Evaluation of chromate reductase activity in the cell-free culture filtrate of Arthrobacter sp. SUK 1201 isolated from chromite mine overburden

Highlights

• First report of extracellular Cr(VI) reductase production by Arthrobacter sp.isolated from chromite mine overburden.

• Extracellular chromate reductase production by the isolate is inducible in nature.

• K_m and V_max of the extracellular chromate reductase indicated its high affinity to Cr(VI).

• Activation energy (Ea) of the extracellular chromate reductase was found to be 36.209 kJ mol⁻¹.

• Extracellular reductase could be effectively utilized to reduce Cr(VI) in mine effluents.

Abstract

Arthrobacter sp. SUK 1201, a chromate resistant and reducing bacterium isolated from chromite mine overburden of Sukinda valley, Odisha, India has been evaluated for its hexavalent chromium [Cr(VI)] reduction potential using cell-free culture filtrate as extracellular chromate reductase enzyme. Production of the enzyme was enhanced in presence of Cr(VI) and its reducing efficiency was increased with increasing concentration of Cr(VI). The Michaelis-Menten constant (K_m) and the maximum specific velocity (V_max) of the extracellular Cr(VI) reductase were calculated to be 54.03 μM Cr(VI) and 5.803 U mg⁻¹ of protein respectively showing high affinity towards Cr(VI). The reducing activity of the enzyme was maximum at pH 6.5–7.5 and at a temperature of 35 °C and was dependent on NADH. The enzyme was tolerant to different metals such as Mn(II), Mg(II) and Fe(III) and was able to reduce Cr(VI) present in chromite mine seepage. These findings suggest that the extracellular chromate reductase of Arthrobacter sp. SUK 1201 has a great promise for use in Cr(VI) detoxification under different environmental conditions, particularly in the mining waste water treatment systems.

Introduction

Hexavalent chromium [Cr(VI)] is a strong oxidizer, which exists as hydrochromate (HCrO⁻⁴), chromate (CrO⁻²) or dichromate (Cr₂O₇⁻²) and is highly soluble at neutral pH. In nature, Cr(VI) is formed as water soluble anions or neutral species (Kumaresan and Riyazuddin, 1999) and is considered as one of the 17 chemicals posing great threat to human health and environment (Marsh and Mc Inerney, 2001). It is readily taken up by the cell via sulphate pathway because of its structural similarity to SO₄. Toxicity of Cr(VI) is mainly attributed to the process of reduction of Cr(VI) to lower oxidation state, leading to the formation of Cr(V) and reactive oxygen species (ROS) causing ultimately to cellular damage (Cervantes et al., 2001, Ackerley et al., 2004). Such damaging actions may be due to defective replication, transcription, sister chromatid exchange, chromosomal aberrations, cell transformation (Dayan and Paine, 2001) and mutations leading to carcinogenic effect (McLean and Beveridge, 2001).

Microbial reduction of toxic Cr(VI) has been identified as a bioremediation tool not only to detoxify chromium, but also to recover the non toxic Cr(III) by physical means. Chromate reductase, the central enzyme involved in bioreduction of Cr(VI) to Cr(III), has been described as an intracellular one in several bacteria (Camargo et al., 2004, Pal et al., 2005, Opperman et al., 2008). The extracellular chromate reductases, on the other hand are advantageous to the cells as they protect the cells from toxic Cr(VI), prevent the entry of insoluble Cr(III) into the cells and damages to DNA. However, reports of extracellular Cr(VI) reductase enzymes from chromate reducing bacteria are few (Gnanamani et al., 2010, Rath et al., 2014, Mala et al., 2015).

Extracellular chromate reductases are usually produced during growth of the bacteria in presence (Mala et al., 2015) or in absence of Cr(VI) and are released into the medium where they carry out the Cr(VI) reduction process. The reduced product, Cr(III) being insoluble at neutral pH is usually precipitated in the environment. Priester et al. (2006) have demonstrated that chromate reductase produced in the cytoplasm of Pseudomonas putida is released in to the external environment following cell lysis or secretion and reduced Cr(VI) extracellularly resulting precipitation of Cr(III) outside the cell. Similarly, McLean and Beveridge (2001) have reported the extracellular chromate reductase activity in soil pseudomonads. Smith and Gadd (2000) also established the extracellular Cr(VI) reduction in sulphate reducing bacteria and 90% of the reduced Cr was detected in the supernatant. Wang et al. (1991) reported that bacteria with membrane bound reductases can also reduce Cr(VI) extracellularly. More recently, extracellular chromate reductase activities, particularly of Bacillus spp. have been demonstrated by several others (Gnanamani et al., 2010, Rath et al., 2014, Mala et al., 2015).

During the course of our survey for bacterial strains capable of tolerating and reducing high concentrations of Cr(VI) from chromite mine overburden, we have isolated a potent chromate reducing strain Arthrobacter sp. SUK 1201 (MTCC 8728, Genbank Accession No. JQ312665). The strain was capable of reducing Cr(VI) during growth (Dey and Paul, 2012), by free whole cells (Dey and Paul, 2014b) as well as immobilized whole cells (Dey and Paul, 2014a) leading to formation and extracellular precipitation of Cr(III). Although preliminary work on the optimization of intracellular crude chromate reductase enzyme of SUK 1201 (Dey and Paul, 2013a) has been done, no attempt has so far been made to characterize the chromate reducing ability of the extracellular enzyme produced by this isolate. The present study, therefore, appears to be the first report on the evaluation of Cr(VI) reduction potential of the crude extracellular enzyme obtained from actively growing culture of Arthrobacter sp. SUK 1201. The conditions for Cr(VI) reduction by crude extracellular enzyme (the cell-free culture filtrate) have been optimized and the ability of the enzyme to reduce Cr(VI) in mine effluent has also been evaluated.