Chemical inhibition of β-galactosidase activities and development of new protocols for β-galactosidase activity assay in microbial cells
Date
2025-04
Authors
Journal Title
Journal ISSN
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Publisher
Federal University of Technology, Owerri
Abstract
Inhibitory effects of surfactants, organic solvents and solvent mixtures on cell-free and cell-bound β-galactosidases were assessed to establish their sub-inhibitory concentrations for the development of methods for cell permeabilization and rapid in situ determination of β-galactosidase activity in Escherichia coli and Kluyveromyces marxianus. The concentration-response relationships of the individual surfactants, solvents and mixtures were fitted to a Gormpertz model to estimate the median inhibitory concentrations (EC50) and No-Observable-Effect-Concentration (NOEC) thresholds. The inhibitory effects of solvents on cell-free β-galactosidase were in the order: N, Ndimethylformamide (DMF) > dimethylsulfoxide (DMSO) > ethanol (E. coli) and DMF > DMSO ≥ ethanol (K. marxianus). The inhibitory effects of surfactants on cell-free β-galactosidases were in the order: 1-cetylpyridinium chloride (CPC) > Cetyltrimethylammonium bromide (CTAB) > sodium dodecyl sulphate (SDS). Triton X-100, sarcosyl, Tween 20, Tween 80 and sodium deoxycholate (SDC) are not inhibitory to β-galactosidases. The established range of concentrations of these permeabilizing agents that are not inhibitory were used for whole cell β-galactosidase activity assay in E. coli and K. marxianus. In this in situ β-galactosidase activity assay, the cells were not washed, and permeabilization agents remained integral part of the reaction mixtures. Some of the selected concentrations of permeabilizing agents used for both organisms were compared with the classical methods in the literature. For E. coli, assay with a 5% 9:1 ethanolchloroform mixture and 0.0008% CPC resulted in higher β-galactosidase activity than Miller’s SDS-chloroform treatment. For K. marxianus, the β-galactosidase activity with 0.15% sarcosyl, 0.4% SDC and 15% of 9:1 ethanol-chloroform mixture resulted in higher β-galactosidase activity than Kippert’s 0.133% sarcosyl treatment. This current method was found to be less timeconsuming and accurate. The treatments could be used as an alternative procedure for in situ assay of β-galactosidase activity in E. coli and K. marxianus cells.
Description
This thesis is for the award of Master of Science (MSc.) in Food and Industrial Microbiology
Keywords
β-galactosidase, ONPG, toxic index, concentration addition, permeabilization, kluyveromyces marxianus, binary solvent mixtures, Department of Microbiology
Citation
Nwangwu, O. R. (2025). Chemical inhibition of β-galactosidase activities and development of new protocols for β-galactosidase activity assay in microbial cells (Unpublished Master's Thesis). Federal University of Technology, Owerri, Nigeria