Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH

Abstract

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced K at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).

Matter‐tag: A universal immobilization platform for enzymes on polymers, metals, and silicon‐based materials

“…(a) Surface representation of liquid chromatography peak I (LCI; hydrophobic in green, hydrophilic in blue) generated by Discovery Studio Visualizer ( Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016 ); (b) Schematic representation of Matter‐ tag ‐fusion protein consisting of protein/enzyme (here phytase), a rigid linker consisting of 17 amino acids, and the Matter‐ tag (here: LCI); (c) Generation of Matter‐ tag ‐fusion proteins for the immobilization on the polymers (PS, PP, and PET), metals (stainless steel and gold), and silicon wafer. Matter‐ tag ‐fusion proteins are generated by genetically fusion of one Matter‐ tag (Cecropin A; Steiner, Hultmark, Engström, Bennich, & Boman, , LCI, PDB ID: 2B9K; Gong, Wang, Chen, Xia, & Lu, or Tachystatin A2, PDB ID: 1CIX; Fujitani et al, ) via a rigid linker to the target protein (enhanced green fluorescent protein, PDB ID: 2Y0G; Royant & Noirclerc‐Savoye, ) or target enzyme (phytase; Körfer et al, or cellulase; Lehmann, Bocola, Streit, Martinez, & Schwaneberg, ). Immobilization on the selected materials through Matter‐ tags is performed by simple dip‐coating into aqueous protein solution at ambient temperature.…”

“…Purification and desalting of phytase and phytase-LCI YmPh and YmPh-LCI were purified using cation-exchange chromatography (HiTrap® SP High Performance; GE Healthcare, Uppsala, Sweden; ÄKTAprime plus, GE Healthcare) and afterward desalted and concentrated according to the published protocol(Körfer et al, 2018;Shivange et al, 2012). Purified proteins were stored at 4°C.…”

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification

“…The resulting mutants are further screened for improved characteristics (Nannemann et al, 2011). This method has become a valuable tool for ameliorating various enzymes such as xylanases (Xu et al, 2016;Acevedo et al, 2017), laccases (Mateljak et al, 2019), phytases (Shivange et al, 2016;Körfer et al, 2018), amylases (Wu et al, 2018;Huang et al, 2019) and cellulases (Liu et al, 2014;Larue et al, 2016;Lin et al, 2016;Goedegebuur et al, 2017;Yang et al, 2017;Cao et al, 2018). Cao et al (2018) modified β-glucosidase (Ks5A7) for improved thermostability, catalytic efficiency and resistance to glucose inhibition.…”

Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of <em>Escherichia coli</em> Colonies

“…Lack of high-throughput methods for phenotype screening can be a bottleneck for directed evolution ( Zeymer and Hilvert, 2018 ). Chromogenic conversion, pH change, and fluorescence readings can be used for phenotype screening; however, they are often indirect measurements of the desired phenotype ( Körfer et al, 2018 ; Alfaro-Chávez et al, 2019 ; Minges et al, 2020 ). A promising approach is mass spectrometry (MS), which provides label-free measurement of a wide variety of analytes.…”