Janus kinase 3 (JAK3) is a non-receptor tyrosine kinases family of protein which is comprised of JAK1, JAK2, JAK3 and TYK2. It plays an important role in immune function and lymphoid development and it only resides in the hematopoietic system. Therefore, selective targeting JAK3 is a rational approach in developing new therapeutic molecule. In this study, about 116 JAK3 inhibitors were collected from the literature and were used to build four-point pharmacophore model using Phase (Schrodinger module). The statistically significant pharmacophore hypothesis of AAHR.92 with r2 value of 0.942 was used as 3D query to search against 3D database namely Zincpharmer. A total of 2, 27,483 compounds obtained as hit were subjected to high throughput virtual screening (HTVS module of Schrodinger). Among the hits, ten compounds with good G-score ranging from -12.96 to -11.18 with good binding energy to JAK3 were identified.

Laccase belongs to the family of multicopper oxidase and have wide range of biotechnological applications starting from the food industry to bioremediation. However, the use of laccase at commercial scale is hindered by factors like high enzyme cost, low activity and /or stability under given conditions. This study was carried out with aim of screening for extracellular thermostable laccase producing bacteria. Twenty-two (22) laccase positive strains were isolated from the selected environmental samples while extracellular laccase activity was detected only in six strains namely TM1, TMT1, PK4, PS1, TMS1 and ASP3. The laccase enzyme produced from PK4 was found to be more thermostable with a half-life of 60 min at 80°C. The strain PK4 was identified and designated as Bacillus sp. PK4. The laccase production was optimized using one-factor-at-a-time method and maximum enzyme production were observed at the temperature of 37°C, pH 7.5, 10% inoculum size with yeast extract and glucose as the nitrogen and carbon sources, respectively at an agitation rate of 150 rpm. Copper sulphate at 0.1 mM concentration was found to maximize the laccase production among the tested inducers. Among the trace elements, FeSO 4 and ZnSO 4 gave the maximum laccase production for the isolated strain in comparison with the control. The effect of copper-induced time showed that the addition of copper before inoculation effectively increased the laccase production compared to the addition of copper after 2, 4, 6 and 8 h of inoculation. The optimization of the media resulted in 11.8 fold increase in laccase production.

Statistical design of experiments was employed to optimize the media composition for the production of laccase from Bacillus sp. PK4. In order to find the key ingredients for the best yield of enzyme production from the selected eleven variables viz yeast extract, glucose, zinc sulphate, copper sulphate, potassium chloride, magnesium sulphate, calcium chloride, ferrous sulphate, sodium chloride, potassium dihydrogen phosphate (KH 2 PO 4) and dipotassium hydrogen phosphate (K 2 HPO 4), Plackett-Burman design was applied. The MgSO 4, FeSO 4, and CuSO 4 showed positive estimate, and their concentration optimized further. The steepest ascent method and Box-Behnken method revealed that 1.5 mM MgSO 4, 0.33 g/l FeSO 4 and 1.41 mM CuSO 4 were optimal for the laccase production by Bacillus sp. PK4. This optimization strategy leads to enhancement of laccase production from 2.13 U/ml to 40.79 U/ml. Agro-wastes residues replace the carbon source glucose in the optimized media namely sugarcane bagasse, wheat bran, rice husk, and groundnut shell, among these groundnut shells (117 U/ml) was found to enhance the laccase production significantly. The laccase produced by Bacillus sp. PK4 was found to have the potential to degrade persistent organic pollutant benzo[a]pyrene.