Phytase engineering for efficient phosphate recovery from press cakes

  • Phytase-Engineering zur effizienten Phosphatrückgewinnung aus Presskuchen

Herrmann, Kevin Rico; Schwaneberg, Ulrich (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Phosphorus (P) is one of the most essential elements and plays a central role in feeding humankind as a key nutrient in fertilizers. Higher consumption than regeneration rates, precarious mining conditions and the transition to a renewable bioeconomy create a strong social impetus to develop P recovery strategies. Main objective of this thesis is the efficient phosphate recovery from deoiled seeds and nuts using phytase enzymes for valorization of plant byproducts. Quantification of the phosphorus storage form phytate (InsP6) in globally abundant press cakes revealed phytate contents > 3 % (w/w), highlighting the hidden potential for P recovery. The development of a widely applicable and sustainable phosphate recovery process from plant biomass using phytases turned out to be very effective in recovering high amounts of phosphate. One-pot phytase-based hydrolysis of InsP6 in aqueous suspension at moderate temperatures releases phosphate amounts > 20 mg/g press cake and is applicable to more than 20 press cakes. Worldwide industrial application could provide significant amounts of up to 1 million tons, with the recovered phosphate being used to produce premium P-based products such as green fertilizer or polyphosphate as food preservative that enable a circular bioeconomy for P. A bottleneck for complete phytate hydrolysis by phytase enzymes is the drastically reduced degradation of lower phosphorylated reaction intermediates from InsP4 onwards compared to InsP6. Phytate harbors six phosphate groups which are negatively charged under reaction conditions, and the net charge is drastically reduced during hydrolysis to lower InsPs. Therefore, from a protein engineering point of view, the ability to remove all six phosphate groups is very challenging for an enzyme as it has to cope with these substantial changes. Here, the first reported engineering campaign to evolve the E. coli phytase toward improved hydrolysis of InsP4 and InsP3 was conducted. After implementation of a suitable screening setup and isolation of required substrates, KnowVolution-based engineering led to variants with up to 3.8-fold improved hydrolysis of InsP4, while InsP3 hydrolysis was increased up to 2.7-fold. Beneficial substitutions are located within the binding pocket and are involved in substrate binding and orientation. Furthermore, there is a demand for tailored phytases in a wide range of areas such as food, feed or technical applications. By DNA recombination of distantly related genes, chimeras can be generated that combine beneficial properties of the parental enzymes in one protein. A PTRec-based DNA recombination library based on sophisticated computational analysis demonstrated the potential for efficient recombination of six genes with low sequence identities of < 50 % to obtain highly functional chimeras. Two phytase chimeras with up to 32 % improved residual activity (90 °C, 60 min) and retained high specific activities of > 1100 U/mg were identified. In summary, P recycling is an important element for a circular bioeconomy and a building block can be the phytase-based process presented here for application to biomass. Protein engineering approaches offer a variety of capabilities to further tailor the phytases and thus make P recovery processes more economical.