Methods for purification of enzymes

BACKGROUND

Enzymes are substances that operate as catalysts in living things, controlling the pace at which chemical processes take place without changing the substance itself.

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Chemical processes underlie every biological activity in living organisms, and the majority of these reactions are regulated by enzymes. Without enzymes, many of these processes wouldn’t progress at a discernible rate. Every element of cell metabolism is catalysed by enzymes.

For instance, during food digestion, major nutrition molecules (such proteins, carbs, and lipids) are broken down into smaller ones; during chemical energy conversion and conservation; and during the synthesis of cellular macromolecules from smaller building blocks.

Numerous hereditary human disorders, like albinism and phenylketonuria, are brought on by a lack of a specific enzyme.

Purification of enzymes

Since the 1980s, it has been proven that certain nucleic acids, known as ribozymes (or catalytic RNAs), have the potential to catalyse, disproving the presumption that all enzymes are proteins.

The chemical process through which enzymes or other biological catalysts drive interactions between organic materials is known as biocatalysis. Biocatalysis adds a new dimension to synthetic chemistry and provides fantastic chances to create chiral chemicals that are helpful for industry.

According to the intended use of the chemical conversion and the associated costs, Whole cell systems or isolated enzymes, either in free or immobilised form, are used in biocatalyst-driven processes.

The purity of the enzyme preparation can be an important consideration when trying to obtain highly pure products, particularly in the pharmaceutical industry. Over time, a variety of enzyme purification techniques have been created.

Traditional purification techniques make use of the enzyme’s physicochemical characteristics. These techniques were created in the 20th century for deciphering protein three-dimensional structures and enzyme processes, but they also seemed useful for producing extremely pure biocatalysts.

The remarkable advancements in recombinant DNA technology and the ensuing revolutionary advances in enzyme manufacturing, enzyme purification, and enzyme engineering, however, have provided progress in the preparation of biocatalysts the biggest boost.

REQUIREMENTS

S. no.
1	Enzyme sample
2	Standard enzyme sample
3	Glass beaker
4	Ammonium sulphate
5	Β-mercaptoethanol
6	Centrifuge
7	Electrophoresis chamber
8	SDS-PAGE gel
9	Isoelectric focusing gel
10	Glass chamber
11	Glass slide
12	Silica slurry
13	Column washing buffer
14	Elution buffer
15	Dialysis bag
16	PBS Buffer

PROCEDURE

Salting In and Out:

1. Take crude enzyme solution in a beaker.
2. Add ammonium sulphate to it slowly, until precipitation occur
3.  Centrifuge the mixture and collect precipitate.

Gel electrophoresis:

1. Prepare SDS-gel for electrophoresis,
2. Add β-mercaptoethanol to sample
3. Add enzyme sample in gel well along with standard,
4. Run the sample in presence of electric field,
5. Stain the gel for 10-20 min, and use destaining solution to remove excess amount of stain.
6. Observe the bands.

Iso Electric Focusing:

1. Prepare Isoelectric focusing gel by using the mold,
2. Transfer the gel onto the electrophoretic chamber carefully,
3. Place a strip of paper on the side of the gel for sample application,
4. Apply the electrical parameters for separation of the sample contains,
5. Stain the gel with suitable dye for 30 to 60 min with gentle shaking,
6. Destain the gel for 30 min with gentle shaking,
7. Observe the bands against a clear background.

Gel Filtration Chromatography:

1. Prepare a column by using an agarose or agarose beads,
2. Mount the column in vertical position by using a stand,
3. Pour filtration buffer to equilibrate the column,
4. Add sample to top of the column,
5. Add buffer on top of the column,
6. Connect vacuum pump to column and start it,
7. Collect elute fraction’s of, and repeat the process from step 5 to collect different size sample elute.

Ion-exchange Chromatography:

1. Prepare column for chromatography and mount it vertical position.
2. Pour a buffer in column to equilibrate the column and drain the buffer from bottom,
3. Ensure the column resin is settle down,
4. Load sample on it along with wash buffer 2-4 times,
5.  Drain the excess buffer and add elution buffer,
6. Collect elute sample for further use.

Adsorption Chromatography:

1. Take a clean glass jar, add solvent to it,
2. Cover the jar with lid for 30 min to equilibrate the environment,
3. Take a clean glass slide pour silica gel slurry on to it, make uniform layer, allow it to dry,
4. Apply sample spot on the glass slide,
5. Place glass slide in the glass jar,
6. Remove the glass slide when mobile phase uprises up to ¼ of the slide,
7. Stain it with suitable stain observes it.

Affinity chromatography:

1. Prepare the column by using the resins and allow resin to settle it down,
2. Add sample on it along with the affinity binding buffer for 2-3 times,
3. Collect buffer fraction, add elution buffer,
4. Collect elute and add high concentration salt buffer to elute remaining binding molecules,
5. Collect 2^nd elute in another tube.

Dialysis:

1. Cut the proper length of the dialysis bag,
2. Open up the bag by soaking in the dialysis buffer,
3. Tie the one end of the dialysis bag with the help of the clamp,
4. Add sample along with the PBS buffer,
5. Kept dialysis bag in the beaker containing PBS buffer for overnight, change the outer buffer every 2-3 hr,
6. Centrifuge the dialysis bag sample,
7. Collect the supernatant and pellet, and store in cold for further use.

CONCLUSION

By using all above procedure we successfully purify the enzyme by qualitatively and quantitatively also.

REFERENCES

1. Sandhya Anand, “Methods of purification of enzymes”, Biotech Articles, January 2011.
2. Adrie H. Westphal, Willem J. H. van Berkel, “Technique for enzyme purification”, Biocatalysis for practitioners: Techniques, Reactions and Applications, First edition, 2021.
3. https://comis.med.uvm.edu/vic/coursefiles/MD540/MD540-Protein_Methods_Learning_Module_10400_593281210/Protein-methods/Protein_Methods_print.html
4. https://www.britannica.com/science/enzyme/Factors-affecting-enzyme-activity
5. https://user.eng.umd.edu/~nsw/ench485/lab6a.htm
6. https://www.usp.org/sites/default/files/usp/document/harmonization/biotechnology/b03_pf_ira_35_1_2009.pdf
7. https://www.conductscience.com

Also read:

• Microbial Staining Methods
• Preparation of enzyme conjugated antibodies
• Purification of Immunoglobulin by Precipitation
• Microplate based Alamar Blue Assays
• Isolation of Genomic DNA from E. coli
• Estimation of DNA and its purity check using UV spectrophotometer (A260 measurement)
• Isolation of O & H Antigen from Salmonella typhi

FAQs:

Q.1   It is difficult to select the right purification column or media, how can we get quick recommendation?

Ans.  We recommend the Purify app, it’ll help you to quickly select the right purification column.

Q.2   When should I use a desalting procedure?

Ans.  You can desalt before purification, between purification or after purification.

Q.3    My interested protein is absence in the elute?

Ans.  There is probably lots of reasons like protein didn’t express, not soluble in lysis buffer, didn’t bind to column, loosely bind to column etc.

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