Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.04.18.649602

Improving Thermal and Gastric Stability of Phytase via pH Shifting and Coacervation: A Demonstration of Bayesian Optimization for Rapid Process Tuning

Khongkomolsakul, W.·Buathong, P.·Yang, E.·Dadmohammadi, Y.·Zhou, Y.·Li, P.·Yang, L.·Frazier, P.·Abbaspourrad, A.
View original source ↗Export metadata ↓

Abstract

Phytase (phyA) breaks down phytate, which can help with nutrient absorption in a plant-based seed diet or high-phytate food. Unfortunately, it is prone to denaturation at food preparation temperatures and is easily inactivated by pepsin during gastric digestion. To protect phyA for use in high-temperature processes (100 {degrees}C) and gastric digestion, chitosan (CS) was used to complex phyA. Bayesian optimization, a machine learning technique, was used to demonstrate how to expedite the optimization process. Thermal stability of the optimized complex increased from 20% (Control: phyA in the native state) up to 74% at 4:1 CS to phyA (CS-phyA) complex and 52% at the 1:1 CS-phyA complex as measured by phytase activity assay. Chitosan complexation also improved the retention of enzyme activity after thermal and gastric digestion by 13-fold, retaining residual activity at 40% for the 4:1 CS to phyA and 22% for the 1:1 CS-phyA complexes compared to the enzyme itself, which only retained 3% residual activity. Molecular docking and circular dichroism were used to investigate the underlying interaction mechanism between CS and phyA and the secondary structure of the enzyme after heat treatment. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) confirmed the complexation of phyA with CS and revealed complex morphology. With improved enzyme stability, there is great potential for efficiently expanding phytase applications in a high plant-based seed food matrix. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/649602v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@889b76org.highwire.dtl.DTLVardef@969ed3org.highwire.dtl.DTLVardef@953e55org.highwire.dtl.DTLVardef@2f5864_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThermal stability of phytase improved from 20% to 74% using chitosan C_LIO_LIpH shifting increased enzyme complexation efficiency and thermal stability (100 {degrees}C) C_LIO_LIBayesian optimization (BO) is a promising optimization tool for complexation conditions C_LI

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Khongkomolsakul, W., Buathong, P., Yang, E., Dadmohammadi, Y., Zhou, Y., Li, P., Yang, L., Frazier, P., Abbaspourrad, A.. 2025-04-20. Improving Thermal and Gastric Stability of Phytase via pH Shifting and Coacervation: A Demonstration of Bayesian Optimization for Rapid Process Tuning. https://doi.org/10.1101/2025.04.18.649602

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

MgATP/MgADP-dependent conformational dynamics and intrinsically disordered regions of vascular KATP channels revealed by cryoEM

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

biochemistry↗
bioRxiv2026

Probing the sequence variability tolerance in a de novo α-helical barrel biocatalyst

De novo-designed enzymes have recently achieved high catalytic activity and stereoselectivity while demonstrating exceptional thermostability in entirely novel protein scaffolds. Among these, -helical barrel protein scaffolds are attractive structures for biocatalysis due to their structural simplicity, high thermostability, and rationalizable sequence patterning. However, enabling major structural reengineering of these scaffolds while maintaining the structure, stability and catalytic activity while also improving soluble protein production remain major challenges and pose the fundamental question how engineerable a de novo backbone-sequence pair is. Here, we combine deep learning based and classic computational protein design to modify and optimize de novo -helical barrel biocatalysts. Using the previously reported six-helical barrel 6H5L as a model scaffold, AlphaFold2-guided RosettaRemodel enabled the design of a truncated variant, whose crystal structure closely matches the design model. Additional sequence-redesign using ProteinMPNN generated a variant with a tenfold increase of soluble protein yield in Escherichia coli. Biochemical, biophysical, and structural analyses showed that both variants retained the overall barrel architecture, high thermal stability, and catalytic activity for both purified protein and whole-cell systems. Detailed kinetic analysis on the variants showed both variation in kcat and Km, reflecting changes in catalytic turnover and substrate binding. Together, these approaches provide new insights and possibilities for the further engineering of functional de novo -helical barrels, their ability to withstand dramatically large sequence changes and their broader application in biocatalysis and biotechnology.

biochemistry↗
bioRxiv2026

Cytokine-induced nuclear translocation of STAT1 via a non-transferable NLS

The targeting function of nuclear localization signals (NLSs) is generally considered independent of a protein's native sequence or fold and is readily transferable to heterologous cargos. Contrary to this paradigm, rapid nuclear translocation of phosphorylated STAT1 (pSTAT1) following cytokine stimulation requires importin {beta}, Ran-GTP, and the importin 5 isoform, which recognizes a non-transferable NLS. Here, we present cryo-EM structures of pSTAT1 bound to importin 5, revealing an asymmetric 2:1 complex that diverges from canonical NLS-mediated cargo recognition. Importin 5 occupies the DNA-binding groove of the pSTAT1 dimer, with a single STAT1 N-terminal domain positioning the C-terminal Armadillo repeats 9-10 (S1B domain) orthogonal to the DNA-binding interface. This interface is also targeted by the Ebola virus protein VP24, an antagonist of interferon signaling. We further show that Ran-GTP alone is insufficient to trigger nuclear release of pSTAT1, which additionally requires the exportin CAS. A cryo-EM reconstruction of the CAS-Ran-GTP-5 complex, supported by in vitro competition assays, demonstrates that CAS and pSTAT1 are mutually exclusive ligands for importin 5. Together, these findings define the molecular choreography of cytokine-induced STAT1 nuclear translocation and release, establishing a general paradigm for STAT family signaling.

biochemistry↗