A codon-optimized endoprotease Endo-Pro-Aspergillus niger: Over expression and high-density fermentation in Pichia pastoris

• We construct the Pichia pastoris expression system according to a codon-optimized endoprotease Endo-Pro-Aspergillus niger (endoprotease EPR) with high activity.
• We develope the fed-batch strategy for high cell-density fermentation of the endoprotease EPR.
• We study the enzyme kinetic parameters such as Km, Kcat and Kcat/Km values using different strength of the chromogenic peptide.
• We find the recombinant endoprotease EPR had a significantly effective on removing the beer haze protein in our study.

The prolyl endopeptidase (endoprotease Endo-Pro-Aspergillus niger, endoprotease EPR) gene from A. niger was optimized according to the codon usage bias in Pichia pastoris. It was successfully expressed using expression vector pPIC9K with high activity (620 ± 25 U/l). The recombinant optimized endoprotease EPR could work in an extremely broad temperature range and over 40% relative activity were remained in the temperature range of 15–70 °C. The optimal pH value and temperature for activity were 4.0–5.0 and 35–40 °C, respectively. The enzyme kinetic parameters Km, Kcat and Kcat/Km values towards various substrates were also determined. A fed-batch strategy was first developed for high cell-density fermentation and the enzyme activity reached 1890 U/l after cultivation in 7 l fermenter. The broad temperature range and efficient expression made this enzyme possible to apply in the food industry directly. This study also sought to investigate the effect of endoprotease EPR on removing the beer haze protein.

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