| Abstrakt: |
Aims: The use of chemical organophosphorus compounds has been a serious threat to human's health and environment as pesticides and nerve agents. These synthetic toxic compounds deactivate acetyrlcholinesterase activity, which leads to accumulation of acetylcholine at synapse. Enzymatic biodegradation of organophosphorus agents is considered as a promising strategy compared to other decontaminating methods. BChE also can be used in medical researches such as type 2 diabetes mellitus, Alzheimer's disease and neural disorders. In this study, a novel bio-active recombinant enzyme was designed and produced using yeast secretory expression system. Methods:Pichia pastoris GS115 was recruited as the yeast host along with pPIC9K vector for secretory expression of mutant (G117H/E197Q) Butyrylcholinsterase (BChE). In the first step, gene transformation was done using E. coli DH5 host. Following the plasmid extraction, by using electroporation, the gene was cloned into yeast host. The protein expression was done in a secretory yeast-based system. The secreted protein was evaluated after 24, 48, 72 and 96 h of methanol induction. Finally, the cell components were separated including cytoplasm, membrane and supernatant by applying the sonication and then, expression yield and enzyme activity were assessed. The plasmid obtained from E. coli, were used in the next step for transformation of yeast. The BChE expression was done on the grown colonies on MD agar. Results: Different fractions (cytoplasm, membrane, supernatant) were obtained and the study of different fractions revealed that the BChE enzyme was inclusively present in supernatant (using ammonium sulfate precipitation method) with molecular weight of ~73 kDa. By using the Bradford assay and Imagej, the yield of recombinant protein was 9 mg/mL. The highest enzyme activity in parathion hydrolysis was observed at 72 h with 366.666 U/L. According to the results, the production of BChE in the yeast expression system accounts as an achievement in which a desirable yield and proper performance of enzyme may be obtained. Conclusions: Obtained BChE was able to degrade parathion. Due to contribution of this enzyme in diseases including Alzheimer's disease, neural disorders and several diseases, it could be used in medical researches. [ABSTRACT FROM AUTHOR] |
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