Abstract
Atmospheric pressure cold plasma (ACP) is introduced as a useful tool in a variety of biological applications. Proteins are the most abundant macromolecules in living systems with a central role in all biological processes. These organic molecules are modified by ACP exposure that is responsible for many of ACP’s biological effects. This study evaluated the effect of ACP on the production of recombinant phytase in yeast Pichia pastoris (P. pastoris) as well as the structure and function of the phytase enzyme. The results indicated that yeast cells treated with ACP, directly or indirectly, produced higher amounts of recombinant phytase, which was associated with the time of ACP treatment. The exposure of commercial phytase solution with ACP caused a significant increase in the enzyme activity (125%) after 4 hours. Evaluation of the phytase solution by far- and near-UV circular dichroism (CD) and fluorescence analysis indicated that this protein maintained its secondary structure when exposed to ACP while the tertiary structure was slightly unfolded. The effects of heat and H2O2 on the phytase structure and function were compared with the effect of ACP treatment. The modification of Cys, Tyr and Trp amino acids upon reactive oxygen/nitrogen spices was simulated using a molecular dynamics approach. RMSF and RMSD analysis suggested that this structural alteration occurs owing to changes made by reactive species in accessible amino acids.
Highlights
Plasma is described as the fourth state of matter
The results showed that P. pastoris efficiently produced phytase and secreted it using its native signal sequence
The molecular weight of recombinant phytase was more than 100 kDa, which was higher than the molecular weight of commercial phytase derived from A. niger and that of predicted from the A. niger phytase amino acid sequences (48.8 kDa)
Summary
Plasma is described as the fourth state of matter. It is classified into thermal and non-thermal types. Developments in the field of plasma physics led to the production of ACP, which made it operationally more flexible It enabled it to generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) at low temperatures. ACP can affect the protein’s structure and function by oxidizing the surface amino acids and making changes in the secondary and/or tertiary structures[8,9,10,11] These changes may lead to positive or negative effects on the protein stability or activity, which is different by case. The recombinant yeast P. pastoris was constructed and grown to produce phytase protein in a secretory manner and the effects of ACP on the growth and productivity of the bioreactor yeast cells was evaluated. Using CD and fluoresce spectroscopy, the direct effects of plasma on the phytase protein’s activity and structure were investigated
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