Identification of a Marine Bacillus Strain C5 and Parathion-Methyl Degradation Characteristics of the Extracellular Esterase B1

A bacterial strain C5 that can produce new type of marine esterase was isolated and screened from marine sludge. According to 16S rRNA sequence analysis and physiological and biochemical experiments, the strain was identified as Bacillus subtilis. A single isozyme with a molecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE. On this basis, the mechanism of esterase B1 secreted by strain C5 degrading parathion-methyl was explored, and the effects of temperature and pH on the degradation rate were investigated. From the results, p-nitrophenol was one of the degradation products of B1 degrading parathion-methyl, and the best degradation effect could be achieved at the temperature of 40°C and the neutral pH value.


Introduction
In order to adapt to various extreme environments in the ocean, marine microbes can produce corresponding enzymes which have unique physiological functions and enzymology properties [1]. Compared with terrigenous microbial enzymes, the enzymes produced by marine microbes have more potential for further development and application and thus attract more and more attention of the world. Nowadays, developing and looking for enzymes with new properties have become the international hot topics in the study of enzyme preparation [2]. Esterase (EC 3.1) is a kind of hydrolase acting on the ester bond, which can catalyze hydrolysis and synthesis of different substrates. These reactions usually have high substrate specificity and regioselectivity or enantiomorphic selectivity, which make esterase an important biological catalyst in organic synthesis. As a result, esterase has been widely used in food, flavors and fragrances industry, chemical industry, environmental protection fields, and so on [3].
Currently, organic phosphorus is widely used as an ester pesticide, but it is also the main pesticide ingredient that causes poisoning of human beings and animals. There are lots of microbes secreting organophosphorus ester in soil, so that this kind of pesticide is easy to be decomposed in soil [4]. In contrast to the bacteria secreting the enzymes, the organophosphate degradation enzymes are more tolerated to abnormal environmental conditions. In the case of lowconcentration pesticide, their degradation effects on the pesticide are far more obvious than the microorganisms themselves since decomposing bacteria can utilize other carbon sources efficiently rather than pesticides [5]. Therefore, degradation enzyme is an effective means of purification and remediation of pesticide-contaminated environment.
According to previous reports, the bacteria that can produce parathion-methyl hydrolase mainly include Acinetobacter, Pseudomonas, Burkholderia, Arthrobacter, Ochrobactrum, and so on [6][7][8][9][10][11]. However, the application of B. subtilis in pesticide degradation is still very scarce. In this study, a Bacillus strain (numbered as C5) that can produce esterase was isolated and screened from marine sludge. Using microbial routine physiological and biochemical detection and molecular biology method, the strain C5 was identified as Bacillus subtilis, with GenBank login number as JN942155. Meanwhile, the preliminary degradation mechanism of esterase B1 secreted by strain C5 on parathion-methyl was explored, and the degradation effects were also discussed.

Identification of the Extracellular Esterase Producing
Bacterial Strain C5. Identification of the strains referred to the common bacterial system identification method. The 16S rDNA sequence of the strain C5 was also identified from the genomic DNA and amplified by PCR using primers 27F (5 -AGAGTTTGATCCTGG TCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGA CTT-3 ). The amplified product was then purified and sequenced. The nucleotide sequence has been submitted to GenBank under accession number JN942155. The existing sequences of this strain's 16S rRNA sequence were retrieved by BLAST, and the sequence which was genetically close to the testing strain was downloaded. BioEdit software was used to carry on the multiple sequence alignment, and MEGA software was used for phylogenetic analysis by neighbor-joining method.

2.2.
Purification of Esterase B1. The crude esterase B1 was purified from culture broth using the optimized medium. All purification steps were carried out at 4 ∘ C. Esterase B1 in the culture broth was precipitated using a 80% saturated solution of ammonia sulfate. The precipitated protein was dissolved in 0.1 M phosphate buffered solution (pH 7.0) and then dialyzed with the same buffer. Sephadex G-100 (5 gm) gel was added in 0.1 M phosphate buffered solution (pH 7.0) and allowed to swell overnight and column (1.5 × 65 cm) was packed. The column (1.5 × 65 cm) was equilibrated with 0.1 M phosphate buffered solution pH 7.0. Total of 5 mL of ammonia sulfate precipitate was loaded onto a Sephadex G-100 column (1.5 × 65 cm). The column was eluted with phosphate buffered solution (pH 7.0) and fractions of 1 mL were collected at a flow rate of 1 mL/min. The esterase-rich fractions were pooled and stored for subsequent analysis.

The Parathion-Methyl Solution Sample
Treatment. The reaction system consisted of 15.6 mL phosphate buffer (pH = 7.0), 0.4 mL parathion-methyl solution (with a concentration of 10 mg/mL, dissolved by methyl alcohol), and 4 mL enzyme solution containing 200 U/mL esterase. This reaction system firstly ran in the shake culture water bath pot for a certain time at 40 ∘ C and then suffered a reduced pressure distillation until drying. Afterwards, 20 mL chromatographically pure acetonitrile was added to redissolve the distilment, followed by a membranous examination. The control sample was inactivated enzyme solution.

HPLC and Mass Spectrometry
Detection. HPLC detection conditions: the mobile phase was the mixture of methyl alcohol and water with a volume ratio of 80/20; the flow rate was 1.0 mL/min; and the detection wavelength was 274 nm. By an external standard method, pesticide parathion-methyl concentration could be calculated according to the sample peak area, the reference sample peak area, and the sample size.
Mass spectrometry chromatographic conditions: temperature of injection port was 250 ∘ C, and detector temperature was 280 ∘ C; carrier gas was high purity helium gas, with a flow rate of 1.0 mL/min; column temperature was raised from 50 ∘ C to 260 ∘ C by program with a rate of 10 ∘ C/min and then kept for 3 min. TOF mass spectrometry conditions: quality scan range was 50-500, and full scan mode was used for the detection.

Effect of Temperature and pH on the Degradation Rate.
According to the treatment method mentioned above, the degradation effect was detected at the temperatures of 20 ∘ C, 30 ∘ C, 40 ∘ C, and 50 ∘ C, respectively. The degradation effect of the enzyme solution on the parathion-methyl was detected in the pH range of 5-8 at 40 ∘ C.
2.6. Esterase Assay. Esterase activity was assayed by following the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate (pNPP). The standard assay was carried out in 1 mL reaction mixture containing 0.04 M PBS buffer (pH 7), 3 mM substrate pNPP, and enzyme. After incubation at 30 ∘ C for 15 min, the reaction was terminated by adding equal volume of 1 M NaOH, and the released pNP in the reaction mixture was measured at 400 nm using a spectrophotometer. One unit of enzyme activity represents the hydrolysis of 1 umol pNP from pNPP per min under the standard assay.

Identification of the Extracellular Esterase Producing Bacterial Strain C5.
Colony of strain C5 on the beef extract peptone medium was white, large, and flat, with rough surface and irregular edges, as shown in Figure 1. The bacterial strain was gram positive and rod shaped, single or arranged as short chain. The size of its thalli was 0.45∼0.7 m × 2.0∼3.8 m, and the spores were centrally or partially accrete, which enlarged the thalli. Electron microscope observation showed that its flagella grew by means of adnation.
Physiological and biochemical identification of the strain C5 were conducted, and the results were shown in Table 1. According to Bergey's Manual of Systematic Bacteriology Eighth Edition, all the physiological and biochemical characteristics of Bacillus strain C5 were basically the same with  Based on the homology of 16S rRNA sequence, strains with different sequence similarity from relative species were selected, and multiple sequence alignment comparison was carried out by BioEdit program. Then, bootstrap values (which were labeled in the figures) were calculated by neighbor-joining analysis method using MEGA software, and phylogenetic tree was constructed as shown in Figure 2. It can be seen that strain C5 had the highest homology up to 99% with Bacillus subtilis subsp. inaquosus strain NRRL B-23052. Bacteria taxonomists widely believe that when the homology of 16S rRNA sequence is above 97%, the strains can be considered as the same species and genus, whereas when the homology is lower than 93%-95%, they are likely the members of different genera [12]. Therefore, according to the phylogenetic analysis of strain C5, it could be preliminarily identified as Bacillus subtilis.

Purification of Esterase B1.
Purification and gel electrophoresis of esterase B1 that was purified by (NH 4 ) 2 SO 4 precipitation and gel filtration chromatography are summarized in Table 2. The purified esterase B1 was analyzed by SDS-PAGE and native-PAGE, and one major band was observed after PAGE under denaturing and nondenaturing conditions ( Figure 3). The results indicated a molecular weight of esterase B1 of 86 kDa. Figure 4 showed the HPLC of parathion-methyl enzymolysis before and after 5 minutes. From the figure, it was obvious that there was pNP generated, along with other new products. GC-TOF mass spectrometry was used to further determine the structure of this product, and the results were shown in Figure 5. Undegraded parathion-methyl and generated pNP were detected. Besides, there were peaks of other materials that needed further research.

Effect of Temperature and pH on the Degradation Effect.
As shown in Figure 6, the degradation rate of enzyme solution at 40 ∘ C was obviously higher than that at other temperatures, and the pesticide parathion-methyl with a concentration of 200 g/mL could be degraded completely within 20 minutes. However, almost no degradation took place at 50 ∘ C, perhaps because the enzyme had been disabled at such high temperature.
As shown in Figure 7, when the pH value of the enzyme solution was 7, the degradation rate was the highest. When the enzyme solution was under acidic or alkaline condition, the hydrolysis of parathion-methyl was accelerated but the degradation rate was reduced. From the results, the pH value had great effects on the degradation rate during the degradation of pesticides, and the degradation rate was the highest under the neutral condition.  there were also reports of different metabolic pathways. Hou studied the parathion-methyl degradation effect of detoxification enzyme from metabolism of Pseudomonas alcaligenes and concluded that parathion-methyl was degraded by detoxification enzyme via P-O bond [16]. Meanwhile, they found that pNP and DMTP were produced during the degradation process, but there was no excessive accumulation of pNP, indicating that there might be further degradation pathways.  [17]. Arthrobacter sp. isolated by Liu et al. could degrade both parathion-methyl and pNP, and almost no pNP was detected during the degradation [18]. Hydrolytic enzyme extracted by Liu et al. from parathion-methyl degradation bacteria could degrade the parathion-methyl into pNP [6]. Pseudoxanthomonas japonensis isolated by Feng et al. could degrade both parathion-methyl and pNP [19]. DM-1 bacteria isolated by Dong could degrade parathion-methyl into amino parathion-methyl with low toxicity [20]. Esterase B1 secreted by strain C5 could degrade parathion-methyl, and HPLC and GC-TOF mass spectrometry analysis of the products confirmed that pNP was one of the degradation products. As for the other product, it might be DMTP, but it still needed further identification.

Conclusions
In this study, a high activity esterase B1 was obtained by marine bacterium C5. The strain was identified as Bacillus subtilis. A single isozyme with a molecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE. The mechanism of esterase B1 degrading parathion-methyl was explored, and the effects of temperature and pH on the degradation rate were investigated. p-Nitrophenol was one of the degradation products of B1 degrading parathion-methyl, and the best degradation effect could be achieved at the temperature of 40 ∘ C and the neutral pH value.