br Materials and methods br Results and

Materials and methods

Results and discussion

Conclusion
Waste materials cause a problem all over the world; they always need a new strategy to get rid of it with a benefit target. Millions tons of corn cob and corn bran are accumulated every year as byproducts of industrial work without considerable benefits. In our study, we open a new arena of research in the production of P. purpurogenum GE1 phytase by using corn cob and corn bran as substrates. The enzyme was produced under solid state fermentation (SSF) and the conditions for enzyme production were optimized by using Box–Behnken design. We suggest that our work will have great benefit in solving corn cob and corn bran waste problem. Also, our research introduced a low cost medium and very simple technique in phytase production which is considered as one of the most important enzymes.

Acknowledgements

Introduction
Rubisco [ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase; EC 4.1.1.39] is the most abundant protein in plants (Ellis, 1979), and is an enzyme that catalyzes the CO2 fixation reaction in photosynthesis forming phosphoglycerate (PGA) with the reaction of RuBP and CO2, and also catalyzes the photorespiration forming the phosphoglycolate and PGA with the reaction to O2 (Parry et al., 2003). The removal of a tightly bound inhibitor such as CA1P (2-carboxyarabinitol-1-phosphate) from the catalytic site of the carbamylated and decarbamylated forms of rubisco requires rubisco activase (Parry et al., 2008). Rubisco activase is one of a new type of chaperones, which functions to promote the catalytic activity of rubisco (Portis, 2003) in the presence of ATP and RuBP (Portis, 1990). The rubisco activation by rubisco activase is affected by environmental factors such as light intensity (Perchorowicz et al., 1981), content of O2 and CO2 (Sage et al., 1988; Schnyder et al., 1986), and temperature (Schnyder et al., 1984). ATP/ADP ratio affects the activation of rubisco activase (Robinson and Portis, 1989), which is regulated by the extension of C-terminal in the large isoform of rubisco activase (Shen et al., 1991).
Some of the heavy metals are used as essential trace elements in plants (Thormalley and Vasak, 1985), but their existence above a specific concentration in g protein coupled receptors hinders the physiological metabolism in plants (Jarvis et al., 1976). In addition, the heavy metals accumulate either within vacuoles (Okorokov et al., 1980) or occur as cytoplasmic granules in plants (Christie and Costa, 1984). Among heavy metals cadmium (Cd), a soil pollutant with a strong toxicity (Waalkes, 2000), inhibits photosynthesis (Qian et al., 2009) and prevents the growth of roots and stem. Cd inactivates some enzymes by a strong affinity with the thiol group (Mendoza-Cozatl et al., 2005) and it forms the active oxygens such as hydrogen peroxide (H2O2), superoxide anion (), and hydroxyl radical (OH) (Romero-Puertas et al., 2004) together with lipid peroxide, causing damage to biopolymer and cell membrane by inducing the oxidative stress (Heyno et al., 2008).
Copper (Cu) is absorbed mainly through roots and causes the physiological disturbances in plants (Påhlsson, 1989). The excessively-absorbed Cu not only decreases the biomass by inducing the chlorosis (Quartacci et al., 2000), but also interferes with the electron transport system of photosynthesis (Pätsikkä et al., 2002). Zinc (Zn), chemically similar to Cd, is one of the trace elements essential for the growth of plants. Zn interferes with the diverse and essential physiological processes such as the inhibition of plant growth and formation of the toxic lipid peroxide (Panda et al., 2003) in a high concentration.
Glutathione, belonging to non-protein thiol, is synthesized through two phases from glutamate, cysteine, and glycine (Hell and Bergmann, 1990). In plants, glutathione exists mostly in a reduced form (GSH), and also exists in a small proportion of an oxidized form (GSSG) (Hell, 1997). Glutathione is present at relatively high concentrations and occurs as an antioxidant in all plant cells (Dixon et al., 1998). It acts as an antioxidant, protects cell constituents against oxidation and eliminates the oxygen radicals formed by products of photosynthesis together with ascorbate (Mittler, 2002). In addition, glutathione has a protective function for the plant in forming conjugates with xenobiotics (Coleman et al., 1997), and acts as a precursor for the synthesis of phytochelatins, which are involved in the detoxification of heavy metals (Cobbett and Goldsbrough, 2002). Although there are many reports regarding the inhibitory effects of heavy metals in plants (Márquez-García et al., 2012), the study of glutathione on effect of heavy metals affecting the rubisco and rubisco activase which are the photosynthesis enzymes has not been reported yet. In this research work, the study on effectiveness of glutathione reducing the inhibitory effects of Cd, Cu, and Zn was carried out by measuring the growth, content of chlorophyll, content and activity of rubisco and rubisco activase, and activity of denaturing agent in tobacco plant cultured in vitro.