Sample collection for bacterial strains
Bacterial strains were isolated from different soil samples of Government College University, Lahore, Pakistan Botanic Garden, Tolinton Market Lahore, and a water sample from a hot spring in Azad Kashmir. Soil solution (1%) was prepared for serial dilution. It have been confirmed that the experimental samples of bacteria, including the collection of bacteria, complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from authorities of the Government College University, Lahore, Pakistan Botanic Garden; Tolinton Market Lahore, and from authorities of ‘Azad Kashmir’ Pakistan. Soil solution with suitable dilution (10-5-10-6) was transferred aseptically on prepared Petri plates. Prepared Petri plates were placed in a 37o centigrade incubator for 48 h selected strains were transferred to prepared slants and kept in the incubator at 37 °C.
Preparation of fermenting media for an anti-viral drug
For the synthesis of a novel anti-viral drug from Bacillus subtilis-M15, the following sub-merged biotechnological method of fermentation was adopted. 2 g of soybean meal, 1.5 g of glucose, and 2 g of peptone, 0.1 g (NH4)2SO4, 0.1 g KH2PO4, and 0.5 g sodium carbonate was weighed and mixed in 70 ml of distilled water. Water was mixed continuously until marked with 100 ml of that solution. Then it was autoclaved for 15 min. for 15 lb/inch2 pressure at 121 °C. Then 0.5 ml of inoculum was mixed in fermentation media. The flask was placed in a shaker for 48 h at 37°C with a 200 rev/min revolution speed. Then after 48 h, the sample was centrifuged with 6000 revolutions per minute for 10 min. Supernatant and protein precipitation was kept in a safe place.
Assay for Protease Inhibitory Activity of the anti-viral drug
Protease Inhibitory activity of the anti-viral drug was calculated with few amendments by Kunitz's (1947)18 methods. 1 ml of Trypsin/Pepsin was mixed with 1 ml of Protease Inhibitor for 15 min with an optimum temperature of 37°C. Then 2ml of casein (1%) was transferred to the test as mentioned above tube for 30min. 2.5ml 0f TCA (0.44M) was added to the test tube for the reaction termination. In this method, the TCA soluble fractions were formed by the action of trypsin/pepsin on the protein substrate. Hammerstein casein was measured by the change in absorbance at 280 nm. The residual caseinolytic activity of the trypsin/pepsin in the presence of an inhibitor at 37 °C was used to measure inhibitory activity. Appropriate blanks for enzyme inhibitors and substrate were included in the assay. The mixture was further processed for 15 min in a centrifugation machine with a speed of 1000rpm. Then absorbance of the supernatant was taken at 280nm. The highest protease inhibitory activity of the anti-viral drug depicted the high efficacy and good potency of a drug against viral protease.
Partial purification of the anti-viral drug (Biological PI)
Ammonium sulphate (NH4)2SO4 was added with the enzyme at a temperature of 4°C. The continuous addition of (NH4)2SO4 was confirmed and marked up to 70% of solution saturation. Then this solution was placed at 4 °C for 10 min. After 30 min, the test tube sample was centrifuged at 10000 revolutions for 10 min. The precipitated enzyme (pellet) was dissolved in a minimum amount of 0.1 M Tris-HCl buffer solution, and then it was dialyzed. Then 10 ml of dissolved pellets were added to a given dialysis tube of length 10 cm and width of 25 mm and kept in 1000 ml of 0.1 M Tris-HCl buffer solution with continuous stirring for 24 h at 4°C buffer was refreshed 3-4 times during this period19.
The immobilizing technique (Physical adsorption)
5 ml of partially purified protease inhibitor was taken, and 0.5 g of each adsorbent denoted as S1 to S7 will be continuously stirred and placed in the water bath at 37° centigrade with the speed of 100 rpm for 60 min. Then it was centrifuged for 10 min with 6000 revs. Then the supernatant was used to calculate the activity of protease inhibitor20.
Characterization of Antiviral Drug
Effect of oxidizing agent on Protease Inhibitory activity of anti-viral drug
The impact of oxidizing agents was measured through incubation of anti-viral drug (biological protease inhibitor) with 1, 2, 3, 4, and 5% (v/v) of hydrogen peroxide and dimethyl sulfoxide (DMSO) for 30 min. Protease Inhibitory Activity was measured as mentioned before.
Effect different supports on Protease Inhibitory activity of anti-viral drug
Different immobilizing supports/ion-exchange resins like Amberlite, Doulite, Lewatit, Dowex 66, Dowex 80, IR 401 and IR100 were used to enhance the Protease Inhibitory activity anti-viral drug. Different ion exchanges and resins were checked using the immobilizing technique mentioned above. Protease Inhibitory Activity was measured.
Effect of Acid Treatment on Protease Inhibitory activity of anti-viral drug
The sensitivity of anti-viral drugs in an acidic environment was evaluated by incubating purified protease inhibitors with different concentrations of HCI ranging from 0.02, 0.04, 0.06, 0.08 and 0.I M (pH 2.0) for 30 min. After the incubation, pH was neutralized with 1 m1 of 0. l M Tris-HCl buffer pH 9.0. Protease Inhibitory Activity was estimated.
Effect of Protease Treatment on Protease Inhibitory activity of anti-viral drug
The sensitivity of anti-viral drugs to gastric enzymes like trypsin/pepsin was assessed by incubating the partially purified protease inhibitor with different concentrations of trypsin/pepsin ranging from 0.2, 0.4, 0.6, 0.8, and 1% for 30 minutes at 37 °C. Protease Inhibitory Activity was estimated.
Comparison of Protease Inhibitory activity of biological anti-viral drug and ritonavir (synthetic drug)
Protease Inhibitory Activity of Ritonavir (synthetic Protease Inhibitor of HIV-AIDS, Cancer and HCV) was calculated through standard assay technique as described above for anti-viral drug as Protease Inhibitor. The value of the Inhibitory Activity of Ritonavir was compared with the Inhibitory Activity of a novel anti-viral drug.