Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

Enny Ratnaningsih; Sulistiya Nirta Sunaryo; Idris Idris; Rindia Maharani Putri

doi:10.14710/reaktor.21.2.59-64

DOI: https://doi.org/10.14710/reaktor.21.2.59-64

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

*Enny Ratnaningsih - Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Indonesia, Indonesia

Sulistiya Nirta Sunaryo - , Indonesia

Idris Idris - , Indonesia

Rindia Maharani Putri - , Indonesia

Received: 5 Jun 2021; Published: 1 Aug 2021.

BibTex Citation Data :

@article{Reaktor38880,
    author = {Enny Ratnaningsih and Sulistiya Sunaryo and Idris Idris and Rindia Putri},
    title = {Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1},
    journal = {Reaktor},
  volume = {21},
    number = {2},
    year = {2021},
    keywords = {},
    abstract = { In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.    Keywords:   recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification  },
   issn = {2407-5973},   pages = {59--64}  doi = {10.14710/reaktor.21.2.59-64},
    url = {https://ejournal.undip.ac.id/index.php/reaktor/article/view/38880}
}

Citation Format:

Abstract

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.

Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

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Section: Research Article

Language : EN

In Volume 21 No. 2 June 2021

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