers.

CM cellulosecation exchangerCH2-COO-CH2-COO-++++Positively charged (basic) protein or enzymeIon Exchange Chromatography38If these groups are basic in nature, they interact with negatively charged molecules are called anion exchangers.

DEAE celluloseanion exchangerCH2-CH2 -NH+(CH2CH2)----Negatively charged (acidic) protein or enzymeCH2-CH2 -NH+(CH2CH2)Ion Exchange Chromatography39For protein binding, the pH is fixed (usually near neutral) under low salt conditions. Example cation exchange columnCM cellulosecation exchangerCH2-COO-CH2-COO-++++Positively charged protein or enzyme bind to the column--------Negatively charged proteins pass through the columnIon Exchange Chromatography40To elute enzyme of interest, add increasingly higher amount of salt (increase the ionic strength). Na+ will interact with the cation resin and Cl- will interact with our positively charged protein to elute off the column.CM cellulosecation exchangerCH2-COO-CH2-COO-++++CM cellulosecation exchangerCH2-COO-CH2-COO-++++Na+Na+Cl-Na+2Cl-Cl-Cl-Na+2+ Increasing [NaCl] of the elution bufferIon Exchange Chromatography41

Low saltHigh saltIon Exchange ChromatographyExamplesNameIonizable groupTypeDEAE-SephadexDiethylaminoethylWeakly basicSP-SepharoseMethylsulfonateStrongly acidicBio-Rex 70Carboxylic acidWeakly acidicP cellulosePhosphateStrongly & weakly acidicAffinity ChromatographyMobile phaseUsually liquid

Stationary phaseAntibody/substrate/ligand bound to inert bead

Immunoaffinity columnAffinity chromatography is a method of separating enzyme / protein mixtures based on a highly specific interaction between : antibody and antigen substrate and enzyme ligand and receptor

Affinity chromatography

Possible elution strategies:pHIon strenghDenatureCompetitor ligand or analog

Affinity chromatographyAb affinity column

48Include Protein A-agarose/sepharoseMonitoring Progress of Purification ProtocolTotal protein (mg)Quantity of protein present in fraction

Total activity (units of activity)Use a portion of sample to determine activity.

Multiply activity by total volume to determine total activity.Monitoring Progress of Purification ProtocolSpecific activity (units of activity/mg)

% yield: measure of activity retained after each step in procedure.

% yield =Total activity at particular stepTotal activity of initial extract

S.A. =Total activityTotal protein

Monitoring Progress of Purification ProtocolPurification level: Measure of increase in purity of protein throughout procedure.

Purification level =Specific activity at particular stepSpecific activity of initial extract

Monitoring Progress of Purification ProtocolStepTotal protein (mg)Total activity (units)Specific activity (units/mg)Yield (%)Purification levelInitial extract15,000150,000101001(NH4)2SO4 precipitation4,600138,00030923Ion-exchange1,278115,50090,3779,03Size exclusion68.675,0001093,350109,3Affinity column1.7552,50030000353000(Berg, Tymoczko, & Stryer. (2002) Biochemistry, 5th ed. W.H. Freeman & Co., New York, NY, p. 86)SDS PAGE of Purification

Complete mix of proteinsHigh SaltIon exchangeGel-filtrationAffinity10micrograms loaded in each lane 53Purification SummarySTEPTotal Activity (Unit)Total Protein (mg)Specific Activity (Unit/mg)Yield (%)Purification factor (Fold)Culture Filtrate39.42030.191001Colloidal Chitin Affinity8.007.081.1220.35.89DEAE-Toyopearl M 6506.454.111.5716.38.21Butyl-Toyopearl M 6501.150.284.212.9122.15

Ra-ChiAMSPSP97(kDa)674330

SDS-PAGE ZYMOGRAMA D P Y L K V A Y Y PA D S Y K I V D Y Y PRa-ChiABa-ChiA1N-terminal amino acid sequences alignment of Ra-ChiA and Bc-ChiA154Molecular mass Optimum activity in various conditions(pH, temperatur, ionic strengh etc) Enzyme specificity Kinetic parameter Mechanism of catalysis Structure (X-ray crystallography) Regulation

Characterization of EnzymeElectrophoresisSeparates molecules based on molecular mass and/or charge.

http://www.science.fau.edu/chemistry/Mari/biochemlab/manual.htmlSDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis

Separation based on molecular mass.

Coat samples with SDS to give uniform charge to mass ratio.Makes all proteins negatively charged.

Protein separation using SDS-PAGE(Laemmli system)StackinggelResolvinggel1. Apply protein/dye samples into polyacrylamide gel wells2. Run the electrophoresis until dye reaches the end of the gel3. Remove the gel from the apparatus and stain for proteins58Isoelectric FocusingSeparation based on charge.Polyampholite (small multicharged polymers) used to prepare pH gradient in the gelCan be used to experimentally determine the pI values.

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