Evaluation of wound healing activity of alkaline proteases gel formulation in wistar rats.

Evaluation of wound healing activity of alkaline proteases gel formulation in wistar rats.

Registration ID: IJNRD_224250

Published ID: IJNRD2406467

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Mr.Abhijeet Mahadev Pawar , Mr.Rishikesh Chakor , Mr.Rohit Phule , Mr.Sitaram Kale , Dr.S.A Phade

Wound healing gel formulation.

A substance produced by a living organism which acts as catalyst to bring about a specific biochemical reaction. Enzyme-substrate reaction. 1.2 Enzymatic reaction Enzymes are the catalytic cornerstones of metabolic activities of living being and catalyse most of the reactions in living organisms. Classically enzymes work by providing an alternative path of lower activation energy for reactions and dramatically accelerating its rate. The term enzyme was coined by German physiologist Wilhelm Kuhne and was derived from the Greek word ενζυμον. Various workers like Buchner, Sumner, Northrop, Stanley, Phillips and several others showed that enzymes are specialized proteins. Enzyme focused research is of importance not only to biological community but also to process designers/engineers, chemical engineers and researchers working in other scientific fields. Enzymes being proteins possess properties such as specificity towards the reactions they catalyse and the substrates on which they act upon. Apart from basic approach, enzymes are widely studied from an industrial point of view. Enzymes have been used since the dawn of mankind in cheese manufacturing and indirectly via yeasts and bacteria in food manufacturing. The past few decades of the twentieth century have witnessed spectacular advances and betterment of living standards due to the beneficial integration of enzyme technology with scientific progress and rapid translation of laboratory findings into practical technologies and commercial scale manufacturing processes. 1.3 Enzyme Nomenclature Enzymes are identified by a common nomenclature system based on the description of what function it performs in the cell and ends with a common phrase. The International Union of Biochemistry and Molecular Biology and the International Union of Pure and Applied Chemistry developed a nomenclature system wherein each enzyme is given an Enzyme Commission Number called as the EC number. Accordingly the top level classes based on the mechanism of operation of an enzyme are: 1. Oxidoreductases: catalyse oxidation/reduction reactions 2. Transferases: transfer a functional group 3. Hydrolases: catalyse the hydrolysis of bonds with addition of water 4. Lyases: remove groups from their substrates 5. Isomerases: catalyze isomerisation changes within a single molecule 6. Ligases: join two molecules with covalent bonds From an industrial standpoint, only a limited number of enzymes are commercially available and few of them have found applications in large quantities. More than 75% of industrial enzymes are hydrolases (Rao et al., 1998). More than fifty commercial industrial enzymes are available and their number is being increased steadily. 1.4. Proteases Proteases are among the oldest and most diverse families of enzymes known and are involved in every aspect of organism‟s function. They constitute a very large and complex group of hydrolytic enzymes that degrade proteins into small peptides and amino acids. Proteases catalyze the addition of water across amide (and ester) bonds to cleave using a reaction involving nucleophilic attack on the carbonyl carbon of the sessile bond. They differ widely in their properties such as substrate specificity, active site and catalytic mechanism and possess different profiles for mechanical stress, chemical environment, pH and temperature for stability and activity. Because of their broad substrate specificity, proteases have a wide range of applications such as in leather processing, detergent formulations, baking, brewing, meat tenderization, peptide synthesis, cheese manufacture, soy sauce production, protein hydrolysate, pharmaceutical industry, waste treatment, silk industry, organic synthesis, recovery of silver from waste photographic film, as well as analytical tools in basic research and have high commercial value (Godfrey and West, 1996). Among the bulk of industrial enzymes, proteases from plant, animal, and microbe constitute around 60 % of the total worldwide enzyme sales (Kunamneni et al., 2003; Merheb-Dini et al., 2009). Currently, the largest share of the enzyme market has been held by detergent proteases which are active and stable at alkaline pH. They are also important from a physiological point of view, as they are involved in many cellular processes like protein turn over and digestion as well as fungal morphogenesis, spore formation and spore germination. Yet, there is a continued search for proteases having novel properties with known and newer applications. Proteases are enzymes occurring everywhere in nature be it inside or on the surface of living organisms such as plants, animals and microbes These enzymes carry out proteolysis i.e. Break down proteins by hydrolysis of the peptide bond that exists between two amino acids of a polypeptide chain. The proteases available today in the market are derived from microbial sources. This is due to their high productivity, limited cultivation space requirement, easy genetic manipulation, broad biochemical diversity and desirable characteristics that make them suitable for biotechnological applications [1]. The world enzyme market is currently at $5.1 billion and is expected to rise by 6.3% annually by 2013. Since proteases share a major part of the global enzyme market, an upward trend in their demand is 1.5SOURCES OF ALKALINE PROTEASES Alkaline proteases are obtained from various microbial sources such as bacteria, fungi and certain yeasts. Of all the microbial sources, bacterial proteases are of particular interest due to their various applications in industries such as detergent, textile, leather, food and feed industry. A major source of bacterial alkaline proteases is Bacillus species, which has been studied extensively. Some fungal species are also known to produce alkaline proteases of industrial use, of which Aspergillus species has been extensively studied. A very few studies exist on yeast species. Alkaline proteases from Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis. 1.6STUDIES ON PRODUCTION, CHARACTERIZATION AND APPLICATIONS OF MICROBIAL ALKALINE PROTEASE For industrial use, only large scale production of alkaline proteases can suffice the requirement. Industrial production of alkaline proteases can be carried out by solid-state and submerged fermentations. Media composition particularly carbon and nitrogen source and process parameters such as temperature, pH, agitation speed , greatly influence the enzyme production. Each micro-organism producing alkaline protease requires a different medium and process conditions. The effect of carbon and nitrogen sources on the enzyme production has been studied extensively. Akhavan Sepahy and Jabalameli studied the effect of various culture conditions on the production of an extracellular protease by Bacillus sp. and reported that sucrose and corn steep liquor are the best substrate for enzyme production. Some alkaline proteases also require metal ions in the form of salts in the production media. It has been shown that FeSO4.7H2O and MgSO4.7H2O enhanced the protease production by Bacillus subtilis RSKK96 Since cost of fermentation media is considerable, production of alkaline proteases have also been carried out using different agroindustrial wastes (green gram husk, chick pea, wheat bran, rice husk, lentil husk, cotton stalk, crushed maize, millet cereal), tannery wastes, shrimp wastes, date wastes etc.. Most of the microorganisms produce alkaline proteases at pH 8-9 and temperature of 32-450C.presents different physiochemical parameters for maximum production of alkaline proteases. Researchers are in a continuous process of optimizing production to achieve maximum yield and economical use of available resources. Traditionally, scientists have adopted “one variable at a time” strategy where each variable is optimized independently. This is very time consuming, expensive and does not reflect true optimum when a large number of variables are involved because of interference by interaction between them. Recently, a number of statistical methods have been developed such as Taguchi methodology, Plackett–Burman design and response surface methodology (RSM) for optimization to achieve rapid and better understanding of interaction between various variables using a minimum number of experiments [30]. 1.7CHARACHTERIZATION OF ALKALINEPROTEASES Alkaline proteases from different sources have been characterized by various workers in order to use them for specific purposes. For example alkaline proteases with broad pH range activity, high thermostability and bleach stable find application in detergent and leather industry. The alkaline protease isolated from Pseudomonas aeruginosa is active at a broad pH range of 6–11 and a temperature range of 25–650C. The studies showed that the purified enzyme retains its activity in surfactants and bleaching agents. These properties indicate its possible use in the detergent industry . 1.8IMMOBILIZATION OF ALKALINE PROTEASES Despite of various advantages, the use of enzymes in industrial applications has been limited due to their high cost of production, instability and expensive recovery which restrict the repeated use of the enzyme. This led to technological developments in the field of immobilized enzymes/whole cells systems which offer the possibility of a wider and more economical use of enzymes. Several attempts have been made to increase stability to temperature, pH, and organic recovery and reuse of enzymes by using various techniques of enzyme immobilization. Whole cell immobilization technique has been employed for higher productivity of alkaline proteases by protecting the cells from shear forces, and advantage .and easy recovery of products .Cell free immobilization techniques have also been widely used for the production of proteases. The alkaline proteases are immobilized on solid support matrix by adsorption, covalent binding, ionic binding, cross linking and entrapment. A few examples of the immobilization of whole cells as well as cell free supernatants using various matrices are listed in Table 6. 1.9MOLECULAR CHARACTERIZATION OF ALKALINE PROTEASES Attempts have been made to deduce the nucleotide and amino acid sequences of alkaline proteases from various sources. These sequences not only help in deriving the primary structure, but also in identifying various functional regions of the proteases. Studies of DNA and protein sequence homology are important for a variety of purposes and have therefore become routine in computational molecular biology. They serve as a prelude to phylogenetic analysis of proteins and assist in predicting the secondary structure of DNA and proteins, and elucidating the structurefunction relationship of proteases. Alkaline protease from marine bacterium strain YS-80-122 with 463 amino acid residues. 1.10USES OF ALKALINE PROTEASES Alkaline proteases are one of the most important classes of proteases from an industrial point of view, occupying a major share of the total enzyme market. Use of alkaline proteases as active ingredients in detergents is the largest application of this enzyme. They are also widely used in leather industry, medical diagnostics, recovery of silver from X-ray films, silk degumming, food and feed industry etc. Due to their vast applications in the industrial processes, many companies started manufacturing them at commercial level. The table 7 gives the commercial manufacturers of alkaline proteases with their product trade name and different applications. 1.11Detergent industry Alkaline proteases have contributed greatly to the development and improvement of modern household and industrial detergents. They are effective at the moderate temperature and pH values that characterize modern laundering conditions in industrial & institutional cleaning. enzymes used in laundry industry are proteases, lipase, cellulases, amaylses etc. Of these, alkaline protease find a major application as 1.12Leather industry Soaking, dehairing of hides and skins and bating have traditionally being carried out by using different chemicals which poses a high tannery waste pollution threat. Hence, proteases with a pH optimum around 9–10 are widely used in soaking to facilitate the water uptake of the hide or skin. Alkaline proteases with elastolytic and keratinolytic activity are used for dehairing and bating process to obtain a desired grain, softness and tightness of leather in a short time. Alkaline proteases with keratinolytic activity have been reported for remarkable dehairing properties . A novel protease showing keratinolytic activity from B. subtilis has been studied as a potential for replacing sodium sulfide in the dehairing process of leather industry. Verma et al. [31] showed the use of protease from Thermoactinomyces sp. RM4 for dehairing goat hides. 1.13Chemical Industry A high stability in the presence of organic solvents is a feature which is highly desired in applications involving biocatalysis in non-aqueous medium for peptide synthesis. Alkaline proteases from Aspergillus flavus, Bacillus pseudofirmus SVB1, Pseudomonas aeruginosa PseA have shown promising results for potential of peptide synthesis due to their organic solvent stability. 1.14Medical Uses The use of immobilized alkaline protease from Bacillus subtilis possessing therapeutic properties has been studied for development of soft gel-based medicinal formulas, ointment compositions, gauze, non-woven tissues and new bandage materials . Oral administration of proteases from Aspergillus oryzae has been used as a diagnostic aid to correct certain lytic enzyme deficiency syndromes . Alkaline-fibrinolytic protease have been reported to preferentially

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"Evaluation of wound healing activity of alkaline proteases gel formulation in wistar rats.", IJNRD - INTERNATIONAL JOURNAL OF NOVEL RESEARCH AND DEVELOPMENT (www.IJNRD.org), ISSN:2456-4184, Vol.9, Issue 6, page no.e721-e759, June-2024, Available :https://ijnrd.org/papers/IJNRD2406467.pdf

Volume 9 Issue 6, June-2024

Pages : e721-e759

Paper Reg. ID: IJNRD_224250

Published Paper Id: IJNRD2406467

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Research Area: Pharmacy

Country: Solapur, Maharashtra, India

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ISSN: 2456-4184 | IMPACT FACTOR: 8.76 Calculated By Google Scholar | ESTD YEAR: 2016

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