**3.2. Effect of copper sulphate and Nitrate in azurin synthesis**

Earlier studies showed that azurin production by different bacterial strains were similar to the azurin produced by *P. aeruginosa* MTCC 2453 but with more yield than previous procedures. Four strains were tested for high yield of azurin productions were *P. aeruginosa*  2453, 741, 1942, and 1934*.* We observed a significant increase in the yield of azurin secreted by *P. aeruginosa* 2453 than genetically engineered strains and other strains. This remarkable increase in the yield of azurin was obtained by addition of CuSO4 and KNO3 in the medium with specific facultative anaerobic cultural conditions. In contrast to earlier studies, adding both CuSO4 (4-5 µg/ml) and KNO3 (0.02 µg/ml) in the medium under facultative anaerobic conditions generate high amount of azurin (Figure 2.), rather adding either CuSO4 or KNO3 (Table 1.).

**Figure 1.** (a) Bacterial culture medium incorporated with CuSO4 and KNO3 (b) Green colour colonies a unique characteristic of *P. aeruginosa* colonies

**3.1. Growth of P. aeruginosa strains** 

unique characteristic of *P. aeruginosa* colonies

performed [17].

**3. Results** 

(Table 1.).

and carbon dioxide in the optical path. The curves were deconvoulted and imported into Omnic's peak fit software (Thermo scientific, Illinois, USA) and a Gaussian curve fitting

The inoculated growth of *P. aeruginosa* MTCC strains 2453, 741, 1934 and 1942 under facultative anaerobic conditions, yields total dry cell protein in the range of 150- 170 g/l medium (Fig. 2.1a). We observed *P. aeruginosa* 2453 produces lesser amount of cellular proteins than other strains. The quality assay was performed after incubation for contamination of any other unwanted organisms. A unique green colour colony in nutrient agar medium was observed and hence we confirmed it as *P. aeruginosa* colonies (Fig 1.).

Earlier studies showed that azurin production by different bacterial strains were similar to the azurin produced by *P. aeruginosa* MTCC 2453 but with more yield than previous procedures. Four strains were tested for high yield of azurin productions were *P. aeruginosa*  2453, 741, 1942, and 1934*.* We observed a significant increase in the yield of azurin secreted by *P. aeruginosa* 2453 than genetically engineered strains and other strains. This remarkable increase in the yield of azurin was obtained by addition of CuSO4 and KNO3 in the medium with specific facultative anaerobic cultural conditions. In contrast to earlier studies, adding both CuSO4 (4-5 µg/ml) and KNO3 (0.02 µg/ml) in the medium under facultative anaerobic conditions generate high amount of azurin (Figure 2.), rather adding either CuSO4 or KNO3

**Figure 1.** (a) Bacterial culture medium incorporated with CuSO4 and KNO3 (b) Green colour colonies a

**3.2. Effect of copper sulphate and Nitrate in azurin synthesis** 

**Figure 2.** Quantification of azurin synthesis by different strains of *P. aeruginosa* MTCC 741, 1934, 1942 and 2453 and impact of CuSO4 and KNO3: 1-5 µg/ml range of CuSO4 concentration with 0.004-0.02 µg/ml of KNO3 was added in the culture medium to study the impact of azurin synthesis.


**Table 1.** Azurin yield from different strains (*P. aeruginosa* MTCC 741, 1934, 1942 and 2453) in addition of 5 µg/ ml CuSO4 and 0.02 µg/ml KNO3 in the culture medium

## **3.3. Chromatography methods for azurin Purification**

DEAE and G-25 are gel filtration columns which remove positively and negatively charged proteins respectively. The unwanted flavo proteins and positively charged proteins were removed during DEAE chromatography. The collected fractions from G-25 were quantified for protein concentration in the UV-Spectrophotometer at 280nm wavelength. Azurin and other proteins more than 5 kDa were eluted immediately after void volume is plotted as graph (Figure 3.).

Peak (a) from G-25 was loaded on G-75 for further purification. The G-75 fractions were quantified for protein concentration in the UV-Spectrophotometer at 280nm wavelength. Azurin a 14 kDa protein will elute after binding in to the beads when the elution buffer elutes it. Thus, azurin and some other proteins will elute very lately, was confirmed from the OD values of the spectrometer, when plotted as graph (Figure 4). The azurin will form a thick band when passages through CM cellulose column which was eluted by ammonium acetate buffer pH 4.65. Ten fractions were collected and absorbed under UV spectrometer at 280nm wavelengths for azurin concentration (Figure 5.).

Purification of Azurin from *Pseudomonas Aeuroginosa* 319

**Figure 5.** Azurin purified from various strains of *Pseudomonas aeruginosa*: **a** Azurin forms thick bands in CM cellulose column chromatography during their purification process. Later this was eluted by ammonium acetate buffer pH 4.65. All *Pseudomonas aeruginosa* strains, particularly *Pseudomonas aeruginosa* 2453 strain shows more significant amount of azurin production. **b** the mean of the azurin production by various *Pseudomonas.aeruginosa* strains: The production of azurin was enhanced by the copper sulphate (5µg/ml) and KNO3 (0.02 µg/ml) containing media under facultative anaerobic condition. The bar graph shows *Pseudomonas aeruginosa* 2453 secrets more azurin than any other strains

**3.4. Characterization of Azurin (Purified from P. aeruginosa MTCC2453)** 

In this study we profiled our purification process at every step by MALDI-ToF (Figure 6.) and SDS-PAGE (Figure 7.) and to confirm the azurin presence in our experiments. Cellular proteins loaded in lane 2 of SDS-PAGE reveals whole cell proteins of *P. aeruginosa* MTCC 2453*.* Fraction (a) collected from G-25 gel filtrations were loaded in lane 3; it shows proteins above 5 kDa in SDS-PAGE which was also confirmed in MALDI-ToF results. Most unwanted proteins were deduced during G-75 gel filtration. Our SDS and MALDI results shows fraction (e) from G-75 contains azurin (14 kDa). The azurin was again purified and

like *Pseudomonas aeruginosa* 741, 1942, 1934 strains tested.

concentrated in CM cellulose.

**Figure 3.** Elution on sephadex G-25: The active fraction from DEAE was loaded on G-25 column. Upon thirty fractions only peak (a) collected for further purification.

**Figure 4.** Elution on sephadex G-75: Fraction (a) collected from the G-25 was loaded with eluent buffer (PBS) to elute bounded proteins. Seventy five fractions were collected at 1 ml/6minutes flow rate. Peak (e) collected for further purification.

280nm wavelengths for azurin concentration (Figure 5.).

thirty fractions only peak (a) collected for further purification.

(e) collected for further purification.

Peak (a) from G-25 was loaded on G-75 for further purification. The G-75 fractions were quantified for protein concentration in the UV-Spectrophotometer at 280nm wavelength. Azurin a 14 kDa protein will elute after binding in to the beads when the elution buffer elutes it. Thus, azurin and some other proteins will elute very lately, was confirmed from the OD values of the spectrometer, when plotted as graph (Figure 4). The azurin will form a thick band when passages through CM cellulose column which was eluted by ammonium acetate buffer pH 4.65. Ten fractions were collected and absorbed under UV spectrometer at

**Figure 3.** Elution on sephadex G-25: The active fraction from DEAE was loaded on G-25 column. Upon

**Figure 4.** Elution on sephadex G-75: Fraction (a) collected from the G-25 was loaded with eluent buffer (PBS) to elute bounded proteins. Seventy five fractions were collected at 1 ml/6minutes flow rate. Peak

**Figure 5.** Azurin purified from various strains of *Pseudomonas aeruginosa*: **a** Azurin forms thick bands in CM cellulose column chromatography during their purification process. Later this was eluted by ammonium acetate buffer pH 4.65. All *Pseudomonas aeruginosa* strains, particularly *Pseudomonas aeruginosa* 2453 strain shows more significant amount of azurin production. **b** the mean of the azurin production by various *Pseudomonas.aeruginosa* strains: The production of azurin was enhanced by the copper sulphate (5µg/ml) and KNO3 (0.02 µg/ml) containing media under facultative anaerobic condition. The bar graph shows *Pseudomonas aeruginosa* 2453 secrets more azurin than any other strains like *Pseudomonas aeruginosa* 741, 1942, 1934 strains tested.

### **3.4. Characterization of Azurin (Purified from P. aeruginosa MTCC2453)**

In this study we profiled our purification process at every step by MALDI-ToF (Figure 6.) and SDS-PAGE (Figure 7.) and to confirm the azurin presence in our experiments. Cellular proteins loaded in lane 2 of SDS-PAGE reveals whole cell proteins of *P. aeruginosa* MTCC 2453*.* Fraction (a) collected from G-25 gel filtrations were loaded in lane 3; it shows proteins above 5 kDa in SDS-PAGE which was also confirmed in MALDI-ToF results. Most unwanted proteins were deduced during G-75 gel filtration. Our SDS and MALDI results shows fraction (e) from G-75 contains azurin (14 kDa). The azurin was again purified and concentrated in CM cellulose.

Purification of Azurin from *Pseudomonas Aeuroginosa* 321

(b) Protein purification was assayed at each step of chromatography. Peak (a) from G-25 was analyzed in MALDI-ToF using Nitrogen laser at 337 nm, confirming the 14 kDa molecular weight of azurin.

**Figure 7.** Protein purification profile further was confirmed by SDS-PAGE analysis: Lane 1: Molecular weight markers 6.5-240 kDa (Bangalore Gene, India), Lane 2: Total cellular proteins, Lane 3: G-25 Fraction [peak (a)], Lane 4: G-75 fraction [peak (e)], Lane 5: CM cellulose purified azurin.

The functional groups of azurin were studied using FTIR spectrum. The presence of the amide I band was indicated by the peak around 1650 cm-1 region, which arises primarily because of the stretching vibration of the main chain of carbonyl groups in the protein backbone coupled with the in-plane N-H bending and C-N stretching modes. Furthermore, the presence of an amide band around 1650 cm-1 signifies α-helix secondary structure of azurin. Azurin synthesized from all strains showed a significant shift in the amide I band with one another, indicating differences in their helix secondary structure of azurin. The most prominent among all strains is *P. aeruginosa* 2453 which showed peak around 1646.936 whereas, others showed peak around 1642.269, 1639.446, 1637.873 for *P. aeruginosa* 741, 1942, 1934 respectively (Fig. 8.). The peaks at 3695 and 3251 cm-1 are the amide A and B bands, respectively, which arise from a Fermi resonance between the first overtone of amide and the N-H stretching vibrations. The 1495 cm-1 peak refers to the amide II band, which arises because of the C-N stretching as well as the C-N-H bending motions. The 1352 peak is the amide III band, which arises predominantly because of the in-phase combination of N-H in

(c) Peak (e) from G-75 was analyzed in MALDI-ToF

**3.5. FTIR analysis** 

plane bending and C-N stretching vibrations.

**Figure 6.** (a) Protein purification was assayed at each step of chromatography. Peak (a) from G-25 was analyzed in MALDI-ToF using Nitrogen laser at 337 nm, confirming the 14 kDa molecular weight of azurin.

(b) Protein purification was assayed at each step of chromatography. Peak (a) from G-25 was analyzed in MALDI-ToF using Nitrogen laser at 337 nm, confirming the 14 kDa molecular weight of azurin. (c) Peak (e) from G-75 was analyzed in MALDI-ToF

**Figure 7.** Protein purification profile further was confirmed by SDS-PAGE analysis: Lane 1: Molecular weight markers 6.5-240 kDa (Bangalore Gene, India), Lane 2: Total cellular proteins, Lane 3: G-25 Fraction [peak (a)], Lane 4: G-75 fraction [peak (e)], Lane 5: CM cellulose purified azurin.

## **3.5. FTIR analysis**

320 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 6.** (a) Protein purification was assayed at each step of chromatography. Peak (a) from G-25 was analyzed in MALDI-ToF using Nitrogen laser at 337 nm, confirming the 14 kDa molecular weight of

azurin.

The functional groups of azurin were studied using FTIR spectrum. The presence of the amide I band was indicated by the peak around 1650 cm-1 region, which arises primarily because of the stretching vibration of the main chain of carbonyl groups in the protein backbone coupled with the in-plane N-H bending and C-N stretching modes. Furthermore, the presence of an amide band around 1650 cm-1 signifies α-helix secondary structure of azurin. Azurin synthesized from all strains showed a significant shift in the amide I band with one another, indicating differences in their helix secondary structure of azurin. The most prominent among all strains is *P. aeruginosa* 2453 which showed peak around 1646.936 whereas, others showed peak around 1642.269, 1639.446, 1637.873 for *P. aeruginosa* 741, 1942, 1934 respectively (Fig. 8.). The peaks at 3695 and 3251 cm-1 are the amide A and B bands, respectively, which arise from a Fermi resonance between the first overtone of amide and the N-H stretching vibrations. The 1495 cm-1 peak refers to the amide II band, which arises because of the C-N stretching as well as the C-N-H bending motions. The 1352 peak is the amide III band, which arises predominantly because of the in-phase combination of N-H in plane bending and C-N stretching vibrations.

Purification of Azurin from *Pseudomonas Aeuroginosa* 323

*P. aeruginosa* strains. The FTIR investigation showed azurin has C=O (protein backbone) stretching, which is the unique nature of the amide I band. The presence of the amide band at 1650 cm-1 signifies the α-helix secondary structure of azurin. The significant shift among four strains synthesized azurin implies that there was a difference in their secondary structure which may be due to their physiological or genetic variations among strains. The impact of the differences in the secondary structure of azurin synthesized from all four

MALDI-Matrix-Assisted Laser Desorption/Ionization, SDS-PAGE-Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, FTIR- Fourier Transform Infrared Spectroscopy, CuSO4 – copper sulphate, KNO3 **–** Potassium nitrate, MTT -3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium, PI-Propidium Iodide, DMSO-dimethylsulfoxide, MALDI-ToF-Matrix-Assisted Laser Desorption/Ionization-Time of Flight, MTCC- Microbial Type Culture

[1] Fleming A. On the antibacterial action of cultures of a penicillium with special reference to their use in the isolation of *B. influenzae*. British Journal Experimental Pathology

[2] Ambler RP, Brown LH. The Amino Acid Sequence of Pseudomonas fluorescens azurin.

[3] Chakrabarty AM. Microorganisms and Cancer:Quest for a Therapy. Journal of

[4] Apiyo D, Wittung-Stafshede P. Presence of the cofactor speeds up folding of

[5] Yamada T, Goto M, Punj V, Zaborina O, Chen ML, Kimbara K, Majumdar D, Cunningham, E, Das Gupta TK, Chakrabarty AM. Bacterial redox protein azurin, tumor

suppressor protein p53, and regression of cancer. PNAS 2002;99 14098-14103. [6] Pozdnyakova I, Guidry J, Wittung-Stafshede P. Copper stabilizes azurin by decreasing the unfolding rate. Archives of Biochemistry and Biophysics 2001;390 146-148.

*Desulfovibrio desulfuricans* flavodoxin. Protein Science 2002;11 1129-1135.

strains tested, were also reflected in the apoptosis generation of all strains.

Collection center, CM-carboxymethyl, DEAE-Diethylaminoethyl Cellulose

Sankar Ramachandran1,2,\*, Moganavelli Singh2 and Mahitosh Mandal1

*Indian Institute of Technology, Kharagpur, West Bengal, India* 

*University of KwaZulu-Natal, Westville, Durban, South Africa* 

*2Department of Biochemistry, School of Life sciences,* 

Journal of Biochemistry 1967;104 784-825.

bacteriology 2003;185 2683-2686.

**Abbreviations** 

**Author details** 

**5. References** 

 \*

Corresponding Author

1929;10 226-236.

*1School of Medical Science and Technology,* 

**Figure 8.** FTIR analysis showed peak around 1646.936 in *P. aeruginosa* 2453 whereas, others showed peak around 1642.269, 1639.446, 1637.873 for *P. aeruginosa* 741, 1942, 1934 respectively.
