Characterization of a novel bacterial dehalogenase from cow dung

Abstract

The large quantities of the halogenated compound for example 2,2- dichloropropionic acid (2,2DCP) in the environment may lead to health problems in humans and pollution due to their toxicity and recalcitrance, respectively. Interestingly, previous studies have indicated that cow dung was proven to degrade pollutants. Hence, such animals feeding on a daily diet of halogen contaminated forage may influence the microflora in their digestive tract. Bacterial species from cow dung able to utilize 2,2DCP is yet to be reported. Therefore, the purpose of this study was to isolate, identify and characterize dehalogenase bacteria from cow dung. Four bacteria were isolated which are SN1, SN2, SN3, and SN4. Strain SN1 was observed with rapid growth in 20 mM 2,2DCP liquid minimal media, and was used for further experiments such as growth in different concentration of 2,2DCP, High-performance liquid chromatography (HPLC), Biolog GENIII, 16S rRNA analysis, characterization of purified enzyme, kinetic analysis, Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and amplification of dehalogenase gene. The growth of strain SN1 in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2DCP with the highest chloride ion released (39.5 μmolCIˉ/mL) while exhibiting a remarkably short doubling time of 3.85 h. The utilization of 2,2DCP was also confirmed by detection of 20 mM 2,2DCP depletion in the growth medium containing strain SN1 measured using HPLC. The result showed 98.6 % utilization of 2,2DCP in the growth medium. Species identification via Biolog GENIII system and 16S rRNA analysis was performed and identified strain SN1 as Bacillus cereus. Further investigations on dehalogenase enzyme were done by using purified enzyme of Bacillus cereus SN1. The molecular weight of the purified enzyme was 25 kDa by SDS-PAGE. The enzyme characteristics revealed it was optimum at pH 6 and 30 °C. It also has low Km value of 0.2 mM. The dehalogenase peptide was identified by LC-MS/MS with 18% sequence coverage to haloacid dehalogenase, Bacillus cereus (strain 03BB102). Moreover, Group I and Group II dehalogenase primers were used to amplify dehalogenase gene and the band only appeared for Group I. The dehalogenase gene fragment amplified was designated “DehSN1” and belongs to Group I dehalogenase since it has 75 % similarity with Group I dehalogenase (DehE). Five conserved residues were identified as Asn33, Tyr117, Cys42, Ala120 and Asp136. As a conclusion, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2DCP. Therefore, further assessment of its ability to degrade other types of haloalkanoic acids merits special consideration.