Enzymolysis characteristics of a complex enzyme system on solid phase tobacco matrix and synergistic effect of an additive

The aim of the study was to investigate the enzymolysis kinetics of a complex enzyme system on a tobacco solid phase matrix, to elucidate the mechanism of the barrier effect of natural tobacco matrix and the synergistic effect and mechanism of an exogenous additive on enzymolysis efficiency and tobacco leaf quality. Using self-constructed composite artificial tobacco leaves, kinetic benchmark parameters for the degradation of starch, protein, and cellulose by a composite enzyme system were established through nonlinear regression fitting of first-order kinetics and Michaelis-Menten integral models. The inhibition patterns of the natural matrix were analyzed by comparing the kinetic parameters between the artificial tobacco leaf and natural tobacco leaf. On this basis, the synergistic effect of a complex enzyme and additive strategy was systematically investigated through kinetic analysis, principal component analysis (PCA) of main chemical compositions, and sensory quality evaluation. Finally, the regulatory mechanism of the additive on enzymatic kinetic parameters was quantitatively analyzed by using the composite artificial tobacco leaves. The results showed that the complex enzyme system exhibited distinct "efficiency-affinity" catalytic effect for the three substrates in composite artificial tobacco leaves. ;Compared to the composite artificial tobacco leaves, starch degradation in natural tobacco leaves was accessibility-limited; rotein degradation suffered from dual-inhibition, with K_m increasing to 1.549 7% and V_max decreasing to 0.015 5%/h; cellulose degradation was conformation/steric-hindrance-limited, with K_m decreasing to 22.724 5% and V_max decreasing to 0.196 3%/h. The additive significantly increased the degradation rate constants (k) of starch, protein, and cellulose in natural tobacco leaves by 199.6%, 695.0%, and 86.0% respectively and effectively improved the sensory quality of the treated tobacco leaves. The additive exerted its synergistic effect through three different kinetic regulation modes which were simultaneous improvement of V_max and K_m for protein, enhancing substrate accessibility for starch, and affinity-driven for cellulose. The natural tobacco matrix barrier presents diversified inhibition patterns for different macromolecules which are decoupled by kinetic parameters. The exogenous additive acting as a micro-environment regulator can overcome these barriers through multiple targeted regulation mechanisms, achieving synergistic regulation of targeted macromolecule degradation and interfacial microenvironment in tobacco.