Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1

In recent years, attention to microbial dehalogenase has continually increased due to its potential application, both in bioremediation and in the biosynthesis of fine chemicals. Many microbial recombinant strains carrying dehalogenase gene have been developed, particularly to increase the dehalogenase production and its quality. In this study, we aimed to find the optimum condition for the production of active haloacid dehalogenase by E. coli BL21 (DE3) harboring recombinant plasmid pET-bcfd1 that carried haloacid dehalogenase gene from Bacillus cereus IndB1 local strain. This would be examined by assessing the ability of whole cell life culture to degrade monochloroacetic acid (MCA) and quantifying the chloride ion released into the medium. Several variables were evaluated to find this optimal condition. We found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG), 6 hours of pre-induction incubation at 37oC with shaking, 2 hours IPTG induction at 30oC with shaking, at pH 7 in Luria Bertani (LB) liquid medium without NaCl, which produced about 0.056 mM chloride ions. Inducer concentration, pre-induction incubation time and temperature, as well as induction time and temperature were apparent to be associated with the expression of the protein, while the MCA concentration and the pH of the medium influenced the ability of the recombinant E. coli BL21 (DE3)/pET-bcfd1 to grow in toxic environment. Our findings laid the foundation for exploration of dehalogenases from local Bacillus strains through genetic engineering for MCA biodegradation.


Introduction
Monochloroacetic acid (MCA) is a derivative of carboxylic acid, produced by acetic acid chlorination. MCA is an industrially important organic compound, widely used as building blocks in the production of carboxymethyl cellulose (CMC), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) herbicides, for paint and graffiti removal, plastic synthesis, and other forms as its ester or amide derivatives (European Commission, 2005). Nevertheless, these compounds are known to cause environmental pollution and harm to human health due to its toxicity, corrosive, persistence, and carcinogenic properties. These compounds along with other halogenated organic compounds, including MCA, are commonly found as pollutants in drinking water and wastewater (Islam et al., 2020). High concentrations of MCA cause hazard to living organisms including humans (McRae et al., 2004).
The presence of halogen in carboxylic acids increases its stability as well as its solubility in water. Naturally, carbon-halogen bond could be cleaved through some chemical processes, such as oxygenation, oxidation, reduction, hydrolysis, and conjugation, which are catalyzed by enzymes (Guengerich, 1984). Dehalogenase is one group of enzyme that catalyzes the cleavage of carbon-halogen bonds in halogenated organic compounds (Slater et al., 1997;Wang et al., 2018). Some microorganisms have been found to be involved in this halogen removing process from haloalkanoic acid compounds in the environment by producing dehalogenase (Ismail et al., 2017). The bioremediation of halogenated compounds by microorganisms was considered as an environmentally friendly approach, particularly in comparison to chemical processes (Abatenh et al., 2017;Zhang et al., 2014).
Attention to these microbial dehalogenases has increased continually due to their potential application, not only in bioremediation but also in biosynthesis of industrial fine chemicals (Adamu et al., 2020;Ang et al., 2018;. Furthermore, dehalogenases have also been developed for biosensor application, which is considered as a promising tool for halocarbon detection (Gul et al., 2021). To increase the dehalogenase production and quality, the microbial recombinant strains harboring gene encoding dehalogenase have been developed (Ang et al., 2018), particularly by cloning the target gene into a strong expression vector.
E. coli has been considered as the most suitable host for expressing stably folded globular proteins from various organisms (Rosano & Ceccarelli, 2014). Among available E. coli hosts, E. coli BL21 (DE3) is a superior host for recombinant protein expression (Shiloach et al., 1996), which could produce the highest amount of specific recombinant proteins (Marisch et al, 2013) with a simple set-up and low protease production. Expression of recombinant protein in this host relies on T7 RNA polymerase under the control of lacUV5 promoter, which prevents any gene expression until induction is performed by adding IPTG into the cultures (Rosano & Ceccarelli, 2014). However, producing a large amount of soluble and functional recombinant proteins in E. coli host still presents challenges. Upon overexpression, the produced protein generally accumulates as insoluble aggregates known as the inclusion bodies in the bacterial cells, which is an inactive form of the enzymes. Therefore, to obtain a soluble active protein, finding an optimal condition that mimics its bacterial life's native condition is very important. Several experimental approaches have been developed to prevent the formation of aggregates, including reducing the inducer concentration (Winograd et al., 1993), increasing the intracellular concentration of chaperon molecules (Mogk et al., 2010), performing induction in mid-exponential phase or final exponential phase (Galloway et al., 2003), lowering the induction temperature (Schein & Noteborn, 1988), and engineered the host cell (Rosano et al., 2019).
In this study, we aimed to find an optimal condition required by the recombinant E. coli BL21 (DE3)/pET-bcfd1 to catalyze MCA biodegradation. The optimal condition was studied by varying inducer and substrate concentrations, pre-induction incubation time and temperature, induction time and temperature, and pH of the utilized medium. Degree of MCA biodegradation was examined by determining the chloride ions released into the medium using a UV-Vis spectrophotometer according to Bergmann and Sanik method (Bergmann & Sanik, 1957). We expected that the optimal condition would promote the production of dissolved and active haloacid dehalogenase, hence the clone would be performing its best in degrading MCA.

Source of the recombinant microorganism
The recombinant clone of E. coli BL21 (DE3) carrying the haloacid dehalogenase gene from Bacillus cereus IndB1 local strain in pET-30(a) expression vector, termed as E. coli BL21 (DE3)/pET-bcfd1, was obtained from our previous research (Ratnaningsih & Idris, 2018). The bcfd1 haloacid dehalogenase gene in this recombinant clone has been sequenced and the sequence has already been deposited in GenBank (accession number KU49803). Initially, this gene was cloned into pGEM-T vector in E. coli TOP10 host for analysis, which then subsequently sub-cloned into pET-30a(+) expression vector in E. coli BL21 (DE3) for expression with T7 RNA polymerase under the control of lacUV5 promoter. The clone has been stored at -80ºC in 20 % glycerol-LB medium (v/v) as a stock culture.

Regenerating the E. coli BL21 (DE3)/pET-bcfd1 culture
The glycerol stock culture of the recombinant E. coli BL21 (DE3)/pET-bcfd1 was inoculated into 2 mL of LB liquid medium (10 g/L tryptone and 5 g/L yeast extract, without NaCl) containing 50 µg/mL kanamycin. The culture was incubated overnight at 37ºC with shaking. A single colony for further experiments was obtained by streaking this fresh culture onto a solid LB medium containing 50 µg/mL kanamycin and followed by overnight incubation at 37ºC. PCR analysis employing the primers used for initial gene isolation (which are 5'-ATGGATGGAACACTACTATC-3' as forward primer and 5'-TTATTTACTAGATGAAGTTTG-3' as reverse primer) as well as restriction analysis using EcoRI and HindIII were accordingly performed for clone confirmation.

Growth of E. coli BL21 (DE3)/pET-bcfd1 in MCA containing medium
Growth of recombinant E. coli BL21 (DE3)/pET-bcfd1 in the medium containing various MCA concentrations was studied by culturing the clone in 2 mL LB broth (without NaCl) containing 50 µg/mL kanamycin. The MCA concentration was varied from 1 mM -10 mM. Inoculation was performed using 10 µL of E. coli BL21 (DE3)/pET-bcfd1 overnight fresh culture, followed by 6 hours incubation at 37ºC with shaking. Ability of the clone to grow in MCA containing medium was observed by measuring culture optical density at 600 nm, called as OD600. The absence of MCA in the medium was used as the growth control.
Our standard condition in performing optimization for MCA biodegradation was as follows. All experiments were carried out in 0.2 mM of MCA, except for IPTG optimization, which was performed with 1 mM MCA. Prior to induction, the culture was incubated at 37ºC for 6 hours with shaking. At this step, 1 mL of this culture was drawn into a new sterile tube and stored at 4ºC to be used as a control. Then, the IPTG inducer was added to the culture at a 10 µM final concentration and the incubation was continued at 30ºC for 2 hours. The culture was then placed on ice for 10 minutes, centrifuged, and the supernatant was collected. This centrifugation and supernatant collection was also performed identically using the control. Then, 1 mL of the supernatant was transferred into a new tube and the amount of chloride ion released into the medium was quantified. The optimum condition of MCA biodegradation was determined by varying one particular variable, while others were set at the standard conditions. As shown in Fig.1, the varying variables were inducer (IPTG) concentration, substrate (MCA) concentration, preinduction incubation period and temperature, induction period and temperature, and pH of the medium. All experiments were conducted in two replicates.

Chloride ion determination
Chloride ion determination was performed according to the method introduced by Bergmann and Sanik (Bergmann & Sanik, 1957) as follows. Into 1 mL of the culture supernatant, 0.1mL of 0.1% (w/v) Hg(SCN)2 was added, mixed well by inverting the tube several times, and incubated for 5 minutes at room temperature. Then, 0.1 mL of 0.25 M FeNH4(SO4)2 in 9 M HNO3 was added into the mixture, mixed well by inverting the tube, and further incubated for 5 minutes at room temperature. Absorbance of the resulted coloured solution was measured at 460 nm, and the chloride ion concentration was determined using NaCl standard curve.

Growth of E. coli BL21 (DE3)/pET-bcfd1 in MCA containing medium
The growth of E. coli BL21 (DE3)/pET-bcfd1 in LB liquid medium (without NaCl) containing various MCA concentrations was evaluated prior to optimization of MCA biodegradation. This assay was conducted in order to observe the highest MCA concentration that can be tolerated by the recombinant E. coli BL21 (DE3)/pET-bcfd1. The result of growth is shown in Fig. 2(a) and the cells' turbidity of the culture was measured by determining culture's optical density at 600 nm (OD600) as shown in Fig. 2(b). It could be seen in Fig. 2(a) that culture's turbidity tends to decrease with incremental increase of MCA concentration, indicating that MCA was toxic to the cells. This observation was supported by culture's OD600, as shown in Fig. 2(b). At 1mM and 2 mM of MCA, the recombinant E. coli BL21 (DE3)/pET-bcfd1 was still apparent to grow quite well, but it already lost half of its growth upon increasing the MCA concentration to 3 mM. There was no growth observed when the MCA concentration was ³ 6 mM. Other previous research has reported that Bacillus sp. TW1 was able to grow in a medium containing 0.5 mM of MCA (Zulkifly et al., 2010), which was lower compared to Arthrobacter sp. D2 that was able to tolerate up to 20 mM of MCA (Alomar et al., 2014). These reports indicated that tolerable MCA concentration could be different from strain to strain. It was also reported that MCA dehalogenase activity was found to be inducible, either in wild type of Pseudomonas sp. R1 (Ismail et al, 2008) or in its recombinant clone of Burkholderia spesies MBA4 (Su et al, 2013). Based on our result, we chose 1 mM of MCA concentration for initial optimization of inducer (IPTG) concentration.

Optimization of inducer concentration
Inducer concentration was optimized by performing experiments at various concentrations of inducer (IPTG, in the range of 0 mM to 1 mM). The result is presented in Fig. 3(a). Biodegradation was represented as chloride ion concentration released into the medium. It was apparent that the amount of IPTG influenced MCA biodegradation. Interestingly, a higher MCA biodegradation occurred at a lower inducer concentration, hence a high inducer concentration did not appear to promote MCA biodegradation. This finding was attributed to the overexpression of recombinant protein in E. coli BL21 (DE3)/pET-bcfd1 that was performed by T7 RNA polymerase under the control of lacUV5 promoter (Ma et al., 2020), which was regulated by the inducer (IPTG). Though protein expression might have been increased at higher inducer concentration, overexpression of the protein would instead lead to inclusion bodies formation, which rendered the enzyme inactive (Ma et al., 2020;Rosano et al., 2014).
It is well known that the slow rate of protein synthesis in intracellular expression system might reduce the formation of insoluble nonfunctional recombinant proteins (inclusion bodies). Hence, reducing the speed of protein synthesis was the main strategy to obtain functional recombinant proteins (Rosano & Ceccarelli, 2014). Therefore, the lowest inducer concentration is much better to express such functional recombinant proteins (Riggs et al., 1994). This fact is in good agreement with our findings. We obtained that the highest MCA biodegradation performed by E. coli BL21 (DE3)/pET-bcfd1 was achieved at 10 µM of IPTG, which was used for further analysis.

Optimization of MCA concentration
Optimal MCA concentration for biodegradation was investigated in LB liquid medium (without NaCl) induced by 10 µM IPTG. The MCA concentrations were varied from 0 mM to 1 mM, and other variables were kept constant as stated in materials and methods. The result is presented in Fig. 3(b). The optimum MCA concentration for best biodegradation by E. coli BL21 (DE3)/pET-bcfd1 was found to be 0.2 mM, as indicated by the highest concentration of chloride ion released into the medium. The MCA biodegradation seemed to decrease with the increase of MCA concentration. Although the growth of E. coli BL21 (DE3)/pET-bcfd1 was still observed at 1 mM of MCA, no MCA biodegradation occurred under this condition, indicating that 1 mM of MCA was already toxic to the cells. Though the bacteria could take up MCA as additional carbon source, a high MCA concentration could become toxic to the cells (Torz & Beschkov, 2005).

Optimization of pre-induction incubation time
Under the optimal concentration of inducer and substrate, which were 10 µM of IPTG and 0.2 mM of MCA respectively, MCA biodegradation was investigated by varying the pre-induction incubation time while keeping all other variables constant (as stated in materials and methods). The result is presented in Fig. 4(a), which clearly indicated that pre-induction incubation period influenced MCA biodegradation. This incubation period was associated with bacterial growth phase prior to induction for expressing the haloacid dehalogenase gene. Our result indicated that the best pre-induction incubation time was 6 hours at 37ºC. Prolonged preinduction incubation time at this condition did not appear to increase the MCA biodegradation. The number of cells and stages of cell growth would affect the level of inducible gene expression. Cells in the middle exponential phase are ideal for expression because in this stage the cells are actively dividing. It was known that enzyme production will increase by increasing the cell concentration until it reached the stationary phase, and addition of inducer at the final exponential phase (close to the stationary phase) did not have any significant effect on enzyme production (Yildir et al., 1998). In addition, when the synthesis of recombinant protein and cell growth simultaneously occurred, which happened if the induction was carried out at early incubation time, protein synthesis and cell growth would tend to be slow because growth resources have to be divided to support both processes (Fazaeli et al., 2019). In our experiments, this fact could be seen at the 1 st to 4 th hour of incubation, where the amounts of the released chloride ions were low. In agreement with a previous report, our experiment showed that after 6 hours of incubation, the culture OD600 has reached 0.6 -0.8, which was optimum for the induction (Galloway et al., 2003).

Optimization of pre-induction and induction temperature
To study the effect of incubation temperature prior to induction and after IPTG induction on MCA biodegradation, experiments were performed by varying these two variables at a standard biodegradation condition as stated in the materials and methods. Duration of pre-induction and after induction incubation was kept to 6 hours and 2 hours respectively. The result is presented in Fig. 4(b). The best MCA biodegradation was observed at 37 ºC pre-induction incubation followed by shifting to 30 ºC incubation after induction. Incubation temperature prior to IPTG induction was clearly associated to bacterial growth rate, whereas condition after the addition of IPTG was related to the rate of recombinant protein expression. This result was consistent with previous report on the optimal temperature growth for E. coli cells, which is 37ºC (Noor et al., 2013). Conversely, lower incubation temperature after the induction would reduce the rate of protein synthesis, thus reducing the accumulation of recombinant protein and increasing protein solubility (Pacheco et al., 2012;Sahdev et al., 2008). This condition might promote correct protein folding and therefore increase MCA biodegradation.

Optimization of induction incubation time
Induction incubation time was defined as the incubation period after IPTG addition. The induction incubation was varied from 1 to 7 hours by keeping all other variables constant. The result is presented in Fig.4(c), which clearly indicated that the best induction incubation time for MCA biodegradation by E. coli BL21 (DE3)/pET-bcfd1 is 2 hours. Prolonged incubation after the induction did not increase MCA biodegradation. Extending induction period might have triggered the formation of inclusion bodies as the recombinant proteins accumulated in the cells. Reducing the duration of the induction time is a strategy to obtain recombinant protein in its active state (Gutiérrez-González et al., 2019).

Optimization of medium pH
The effect of pH in MCA biodegradation was studied at a standard condition (as stated in materials and methods) by varying the medium pH from 6 to 10. The result is presented in Fig. 4(d). It could be seen that highest MCA biodegradation occurred at pH 7 which released ~0.056 mM chloride ions to the medium. Apparently, the E. coli BL21 (DE3)/pET-bcfd1 drastically lost its ability to degrade MCA when the pH of the medium was acidic (pH 6) or alkaline (pH 9). We attributed this finding to the growth difficulty of E. coli BL21 (DE3) in acidic or alkaline environment. It was known that the most common pH condition that supports the growth of E. coli BL21 (DE3) was in the range of 6.5 to 7.5 (Wang et al., 2014).

Conclusion
The whole-cell culture of the recombinant E. coli BL21 (DE3)/pET-bcfd1 carrying the gene from Bacillus cereus IndB1 local strain was able to degrade MCA. Our studies found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 µM of IPTG, 6 hours of pre-induction incubation at 37ºC with shaking, followed by 2 hours IPTG induction at 30ºC with shaking, at pH 7 in LB liquid medium without NaCl, which produced ~0.056 mM chloride ions. The reported findings provide useful foundation for recombinant enzyme production to facilitate further studies on engineered haloacid dehalogenase for MCA biodegradation. In particular, our result provides optimum condition to utilize whole cell culture of recombinant E. coli BL21 (DE3)/pET-bcfd1 for MCA biodegradation. Enzyme availability and enzyme activity within the cells still require further studies. The production of haloacid dehalogenase in this recombinant clone might be further increased by cloning the gene into a stronger expression vector or utilizing other E. coli host with more favourable characteristics.