Functional expression of recombinant goat chymosin in Pichia pastoris bioreactor cultures: A commercially viable alternate

• The paper described for the first time high level inducible expression of goat prochymosin gene.
• Also, first report of bioreactor level functional expression of goat prochymosin which is not glycosylated.
• The recombinant goat prochymosin has residual activity at broad range of pH and temperature.
• The recombinant strain could be an excellent candidate for exploitation in the cheese-making industry.

Recombinant goat chymosin (RGC), a promising alternate of bovine/calf chymosin, has better catalytic, proteolytic and thermostable characteristics. Limited information exists about its expression in different hosts. Pichia pastoris (P. pastoris) has ability to grow to very high cell densities and improved protein production has been demonstrated in scale up studies. A modified methanol feeding strategy based on flask level optimizations was successfully used for expressing the goat prochymosin in recombinant methylotrophic yeast P. pastoris Mut+ bioreactor cultures. Additionally, a comparative analysis of RGC expression in flask versus bioreactor cultures was performed. Bioreactor cultures yielded an approximate 8 times increase in cell density compared to flask cultures prior to induction and an increase of 24% in expression level of chymosin was also recorded. Functional characterization demonstrated that the enzyme produced even at early stages was found to be correctly folded, intact and functional. The recombinant enzyme preparation has 42.4% residual activity at 60 °C and retained 69% activity at pH 6.5. An expression strategy for scale up production of RGC, which can act as a lead for production of industrially viable enzyme preparation, is outlined in the present study.