ANALYSIS OF ENZYME PURITY.pptx

enzymes

1. ANALYSIS OF YIELD, PURITY AND ACTIVITY OF ENZYMES PRESENTED BY: P. ANUSIYA (19MTBT03) M.TECH IIIrd year(BIOTECHNOLOGY) BHARATHIDASAN UNIVERSITY

2. CONTENTS Introduction Issues from poor protein Criteria of purity Different methods for Analysis Quantification Analysis Conclusion.

3. INTRODUCTION: Methodology of Enzyme production: Extraction< Purification< Analyzing For Analyzing: By using different methods Quantitative analyze using different parameters Enzyme assays What is the need of analysis? During purification, protein mixtures are subjected to series of separation under different techniques in order to achieve a pure protein of interest. Analysis is a kind of quality report of purified protein. 3 IMPORTANT STEPS IN PROTEIN PURIFICATION: 1) Measure the volume at each step 2) Unit of Enzyme 3) Total amount of protein.

4. ISSUES FROM POOR PROTEIN: If our protein is in impure stage it tends to lot’s of implications and results in the failure of experiments. Proteins can misfold, aggregate, and precipitate out of solution, which is often an irreversible process when not carefully controlled  Aggregation and degradation also causes proteins to not ‘behave’ as they would in their native state, so they may not bind to other proteins or molecules correctly. When enzymes become inactivated or misfolded, enzymatic assays fail or result in poor reproducibility. This can be of particular importance if you are using enzymatic assays as indicators for other proteins of interest. So, it’s mandatory to h ave some control over storage conditions, including pH, buffer, salt concentration, protein concentration, and storage temperature. 

5. CRITERIA OF PURITY: At each and every step of purification, the enzyme mixture should undergoes analysis of specific level of purification. Among different quantifying parameters, Calculating total protein at each step is mandatory. It was due to 2 reasons, - Will denote whether protein content is lost from the system or not - It will indicate the specific activity of important enzyme in each fraction. Successive purification results in the increased specific activity but it does not assure that our protein is completely pure. The simplest method to determine total protein is Spectrophotometry @ 280nm. In this range of wavelength, there is a presence of nucleic acid in crude extract. It was adjusted by using the formula, Total protein = 1.55(A280nm)- 0.75(A260nm)

6. METHODS OF PURIFICATION: Protein purification is based on the different parameters 1) BASED ON IONIC PROPERTY: Salting in and salting out Ion exchange chromatography 2) BASED ON ABILITY TO GEL ABSORBED: Absorption chromatography Affinity chromatography 3) BASED ON SIZE: Gel filtration chromatography

7. QUANTITATIVE ANALYSIS: There are some important quantities, which shout keep racking towards the end of the process from each and every steps in purification. 1) TOTAL PROTEIN : The total amount of protein present in each fraction is determined by the determining the protein concentration in different fraction. Unit is mg or microgram 2) ENZYME ACTIVITY: This describes the ability of the enzyme to promote the particular reaction It tells us the micromole of product produced per minute( U) in the mixture. Unit is represented as U 3) SPECIFIC ACTIVITY: It gives us overall idea of purity of our enzyme in the mixture

8. It is determined by the ratio of enzyme activity to the total protein. Good result: high specific activity Unit is U/mg 4) YIELD: It is the measure of how much protein retained after the each step of purification. It is calculated via by determining the enzymatic activity retained in every purification step divided by the total initial activity of the crude extract. It is expressed in %. 5) PURIFICATION LEVEL: Also mentioned as purification fold It is a index of how much specific activity of the target protein has increased in the comparison to the specific activity of the target protein the crude extract.

9. It is determined by dividing the specific activity by the specific activity of the initial crude extract. Higher the purification fold, grater is the extend of protein purification that has been achieved. It has no units. SUMMARY: Total protein = how much protein present in mixture Enzyme activity = ability of enzyme to promote particular reaction Specific activity = Enzyme activity Total protein Yield = Enzyme activity in fraction Initial activity Purification level = Specific activity of fraction specific activity of crude (100)

10. QUANTITATIVE ANALYSIS OF PURIFIED ENZYME USING DIFFERENT PARAMETERS To quantify Enzyme activity, Total protein, Specific activity, yield, purification level from the given different fractions of sample. SAMPLE A- Original crude mixture of enzyme solution SAMPLE B- By Salting out SAMPLE C- By Ion exchange chromatography SAMPLE D- By Gel filtration chromatography

11. S.NO TOTAL PROTEIN ENZYME ACTIVITY SPECIFIC ACTIVITY YIELD PURIFICATION LEVEL SAMPLE-1 20,000 2,00,000 SAMPLE-2 5000 1,75,000 SAMPLE-3 1500 1,50,000 SAMPLE-4 200 85,000

12. SOLUTION S.NO TOTAL PROTEIN ENZYME ACTIVITY SPECIFIC ACTIVITY YIELD PURIFICATION LEVEL SAMPLE-1 20,000 2,00,000 10 100 1 SAMPLE-2 5000 1,75,000 35 87.5 3.5 SAMPLE-3 1500 1,50,000 100 75 10 SAMPLE-4 250 85,000 340 42.5 34