Expression of soybean peroxidase from soybean in Pichia pastoris

Abstract:

[Objective] Soybean peroxidase (SBP) will be widely used in immunoassay, and wastewater treatment and so on, due to its wide substrates, high specific activity, and good thermal stability. Nowadays, it was obtained mainly by extracted from soybean hull. However, it cannot meet the requirements of industrial applications for its low yield, high cost. In this study, SBP will be expressed in Pichia pastoris. [Methods] Both the genes of SBP and truncated C-terminal 20 amino acid SBP were cloned into pPIC-9K. These constructed expression vectors were transformed into Pichia pastoris X-33, and then be used to express SBP. Furthermore, the effects of asparagine glycosylation on SBP expression were also investigated by mutating asparagine into glutamine. [Results] Full length of SBP is inactive in Pichia pastoris. But the truncated C-terminal 20 amino acid SBP showed 23.5 U/mL. Our results indicated that glycosylation site of 144, 185, 197 have a great effect on the enzyme activity. These mutants were almost inactive; Whereas 211 and 216 deglycosylation sites had little effect on activity of SBP, can not be deglycosylation. [Conclusion] The highest activity of SBP was 510 U/mL in fermentation, which is the highest level of the reported.

Key words:Soybean peroxidase, Pichia pastoris, Deglycosylation