NISCAIR Online Periodicals Repository Collection: IJBB Vol.49(4) [August 2012]

Refolding of recombinant human granulocyte colony stimulating factor: Effect of cysteine/cystine redox system

Title: Refolding of recombinant human granulocyte colony stimulating factor: Effect of cysteine/cystine redox system

Authors: Tiwari, Krishnanand; Shebannavar, Sunil; Kattavarapu, Krishna; Pokalwar, Santosh; Mishra, Maheshwari K; Chauhan, Ugam Kumari

Abstract: Granulocyte colony-stimulating factor (G-CSF) is a multi-functional cytokine which is widely used for treating neutropenia in humans. Evaluation of alternative to expensive components of redox buffer (reduced and oxidized glutathione) is an important step in reducing the cost of production of human biotherapeutic proteins. In the present study, refolding of recombinant human G-CSF expressed as inclusion bodies (IBs) in E. coli was optimized using cysteine and cystine redox agents. The refolding to correct native form of G-CSF was assessed by reverse phase high performance liquid chromatography (RP-HPLC). The optimized concentrations of cysteine and cystine for correct refolding of G-CSF were found to be 2 mM and 1 mM, respectively. The correctly refolded G-CSF was detected as early as 4 h of incubation in renaturation buffer containing optimized concentrations of cysteine (2 mM) and cystine (1 mM) redox agents. Refolding of G-CSF in optimized redox system increased with increase in shuffling time. Overall, the results suggested the use of cysteine/cystine redox pair could be an alternative to the costlier redox pairs for successful refolding of G-CSF and possibly other human biotherapeutic proteins of importance.

Page(s): 285-288

Effect of acute exposure of N, N-Dimethylformamide, an industrial solvent on lipid peroxidation and antioxidants in liver and kidney of rats

Title: Effect of acute exposure of N, N-Dimethylformamide, an industrial solvent on lipid peroxidation and antioxidants in liver and kidney of rats

Authors: Jyothi, Kanagaraj; Kalyani, Durai; Nachiappan, Vasanthi

Abstract: N, N-Dimethylformamide (DMF), an industrial solvent widely used throughout the world is a known toxic compound. Here, we studied the effects of acute exposure of DMF on liver and kidney in rats. Rats were treated intraperitoneally with a single dose of DMF (1.5 g/kg) for 24 and 48 h. Hepatic and nephrotoxicity was confirmed based on the significant increase in the serum levels of aspartate amino transferase, alanine amino transferase, alkaline phosphatase, g-glutamyl transferase, urea, creatinine and electrolytes. Oxidative stress was assessed by determining the levels of lipid peroxidation (LPO) and antioxidants in liver and kidney. The LPO levels were elevated in both the tissues upon DMF exposure, whereas the activities of enzymatic antioxidants SOD, CAT and Gpx and non-enzymatic antioxidants (glutathione and vitamin C) were declined. The hepatic- and nephrotoxicity was further confirmed by the increasing incidence of inflammation in the histopathological studies. The findings indicate that acute exposure of DMF results in oxidative stress, antioxidant deficiency, attenuating liver and kidney marker enzymes, resulting in tissue inflammation and damage.

Page(s): 279-284

Optimization of media composition for D-amino acid oxidase production by Trigonopsis variabilis using biostatistical analysis

Title: Optimization of media composition for D-amino acid oxidase production by Trigonopsis variabilis using biostatistical analysis

Authors: Gupta, Neeraj; Gundampati, Ravi Kumar; Debnath, M

Abstract: D-amino acid oxidase (DAAO) is biotechnologically relevant enzyme that is used in various food and pharmaceutical industries. DAAO from the yeast Trigonopsis variabilis is an important agent for use in commercial applications because of its high activity with cephalosporin C and is reasonable resistant to the oxidants O₂and H₂O₂ byproducts of reaction. In this study, response surface methodology (RSM) in shake flask culture was used to enhance the production of DAAO from T. variabilis by optimization of fermentation media composition. The effects of six factors (DL-alanine, glucose, pH, ZnSO₄, (NH₄)₂SO₄ and temperature) were evaluated on DAAO production. Results of Placket-Burman design showed that DL-alanine, pH, glucose and ZnSO₄ were significant factors for DAAO production (P<0.05). The optimum values of media components as predicted by the central composite design were inducer (DL-alanine) concentration 3 g/L, pH 7.7, glucose 17 g/L and ZnSO₄34 mg/L. At these optimum values of media composition, maximum production of DAAO was 153 U/g yeast dry weight. Two-fold increase in DAAO production was achieved after optimization of the physical parameters by RSM.

Page(s): 272-278

Purification, characterization and properties of phytase from Shigella sp. CD2

Title: Purification, characterization and properties of phytase from Shigella sp. CD2

Authors: Roy, Moushree Pal; Poddar, Madhumita; Singh, Kamal Krishna; Ghosh, Shilpi

Abstract: Phytases catalyze the release of phosphate from phytic acid. In this study, a phytase producing bacterial strain Shigella sp. CD2 was isolated from the wheat rhizosphere. Phytase production started from the exponential phase of bacterial growth, showing the highest activity during the stationary phase. The enzyme activity was detected in both periplasmic and intracellular fractions. The enzyme was purified by about 133-fold with specific activity 780 U mg^-1 protein. The optimum pH and temperature of the enzyme was 5.5 and 60^oC, respectively. The enzyme was thermostable and retained 100% and 75% of its activity on pre-incubation at 70^o and 80^oC for 30 min, respectively. The K_m value for the substrate sodium phytate was 0.25 mM. The enzyme was highly specific to substrate phytate, and no activity was detected in presence of other phosphorylated substrates, such as ATP, ADP, glucose 6-phosphate, fructose 6-phosphate and p-nirophenyl phosphate. The activity declined dramatically in presence of Cu²⁺, Zn²⁺ and Fe²⁺ and SDS, whereas Mg²⁺ and Co²⁺ slightly enhanced the enzyme activity. The addition of other metal ions or chemicals had little or no effect on phytase activity. The enzyme was resistant to both pepsin and trypsin. Due to high specific activity, substrate specificity, good pH profile, protease insensitivity and thermostability, phytase encoding gene from Shigella sp. CD2 could be an interesting candidate for industrial applications. Further studies on cloning and expression of Shigella phytase gene are currently in progress.

Page(s): 266-271