Organomercury Captured by Lyase Overexpressed Escherichia coli and Its Evaluation by In-Cell Radiometry*

Organomercury lyase (MerB) overexpressed in Escherichia coli captured and decomposed organomercury compounds, and it has been detected by radioactive analysis with neutron irradiation. Genetically modified E. coli captures a lot of mercury from a cultivation solution with about 80% recovery, when the bacteria are growing during 24 to 72 hours. Since the modified E. coli has no additive gene for mercury metabolism, the bacteria could hold mercury tightly by the MerB enzyme in their cell and do not release them into medium. In the later, 72 hours after, bacteria have less recovery ratio; it may be affected by undecompsed mercury compounds in bacteria growth. The recovery ability of the bacteria would not be changed by addition of the MerB producing reagent (IPTG). A quantitative value of mercury atom is estimated by an emission of γ-ray by reactor neutron from a dried cell or solution on a filter paper, which is available for nondestructive testing of bacteria holding mercury atoms. In this method an efficient recovery system of toxic mercury from a polluted solution has been archived without destruction of samples, so called in-cell analysis.


Introduction
Mercury, especially some organic mercury compounds such as methyl, poses a risk to the environment and human health [1]. Currently, there is an urgent challenge to prevent mercury pollution on a global scale. In Japan, pollution by *Organomercury captured and evaluated by in-cell radiometry.
How to cite this paper: Morimoto, Y. and Takamiya, K. (2020) Organomercury Captured by Lyase Overexpressed Escherichia coli and Its Evaluation by In-Cell Radiometry. Advances in Enzyme Research, 8, mercury has been investigated for a long time, and lessons have been learned from the mercury poisoning disaster that occurred in Minamata, Japan (i.e. Minamata disease [2]). Moreover, Mukai et al. [3] reported on a method that permits the almost complete removal of inorganic mercury in waste liquid (termed coprecipitation) by the precipitation of methylmercury using chlorine gas. Later, scientists focused on bioremediation and the development of genetic engineering. Many research efforts have been made to develop complicated chemical reactions and measuring devices to determine the amount of residual mercury; however, the conversion method (from organic mercury to inorganic mercury) and its application methodology have yet to be developed. The utilization of microorganisms [4] capable of decomposing the mercury compounds is essential to combat mercury pollution. Achieving this effectively and using synthetic purification agents as an economical environmental protection technology is important.
The most important issue to be solved is to identify whether the overexpressing bacterial cell lines "actively" take in organic mercury in a solvent. In general enzymatic reactions in the living cell, systems that induce the uptake of substrates are present when the uptake of a substrate in a solution exceeds its diffusion coefficient. In mercury-resistant bacterial strains, organic mercury compounds in a solution are carried to the cells by gene products (proteins) MerP and MerG located on the extracellular membrane and MerE and MerT present on the inner membrane [5]. The organic mercury degrading enzyme (MerB) is degraded "by free diffusion" within the cells. After being translocated within a complex-forming structure, MerA produces inorganic mercury and excretes it outside the cells.
This study focuses on the MerB protein, which was first isolated as a bacterial enzyme [6]. They also studied its various physical properties. Che et al. [7] suggested a route of detoxification and release, where MerB catalyzed the removal of Hg(II) from methylmercury, and the MerA was then converted to Hg(0) inorganic mercury. After this groundbreaking research, the scientists significantly progressed using mercury-resistant bacteria. For example, Di Lello P. et al. [8] proposed the break and removal resistance mechanisms by the MerA-MerB complex and interlinking by a nuclear magnetic resonance (NMR) method. However, the details of the main reaction mechanism (i.e. the relationship between the active portions and mercury compound substrate, reaction pathway, etc.) were not clarified. In 2009, two Canadian teams [9] [10] independently presented a crystal structure analysis of MerB, suggesting a reaction scheme for breaking the carbon-mercury bond. This mercury-resistant strain was isolated into inorganic mercury by a series of expressed proteins of the Mer gene group and then released outside the bacterial cell walls causing subsequent mercury contamination. Therefore, it is essential to improve the collection ratio of mercury so that it is kept within the bacterial cells; that is, in the state, where the components are retained inside and outside of the cell membrane. Quantitative evaluation methods are also to be developed. ICP mass spectrometry is an accu- trons were used to activate the mercury atoms as bacteria cells, and the activation analysis was applied to detect the emitted gamma rays in the cells.

Enzyme Expression and Cultivation
The MerB genes derived from the mercury-resistant region (R831b plasmid) bacteria were chemically synthesized (GenScript Co. Ltd.

Activation Analysis
The obtained samples were adsorbed on a filter membrane on the cell and 1 cm 2 of a filter paper on the solution. The paper with samples was dried. Then, the activation analysis was performed by Pn-2 Port of the research reactor (KUR).
Neutron activation analysis was conducted to determine the amount of mercury (Hg) in each cell sample by means of the following procedure. The cell samples were irradiated by neutrons using the pneumatic sample transport system (Pn-2) [11] for 20 min at KUR with thermal power of 5 MW. After 12 to 14 days from the neutron irradiation, the gamma-ray spectrum was measured for the irradiated samples using the Ge-detector (CANBERRA GC4020). The measurement time varied from approximately 4000 to 150,000 sec, depending on the counting ratio of gamma-rays. The photo-peak area corresponded to the 279 keV gamma-rays, which were emitted subsequent to the beta-decay of 203 Hg (t 1/2 : 46.6 d).
The area was estimated by the Covell method [12]. The cell/sup ratio was calculated as a ratio between the estimated photo-peak area of the cell and sup samples after applying the decay correction.

Activation Analysis for Neutron Irradiation
The values obtained from each sample during the activation analysis are shown in Table 1.

Enzyme Holds Mercury
It is important that we have an evidence for a capture of mercury in the MerB enzyme even Figure 2 shows mobility of mercury is high for a cell part. It was also shown that mercury is retained in the form of mercury atoms in the active site of enzyme molecule after the decomposition of compounds by the mixed crystal analysis of this enzyme and CH 3 HgC1 2 ( Figure 3).
Crystallization was carried out by use of the overexpressed MerB and mixing with organomercury compound (CH 3 HgC1 2 ). Crystals were obtained within one week, and the data collection was done at the SPring-8, Japan. Figure

Conclusions
Herein, the bacterial cells with a gene expression of organic mercury degrading enzyme (MerB) were cultured in a medium. Mercury uptake into these bacterial cells was evaluated by activation analysis. Significant uptake was shown by the MerB-expressing strain. We also have afforded a proof of mercury capture in the  enzyme by a determination of crystal structure analysis of the enzyme with organomercury compound. As such, the uptake detection method and in-cell analysis, which neither destroy the bacterial cells nor require any pretreatment, are suggested. Although some research, especially manganease capture [13] [14] by bactreria, are succeeded in the recent, such an in-cell radioactive analysis is simple and nondestructive evaluation technique for bacteria holding variable metals rather than before spectroscopy.
In this study, the mercury atoms were retained in the enzyme molecule and were not mineralized or released outside of the bacterial cells by interlinking with MerA as only the bacteria cells that express MerB were used. Consequently, the collection of equimolar was possible as one mercury atom was bound to one molecule of the enzyme. This enzyme had a 6xHis tag fused at the N-terminus and could be supplemented with a Ni gel. Hence, mercury atoms can be easily collected with the growth of the bacteria cells from a mercury-contaminated solution or soil solution using affinity column chromatography, which is expected to be effective for soil improvement.