Isolation and Purification of enzymes

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Isolation and Purification of Enzymes Enzyme purification is a fine art. It needs skill and trial and error. Once if we want to purify a particular enzyme of our interest the source of the enzyme like plant, microbial or animal has to be selected. After selecting the system a suitable assay procedure has to be developed. The assay procedure has to be standardized in our laboratory conditions. Unless the assay procedure is not reliable the whole purification will lead to wrong conclusions. Before isolation one must understand the properties of the enzyme like substrate specificity, pH, temperature and assay method [protein turnover number (or) enzyme unit]. Since enzymes are commercially important in diagnosis, cure, pharmaceutical industries, veternary field, food industries, leather industries and in the recent PCR, RFLP techniques and recombinant DNA technological industries.

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No definite procedure is available for all enzymes. More than 3000 enzymes and its isolation and purification procedure have been explained "Methods In Enzymology of CALOWICK AND KAPLAN" series ranging from volume 1-volume 196. The common steps involved is explained below, I. PROTEIN ISOLATION Selection of source Methods of solubilization Stabilization Assay General Strategy of Purification II. SOLUBILITIES OF ENZYME Effect of salt concentration Effect of organic solvents Effect of pH Crystallization

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III. CHROMATOGRAPHIC SEPERATION Ion-exchange chromatography Paper chromatography Gel filtration chromatography Affinity Chromatography Other Chromatographic Techniques. IV. ELECTROPHORESIS Paper Electrophoresis Gel Electrophoresis SDS-PAGE Isoelectric focussing Capillary Electrophoresis V. ULTRA CENTRIFUGATION Sedimentation Preparative Ultra centrifugates . VI. CRITERIA OF PURITY PAGE SDS-PAGE Isoelectric focussing Ouchterlony immunodiffusion -technique Rocket-electrophoresis.

I Selection of Source:

I Selection of Source 1. Selection of source Commonly animal material, plant material (or) microbial source is used. For metabolic enzymes liver is the source. For insulin pancreas is the source. For ATPase mitochondria is the source. For protein synthesis ribosomes is the source. For carbohydrate synthesis plant material is the source. For industrial and commercial enzymes microbes are the source.

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II. SOLUBILITIES OF PROTEIN Since enzyme is a multiple acid-base group, its solubility properties depends on the concentration of the salt pH, polarity of the solvent and temperature. This is used for precipitating enzymes. 1.Effect of salt concentration The salt concentration is expressed in terms of ionic strength I = ½ CiZi 2 where I-ionic strength C-molar concentration Z - ionic charge Usually ammonium sulphate is used in all laboratories by the process of salting-in and salting out. Salting in is the phenomenon that as the salt concentration of protein solution increases, the additional counter ions more effectively shield the protein molecules multiple ionic charges and thereby increases protein solubility. At high ionic strength the solubilities of proteins as well as those of most other substances decreases. This effect is known as salting out. In addition to ammonium sulphate , NaCl , KCl , MgSO 4 , K 2 SO 4 are also used for precipitation.


Solubilization 2. Methods of solubilization Before isolation the enzyme source should be in soluble form. The material has to be break open ( lysis ) in a hypotonic (homogenizer) or sonication (breaking the cell through ultrasonic vibration). If the enzyme is in organelle differential centrifugation followed by the use of detergent solutions (or) Butanol to get the enzyme.

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3. Stabilization Care has to be taken from denaturation of the enzyme by solvent, temperature and pH. 4. Assay of proteins A suitable assay method and enzyme unit has to be standardized before attempting isolation. 5. General strategy of enzyme purification After studying the characters of the enzyme based on their charge, polarity, size, specificity any of the chromatographic methods like ion-exchange chromatography, electrophoresis, isoelectric focussing , adsorption chromatography, paper chromatography, reverse phase chromatography, hydrophobic chromatography, dialysis, ultra filtration, gel electrophoresis, gel filtration chromatography, ultra centrifugation and affinity chromatography are used.

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2. Effect of organic solvent Sometimes acetone, ethanol, DMSO ( Dimethyl sulphoxide ), or N, N' dimethyl formamide (DMF), are used for precipitation. 3. Effect of pH Sometimes changes of pH and isoelectric point ( pI ) is used for precipitation. 4. Crystallization When the enzyme is somewhat pure it can be crystallized and then used. 5. Ammonium Sulphate precipitation The standard (NH 4 ) 2 SO 4 precipitation is carried out by the standard chart made with various percentage like 0-20%,20-40%,40- 60%,60-80%,80-100%

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HOMOGENISATION The particular cell from a plant or animal or a microbe has to be ground well in a mortar or in a virtishomogenicer. It has to be ground well till a mixture of homogenised solution is obtained. In all the subsequent steps the pH, ionic strength and temperature has to be maintained.

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PRECIPITATION The enzymes are charged molecules which are precipitated by charge breaking chemicals, acids, bases, salts and organic solvents. The procedure of precipitation of enzymes by the addition of concentrated solution of salts is known as "Salting out". It is a complex process and involves disruption of various physical forces involved in enzyme solubilization . The added salts alter the structure of the solvents which can lead to large changes in enzyme configuration by altering the electrostatic interaction of charged group on enzyme surfaces and solvation of polar uncharged residues exposed to the solvent. The salt may also interfere with the formation of Vanderwaal's forces between hydrophobic groups of the aminoacids . Further, it may compete with the enzyme molecules thereby lower its solvation . In other words salt dehydrates enzyme molecules which then form aggregates and precipitate out. Commonly ammonium sulphate is used for precipitation. By measuring the amount of enzyme solution various concentration ranging from 0 to 30, 30 to 60, 60 to 90 and 90 to 100 percent of ammonium sulphate is used. Out of various percentages, with the available assay procedures one can estimate in which percentage of precipitation the desired enzyme is present. Once it is established, from the susbequent precipitation directly the required percentage and amount of ammonium sulphate can be taken. Care should be taken by adding ammonium sulphate from 0 to 4 degree centigrade and high degree of purity of chemicals. While adding ammonium sulphate magnetic stirring is done to prevent air entrainment which might inactivate the enzyme.

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Ammonium Sulphate precipitation chart of an enzyme -------------------------------------------------------------------------------------- S.No % of (NH 4 ) 2 SO 4 Volume(ml) Enzyme Protein Specific content activity  g/ml activity units/ml -------------------------------------------------------------------------------------- 1 Culture filtrate 2 35% 3 70% 4 100% -------------------------------------------------------------------------------------- The suitable precipitation percentage is carried in the next step.

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III. Dialysis Dialysis is carried out in dialysis bag made up of cellophane. The precipitated enzyme solution is poured in the dialysis bag and make a knot at the other end. It is kept in a beaker containing the buffer solution.

Chromatographic methods :

Chromatographic methods



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IV. CHROMATOGRAPHIC SEPERATION The dialysed enzyme is further purified in the affinity (or) ion-exchange chromatography. The ion-exchange chromatographic set up is made up of a peristatic pump, a column filled ion-exchange resins like CMC (Carboxyl methyl cellulose) (or) DEAE-Cellulose, a fraction collector which collects 5 ml of the elutionn rotates in 5 minutes and a recorder which is used to record the measurement of the eluent absorption at 280 nm. After standardizing the column with buffer the enzyme is loaded on the column, after the enzyme binding the column is washed with the buffer and then eluted the enzyme by 0.1m to 0.3m NaCl either by stepwise gradient or concentration gradient.

ENZYMES Isolation and Purification of Enzymes:

ENZYMES Isolation and Purification of Enzymes



ENZYMES Isolation and Purification of Enzymes:

ENZYMES Isolation and Purification of Enzymes

Schematic of pore vs. analyte size :

Schematic of pore vs. analyte size

A typical Waters GPC instrument including A. sample holder, B.Column C.Pump D. Refractive Index :

A typical Waters GPC instrument including A. sample holder, B.Column C.Pump D. Refractive Index

The inside of sample holder of Waters GPC instrument :

The inside of sample holder of Waters GPC instrument

GPC Chromatogram; Vo= no retention, Vi= complete retention, A and B = partial retention :

GPC Chromatogram; V o = no retention, V i = complete retention, A and B = partial retention

Standardization of a size exclusion column. :

Standardization of a size exclusion column.

Metrohm 850 Ion chromatography system :

Metrohm 850 Ion chromatography system

Dionex ICS-5000 world first capillary IC system (UP) and other Dionex IC devices :

Dionex ICS-5000 world first capillary IC system (UP) and other Dionex IC devices

Column chromatography :

Column chromatography



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ELUTION PROFILE OF AN ENZYME Further step of purification is carried out after gel electrophoresis to see whether there is a single obtained,if it is not obtained gel chromatography is obtained. Gel Chromatography. Here also the same set of peristatic pump, column, fraction collector and recorder is there. The elution technique also is done in same way like the linear concentration gradient except that the column is filled with sephadex G-50,Sephadex G-500 (or) Sephacryl based on the molecular weight of the enzyme. The elution profile of the enzyme by gel chromatography:

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LYOPHILISATION The pure enzyme purified of the gel chromatography is pooled into one fraction and poured on to a round bottom flask. This is cooled on the ice mixed with NaCl . When the solution is freezed and it is filled into the lyophilizer and powdered. This powder form is used for various tests of purity (or) homogenity . The entire purification steps, protein content, enzyme activity, specific activity, purification fold and maximum yield is listed in the below table.

Concentration of the purified protein:

Concentration of the purified protein

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-------------------------------------------------------------------------------------------------------------- Purification Volume (ml) Total protein Total enzyme yield steps  g activity -------------------------------------------------------------------------------------------------------------- Culture filtrate (NH 4 )2SO4 precipi - tation DEAE-Cellulose Column Gel column ------------------------------------------------------------------------------------------------------------









Protein Purification & Enzyme Assay:

Protein Purification & Enzyme Assay Activities


Practice Pipetman and tips Vortexer Microfuge tubes 13x100 mm borosilicate glass test tubes

Protein Purification:

Protein Purification Label four microfuge tubes: S, FT, LS, & HS Transfer 300ul of sample to tube S Collect 1ml of flow thru Add 2 ml of elution buffer (Wash) Collect 1ml of LS Collect 1 ml of HS

SDS Polyacrylamide Gel Electrophoresis:

SDS Polyacrylamide Gel Electrophoresis Add Sample S to well 3 Add Sample FT to well 4 Add Sample LS to well 5 Add Sample HS to well 7



Creatine Kinase Assay:

Creatine Kinase Assay

Protein Content:

Protein Content



Computer Data Analysis and Graphing:

Computer Data Analysis and Graphing

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