Available online 26 July 2022

The aim of the present study was to investigate the effect of substrate conformational structure changes on the laccase-induced protein cross-linking. The effects of laccase amount, pH, and ferulic acid (FA) on the enzymatic cross-linking of substrate, Cyt C, were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. High performance size exclusion chromatography, laser particle size analysis and isothermal titration calorimetry (ITC) were also applied to investigate the cross-linking product and enthalpy changes. Structural changes of Cyt C at different pH values were analyzed by ultraviolet-visible (UV-Vis), fluorescence, and circular dichroism (CD) measurements.

Complete cross-linking, partial cross-linking, minute cross-linking, and no cross-linking occurred at pH 2.0, 4.0, 6.0, and 8.0, respectively. ITC analysis demonstrated that the enzymatic cross-linking of Cyt C was an endothermic process. The UV-Vis, fluorescence, and CD measurements exhibited that the tertiary structure of Cyt C was disrupted, and part of the α-helical polypeptide region unfolded at pH 2.0. The structural flexibilities decreased and the tertiary structure of Cyt C became increasingly compact with the increase in pH values from 4.0 to 8.0. The gradual changes in the structure of Cyt C at different pH values were in accordance with the cross-linking results of Cyt C catalyzed by laccase.

The results demonstrated that minute structure changes of substrate had a remarkable effect on the laccase-induced cross-linking. The findings promote the understanding of the substrate requirement of laccase in protein cross-linking and are instructive for the modulation of laccase-induced protein cross-linking.

Conformational structure

Cytochrome c

High performance size exclusion chromatography

Isothermal titration calorimetry

Laccase

Laser particle size analysis

pH value

Protein cross-linking

Substrate requirement

2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt

high performance size exclusion chromatography

isothermal titration calorimetry

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

ultraviolet-visible

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