复合酶体系在烟草固相基质上的酶解特性及助剂增效作用OA

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 obtained through nonlinear regression fitting of first-order degradation kinetics and Michaelis-Menten kinetics empirical approximation models.The inhibition patterns of the natural matrix were analyzed by comparing the kinetic parameters between the artificial tobacco leaves and natural tobacco leaves.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 compared and analyzed by using the composite artificial tobacco leaves.The results showed that the degradation of the three substrates by complex enzyme system in composite artificial tobacco leaves exhibited different kinetic characteristics.Compared to the composite artificial tobacco leaves,starch degradation in natural tobacco leaves was accessibility-limited and protein degradation suffered from dual-inhibition,with Michaelis-Menten constant(Km)increasing to 1.549 7%and maximum reaction rate(Vmax)decreasing to 0.015 5%/h.Cellulose degradation was conformation/steric hindrance-limited,with Km decreasing to 22.724 5%and Vmax decreasing to 0.196 3%/h.The additive increased the first-order 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 the change of Vmax and Km for protein,the enhancement of substrate accessibility for starch,and the change of apparent kinetic parameters for cellulose.The natural tobacco matrix barrier presents differential inhibition patterns that can be characterized by kinetic parameters for different macromolecules.The exogenous additive acting as a micro-environment regulator can overcome these barriers through multiple possible regulatory pathways,achieving synergistic regulation of effective macromolecule degradation and interfacial microenvironment in tobacco.