**1. Introduction**

310 Chromatography – The Most Versatile Method of Chemical Analysis

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6.pdf

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The use of microorganisms and their products as possible therapeutic agents in the control of cancer begins at the latter part of the nineteenth century. The search of new drugs from microbial sources against infectious disease has been augmented when Alexander Fleming (1928) discovered penicillin [1]. The secondary metabolites from microorganisms play a vital role in developing antibiotics and chemotherapeutics [2, 3]. Several researchers have reported various anticancer molecules from different microbial sources [4]. Even though chemotherapy is efficient in enhancing patient survival with primary tumors continue to have deprived prognosis. The rapid advances in the field of antibiotics have inspired new hope that the search among biological systems will disclose a chemical agent which will exert a destructive effect upon neoplastic growth without seriously affecting normal cells. Using live or attenuated pathogenic bacteria or its metabolites in treatment of cancer excretes toxic effects among patients. Azurin, a redox protein recently fascinated biomedical researcher's immense interest as an anti cancer therapeutic agent which enters human breast cancer cells and induces apoptosis without any adverse effects in cancer patients [5]. Azurin, a secondary metabolite derived from bacterial species especially from *P. aeruginosa* function as a donor in terminal electron transfer process [6]. Azurin also termed as blue small copper proteins highly stable in nature. The presence of copper ion in the polypeptide chain contributes to the azurin stability [6-8]. Azurin reported as a potential anticancer protein against breast cancer cell lines, evoked the researchers of novel methods for enhanced synthesis of azurin has initialized. *P. aeruginosa* a common gram negative opportunistic pathogenic bacterium found naturally [9]. They are considered as facultative anaerobic grow in partial or total oxygen depletion cultural conditions. This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor. *P. aeruginosa* secretes a variety of pigments, including pyocyanin, pyoverdine and pyorubin.

© 2012 Ramachandran et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ramachandran et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Purification of Azurin from *Pseudomonas Aeuroginosa* 313

observed by adding different concentration of copper sulphate (1µg/ml- 5µg/ml) and potassium nitrate (5µg/l – 20µg/l) at different flasks for each concentration distinctly. The azurin protein optimization and quantification was studied in UV spectrometer (Perkin Elmer, Massachusetts, USA) at 595 nm by Bradford's method. The azurin synthesized from P.

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

The Crude (supernatant) was saturated to 45% (277g/l) by slowly adding ammonium sulfate salt at 40C for precipitation, kept it for overnight [5,6]. After precipitation the solution was centrifuged at 20,000g for 25 minutes [6,]. Collected the yellow supernatant saturated again to 95% by adding (NH4)2SO4 (372g/l) slowly and kept at 40C for overnight. The overnight precipitated solution was centrifuged at 23000g for 45 minutes. Pale supernatant was discarded. Precipitate (contains azurin) were collected and resuspended in 0.02M Potassium

Azurin suspended in 0.02M potassium phosphate buffer pH 7 was dialysed by standard dialyses bag purchased from Sigma-Aldrich, (Kolkata, India) having 3 kDa molecular weight cut off at 40C for 20 hours on the same buffer for overnight with continuous gentle stirring. Dialysis was done until the solution attains its buffer pH. The solutions were kept at

Dialysate (contains azurin) were initially treated with DEAE. 100 ml slurry of DEAE cellulose equilibrated in 0.02M potassium phosphate buffer pH 7 were treated with the

aeruginosa MTCC2453 is significantly higher than other strains [11, 12].

**2.4. Extraction of cellular protein** 

previous extracts [11, 12].

Phosphate buffer pH 7[11, 12].

*2.6.1.1. DEAE cellulose treatment* 

**2.6. Dialysis of the supernatant** 

40C after dialysis for further purification [11, 12].

**2.6.1. Purification of Azurin on Ion – Exchange chromatography** 

**2.5. Ammonium sulfate precipitation of proteins** 
