Neutral protease expression and optimized conditions for the degradation of blood cells using recombinant Pichia pastoris

• This study offers an environmental friendly solution to the degradation of blood waste.
• This study innovatively to degrade blood cells using genetic engineering methods.
• Two statistical methods were applied to optimize the liquid-state fermentation conditions.

Significant amount of blood waste is generated by the livestock industry. However, the poor utilizability of blood cells limited the application of blood. In our experiment, to degrade blood cells effectively, a recombinant Pichia pastoris strain with the Aspergillus oryzae neutral protease gene was generated using genetic engineering methods. The recombinant strains were screened for high protease activity on agar plates and blood cells fermentation medium using blood cells as the nitrogen source. Single-factor experiments and response surface methodology (RSM) were applied to predicted the degree of hydrolysis (DH) of blood cells and the predicted maximal DH appeared at the region where the pH, time, and temperature were 8.1, 116.6 h, and 33.9 °C, respectively. Under the proposed optimized conditions, the experimental DH value for recombinant pGAPZαA-NpI-GS115 was 49.05%. Therefore, the efficient degradation of blood cells by this recombinant stain may offer an environmental-friendly solution for the degradation of blood waste and other organic matter of similar molecular composition and more meaningfully may supply a high-quality and low-cost protein source for feed industry.