INTRODUCTION:

In the era of industrialisation, there is a massive demand for some novel or enhanced enzymes for various industrial processes¹. These enzymes are commonly sourced from microorganisms, and quite often from fungi^2,3,4. Proteaseshydrolyse the peptide bonds and produce the amino acids along with ammonia. The name protease does not correspond to one enzyme, but it comprises amidases, proteinases, and peptidases⁵. Plant and animals are also protease-producers but are unable to meet the current demand. Thus, microbial enzymes were exploited as they grow at a high rate and easily amendable by genetic engineering. So extensive research is being done in this direction to meet the demand for detergent, food, leather, and pharmaceutical industries^6,7. Proteases also find applications as anti-inflammatory enzymes. Such proteases have been extracted from plants previously⁸.

Different bacteria, fungi and actinomycetes have been documented as producers of proteases⁹. Out of these, Aspergillus, Penicillium, Rhizopus and Trichoderma are the potential ones¹⁰. The fungal strains favour the moist environment, and thus, solid-state fermentation favours the growth and development of filamentous hyphae and mycelia which adhere to the solid surface and remains attached to it¹¹. These strains have not only found applications in enzyme production, but also decolorization and degradation of dyes¹²), degradation of natural rubber¹³ and Biosorption of copper¹⁴. There are several reports which have used the different types of substrates such as corn stover, pre-filtered palm oil effluent, mustard seed oil et cetera^15,16,17. Aspergillus terreus, is one predominant fungal species which secretes protease enzyme at a highconcentration through the fermentation process¹⁸. Aspergillus terreus has been known to show anti-microbial activity against the pathogenic Streptococcus mutans. So, having one fungus with multiple biotechnological applications is considered an advantage¹⁹.

The current study focusses on the isolation of Aspergillus terreus and the production of protease enzyme by utilising Okara, Broken wheat, Chickpea and Black gram as a substrate along with the optimisation of solid-state fermentation for enzyme production.

MATERIAL AND METHODS:

Soil Collection:

The soil samples were collected from the fields of village Birring, Jalandhar City [Longitude: 75.5761679; Latitude: 31.3260302] from the top soil and the soil was collected in the sterile zipped polybags.

Isolation and Screening of the sample:

For experimenting, the 10^-4-fold diluted culture was spread-plated on Skimmed-Milk Agar and Czapek Dox Agar, supplemented with or without Amphotericin B and Tetracycline. The cultures were grown at 30ºC for 4-5 days²⁰. On observing the fungal growth, Aspergillus terreus was identified based on morphological characteristics²¹. Further, the culture was maintained by streaking it on the Czapek Dox agar+ 0.75%

Preliminary Screening of Proteolytic Fungi by Plate Assay

The culture was screened for protease production by plate-assay with modifications in Kamath et al. (2010) protocol²². The fungal isolate was grown on Czapek Dox Agar supplemented with 0.75% casein for three days at 30ºC. The zone of hydrolysis was observed for protease production.

Selection of Substrate for Solid-State Fermentation

Substrates like broken wheat (BW), Okara (OK), Chickpea (CP) and Black gram (BG) were cleaned with 0.2% H₂SO₄as well as distilled water and dried in a hot air oven at 60ºC for 24 hours. The substrates were then tested individually for optimal production of the protease²³.

Inoculum Preparation:

The 7-day old culture of isolated Aspergillus terreus, preserved on the Czapek Dox Agar supplemented with 0.75% casein was suspended in a 10% Tween-80 solution. The fungal spores were inoculated with a needle into the solution for preparing the pre-fermentation culture and was kept for incubation of 5 days at 30ºC²⁴.

Solid-State Fermentation Condition and Enzyme Extraction:

Twenty grams of each substrate, i.e. BW, OK, CP and BG were added to 50mL of culture medium and incubated overnight before inoculation. Excess water was drained using the muslin cloth, and the substrate was transferred to 250ml flasks. Next, the flasks were sterilised by autoclaving and 4mL of spore-suspension was added for conducting the solid-state fermentation and flasks were incubated at 30ºC.

Additionally, vegetative cells were also inoculated to enhance fungal growth. The substrate showing no growth of Aspergillus terreus was discarded. Further, the assessment of protease production by solid-state fermentation was done after 5 and 19 days²⁵. The protease was extracted by adding the 60ml of NaCl (1%), in each of the flasks. The flasks were then incubated at 27ºC at 110rpm with shaking for two hours. Next, residues were removed with the help of muslin cloth and the filtrate was sedimented at 8000rpm/10minutes. The crude enzyme extract (supernatant) was collected, whereas the pellet was dissolved in 200µL of lysis buffer and then both were stored at 4ºC for further analysis¹¹.

Determination of localisation of protease by Gel Diffusion Method:

For determination of the enzyme localization, Czapek Dox Agar supplemented with 0.75% casein was poured in petri plates, 10mm holes were bored and 100µL of crude enzyme extract (supernatant and lysate) was added into the wells. The plates were incubated in the upright position at 30ºC for 24 hours. After incubation, the zone of clearance was observed for the presence of protease enzyme²⁶.

Assessment of crude protease activity:

For estimating the protease activity, the modified protocol of Anson was followed where in casein protein was used as substrate. The enzyme dilutions were prepared by dissolving the crude enzyme in a mixture of 10mM sodium acetate plus 5mM calcium acetate (pH 7.5) and 5ml of 0.65% buffered casein solution was added and incubated at 37ºC for 30 minutes. After the incubation, 5ml of TCA (Trichloroacetic acid) was added to cease the reaction, followed by 30 minutes incubation at 37ºC. The solution was filtered with 0.45µm polyethersulfone filters. Next, 5mL sodium carbonate and 1mL of Folin’s reagent was mixed with 2mL of the filtrate. Enzyme activity was calculated through absorbance measurement at 660nm and comparison with the tyrosine standard. The unit definition of protease implies the concentration of enzyme needed for the liberation of 1µg of tyrosine per ml per minute²⁷.

Determination of Protein Concentration:

Total protein in the enzyme preparation was estimated using the Bradford method²⁸. The concentration of protein was estimated by measuring the absorbance at 595nm and comparing it with the BSA standard curve.

Effect of pH on Protease Activity:

Effect of pH on protease activity was determined by measuring its activity with varying pH. The buffers used for analysis were sodium acetate (pH 3, 4, 5), potassium phosphate (pH 6, 7, 8) and sodium carbonate (9, 10). The reaction mixtures containing 300µL of the buffer, 500µL of casein and 200µL of the enzyme were incubated at 37°C for 30 minutes. The protease activity was determined using the standard Lowry’s method^22,29.

Variation of Enzyme Activity with Temperature:

The dependence of enzyme activity on temperature was determined by measuring its activity upon varing temperature ranging from 6ºC, 25ºC, 35ºC, 45ºC, 55ºC, 65ºC, 75ºC and 90ºC. The reaction mixtures containing 300µL of sodium carbonate buffer, 500µL of casein and 200µL of enzyme were incubated at 37ºC for 30 minutes. The protease activity was determined using the standard Lowry’s method^22,29,30.

Determination of Metal Ion Cofactor:

Effect of metal on protease activity was determined by measuring the enzyme activity with or without metal ions like Mg²⁺, Mn²⁺, Cu²⁺ and Ca²⁺. The reaction mixtures were then incubated at 55°C for 60 minutes and the protease activity was determined by standard Folin’s protocol^22,29,30.

RESULTS:

Isolation, Identification and Screening of Protease-producing Aspergillus strain:

The culture plate of both, Skim milked agar and Czapek Dox agar (with and without Tetracycline and Amphotericin B) were observed for the growth of fungal hyphae. Based on the size and morphology, one distinct colony of cinnamon brown colour with white-creamish vegetative part was selected as shown in Figure 1a and b. On visualizing the fungal colony, at 40X the biseriate, compact, dense columnar and smooth conidiophores were observed. The characteristics were similar with the Aspergillus terreus²¹. The fungal strain showed the zone of clearance on the Czapek Dox agar supplemented with 0.75% casein as shown in Figure 1c, which confirmed the production of protease enzyme.

Selection of Substrate and inoculum preparation for Solid-state fermentation:

The substrate flasks inoculated with the spores of Aspergillus terreus were observed for fungal growth. It was observed that OK, CP and BG showed the growth and no growth was observed for BW. Thus, fresh inoculum of Aspergillus terreus was prepared for inoculation of OK, CP and BG for fermentation.

Solid-State Fermentation for Protease Extraction:

In SSF, the substrate not only served the purpose of anchoring material but also supplements as the nutrient media. Hence, the selected substrates OK, CP and BG were inoculated with prepared inoculum for 5 and 19-days long fermentation process. After the 5^th and 19^th day, the supernatant and cell-free extract were collected for each substrate for further analysis.

Determination of localization of Protease Enzyme by Gel Diffusion Method:

The protease localization was studied using the standard agar diffusion assay and it was found that the culture supernatant showed the enzyme activity after 5^th as well as 19^th day of fermentation. The zone of clearance as shown in Figure 1d proved the enzyme to be extracellular.

Fig 1: a) Czapek-Dox Agar Plate showing the fungal isolate of ‘Aspergillus terreus’,b) Pure culture of Aspergillus terreus, c) Isolated Aspergillus terreus showing the zone of hydrolysis on Czapek Dox Agar supplemented with 0.75% casein, d) Czapek-Dox-Casein Agar plate showing the localization of protease enzyme

Assessment of Crude Protease Activity and Protein Estimation:

The crude protease activity was estimated by using casein as the substrate and measuring the absorbance at 660nm. The enzyme activity was determined by comparison with the tyrosine standard and using the following formula:

Enzyme Activity = {(551.9µM/0.903) x Absorbance of Unknown Sample}/30

The enzyme activity of sample is illustrated in Table 1 and Figure 2a and 2bfor both 5^th and 19^th day extract. In both cases, the maximum protease activity was observed for the substrate OK in comparison to CP and BG as illustrated in Table 1. Protein estimation was done using the BSA standard curve and calculated as per the following formula:

Protein content = (500 µg/ml/0.044) x Absorbance of Unknown Sample

The Protein content of all the substrate is illustrated in Table 2 for both 5^th and 19^th day extract. In both cases, the maximum value for protein content was found for substrate OK as illustrated in Table 2.

Effect of pH on Protease Activity:

The effect of pH on enzyme activity was studied by carrying out protease assay in buffers of different pH. The protease showed the highest activity of 62.40µM/ml/min at the alkaline pH of 10. This confirmed the protease was an alkaline protease and the results were plotted in bar graph as in Figure 2c.

Variation of Enzyme Activity with Temperature:

The effect of temperature was studied by performing the standard protease assay at varying temperatures, followed by quantification of tyrosine released. It was recorded that protease showed the maximum activity of 34.22µM/ml/min at 55^oC as illustrated in Figure 2d.

Determination of Metal ion Cofactor:

The optimum cofactor metal ion for the protease was identified through protease assay in presence or absence of (2mM and 5mM) different metal ions. Whereas Mg²⁺, Ca²⁺ and Cu²⁺ also showed the increased activity, it was found that the most favourable ion for our protease was Mn²⁺ at a concentration of 5mM. The activity was found to be 94.73µM/ml/minas illustrated in Figure 2e.

DISCUSSION:

Many fungal genera like Aspergillus, Penicillium, Rhizopus and Trichoderma have reported to be the potent producers of protease. Fungal proteases have also been isolated from fungal strains previously³¹. Amongst these, very less information is available on Aspergillus terreus for producing protease enzyme which is supported by the previous reports. The standard protocols were followed for the isolation of Aspergillus terreus by referring the method followed by (Gaddeyya et al., 2012)³². The morphological characteristics of the fungal strain were in accordance with the report by Balajee et al (2007)²¹. The plate assay showed positive results for protease production on the Czapek Dox Agar supplemented with 0.75% casein similar to results obtained by Pilli and Siddalingeshwara (2016)³³. The zone of hydrolysis was the confirmation for protease production.

Various substrates like corn-stover, pre-filtered palm oil and mustard oil have been used for the production of protease. But, low-cost substrate for protease production are required. Thus, Broken wheat (BW), Okara (OK), Chickpea (CP) and Black gram (BG) were used as the substrate for the growth of Aspergillus terreus. The OK, CP and BG favoured the substrate for their growth and proliferation. Various studies have proposed that the solid-state fermentation is an effective method for the enzyme production^34,35,36. Therefore, the selected substrates were used for the solid-state fermentation. The supernatant and cell-free extract were collected for each substrate for the determination of localization of the protease enzyme by agar-diffusion assay³⁷. Generally, enzymes like lipase, cellulase, xylanase, protease are extracellular in nature^38,39,40.

Several reports claims that pH 7.0-7.5 is optimum for the protease production. But, the Aspergillus terreus isolate showed the highest activity at pH 10.0 which is in concordance with activity reported for Bacillus pumillus⁴¹. Literature review has revealed that most of fungi like Aspergillus oryzae, Beauveria feline, Penicillium sp. produce protease under SSF condition within the range of 25-30°C^42,43,44,45. Aspergillus oryzae has been reported to exhibit protease activity at 57°C which is quite close with our result⁴⁶. According to reults published previously, Ca²⁺, Mn²⁺, Mg²⁺ ions aid in the protease production. Similar result has been recorded for Pseudomonas thermaerum GW1⁴⁷. Thus, our study reports about the protease producing strain of Aspergillus terreus, which could play essential role in the enzyme industry. Alkalophilic protease have also been isolated from different soils and show enhanced activity in presence of calcium and manganous ions⁴⁸.

CONCLUSION:

In the era of biotechnology, the usage of biocatalysts has gained considerable attention. Thus, there is need to explore the microbes which have ability to serve the industrial demand. The major challenges which need attention are high production cost, specific growing condition in SSF and loss of enzyme activity with respect to time. Therefore, to confer these challenges we need to isolate and characterize the new strains of microbes which can grow on the low-cost substrate in a continuous process. There are various factors that influence the protease activity like pH, ionic strength, temperature and handling. Isolation of newer enzymes can increase the rate of industrial process in comparison to the current enzyme. The protease from the Aspergillus terreus isolate worked best at 55^oC and at a pH of 10.0, which reflects the advantage of production of a highly effective enzyme using low-cost substrates. Further, genetic manipulations in enzyme-coding genes can aid in enhancing the production of enzyme as well as make them ready to work under harsh condition in the industrial process.

ACKNOWLEDGEMENT:

The authors thank the senior administration of Lovely Professional University for providing support for completion of the project.

REFERENCES:

1. Panesar PS, Kaur R, Singla G and Sangwan RS. Bio-processing of agro-industrial wastes for production of food-grade enzymes: progress and prospects. Applied Food Biotechnology. 2016; 3(4): 208-27.

2. Chand T, Pandey FK, Dhingra S, Sharma MK. Production of Industrially Significant Enzymes from Bio-Wastes Using Aspergillus niger by Solid State Fermentation. Research Journal of Pharmaceutical Dosage Forms and Technology. 2014;6(1):33-6.

3. Utharalakshmi N, Kumar AG, Narendra kumar G. Optimization of Cellulase Producing Aspergillus flavus SB4 by Solid State Fermentation using Response Surface Methodology (RSM)-CCD. Research Journal of Pharmacy and Technology. 2015;8(4):349-54.

4. Jagathy K, Kalpana K, Rajesh kumar S. Production, optimization, Characterization and Immobilization of Glucose oxidase from Aspergillus species. Research Journal of Pharmacy and Technology. 2017;10(6):1924-8.

5. Mótyán J, Tóth F and Tőzsér J. Research applications of proteolytic enzymes in molecular biology. Biomolecules. 2013; 3(4): 923-42.

6. Robinson PK. Enzymes: principles and biotechnological applications. Essays in Biochemistry. 2015; 59: 1-41.

7. Ravindran R and Jaiswal A. Microbial enzyme production using lignocellulosic food industry wastes as feedstock: a review. Bioengineering. 2016; 3(4): 30.

8. Chanda I, Chanda SR, Dutta SK. Anti-inflammatory Activity of a Protease Extracted from the Fruit Stem Latex of the Plant Artocarpus heterophyllus Lam. Research Journal of Pharmacology and Pharmacodynamics. 2009;1(2):70-2.

9. Shahina Z, Hossain MT and Hakim MA. Variation of Protease Production by the Bacteria (Bacillus fastidiosus) and the Fungus (Aspergillus funiculosus). Journal of Microbiology Research. 2013: 135-42.

10. Nigam P. Microbial enzymes with special characteristics for biotechnological applications. Biomolecules. 2013; 3(3): 597-611.

11. Benluvankar V, Jebapriya GR and Gnanadoss JJ. Protease production by Penicillium sp. LCJ228 under solid state fermentation using groundnut oilcake as substrate. Life. 2015; 50: 12.

12. Muthukumaran PM, Mathumitha M. Decolorization and degradation of reactive dyes by Aspergillus niger. Research Journal of Engineering and Technology. 2013;4(4):235-8.

13. Singh S, Farooq D, Thakur R. Screening of Natural Rubber Degradation by Fungi; Aspergillus and Phlebia Sp. and Bacteria Pseudomonas and Streptomyces sp. Research Journal of Pharmacy and Technology. 2017;10(11):3939-44.

14. Chatterjee A, Abraham J. Biosorption of Copper using Oryza sativa and Aspergillus oryzae. Research Journal of Pharmacy and Technology. 2016;9(6):664-70.

15. Wu TY, Mohammad AW, Jahim JM and Anuar N. Investigations on protease production by a wild-type Aspergillus terreus strain using diluted retentate of pre-filtered palm oil mill effluent (POME) as substrate. Enzyme and Microbial Technology. 2006; 39(6):1223-9.

16. Gao J, Weng H, Zhu D, Yuan M, Guan F and Xi Y. Production and characterization of cellulolytic enzymes from the thermoacidophilic fungal Aspergillus terreus M11 under solid-state cultivation of corn stover. Bioresource Technology. 2008; 99(16): 7623-9.

17. Sethi BK, Rout JR, Das R, Nanda PK and Sahoo SL. Lipase production by Aspergillus terreus using mustard seed oil cake as a carbon source. Annals of Microbiology. 2013; 63(1): 241-52.

18. Souza PM, Bittencourt ML, Caprara CC, Freitas MD, Almeida RP, Silveira D, Fonseca YM, Ferreira Filho EX, Pessoa Junior A and Magalhães PO. A biotechnology perspective of fungal proteases. Brazilian Journal of Microbiology. 2015; 46(2): 337-46.

19. Jain P, Pundir RK. Antagonistic activity of soil fungal metabolite against Streptococcus mutans strains. Research Journal of Pharmacy and Technology. 2010;3(2):417-9.

20. Cappuccino JG and Sherman N. Microbiology: a laboratory manual. San Francisco: Pearson/Benjamin Cummings; 2005.

21. Balajee SA, Houbraken J, Verweij PE, Hong SB, Yaghuchi T, Varga J and Samson RA. Aspergillus species identification in the clinical setting. Studies in Mycology. 2007; 59: 39-46.

22. Kamath P, Subrahmanyam VM, Rao JV and Raj PV. Optimization of cultural conditions for protease production by a fungal species. Indian Journal of Pharmaceutical Sciences. 2010; 72(2): 161.

23. Almalki MA. Solid state fermentation of agro-residues for the production of amylase from Bacillus subtilis for industrial applications. International Journal of Current Microbiology and Applied Sciences. 2018; 7(3): 1341-8.

24. Radha S, Sridevi A, Himakiran Babu R, Nithya VJ, Prasad NB and Narasimha G. Medium optimization for Acid protease production from Aspergillus sp under solid state fermentation and mathematical modelling of protease activity. Journal of Microbiology and Biotechnology Research. 2012 ;2: 6-16.

25. Paranthaman R, Alagusundaram K and Indhumathi J. Production of protease from rice mill wastes by Aspergillus niger in solid state fermentation. World Journal of Agricultural Sciences. 2009; 5(3): 308-12.

26. Vijayaraghavan P and Vincent SG. A simple method for the detection of protease activity on agar plates using bromocresol green dye. Journal of Biochemical Technology. 2013; 4(3): 628-30.

27. Oliveira AN, Oliveira LA and Andrade JS. Production and some properties of crude alkaline proteases of indigenous Central Amazonian Rhizobia strains. Brazilian Archives of Biology and Technology. 2010; 53(5):1185-95.

28. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976; 72(1-2): 248-54.

29. Classics Lowry O, Rosebrough N, Farr A and Randall R. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 1951; 193: 265-75.

30. Fan Y, Tian L, Xue Y, Li Z, Hou H and Xue C. Characterization of protease and effects of temperature and salinity on the biochemical changes during fermentation of Antarctic krill. Journal of the Science of Food and Agriculture. 2017; 97(11): 3546-51.

31. Vidhya CV. Production and Optimization of Extra-cellular protease from Ganoderma sp. Research Journal of Pharmacy and Technology. 2019;12(4):1832-8.

32. Gaddeyya G, Niharika PS, Bharathi P and Kumar PR. Isolation and identification of soil mycoflora in different crop fields at Salur Mandal. Advances in Applied Science Research. 2012; 3(4): 2020-6.

33. Pilli R and Siddalingeshwara KG. Rapid confirmation and molecular identification of alkaline protease producing Aspergillus awamori through submerged fermentation. International Journal of Current Microbiology and Applied Sciences. 2016; 5: 1114-24.

34. Rao GG, Patel PM, Rao BV and Pancha RR. Alkaline protease production by Aspergillus terreus BAB-346 using poultry litter waste. International Journal of Current Microbiology and Applied Sciences. 2016; 5(10): 174-84.

35. Sethi BK, Jana A, Nanda PK, Das Mohapatra PK and Sahoo SL. Thermostable acidic protease production in Aspergillus terreus NCFT 4269.10 using chickling vetch peels. Journal of Taibah University for Science. 2016; 10(4): 571-83.

36. Rai MP and Nigam S. Cost Effective Method of Protease Production in Solid State Fermentation Using Combined Substrate Corn Cob and Lentil Husk. Research and Reviews: A Journal of Life Sciences, 2018; 3(1): 1-7.

37. El-Safey EM and Abdul-Raouf UM. Production, purification and characterization of protease enzyme from Bacillus subtilis. In: International Conferences for Development and the Environment in the Arab World 2004 (p. 14). Assiut University.

38. Archna S, Priyank V, Nath YA and Kumar SA. Bioprospecting for extracellular hydrolytic enzymes from culturable thermotolerant bacteria isolated from Manikaran thermal springs. Research Journal of Biotechnology. 2015; 10(4): 33-42.

39. Jugran J, Joshi GK, Bhatt JP and Shanker A. Production and partial characterization of extracellular protease from Bacillus sp. GJP2 isolated from a hot spring. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences. 2016; 86(1): 171-8.

40. Sachan S, Iqbal MS and Singh A. Extracellular lipase from Pseudomonas aeruginosa JCM5962 (T): Isolation, identification, and characterization. International Microbiology. 2018; 21(4): 197-205.

41. Gomaa EZ. Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes. Brazilian Journal of Microbiology. 2013; 44(2): 529-37.

42. Agrawal D, Patidar P, Banerjee T and Patil S. Production of alkaline protease by Penicillium sp. under SSF conditions and its application to soy protein hydrolysis. Process biochemistry. 2004; 39(8): 977-81.

43. Agrawal D, Patidar P, Banerjee T and Patil S. Alkaline protease production by a soil isolate of Beauveria felina under SSF condition: parameter optimization and application to soy protein hydrolysis. Process Biochemistry. 2005; 40(3-4): 1131-6.

44. Sumantha A, Sandhya C, Szakacs G, Soccol CR and Pandey A. Production and partial purification of a neutral metalloprotease by fungal mixed substrate fermentation. Food Technology and Biotechnology. 2005; 43(4): 313-9.

45. Sharma KM, Kumar R, Panwar S and Kumar A. Microbial alkaline proteases: Optimization of production parameters and their properties. Journal of Genetic Engineering and Biotechnology. 2017; 15(1): 115-26.

46. De Castro RJ and Sato HH. Protease from Aspergillus oryzae: biochemical characterization and application as a potential biocatalyst for production of protein hydrolysates with antioxidant activities. Journal of Food Processing. 2014; 2014.

47. Gaur S, Agrahari S and Wadhwa N. Purification of protease from Pseudomonas thermaerum GW1 isolated from poultry waste site. The Open Microbiology Journal. 2010; 4: 67-74.

48. Sitoke A, Chopra RS, Kumar GP, Chopra C. Identification and Characterization of an Alkalophilic Protease from Bacillus Mycoides strain isolated from Industrial Soil of Phagwara, India. Research Journal of Pharmacy and Technology. 2017;10(10):3435-8.

Received on 20.01.2020 Modified on 24.03.2020

Research J. Pharm. and Tech. 2021; 14(1):219-224.

DOI: 10.5958/0974-360X.2021.00038.X

Substrate		BG		CP		OK
Enzyme Activity (µM/ml/min)	Dilution	Day 5	Day 19	Day 5	Day 19	Day 5	Day 19
	0.4	0.91	1.95	5.19	4.91	9.41	16.0
	0.6	3.5	2.56	5.43	7.51	10.5	19.67

Substrate	Protein Content (µg/ml)
Substrate	Day 5	Day 19
BG	613.66	1602.27
CP	886.36	1193.18
OK	2613.63	5352.27