ISOLATION, EXTRACTION, PURIFICATION AND CHARACTERIZATION OF ALKALINE PROTEASE FROM NEUROSPORA CRASSA AND ENZYME ASSAY
S.R.T.M. University Nanded,
The present invention relates to a filamentous fungus useful for the production of heterologous polypeptides, having been modified by recombinant DNA technology in a manner by which the expression of alkaline proteases have been completely or partially inactivated. The invention also encompasses processes for the production of proteins of interest in high yields by using the fungi of the invention. The invention furthermore relates to methods for producing such fungi and DNA constructs to be used in these methods. Extracellular alkaline proteases from Neurospora crassa is produced for the commercial requirement of proteases. The purification procedure consisted of an ammonium sulfate precipitation, dialysis, and anion-exchange chromatography, and gel filtration. We found only one enzyme identical by polyacrylamide gel electrophoresis. Optimization was done with characterization of enzyme. With a pH value of 10.0 and temperature 60 c the enzyme production was increased.
Reference Id: PHARMATUTOR-ART-1575
INTRODUCTION TO PROTEASES
Proteases are the single class of enzymes, which occupy a pivotal position with respect to their applications in both physiological and commercial fields. They perform both degradative and synthetic functions. Proteolytic enzymes catalyze the cleavage of peptide bonds in other proteins. Advances in analytical technique have demonstrated that proteases conduct highly specific and selective modifications of proteins.
Proteases are classified on the basis of three major criteria: (1) Type of reaction catalyzed (2) Chemical nature of the catalytic site and (3) Evolutionary relationship with reference to structure. Proteases are grossly subdivided into two major groups’ i.e., exopeptides and end peptides, depending on their site of action. Based on the functional group present at the site active site, proteases are further classified into 4 prominent groups i.e. serine proteases, cysteine proteases, aspartic proteases and metallo proteases. Proteases are also divided into acid, neutral and alkaline proteases on the basis of pH range in which their activity is optimum.
Proteases occur ubiquitously in a wide diversity of sources such as plants, animals and microorganisms. Microbes are the attractive sources of proteases and have gained much popularity than any other sources because of their broad biochemical diversity. The inability of the plant and animal proteases to meet current world demands has lead to an increased interest in microbial proteases. Proteases execute a large variety of functions, extending from the cellular level to the organ and organism level. Their involvement in the life cycle of disease organisms has lead them to become a potential target for developing therapeutic agents against fatal disease such as cancer and AIDS.
Proteases have a long history of applications in different industries viz, detergents, food-brewing, meat tenderization, baking, manufacture of Soya products, debittering of protein hydrolysis’s, synthesis of aspartame, dairy, leather, silk and for recovery of silver from used x-ray films. Besides their industrial and medicinal applications, proteases play an important role in basic research. Their selective peptide bond cleavage is used in the study of sequencing of proteins.
A wide range of microorganisms including bacteria, fungi, yeast and also mammalian tissues produces alkaline proteases. The Proteolytic enzymes from Fungi are so far the most important group of enzymes produced commercially.
Technology of immobilization of enzymes was developed to extend the use of this biocatalyst for practical applications. The use of immobilized enzymes in the food, pharmaceutical and chemical industry has increased steadily during the past decade. Their use in the synthesis of chiral drugs, complex organic compounds, fine chemicals, novel specific biosensors and in bioremediation is likely to be expanded through further research and development in enzyme technology. Proteases are a complex group of enzymes, which differ in their properties. Despite the extensive research on several gaps in our knowledge of these enzymes there is still tremendous scope for improving their properties to suit projected applications.
METHOD FOR EXTRACTION, PURIFICATION AND CHARECTERIZATION OF PROTEASE.
1. Screening of Neurospora for Protease Production
2. Extracellular Enzymatic activity of Microorganisms: Preparation of Crud Extract
3. Enzyme assay
4. Purification of Protease
5. Protease Assay after Purification.
6. Estimation of Standard and Purified Protease by Lowry’s Method
7. Enzyme Kinetics: Effect of PH, Temperature and Substrate on activity of Protease.
I. SCREENING OF NEUROSPORA FOR PROTEASE PRODUCTION.
Organism’s shows difference in physiological and biochemical reactions, thus by using this principle organisms were screened by providing the selective medium.
Procedure: 1. Prepare Vogel’s azocasein medium Autoclave at 1210c, 15lbs, for 15min. 2.Transfer the broth into tubes. 3. Azocasein was added after sterilization. 4.Take a loop full of pure culture and inoculate into broth.5. Place the tubes in the orbital shaking incubator for 72 hrs at30°c
Culture grown was checked with utility of azocasein.
II. EXTRACELLULAR ENZYMATIC ACTIVITIES OF MICROORGANISMS:
Because of their large sizes, high-molecular weight nutrients such as polysaccharides, lipids, and proteins are not capable of permeating the cell membrane. These macromolecules must first be hydrolyzed by specific extra cellular enzymes into their respective basic building blocks. These transported into the cells and used for the synthesis of protoplasmic requirements and energy production.
Preparation of Crud Extract:
After 72hours of incubation, the crude extract of the enzyme was prepared by centrifuging the culture broth at 15000rpm for 20 minutes. The supernatant was used as source of extra cellular protease. The crude enzyme extract from Vogel’s broth’s media was prepared by crushing with distilled water using mortar and pestle.
III. ENZYME ASSAY
Quantative assay of protease: Assay of Protease (by Azo-casein digest method):
Protease activity was determined with azocasein as substrate by a modified procedure described by Kreger and Lockwood (1981). For the azocasein assay method 8001.11 of the sample and400 μl of 1.5% azocasein in 0.05 M tris HCI (pH 8.5) was taken in a screw cap tube. The mixture was then incubated in a water bath at 37°C for 30 minutes. Then 2.8 ml or 5% TCA was added to stop the reaction and put on ice for 15 minutes. The solution was centrifuged at 4000 rpm for 5 minutes. In the 2 ml of supernatant 2 ml of 0.5 N NaOH solutions was added and mixed well. Absorbance was measured at 440 nm. Appropriate blank was also included. Then blank was prepared in the same manner except that 2.8 ml of TCA was added before addition of enzyme. One unit of protease activity is defined as the amount of the enzyme that produces an increase in an absorbance of 0.816 under the above assay condition.
Result:10 units of enzyme were produced per 1 ml of crude extract.
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