Extraction of Protease from Soil Microbe

Journal of Research and Development Peer Reviewed International, Open Access Journal. ISSN : 2230-9578 | Website: https://jrdrvb.org Volume-17, Issue-10(II)| October2025 113 Extraction of Protease from Soil Microbe Ishwari Nitesh Mehta 1, Dr.Shilpa Gharat 2, Dhairya Jain3 1 Ph.D Scholar, Department of Biotechnology, Sonopant Dandekar Arts,V.S. Apte Commerce & M.H. Mehta Science College, Palghar. 2Research Guide & Co-ordinator, Department of Biotechnology, Sonopant Dandekar Arts,V.S. Apte Commerce & M.H. Mehta Science College, Palghar. 3Department of Biotechnology, Sonopant Dandekar Arts, V.S. Apte Commerce & M.H. Mehta Science College, Palghar, Email-ishwar[email protected]m Manuscript ID: JRD -2025-171026 ISSN: 2230-9578 Volume 17 Issue 10(II) Pp. 113-117 October 2025 Submitted: 07 Oct. 2025 Revised: 20 Oct. 2025 Accepted: 25 Oct. 2025 Published: 31 Oct. 2025 Abstract Proteases are essential enzymes with wide-ranging applications in fields food processing, detergents and biotechnology. This study focused on the crude extraction of protease from soil bacteria, followed by determining the enzyme activity under different conditions using the Biurret method. Protease activity was assayed by using casein as a substrate by spreading soil sample collected from the garden and then incubated at Room temperature for 24 hrs. Optimization of the enzyme activity was based on temperature stability, enzyme substrate and effect of inhibitor on enzymatic activity. The bacterial isolate isolated from garden soil showed a clear zone around the colony on casein agar plate indicating protease activity. It was observed that proteases extracted from soil microbes showed optimum activity at 55°C. An increase in substrate concentration resulted in higher enzyme activity. It was observed that in presence of 0.5% CuSO₄, the enzyme activity decreased. Keywords: Protease, soil bacteria, Biuret method, Substrate concentration, Inhibitors, CuSo4, Optimum temperature Introduction: Proteases are a group of enzymes that catalyze hydrolysis of bonds in polypeptide chains and split them into smaller polypeptides or else free amino acids. Proteases are the enzymes that perform proteolysis i.e. digestion of proteins (Arun Kumar Sharma et al.,2015). They are widely used in food processing, as a part of detergents to remove the stains and finds its immense use in textile wet processing, including desizing, bioscouring, biobleaching, and biopolishing (Iram Liaqat, 2025). Soil is an excellent source of microbes that produce protease enzyme (Dalal rupali*.,2015) Various bacterial and fungal species in soil are known for their ability to produce proteases, which play a crucial role in organic matter decomposition and nutrient cycling. Soil harbors a rich diversity of microorganisms due to its complex organic matter. Microbes in soil often produce robust and stable enzymes like protease, amylase etc to survive in variable conditions like temperature, pH, and moisture fluctuations. Soil microbes naturally produce proteases to degrade proteins in decaying plants and animals (Anmol Valsange et al., 2012). In the present study, bacteria isolated from garden soil was used as a source for isolation and extraction of protease enzyme. The present study focused on extraction of protease from the easily available source i.e. bacterial isolates isolated from garden soil and determining the for enzyme activity by using casein agar and also studying the optimum conditions such as temperature, substrate concentration and effect of inhibitors to show the maximum enzyme activity. This study can be helpful to scale up the production of proteases using s Rationale of the Study: Proteases are essential enzymes that catalyze the hydrolysis of proteins into peptides and amino acids, playing a oil microbes as well as its industrial applications using the optimal parameters. Quick Response Code: Website: https://jrdrvb.org/ DOI: Creative Commons (CC BY-NC-SA 4.0) This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License, which allows others to remix, tweak, and build upon the work noncommercially, as long as appropriate credit is given and the new creations ae licensed under the idential terms. Address for correspondence: Ishwari Nitesh Mehta,Ph.D Scholar, Department of iotechnology, Sonopant Dandekar Arts,V.S. Apte Commerce & M.H. Mehta Science College, Palghar How to cite this article: Ishwari Nitesh Mehta,Shilpa Gharat,Dhairya Jain,(2025 ),Extraction of Protease from Soil Microbe,Journal of Research & Development, 17(10(II)),113-117 Original Article Journal of Research and Development Peer Reviewed International, Open Access Journal. ISSN : 2230-9578 | Website: https://jrdrvb.org Volume-17, Issue-10(II)| October2025 114 significant role in various biological and industrial processes. They are widely applied in sectors such as food processing, pharmaceuticals, detergents, and biotechnology. Exploring different natural sources of protease can help identify enzymes with desirable properties for specific industrial uses. This study focuses on the extraction of protease enzymes from microbial (soil bacteria) sources. Soil bacteria are known for producing stable, high-yield extracellular proteases capable of functioning under a broad range of temperature. The present study involves optimizing the enzyme activity by determining the optimum temperature, substrate specificity and studying the effects of inhibitors. This study aims to contribute to the development of cost-effective and sustainable enzyme sources, providing valuable insights into their potential for large-scale commercial and biotechnological use. Objectives: The objective of this research is to extract protease enzymes from bacteria isolated from the garden soil and optimize its enzymatic activity using temperature, substrate concentration and study the effect of inhibitor concentration using (CuSO₄). Methodology: 1) Extraction of Protease from Bacteria isolated from garden soil. A) Sample collection :-The soil sample was collected from the botanical garden of Sonopant Dandekar Arts, V.S. Apte Commerce & M.H. Mehta Science College, Palghar. Soil samples were collected in sterile poly bag, labeled with date and site of collection and stored at room temperature till further processing. B) Isolation of protease producing bacteria ● The soil sample collected was serially diluted till 10-5 ● 0.1 ml of 10-5 dilution was spread onto 1% Casein agar plate. ● Plates were incubated at Room Temperature for 24–48 hours. ● Colonies with clear zones were selected (indicating protease activity due to casein hydrolysis) ● Selected colonies were transferred in Sterile Nutrient Broth+ 1% casein for scale up of Protease producers, where Casein solution was added as an Inducer for protease production. ● Flask was incubated at room temperature for 24 hours for the appearance of the dense growth. C) Extraction of Protease from Culture ● Centrifuge the culture broth at 10,000–12,000 rpm for 15–20 min at 4°C to remove microbial cells. ● Collect the clear supernatant, which contains extracellular protease ● Filter through a Whatman filter Paper to remove any remaining debris. ● Crude enzyme(from bacteria) was obtained. Enzyme extract was stored at 4°C to maintain enzyme stability. 2) Study of effect of different parameters on enzyme activity and Optimization of parameters: The Biuret method was used to estimate protein concentration and enzyme activity considering different parameters viz. Optimum temperature, substrate concentration, effect of inhibitors. The absorbance of the system was determined spectrophotometrically at 550 nm. A) Effect of Temperature: ● 2 ml 1% Casein substrate in each of the 5 tubes. ● A 2 ml phosphate buffer was added in the above tubes . ● 1 ml of enzyme extract was added to all tubes. ● The tubes were incubated at 4°C, 28°C, 37°C, 55°C,100°C) for 30 mins. ● Centrifuge the tubes at 4°C at 10,000 rpm for 10-15 mins. ● Supernatant was carefully collected. ● 4 ml Biuret reagent was added in each tube. ● The tubes were incubated for 30 mins at room temperature. ● Take the O.D in colorimeter at 530 nm. B) Effect Of Substrate ● Casein (substrate) 0.5,1.0,1.5, 2.0 ml was added in 5 tubes. ● The working substrate concentration used is 1 % casein prepared in a phosphate buffer. ● 1 ml of enzyme extract was added to all tubes. ● A 2 ml phosphate buffer in the above tubes was added in all tubes. ● The tubes were incubated at 55°C for 30 mins. ● The tubes were centrifuged at 4°C at 10,000 rpm for 10-15 mins. ● Supernatant was carefully collected. ● 4 ml Biuret reagent was added in each tube. ● The tubes were incubated for 30 mins at room temperature. C) Effect Of Inhibitors ● 2 ml 1% Casein substrate in 5 tubes. Journal of Research and Development Peer Reviewed International, Open Access Journal. ISSN : 2230-9578 | Website: https://jrdrvb.org Volume-17, Issue-10(II)| October2025 115 ● A 2 ml phosphate buffer was added in the above tubes . ● 1 ml of enzyme extract was added to all tubes. ● Inhibitor (1% CuSO4) in the following volume 0.2,0.4,0.6 ml was added in respective tubes. ● The tubes were incubated at 55°C for 30 mins. ● The tubes were centrifuged at 4°C at 10,000 rpm for 10-15 mins. ● Supernatant was carefully collected. ● 4 ml Biuret reagent was added in each tube. ● The tubes were incubated for 30 mins at room temperature. Observations: Fig. 1. Zone of Clearance around Fig. 2. Crude enzyme extract Bacterial colony indicating protease activity Colony Characteristics: A. Effect of Temperature: B. Substrate Concentration C. Effect of Inhibitor Results: Size Shape Edge Elevation Opacity Texture Color Gram Staining Morphology Big Irregular Smooth Flat Opaque Rough White Gram Positive Cocci Temperature (°C) O.D at 530 nm BLANK 0.00 4 0.03 28 0.05 37 0.08 55 0.09 100 0.02 1% Casein (Substrate) (ml) O.D at 530 nm BLANK 0.00 0.5 0.04 1 0.05 1.5 0.07 2 0.10 1% CuSO4 (Inhibitor) (ml) O.D at 530 nm BLANK 0.00 0.2 0.08 0.4 0.06 0.6 0.04 0.8 0.03 Journal of Research and Development Peer Reviewed International, Open Access Journal. ISSN : 2230-9578 | Website: https://jrdrvb.org Volume-17, Issue-10(II)| October2025 116 In the present study it was observed that microbial proteases exhibited higher catalytic efficiency at 55°C. This is consistent with research conducted by Dr Sankareswaran Muruganantham et al in 2014 , who reported that microbial proteases, particularly from Bacillus species, have higher enzymatic activity. Studies on bacterial proteases by Akcan & Uyar (2011) also found that higher substrate concentrations enhance enzyme activity until saturation is reached. These results were found to coincide with the present study results where it was observed that protease activity is directly proportional to substrate concentration. It was found in the present study, as the concentration of the inhibitor increased there was a decrease in the enzyme activity. Similar results were obtained by Thirumalai Maruthiah in 2014 where the metal ions decreased and inhibited the activity of microbial proteases. GRAPH 1 GRAPH 2 GRAPH 3 Conclusion: The Protease enzyme was extracted from bacteria isolated from garden soil by chemical and biophysical methods. The enzyme extracted was then processed for determining the optimum conditions where it will prove beneficial to maintain the stability of the extracted enzyme from soil bacteria. The optimum temperature determined was 55 °C, wherein it can be proved that it is a thermostable enzyme and can be used in the food and textile industry. It was always found that as the concentration of Casein that is substrate increases the enzyme activity also increases. From the study above, it can be said the protease extracted from soil bacteria is thermostable. As the volume of substrate increases the protease activity also increases. The Protease activity in presence in 1% CuSO4 solution showed the peak activity at 0.2 ml. Enzyme activity decreased with the increased volume of CuSO4. For inhibitors, it was observed that as the concentration of the inhibitor goes on increasing the activity of protease also decreases in the present study. Recommendations: With grateful recommendations and constant encouragement from Dr. Kiran Save, Principal and Dr.Shilpa Gharat, coordinator, Department of Biotechnology, Sonopant Dandekar Arts, V.S. Apte Commerce & M.H. Mehta Science College, Palghar we at Department of Biotechnology,Sonopant Dandekar Arts, V.S. Apte Commerce & M.H. Mehta Science College, Palghar, are able to carry on the research in this field of Biotechnology under varied topics. References: 1. Anmol Valsange1, Sarang P. Evarkar1, Sachin V. Tawde2 , Basawraj M. Kareppa2 and Rekha S. Gujar 3 International Multidisciplinary Research Journal 2012, 2(6):01-04 ISSN: 2231-6302 Analysis of protease activity of enzyme isolated from compost soil Journal of Research and Development Peer Reviewed International, Open Access Journal. ISSN : 2230-9578 | Website: https://jrdrvb.org Volume-17, Issue-10(II)| October2025 117 2. Arun Kumar Sharma1, Vinay Sharma1,*, Jyoti Saxena2, Bindu Yadav1, Afroz Alam1, Anand Prakash 1 International Journal of Scientific Research in Environmental Sciences, 3(9), pp. 0334-0340, 2015 Isolation and Screening of Extracellular Protease Enzyme from Bacterial and Fungal Isolates of Soil 3. Dalal Rupali* ISSN: 2319-7706 Volume 4 Number 8 (2015) pp. 597-606 Screening and Isolation of Protease Producing Bacteria from Soil Collected from Different Areas of Burhanpur Region (MP) India. 4. Dr Sankareswaran Muruganantham, Dr. Prabhavathi Paulraj; Optimization of production of an extracellular alkaline protease by soil isolated Bacillus species using submerged and solid state fermentation with agricultural wastes; African Journal of Microbiology Research 8(9)(9); February 2014. 5. Thirumalai Maruthiah, Palanichamy Esakkiraj, Grasian Immanuel and Arunachalam Palavesam; Alkaline Serine Protease from Marine Bacillus flexus APCMST-RS2P: Purification and Characterization; Current Biotechnology 3(3), August 2014,