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Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity.

ABSTRACT: During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus the probability of immobilization with reactive surface sites (i.e., ligation efficiency). Here, the connection between ligation reaction efficiency and the retention of enzyme structure and activity was investigated by leveraging the extremely fast reaction of strained trans-cyclooctene (sTCOs) and tetrazines (Tet). Remarkably, upon immobilization via Tet-sTCO chemistry, carbonic anhydrase (CA) retained 77% of its solution-phase activity, while immobilization via less efficient reaction chemistries, such as thiol-maleimide and azide-dibenzocyclooctyne, led to activity retention of only 46% and 27%, respectively. Dynamic single-molecule fluorescence tracking methods further revealed that longer surface search distances prior to immobilization (>0.5 μm) dramatically increased the probability of CA unfolding. Notably, the CA distance to immobilization was significantly reduced through the use of Tet-sTCO chemistry, which correlated with the increased retention of structure and activity of immobilized CA compared to the use of slower ligation chemistries. These findings provide an unprecedented insight into the role of ligation reaction efficiency in mediating the exploration of denaturing hotspots on surfaces by enzymes, which, in turn, may have major ramifications in the creation of functional biohybrid materials.

SUBMITTER: Chaparro Sosa AF

PROVIDER: S-EPMC8574164 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 μg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis-Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 μM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.

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Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity.

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