In times of a constantly growing world population and increasing demand for food, sustainable agriculture is crucial. The rainfastness of plant protection agents is of pivotal importance to reduce the amount of applied nutrients, herbicides, and fungicides. As a result of protective agent wash-off, plant protection is lost, and soils and groundwater are severely polluted. To date, rainfastness of plant protection products has been achieved by adding polymeric adjuvants to the agrochemicals. However, polymeric adjuvants will be regarded as microplastics in the future, and environmentally friendly alternatives are needed. Anchor peptides (APs) are promising biobased and biodegradable adhesion promoters. Although the adhesion of anchor peptides to artificial surfaces, such as polymers, has already been investigated in theory and experimentally, exploiting the adhesion to biological surfaces remains challenging. The complex nature and composition of biological surfaces such as plant leaves and fruit surfaces complicate the generation of accurate models. Here, we present the first detailed three-layered atomistic model of the surface of apple leaves and use it to compute free energy profiles of the adhesion and desorption of APs to and from that surface. Our model is validated by a novel fluorescence-based microtiter plate (MTP) assay that mimics these complex processes and allows for quantifying them. For the AP Macaque Histatin, we demonstrate that aromatic and positively charged amino acids are essential for binding to the waxy apple leaf surface. The established protocols should generally be applicable for tailoring the binding properties of APs to biological interfaces.
Protein engineering campaigns are driven by the demand for superior enzyme performance under non-natural process conditions, such as elevated temperature or non-neutral pH, to achieve utmost efficiency and conserve limited resources. Phytases are industrial relevant feed enzymes that contribute to the overall phosphorus (P) management by catalyzing the stepwise phosphate hydrolysis from phytate, which is the main phosphorus storage in plants. Phosphorus is referred to as a critical disappearing nutrient, emphasizing the urgent need to implement strategies for a sustainable circular use and recovery of P from renewable resources. Engineered phytases already contribute today to an efficient phosphorus mobilization in the feeding industry and might pave the way to a circular P-bioeconomy. To date, a bottleneck in its application is the drastically reduced hydrolysis on lower phosphorylated reaction intermediates (lower inositol phosphates, ≤InsP4) and their subsequent accumulation. Here, we report the first KnowVolution campaign of the E. coli phytase toward improved hydrolysis on InsP4 and InsP3. As a prerequisite prior to evolution, a suitable screening setup was established and three isomers Ins(2,4,5)P3, Ins(2,3,4,5)P4 and Ins(1,2,5,6)P4 were generated through enzymatic hydrolysis of InsP6 and subsequent purification by HPLC. Screening of epPCR libraries identified clones with improved hydrolysis on Ins(1,2,5,6)P4 carrying substitutions involved in substrate binding and orientation. Saturation of seven positions and screening of, in total, 10,000 clones generated a dataset of 46 variants on their activity on all three isomers. This dataset was used for training, testing, and inferring models for machine learning guided recombination. The PyPEF method used allowed the prediction of recombinants from the identified substitutions, which were analyzed by reverse engineering to gain molecular understanding. Six variants with improved InsP4 hydrolysis of >2.5 were identified, of which variant T23L/K24S had a 3.7-fold improved relative activity on Ins(2,3,4,5)P4 and concomitantly shows a 2.7-fold improved hydrolysis of Ins(2,4,5)P3. Reported substitutions are the first published Ec phy variants with improved hydrolysis on InsP4 and InsP3.
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