Improvement of lysozyme activity determination assay and its isolation from natural rubber latex using surfactant precipitation

Natural rubber latex (NRL) of the species Hevea brasiliensis can be separated into three different layers by centrifugation process, which were the rubber-cream phase (the white top layer), the C-serum, and the bottom fraction. All the fractions are considered as waste except the rubber-cream phase. However, the fractions contain lysozyme, a bacteriolytic enzyme, that has huge potential for biotechnological exploitations. So far, there are limited reports on lysozyme recovery from NRL byproducts. Furthermore, a simple, reliable, and accurate assay for quantifying lysozyme activity from NRL is needed, due to its whitish colour, interferes with the spectrophotometric measurement. Therefore, this study aimed to develop, improve, and validate a lysozyme assay for NRL sample based on a lysoplate method using Micrococcus lysodeikticus cells. The lysozyme was then isolated from the NRL fractions using ammonium sulphate ([NH4]2SO4) precipitation (direct and fractional modes) and the novel sodium di-(2-ethylhexyl) sulfosuccinate (AOT) surfactant precipitation. (NH4)2SO4 precipitation is commonly used as preliminary method in protein downstream processing. Meanwhile, surfactant precipitation is poised as alternative method of protein recovery with primary advantage of ease of operation, high yield, and process selectivity. AOT, is an anionic surfactant that dissolve easily in water and solvent and remarkably useful in preparation of microemulsion. The assay developed in this study was high in both accuracy (97.5%) and precision (13.6% relative standard deviation). The detection and quantification limits, as well as the linearity range of the assay were 0.13 U/mL, 0.04 U/mL and 0 - 10,445.4 U/mL, respectively. The incorporation of dye during agarose preparation was also explored to improve contrast. Comparative analysis revealed that non-dyed and bromocresol purple dyed agarose exhibited superior visible contrast in comparison to amido black and methylene blue dye. Particularly, bromocresol purple was identified as the optimal dye, providing enhanced contrast compared to non-dyed agarose. The concentrations of lysozyme in the C-serum and bottom fraction of NRL were determined to be approximately 15.7 μg/mL (628.2 units/mL) and 36.1 μg/mL (1442.7 units/mL), respectively. As for the lysozyme recovery from the C-serum of NRL, the performance of the AOT precipitation via negative mode (activity recovery [AR] = 67.3%; purification factor [PF] = 2.4) was comparable (p > 0.05) to that of (NH4)2SO4 precipitation (direct mode: AR = 88.4%, PF = 2.2; fractional mode: AR = 95.4%, PF = 3.1). Meanwhile, for the lysozyme recovery from the bottom fraction of NRL, the AOT precipitation (AR = 71.9%; PF = 1.7) was shown to be superior than the (NH4)2SO4 precipitation (direct mode: AR = 25.7%, PF = 3.2; fractional mode: AR = 27.9%, PF = 12.9). This study contributes valuable insights into the optimization of agar diffusion assays for improved visualization of lysis zones, with potential applications in various research and diagnostic settings. Furthermore, the establishment of a standard lysozyme gradient becomes crucial for accurately estimating lysozyme concentrations in NRL samples. The surfactant precipitation findings described herein are significant for the development of a downstream processing scheme for lysozyme recovery from NRL.

Text 125067 fulltext.pdf Download (926kB) Official URL or Download Paper: https://ethesis.upm.edu.my/id/eprint/18807

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