Joint Effects of Water-Miscible Solvents on Kluyveromyces marxianus Cell-Free β-Galactosidase Activity

Abstract

Aim: The study aims to assess the inhibitory effects of ethanol-N,N-dimethylformamide (DMF) and ethanol-dimethyl sulfoxide (DMSO) binary mixtures on Kluyveromyces marxianus cell-free β-galactosidase activity. Study design: A cell-free enzyme assay integrating concentration-response modelling with mixture-toxicity analysis was used to characterize the inhibitory effects of ethanol, DMSO, DMF and their binary mixtures on Kluyveromyces marxianus β-galactosidase activity. Methods: No-observable-effect concentration (NOEC) and median inhibitory concentration (EC50) limits were calculated by fitting concentration-response relationships for the individual solvents and mixtures to a Gompertz model. Values generated from the prediction models are presented as mean ± standard deviation. One-way analysis of variance (ANOVA), followed by Duncan's post hoc test, was used to determine significant differences among treatments. Results: The NOEC values for ethanol, DMSO and DMF were 2.651 ± 0.796%, 2.215 ± 0.309% and 0.920 ± 1.161%, respectively. The corresponding EC50 values were 18.930 ± 1.171%, 19.114 ± 0.534% and 13.587 ± 0.489%, respectively, giving an inhibition order of DMF > DMSO ≥ ethanol. Ethanol-DMSO and ethanol-DMF mixtures with higher EC50 and NOEC values showed lower inhibitory effects than the individual solvents, particularly DMF and DMSO. The toxic index (TI) and concentration addition (CA) model were used to predict the inhibitory effects of ethanol-DMF and ethanol-DMSO mixtures. The CA-predicted EC50 values of ethanol-DMSO and ethanol-DMF mixtures ranged from 18.880 ± 1.444 to 18.897 ± 1.604 and from 18.525 ± 0.886 to 22.664 ± 1.099, respectively. TI values for ethanol-DMSO mixtures ranged from 1.018 ± 0.014 to 1.062 ± 0.021. Ethanol-DMF mixtures at ratios of 9:1 and 8:2 indicated synergistic effects, whereas ratios of 7:3 and 6:4 indicated additive effects. For TI values marginally above 1, the joint effect was adjudged additive because the observed EC50 values were not significantly different from the CA-predicted EC50 values. Conclusion: This study provides foundational information for establishing sub-inhibitory concentrations of water-miscible solvents for the in situ β-galactosidase activity assay derived from Kluyveromyces marxianus.