**2.4. Extraction of cellular protein**

312 Chromatography – The Most Versatile Method of Chemical Analysis

**2.2. Cultivation of P. aeruginosa MTCC 2453** 

and stored at -70oC for further study.

**2. Materials and methods** 

**2.1. Chemicals and reagents** 

Previous researchers [10] adopted genetic engineering techniques and other bacterial species for purification of azurin. This study is concerned of enhanced azurin synthesis from different strains of *P. aeruginosa* with lucid homogeneity by customized methods. The growth of different *P. aeruginosa* MTCC strains 1934, 741, 2453, and 1942 for the synthesis of azurin were scrutinized for enhanced azurin synthesis. The enhanced azurin synthesis from *P. aeruginosa*  strains was improved by the CuSO4 and KNO3 containing media under facultative anaerobic condition. The purification of azurin had been performed by ion-exchange and gel-filtration chromatography. High yield was reported in *P. aeruginosa* 2453 strain than other strains.

Growth medium constituents were of analytical grade obtained from Hi-Media laboratories, India. The buffer ingredients were purchased from Merck Chemicals Ltd, India. Sephadex G-25, G-75, diethyl amino ethyl cellulose (DEAE) cellulose and carboxy methyl (CM) cellulose were all obtained from Sigma-Aldrich, USA. The 3, 5-dimethoxy, 4-hydroxy cinnamic acid otherwise called as sinnapinic acid a matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) was also acquired from Sigma-Aldrich. Protein concentrations were measured by Lowry's method with bovine serum albumin as standard. Standard dialysis bag with 3 kDa cutoff was purchased from Sigma-Aldrich. Powder of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), propidium iodide (PI) and dimethylsulfoxide (DMSO) solution were procured from Sigma Aldrich, India. Cell culture media and other constituents of media are purchased from Hi-media Laboratories

Ltd, India. Fetal bovine serum was obtained from Invitrogen Life Technologies, USA.

A freeze dried culture of *P. aeruginosa* strains MTCC2453,741,1934,1942 was obtained from the Microbial type culture collection center, Chandigarh, India and was grown in a medium containing 7g yeast extract, l0g peptone, 20g of KNO3, 6.4g of KH2PO4, 3.6g of Na2HPO4 (anhydrous), 2.5g of NaCl, 5µg/ml of CuSO4 per liter. The initial pH was adjusted to 6.5 with NaOH. The strains were maintained on nutrient broth with 50% glycerol concentration

Inoculums' of *P. aeruginosa* strains was prepared by inoculating a loopful of colonies in individual 100 ml conical flasks with the exact constituents of the above prescribed media and incubated at 370C, in stirring mode at 100/rev for 21 hours. These inoculums was used to seed the bulk 500ml x 4 sterile medium in 2000 liter conical flasks (separate conical flask used for all

All P*. aeruginosa* strains MTCC2453, 741, 1934, and 1942 are inoculated separately in a sterile medium. Impact of copper sulphate and potassium nitrate in azurin synthesis were

four strains) which was also kept at stirred mode (100/rev) for 21 hours at 370C [11].

**2.3. Impact of copper sulphate and potassium nitrate on culture medium** 

After 21 hrs incubation, cells were harvested by centrifugation method at 13200 g for 15-20 minutes by using ultra centrifuge (Eppendorf, Hamburg, Germany). Cell pellets was collected and suspended in the appropriate volume of 0.02M potassium phosphate buffer pH 7 with protease inhibitor and kept in the ice basket for sonication. Cells were sheared by Ultra sonicator (Cole Parmer, USA) of approximately 100 ml batches of cell suspension. All batches were sonicated for 1-2 minutes at 100W. After sonication the samples was stirred vigorously and centrifuged at 10000g for 20 minutes which removes cell wall debris. The green-brown crude supernatant was stored. Resuspended the precipitate in same buffer, stirred it vigorously and centrifuged as before and the supernatant were stored with the previous extracts [11, 12].
