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. 2023 Aug 4;15(8):494.
doi: 10.3390/toxins15080494.

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05

Junhui Teng  1 , Meijie Song  1 , Qianqian Xu  1 , Qianwen Zou  1 , Haiyang Zhang  1 , Chunhua Yin  1 , Xiaolu Liu  1 , Yang Liu  1 , Hai Yan  1
Affiliations

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05

Junhui Teng et al. Toxins (Basel). .

Abstract

Hepatotoxic microcystins (MCs) are produced and released by the harmful bloom-forming cyanobacteria, which severely threaten drinking water safety and human health due to their high toxicity, widespread distribution, and structural stability. The linearized microcystinase (MlrB) further hydrolyses the poisonous linearized MCs produced by the microcystinase-catalysed MCs to form tetrapeptides. Here, the purification and activity of MlrB were investigated. The results showed that the linearized products generated by 12.5 mg/L MC-LR and MC-RR were removed by purified recombinant MlrB at a protein concentration of 1 mg/L within 30 min. The high catalytic activity of MlrB can be obtained via heterologous expression and affinity purification, which lays the foundation for further studies on the properties and mechanism of MCs biodegradation enzymes.

Keywords: activity; biodegradation; linearized microcystinase; microcystins; purification.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis for each fraction during the purification. Lane marker: Molecular weight standards; Lane 1: Precipitation fraction of recombinant cell extract; Lane 3: Supernatant fraction of recombinant cell extract; Lane 5: Flow-through fraction after His-labeled MlrB binding on the resin; Lane 7 and 8: Flow-through fraction after washing the resin; Lane 9: Fluted His-labeled MlrB for the first time; Lane 10: Eluted His-labeled MlrB for the second time; Lane 11: Purified His-labeled MlrB protein after ultrafiltration. The inclusion bodies and enzyme bands of MlrB are marked with black and red arrows, respectively. The black line indicates the location of the enzyme.
Figure 2
Figure 2
High-performance liquid chromatography profiles for linearized MC-LR and MC-RR biodegradation by cleaned linearized microcystinase MlrB. A coordinate system was established based on millimolar absorption units (MAU) as the vertical coordinate and retention time as the horizontal coordinate. (a) Demonstration of the biodegradation of linearized MC-LR by MlrB to produce tetrapeptides at three stages, 0, 5, and 30 min, respectively. (b) Demonstration of the biodegradation of linearized MC-RR by MlrB to produce tetrapeptides at three stages, 0, 5, and 30 min respectively.
Figure 3
Figure 3
Linearized MCs biodegradation kinetics by purified MlrB: (a) For linearized MC-LR, (b) for linearized MC-RR. In the control group, PBS was filled into the reaction system instead of MlrB. The nodes in the control group are marked with black squares. The nodes in the biodegradable group are marked with red dots. Each node was run in parallel three times, and the average of these was taken as the final data. Note: The error bars represent standard deviation (SD)+ standard error of the mean (SE) of three replicates in each group.
Figure 4
Figure 4
The UV spectra profiles of linearized MCs: (a) For linearized MC-LR, (b) for linearized MC-RR. linearized MC-LR or MC-RR (blue) and its product (red). The spectra were taken at intervals of 5 nm.
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