Biochemical Properties of Mercuric Reductase from Local Isolate of Bacillus sp for Bioremediation Agent

Purkan Purkan,Endang Triwahyu Prasetyawati,Yuliana Firdausi Nuzulla,Sofijan Hadi

doi:10.20884/1.jm.2017.12.2.398

Purkan Purkan, Endang Triwahyu Prasetyawati + Show 2 more

Open Access

https://doi.org/10.20884/1.jm.2017.12.2.398

Copy DOI

Abstract

Mercuric reductase is the important enzyme which catalyzes a reduction of a toxic Hg2+ to non-toxic Hg0. The enzyme which has been potentially used as mercury bioremediation agent is produced by mercury resistant bacteria. These research aims are to determinate the resistance level of a local Bacillus sp to HgCl2 in media, to determine the mercuric reductase activity from the bacteria, and to determine the biochemical properties of the mercuric reductase. The Bacillus sp was grown in the Nutrient Broth media with various of 0; 20; 40; 60; 120; and 160 µM HgCl2 to know the response of the bacteria against mercury, The cell growth of Bacillus sp was measured by optical density (OD) method of at λ 600 nm. The mercuric reductase activity was assayed in the solution of MRA (Mercury Reductase Assay), then the oxidized NADPH was observed by the spectrophotometry method at λ340 nm. The result showed that the Bacillus sp has been resistant to media containing mercury at 120 µM, but the microbial growth was decreased by 50% in media containing mercury 80 µM. The Bacillus sp could produce highly the mercuric reductase enzyme at 16 hours of growth time with enzyme activity as 0.574 Unit/µg. The mercuric reductase from the bacteria has an optimum activity at pH 6 and temperature 37 °C

Highlights

The development of industry in Indonesia is so rapid, it followed by the addition of the amount of waste in the environment
The Bacillus sp.was grown on Nutrient Agar (NA) media composed by 0.3% (b/v) Beef extract and 0 . 5% (b/v) peptone, incubated for 24 hours at 37 °C to achieve optical density (OD) 0.1 at λ 600 nm and used as a starter
Assay) solution which composed by 50 mM of Na3PO4 buffer, 0.5 mM EDTA, 0.2 mM MgSO4, 0.1% (v/v) β-mercaptoethanol, 0.1 mM NADPH was reacted with 1 mL 80μM HgCl2 and 0.1 mL of crude enzyme, incubated at 37 °C for 15 min, residual of NADPH was measured at λ 340 nm

Summary

INTRODUCTION

The development of industry in Indonesia is so rapid, it followed by the addition of the amount of waste in the environment. The potency of the bacteria is important to be used as a microbial bioremediation agent to clean mercury contaminant in environments. Mercury-resistant bacteria can reduce mercury ions by involving mercuric reductase enzymes and NADPH via the redox reaction equation (Figure 1). Some bacteria have been reported to produce mercuric reductase enzymes are E. coli, Acinetobacter, Enterobacteriaceae, Pseudomonas sp., Xanthomonas sp., Bacillus sp., and Staphylococcus sp. Has been isolated from Malang agricultural, but its potency has been unknown to produce the mercuric reductase enzyme. In addition to detect the properties of mercuric reductase enzyme which is produced by the bacteria as a bioremediation agent to reduce mercury waste. The mechanism of action of mercuric reductase is carried out by the transfer of electrons from NADPH via FAD to reduce the active part of the cysteine. The instrument used in this research are autoclave, micropipette, pH meter, thermometer, laboratory commonly used glass equipment, UV-Vis spectrophotometer Shimadzu UV-1800, batch ultrasonicator, shaker, incubator, Laminar Air Flow Kottermann 8580, centrifuge (Beckman 201)

Procedures Culturing of Bacteria

RESULTS AND DISCUSSION

CONCLUSION