Optimization of Media and Growth Conditions Improve Overall Quality of Yeast Superoxide Dismutase in Escherichia coli Periplasm (Research Article)

Author(s): Yogesh Suryawanshi*, Surekha Gupta and Shabana Patel

Abstract: Superoxide dismutase (SOD), an antioxidant enzyme is one of the leading required cosmetic industry protein as it prevents skin damage. Small proteins with few disulphide bonds are generally targeted to the oxidizing periplasmic space of Escherichia coli for proper folding. However, it is difficult to achieve good protein concentration without any optimization. In this study, Yeast Cu-Zn Superoxide dismutase is cloned in Escherichia coli to be expressed in periplasm. Yeast Cu-Zn SOD is homodimer in holo form and single subunit is 15.7 kDa in size. For cloning Yeast Cu-Zn SOD, mRNA was isolated from Saccharomyces cerevisiae and cDNA was generated in two-step RT-PCR. PelB was selected as a signal peptide and pET21b as a cloning vector. To resolve problems related to periplasmic expression, techniques such as codon optimization of signal peptide, and media and growth condition optimization were used. SOD activity assay has been used to analyse expression efficiency. SOD protein expressions were done in shake flask and lab scale fermenter. In both the cases, it was found out that slow growth conditions and longer harvesting time results in increased SOD activity. Among various media additives tested, combination of L-arginine, NaCl, and sorbitol were found helpful and caused 64% increase SOD enzyme activity.

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