Abstract: To address the problem that residual pectin methylesterase (PME) can cause turbidity loss and increased microbial safety risks in orange juice during high pressure processing (HPP), this study focused on the regulatory mechanism of HPP at different temperatures combined with microenvironment on PME and pectin methylesterase inhibitor (PMEI). Recombinant PMEI was prepared via molecular cloning, and circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and molecular dynamics simulation were combined to systematically analyze the effects of multi-factor interactions on the enzyme activity and structure. The results showed that Ca2+ concentrations in the range of 0.1–0.5 mol/L could completely inhibit PME activity under HPP/25 ℃ treatment, while pectin concentration (0.1%–0.5%) had no significant regulatory effect on PME activity, but high pectin concentrations could cause physical interference. Under HPP/60 ℃ treatment at pH 7.0, PME activity was decreased by more than 95%, accompanied by changes in its secondary structure and a significant alteration in the polarity of the tryptophan microenvironment. At pH 7.0, the secondary structure of PMEI heterologously expressed in Escherichia coli (E.PMEI) transformed from α-helix to β-sheet, with its inhibitory activity remaining stable. In contrast, the relative content of α-helix structure in PMEI heterologously expressed in Pichia pastoris (P.PMEI) and its inhibitory activity increased under acidic conditions. At the molecular level, these findings clarified that HPP combined with pH adjustment could dynamically regulate the activities of PME and its inhibitor by specifically altering their conformations, providing a theoretical basis for the development of new food processing technologies that allow precise control of pectinase activity.

Key words: high-pressure treatment at different temperatures; pectin methylesterase; storage microenvironment; pH regulation; structure-activity relationship