Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

Kranokpron  MOOLWANG; Sakda  DADUANG; Thidarat  SOMDEE; Theerasak  SOMDEE

doi:10.48048/wjst.2021.9455

Authors

Kranokpron MOOLWANG Protein and Proteomics Research Center for Commercial and Industrial Purposes, Khon Kaen University, Khon Kaen 40002, Thailand
Sakda DADUANG Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand https://orcid.org/0000-0003-2672-7710
Thidarat SOMDEE Faculty of Public Health, Mahasarakham University, Mahasarakham 44150, Thailand https://orcid.org/0000-0002-7247-805X
Theerasak SOMDEE Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand https://orcid.org/0000-0003-4114-3430

DOI:

https://doi.org/10.48048/wjst.2021.9455

Keywords:

Microcystin, MlrA protein, Microcystinase, Cloning, Expression, Structural modeling

Abstract

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1^st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha⁷] MC-LR. [Dha⁷] MC-LR at an initial concentration of 30 mg L^-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha⁷] MC-LR appeared with a decrease in the [Dha⁷] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha⁷] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha⁷] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group.

HIGHLIGHTS

Research focused on characterization of microcystinase (MlrA) protein
First research worked on the degradation of [Dha⁷] MC-LR by MlrA
This work is useful for the applications aimed at the removal of MCs in freshwater environments

GRAPHICAL ABSTRACT

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