Research ArticleIdentification of a Marine Bacillus Strain C5 andParathion-Methyl Degradation Characteristics of theExtracellular Esterase B1

Jianhua Hao Junzhong Liu and Mi Sun

Key Laboratory of Sustainable Development of Marine Fisheries Ministry of Agriculture Yellow Sea Fisheries Research InstituteChinese Academy of Fishery Sciences 106 Nanjing Road Qingdao Shandong 266071 China

Correspondence should be addressed to Mi Sun sunmiysfriaccn

Received 4 June 2014 Accepted 16 September 2014 Published 11 December 2014

Academic Editor Tong Zhang

Copyright copy 2014 Jianhua Hao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A bacterial strain C5 that can produce new type of marine esterase was isolated and screened from marine sludge According to16S rRNA sequence analysis and physiological and biochemical experiments the strain was identified as Bacillus subtilis A singleisozyme with amolecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE On this basis the mechanism of esteraseB1 secreted by strain C5 degrading parathion-methyl was explored and the effects of temperature and pH on the degradation ratewere investigated From the results p-nitrophenol was one of the degradation products of B1 degrading parathion-methyl and thebest degradation effect could be achieved at the temperature of 40∘C and the neutral pH value

1 Introduction

In order to adapt to various extreme environments in theoceanmarinemicrobes can produce corresponding enzymeswhich have unique physiological functions and enzymol-ogy properties [1] Compared with terrigenous microbialenzymes the enzymes produced by marine microbes havemore potential for further development and application andthus attractmore andmore attention of the world Nowadaysdeveloping and looking for enzymes with new propertieshave become the international hot topics in the study ofenzyme preparation [2]

Esterase (EC 31) is a kind of hydrolase acting on theester bond which can catalyze hydrolysis and synthesisof different substrates These reactions usually have highsubstrate specificity and regioselectivity or enantiomorphicselectivity which make esterase an important biologicalcatalyst in organic synthesis As a result esterase has beenwidely used in food flavors and fragrances industry chemicalindustry environmental protection fields and so on [3]

Currently organic phosphorus is widely used as an esterpesticide but it is also the main pesticide ingredient that

causes poisoning of human beings and animals There arelots of microbes secreting organophosphorus ester in soil sothat this kind of pesticide is easy to be decomposed in soil[4] In contrast to the bacteria secreting the enzymes theorganophosphate degradation enzymes are more toleratedto abnormal environmental conditions In the case of low-concentration pesticide their degradation effects on thepesticide are far more obvious than the microorganismsthemselves since decomposing bacteria can utilize other car-bon sources efficiently rather than pesticides [5] Thereforedegradation enzyme is an effective means of purification andremediation of pesticide-contaminated environment

According to previous reports the bacteria that can pro-duce parathion-methyl hydrolase mainly includeAcinetobac-terPseudomonasBurkholderiaArthrobacterOchrobactrumand so on [6ndash11] However the application of B subtilis inpesticide degradation is still very scarce In this study a Bacil-lus strain (numbered as C5) that can produce esterase wasisolated and screened from marine sludge Using microbialroutine physiological and biochemical detection and molec-ular biology method the strain C5 was identified as Bacillus

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 863094 7 pageshttpdxdoiorg1011552014863094

2 BioMed Research International

subtilis with GenBank login number as JN942155 Mean-while the preliminary degradation mechanism of esterase B1secreted by strain C5 on parathion-methyl was explored andthe degradation effects were also discussed

2 Materials and Methods

21 Bacterium Sources and Chemicals Bacterial strainC5 was isolated from China Bohai Sea (N38∘291015840429410158401015840E119∘231015840599010158401015840) and stored in Laboratory of MarineProducts and Enzyme Engineering Yellow Sea FisheriesResearch Institute Chinese Academy of Fishery Sciences

PCR purification kit of UNIQ-10 was purchased fromShanghai Sangon Biological Engineering Technology amp Ser-vices Co Ltd (Shanghai China) and the related reagentsfor PCR amplification were from Takara Biotechnology(Dalian) Co Ltd (Shanghai China) 99 parathion-methylwas purchased from Aladdin (Los Angeles USA) All otherreagents were purchased from Sinopharm Chemical ReagentCorp (Shanghai China) and were of the highest analyticalgrade

211 Identification of the Extracellular Esterase ProducingBacterial Strain C5 Identification of the strains referred tothe common bacterial system identification method The 16SrDNA sequence of the strain C5 was also identified fromthe genomic DNA and amplified by PCR using primers27F (51015840-AGAGTTTGATCCTGG TCAG-31015840) and 1492R (51015840-GGTTACCTTGTTACGA CTT-31015840) The amplified productwas then purified and sequenced The nucleotide sequencehas been submitted to GenBank under accession numberJN942155 The existing sequences of this strainrsquos 16S rRNAsequence were retrieved by BLAST and the sequence whichwas genetically close to the testing strain was downloadedBioEdit software was used to carry on the multiple sequencealignment and MEGA software was used for phylogeneticanalysis by neighbor-joining method

22 Purification of Esterase B1 The crude esterase B1 waspurified from culture broth using the optimized medium Allpurification steps were carried out at 4∘C Esterase B1 in theculture broth was precipitated using a 80 saturated solutionof ammonia sulfateThe precipitated protein was dissolved in01M phosphate buffered solution (pH 70) and then dialyzedwith the same buffer Sephadex G-100 (5 gm) gel was addedin 01M phosphate buffered solution (pH 70) and allowed toswell overnight and column (15 times 65 cm) was packed Thecolumn (15 times 65 cm) was equilibrated with 01M phosphatebuffered solution pH 70 Total of 5mL of ammonia sulfateprecipitate was loaded onto a Sephadex G-100 column (15times 65 cm) The column was eluted with phosphate bufferedsolution (pH 70) and fractions of 1mLwere collected at a flowrate of 1mLminThe esterase-rich fractions were pooled andstored for subsequent analysis

23 The Parathion-Methyl Solution Sample Treatment Thereaction system consisted of 156mL phosphate buffer (pH =70) 04mL parathion-methyl solution (with a concentration

of 10mgmL dissolved by methyl alcohol) and 4mL enzymesolution containing 200UmL esterase This reaction systemfirstly ran in the shake culture water bath pot for a certaintime at 40∘C and then suffered a reduced pressure distillationuntil drying Afterwards 20mL chromatographically pureacetonitrile was added to redissolve the distilment followedby a membranous examination The control sample wasinactivated enzyme solution

24 HPLC and Mass Spectrometry Detection HPLC detec-tion conditions the mobile phase was the mixture of methylalcohol and water with a volume ratio of 8020 the flow ratewas 10mLmin and the detection wavelength was 274 nmBy an external standard method pesticide parathion-methylconcentration could be calculated according to the samplepeak area the reference sample peak area and the sample size

Mass spectrometry chromatographic conditions temper-ature of injection port was 250∘C and detector temperaturewas 280∘C carrier gas was high purity helium gas with a flowrate of 10mLmin column temperaturewas raised from50∘Cto 260∘Cby programwith a rate of 10∘Cmin and then kept for3min TOFmass spectrometry conditions quality scan rangewas 50ndash500 and full scan mode was used for the detection

25 Effect of Temperature and pH on the Degradation RateAccording to the treatment method mentioned above thedegradation effect was detected at the temperatures of 20∘C30∘C 40∘C and 50∘C respectively The degradation effect ofthe enzyme solution on the parathion-methyl was detected inthe pH range of 5ndash8 at 40∘C

26 Esterase Assay Esterase activity was assayed by followingthe release of p-nitrophenol (pNP) from p-nitrophenyl phos-phate (pNPP) The standard assay was carried out in 1mLreaction mixture containing 004M PBS buffer (pH 7) 3mMsubstrate pNPP and enzyme After incubation at 30∘C for15min the reaction was terminated by adding equal volumeof 1M NaOH and the released pNP in the reaction mixturewasmeasured at 400 nmusing a spectrophotometerOne unitof enzyme activity represents the hydrolysis of 1 umol pNPfrom pNPP per min under the standard assay

3 Results

31 Identification of the Extracellular Esterase Producing Bac-terial Strain C5 Colony of strain C5 on the beef extractpeptonemediumwaswhite large andflat with rough surfaceand irregular edges as shown in Figure 1The bacterial strainwas gram positive and rod shaped single or arranged as shortchainThe size of its thalli was 045sim07120583mtimes 20sim38 120583m andthe spores were centrally or partially accrete which enlargedthe thalli Electron microscope observation showed that itsflagella grew by means of adnation

Physiological and biochemical identification of the strainC5 were conducted and the results were shown in Table 1According to Bergeyrsquos Manual of Systematic BacteriologyEighth Edition all the physiological and biochemical char-acteristics of Bacillus strain C5 were basically the same with

BioMed Research International 3

Figure 1 Single colony of strain C5 observed by electron microscope

Table 1 Comparison of features between strain C5 and Bacillussubtilis

CharacteristicsBacillussubtilis

(ATCC 6051)C5

Gram stain + +Flagella Adnation AdnationAnaerobism minus minus

Hydrolysis of casein + +Acid from glucose + minus

Produces gas from glucose minus minus

Voges and Proskauer test + minus

Methyl red test minus minus

Indole minus minus

Utilization of citrate + +Hydrogen sulfide production minus minus

Hydrolysis of starch + minus

+ gt85 positive minus 0ndash15 positive

Bacillus subtilis Further combined with the phylogeneticanalysis the strain C5 was identified as Bacillus subtilis

1462 gene bases (JN942155) from 16S rRNA of strain C5gene were searched for homologous sequences in GenBankAmong the displayed 100 strains with high similarity 75belonged to B subtilis 15 were unidentified 3 belonged toB tequilensis 2 belonged to B mojavensis and the last threebelonged to B amyloliquefaciens B licheniformis and Baxarquiensis respectively

Based on the homology of 16S rRNA sequence strainswith different sequence similarity from relative species wereselected and multiple sequence alignment comparison wascarried out by BioEdit program Then bootstrap values(which were labeled in the figures) were calculated byneighbor-joining analysismethodusingMEGAsoftware andphylogenetic tree was constructed as shown in Figure 2 Itcan be seen that strain C5 had the highest homology upto 99 with Bacillus subtilis subsp inaquosus strain NRRLB-23052 Bacteria taxonomists widely believe that when thehomology of 16S rRNA sequence is above 97 the strains can

be considered as the same species and genus whereas whenthe homology is lower than 93ndash95 they are likely themembers of different genera [12] Therefore according to thephylogenetic analysis of strain C5 it could be preliminarilyidentified as Bacillus subtilis

32 Purification of Esterase B1 Purification and gel elec-trophoresis of esterase B1 that was purified by (NH

4)2SO4

precipitation and gel filtration chromatography are sum-marized in Table 2 The purified esterase B1 was analyzedby SDS-PAGE and native-PAGE and one major band wasobserved after PAGE under denaturing and nondenaturingconditions (Figure 3) The results indicated a molecularweight of esterase B1 of 86 kDa

321 Preliminary Research on the Mechanism of Parathion-Methyl Degradation by Esterase B1 Figure 4 showed theHPLC of parathion-methyl enzymolysis before and after 5minutes From the figure it was obvious that there was pNPgenerated along with other new products GC-TOF massspectrometry was used to further determine the structure ofthis product and the results were shown in Figure 5 Unde-graded parathion-methyl and generated pNP were detectedBesides there were peaks of other materials that neededfurther research

33 Effect of Temperature and pH on the Degradation EffectAs shown in Figure 6 the degradation rate of enzymesolution at 40∘C was obviously higher than that at othertemperatures and the pesticide parathion-methyl with aconcentration of 200120583gmL could be degraded completelywithin 20 minutes However almost no degradation tookplace at 50∘C perhaps because the enzyme had been disabledat such high temperature

As shown in Figure 7 when the pH value of the enzymesolution was 7 the degradation rate was the highest Whenthe enzyme solution was under acidic or alkaline conditionthe hydrolysis of parathion-methyl was accelerated but thedegradation rate was reduced From the results the pHvalue had great effects on the degradation rate during thedegradation of pesticides and the degradation rate was thehighest under the neutral condition

4 BioMed Research International

Table 2 Summary of esterase B1 purification from Bacillus subtilis C5

Purification step Total activity (U) Total protein (ug) Specific activity (Umg) Yield () Purification foldCulture supernatant 755 22162 3407 100 10(NH4)2SO4 precipitation 212 116085 18262 5616 536Sephadex G-100 355 6375 55686 94 1634

Bacillus methylotrophicus strain Mo-Bm (HQ325853)Bacillus pumilus strain 1Re3 (EF178456)

Bacillus amyloliquefaciens strain sdg-31 (FJ436406)Bacillus mucilaginosus strain s-2 (HM849728)

Bacillus velezensis strain BCRC 17467 (EF433407)Bacillus atrophaeus strain LSSC3 (GU994860)Strain C5 (JN942155)

Bacillus subtilis subsp inaquosus strain NRRL B-23052 (EU138467)Bacillus cereus (AB523742)

Bacillus anthracis strain me-12 (EU652063)Bacillus flexus strain PL1 (GU001887)Bacillus levoglucosan-8 (EU661931)Bacillus circulans strain NBY8 (HQ851051)Staphylococcus saprophyticus strain YSY1-5 (GU197534)Bacillus licheniformis strain IV1 (EU366371)Lysinibacillus fusiformis (AB547327)Bacillus sphaericus (AJ311894)

Sporosarcina saromensis strain ML-42 (HQ848095)Lentibacillus halodurans (NR042837)Virgibacillus pantothenticus (AB617570)

89

99

99

100 99

73 88100

100

98

Figure 2 Phylogenetic tree of strain C5 based on the 16S rRNA gene sequence homology (in parentheses were the GenBank login numbers)

M(kDa) 1

116

662

450

350

250

184

144

(a)

2 3

(b)

Figure 3 SDS-PAGE and isozyme analysis of esterase purified from C5 (a) SDS-PAGE gel (b) Native-PAGE gel stained with Fast blue B saltLanes M molecular mass protein marker 1 purified esterase 2 esterase crude extract and 3 purified esterase

BioMed Research International 5

0

002

004

006

008

01

012

014

0 2 4 6 8 10

(AU

)

(min)

minus002

(a)

0

001

002

003

004

005

006

007

0 2 4 6 8 10

(AU

)

(min)

minus001

(b)

0001002003004005006007

0 2 4 6 8 10

(AU

)

(min)

minus001

(c)

Figure 4 HPLC of parathion-methyl enzymolysis (a) Standard sample of pNP (b) before enzymolysis of parathion-methyl (c) afterenzymolysis of parathion-methyl

OO

PS

O

O

N+

OminusCH3

CH3

Parathion-methyl

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

minus5

50 500

(a)

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

Parathion-methyl

p-Nitrophenol

50 500

minus5

OO

PS

O

O

N+

OminusCH3

CH3

O

HO N+

Ominus

(b)

Figure 5 TOF mass spectrogram (a) Before enzymolysis of parathion-methyl (b) after enzymolysis of parathion-methyl

34 Discussions Study on microbial degradation of pesti-cides began in the late 40s and it has always been a hotresearch spot The action modes of microbes degradingpesticides mostly belong to enzymatic reactions which aremainly triggered by a microbial intracellular enzyme Forenzymatic reactions pesticides are firstly adsorbed on thesurface of microbial cells and then the pesticides penetrate

cell membranes into the cell interior Inside the membranethe pesticides can combine with degrading enzyme andtrigger the enzyme catalytic reactions [13ndash15]

Many researches have been carried out on themechanismof parathion-methyl biodegradation all over the world Mostreported biodegradations of parathion-methyl were achievedby producing pNP and dimethyl thiophosphate (DMTP) but

6 BioMed Research International

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

0

50

100

150

200

250

300

20 30 40 50

Deg

rada

tion

rate

(120583g

min

)

Temperature (∘C)

Figure 6 Effect of temperature on the degradation rate ◻20∘C 99877130∘C40∘C and ◼50∘C

0

50

100

150

200

250

300

5 6 7 8pH

Deg

rada

tion

rate

(120583g

min

)

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

Figure 7 Effect of pH on the degradation rate ◼pH 5 998771pH6 epH7 and ◻pH8

there were also reports of different metabolic pathways Houstudied the parathion-methyl degradation effect of detoxifi-cation enzyme from metabolism of Pseudomonas alcaligenesand concluded that parathion-methyl was degraded by detox-ification enzyme via PndashO bond [16] Meanwhile they foundthat pNP and DMTP were produced during the degradationprocess but there was no excessive accumulation of pNPindicating that there might be further degradation pathwaysParathion-methyl degradation enzyme screened by Liu et alcould directly mineralize parathion-methyl and there wereno intermediate metabolites or final degradation products[6] Pseudomonas aeruginosa screened by Zheng et al coulddegrade the parathion-methyl into pNP meanwhile it coulddegrade the metabolic intermediate material pNP whichavoided the secondary pollution caused by accumulation ofintermediatematerials [17]Arthrobacter sp isolated by Liu etal could degrade both parathion-methyl and pNP and almostno pNP was detected during the degradation [18] Hydrolyticenzyme extracted by Liu et al from parathion-methyl degra-dation bacteria could degrade the parathion-methyl into pNP

[6] Pseudoxanthomonas japonensis isolated by Feng et alcould degrade both parathion-methyl and pNP [19] DM-1bacteria isolated by Dong could degrade parathion-methylinto amino parathion-methyl with low toxicity [20] EsteraseB1 secreted by strain C5 could degrade parathion-methyland HPLC and GC-TOF mass spectrometry analysis of theproducts confirmed that pNP was one of the degradationproducts As for the other product it might be DMTP butit still needed further identification

4 Conclusions

In this study a high activity esterase B1 was obtained bymarine bacterium C5 The strain was identified as Bacil-lus subtilis A single isozyme with a molecular weight of86 kDa was observed by SDS-PAGE and native-PAGE Themechanism of esterase B1 degrading parathion-methyl wasexplored and the effects of temperature and pH on thedegradation rate were investigated p-Nitrophenol was one of

BioMed Research International 7

the degradation products of B1 degrading parathion-methyland the best degradation effect could be achieved at thetemperature of 40∘C and the neutral pH value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National High Tech-nology Research and Development Program of China(2011AA090703) and the Qingdao Municipal Science andTechnology Program (11-2-4-20-hz)

References

[1] L Loperena V Soria H Varela et al ldquoExtracellular enzymesproduced by microorganisms isolated from maritime Antarc-ticardquo World Journal of Microbiology and Biotechnology vol 28no 5 pp 2249ndash2256 2012

[2] C Zhang and S-K Kim ldquoApplication of marine microbialenzymes in the food and pharmaceutical industriesrdquo Advancesin Food and Nutrition Research vol 65 pp 423ndash435 2012

[3] A Asoodeh and T Ghanbari ldquoCharacterization of an extra-cellular thermophilic alkaline esterase produced by BacillussubtilisDR8806rdquo Journal of Molecular Catalysis B Enzymaticvol 85-86 pp 49ndash55 2013

[4] K Mandal B Singh M Jariyal and V K Gupta ldquoMicrobialdegradation of fipronil by Bacillus thuringiensisrdquo Ecotoxicologyand Environmental Safety vol 93 pp 87ndash92 2013

[5] G-N Lu X-Q Tao Z Dang and X-Y Yi ldquoAdvances of studyon pesticide-degrading strains and their gene engineeringrdquoBulletin of Mineralogy Petrology and Geochemistry vol 24 no3 pp 258ndash263 2005

[6] F-Y Liu M-Z Hong D-M Liu et al ldquoBiodegradation ofmethyl parathion by Acinetobacter radioresistens USTB-04rdquoJournal of Environmental Sciences vol 19 no 10 pp 1257ndash12602007

[7] B Wang L Xiong Y Zheng et al ldquoCloning and expression ofthempd gene fromanewly isolatedmethylparathion-degradingstrain of bacteriardquo Acta Scientiae Circumstantiae vol 10 pp1969ndash1974 2008

[8] X-X Wang Z Chi S-G Ru and Z-M Chi ldquoGenetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica forremoval of methyl parathion in waterrdquo Biodegradation vol 23no 5 pp 763ndash774 2012

[9] A Ekkhunnatham B Jongsareejit W Yamkunthong and JWichitwechkarn ldquoPurification and characterization of methylparathion hydrolase from Burkholderia cepacia capable ofdegrading organophosphate insecticidesrdquo World Journal ofMicrobiology and Biotechnology vol 28 no 4 pp 1739ndash17462012

[10] K-D Kim J-H Ahn T Kim et al ldquoGenetic and phenotypicdiversity of fenitrothion-degrading bacteria isolated from soilsrdquoJournal of Microbiology and Biotechnology vol 19 no 2 pp 113ndash120 2009

[11] J Tian P Wang S Gao X Chu N Wu and Y Fan ldquoEnhancedthermostability of methyl parathion hydrolase from Ochrobac-trum sp M231 by rational engineering of a glycine to proline

mutationrdquo The FEBS Journal vol 277 no 23 pp 4901ndash49082010

[12] P Vandamme B Pot M Gillis P de Vos K Kersters andJ Swings ldquoPolyphasic taxonomy a consensus approach tobacterial systematicsrdquoMicrobiological Reviews vol 60 no 2 pp407ndash438 1996

[13] A W Boyle C J Silvin J P Hassett J P Nakas and S WTanenbaum ldquoBacterial PCB biodegradationrdquo Biodegradationvol 3 no 2-3 pp 285ndash298 1992

[14] K Sato ldquoEffect of nutrients on interaction between pesti-cide penta-chlorophenol and microorganisms in soil biore-mediation through rhizosphere technologyrdquo in Bioremediationthrough Rhizosphere Technology pp 43ndash55 AmericanChemicalSociety Washington DC USA 1994

[15] V T Gajbhiye N P Agnihotri and H K Jain ldquoCypermethrinresidues on teardquo Pesticide Research Journal vol 1 pp 83ndash871989

[16] Y X Hou J N Zhang L Han et al ldquoDegradation analysis ofmethyl parathion by detoxifying enzymerdquo Journal of Agricul-tural Science and Technology vol 13 pp 110ndash115 2011 (Chinese)

[17] Y L Zheng D L Liu S L Chen et al ldquoStudy on isolation andcharacteristic identification of a novel high-efficiency degradingstrain of methyl parathionrdquo Research of Environmental Sciencesvol 19 pp 100ndash104 2006

[18] P-P Liu Y-C Yan and X-P Xie ldquoThe mechanism of purifica-tion and degradation of methylparathion-degrading bacteriumYL8rdquo China Environmental Science vol 26 no 2 pp 206ndash2092006 (Chinese)

[19] Z Z Feng B Liang J Zhang et al ldquoDegradation characteristicsof methyl-parathion by Pseudoxanthomonas sp XY3rdquo JiangsuAgricultural Sciences vol 4 pp 375ndash378 2010 (Chinese)

[20] M Dong Y L Yuan and X M Guo ldquoIsolation and identifica-tion of methyl parathion degrading strain Pseudomonas spinosaand the characteristics of the degradationrdquo in Proceedings of thePesticide and environment safety International Conference pp148ndash153 2003 (Chinese)

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BioMed Research International 3

Figure 1 Single colony of strain C5 observed by electron microscope

Table 1 Comparison of features between strain C5 and Bacillussubtilis

CharacteristicsBacillussubtilis

(ATCC 6051)C5

Gram stain + +Flagella Adnation AdnationAnaerobism minus minus

Hydrolysis of casein + +Acid from glucose + minus

Produces gas from glucose minus minus

Voges and Proskauer test + minus

Methyl red test minus minus

Indole minus minus

Utilization of citrate + +Hydrogen sulfide production minus minus

Hydrolysis of starch + minus

+ gt85 positive minus 0ndash15 positive

Bacillus subtilis Further combined with the phylogeneticanalysis the strain C5 was identified as Bacillus subtilis

1462 gene bases (JN942155) from 16S rRNA of strain C5gene were searched for homologous sequences in GenBankAmong the displayed 100 strains with high similarity 75belonged to B subtilis 15 were unidentified 3 belonged toB tequilensis 2 belonged to B mojavensis and the last threebelonged to B amyloliquefaciens B licheniformis and Baxarquiensis respectively

Based on the homology of 16S rRNA sequence strainswith different sequence similarity from relative species wereselected and multiple sequence alignment comparison wascarried out by BioEdit program Then bootstrap values(which were labeled in the figures) were calculated byneighbor-joining analysismethodusingMEGAsoftware andphylogenetic tree was constructed as shown in Figure 2 Itcan be seen that strain C5 had the highest homology upto 99 with Bacillus subtilis subsp inaquosus strain NRRLB-23052 Bacteria taxonomists widely believe that when thehomology of 16S rRNA sequence is above 97 the strains can

be considered as the same species and genus whereas whenthe homology is lower than 93ndash95 they are likely themembers of different genera [12] Therefore according to thephylogenetic analysis of strain C5 it could be preliminarilyidentified as Bacillus subtilis

32 Purification of Esterase B1 Purification and gel elec-trophoresis of esterase B1 that was purified by (NH

4)2SO4

precipitation and gel filtration chromatography are sum-marized in Table 2 The purified esterase B1 was analyzedby SDS-PAGE and native-PAGE and one major band wasobserved after PAGE under denaturing and nondenaturingconditions (Figure 3) The results indicated a molecularweight of esterase B1 of 86 kDa

321 Preliminary Research on the Mechanism of Parathion-Methyl Degradation by Esterase B1 Figure 4 showed theHPLC of parathion-methyl enzymolysis before and after 5minutes From the figure it was obvious that there was pNPgenerated along with other new products GC-TOF massspectrometry was used to further determine the structure ofthis product and the results were shown in Figure 5 Unde-graded parathion-methyl and generated pNP were detectedBesides there were peaks of other materials that neededfurther research

33 Effect of Temperature and pH on the Degradation EffectAs shown in Figure 6 the degradation rate of enzymesolution at 40∘C was obviously higher than that at othertemperatures and the pesticide parathion-methyl with aconcentration of 200120583gmL could be degraded completelywithin 20 minutes However almost no degradation tookplace at 50∘C perhaps because the enzyme had been disabledat such high temperature

As shown in Figure 7 when the pH value of the enzymesolution was 7 the degradation rate was the highest Whenthe enzyme solution was under acidic or alkaline conditionthe hydrolysis of parathion-methyl was accelerated but thedegradation rate was reduced From the results the pHvalue had great effects on the degradation rate during thedegradation of pesticides and the degradation rate was thehighest under the neutral condition

4 BioMed Research International

Table 2 Summary of esterase B1 purification from Bacillus subtilis C5

Purification step Total activity (U) Total protein (ug) Specific activity (Umg) Yield () Purification foldCulture supernatant 755 22162 3407 100 10(NH4)2SO4 precipitation 212 116085 18262 5616 536Sephadex G-100 355 6375 55686 94 1634

Bacillus methylotrophicus strain Mo-Bm (HQ325853)Bacillus pumilus strain 1Re3 (EF178456)

Bacillus amyloliquefaciens strain sdg-31 (FJ436406)Bacillus mucilaginosus strain s-2 (HM849728)

Bacillus velezensis strain BCRC 17467 (EF433407)Bacillus atrophaeus strain LSSC3 (GU994860)Strain C5 (JN942155)

Bacillus subtilis subsp inaquosus strain NRRL B-23052 (EU138467)Bacillus cereus (AB523742)

Bacillus anthracis strain me-12 (EU652063)Bacillus flexus strain PL1 (GU001887)Bacillus levoglucosan-8 (EU661931)Bacillus circulans strain NBY8 (HQ851051)Staphylococcus saprophyticus strain YSY1-5 (GU197534)Bacillus licheniformis strain IV1 (EU366371)Lysinibacillus fusiformis (AB547327)Bacillus sphaericus (AJ311894)

Sporosarcina saromensis strain ML-42 (HQ848095)Lentibacillus halodurans (NR042837)Virgibacillus pantothenticus (AB617570)

89

99

99

100 99

73 88100

100

98

Figure 2 Phylogenetic tree of strain C5 based on the 16S rRNA gene sequence homology (in parentheses were the GenBank login numbers)

M(kDa) 1

116

662

450

350

250

184

144

(a)

2 3

(b)

Figure 3 SDS-PAGE and isozyme analysis of esterase purified from C5 (a) SDS-PAGE gel (b) Native-PAGE gel stained with Fast blue B saltLanes M molecular mass protein marker 1 purified esterase 2 esterase crude extract and 3 purified esterase

BioMed Research International 5

0

002

004

006

008

01

012

014

0 2 4 6 8 10

(AU

)

(min)

minus002

(a)

0

001

002

003

004

005

006

007

0 2 4 6 8 10

(AU

)

(min)

minus001

(b)

0001002003004005006007

0 2 4 6 8 10

(AU

)

(min)

minus001

(c)

Figure 4 HPLC of parathion-methyl enzymolysis (a) Standard sample of pNP (b) before enzymolysis of parathion-methyl (c) afterenzymolysis of parathion-methyl

OO

PS

O

O

N+

OminusCH3

CH3

Parathion-methyl

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

minus5

50 500

(a)

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

Parathion-methyl

p-Nitrophenol

50 500

minus5

OO

PS

O

O

N+

OminusCH3

CH3

O

HO N+

Ominus

(b)

Figure 5 TOF mass spectrogram (a) Before enzymolysis of parathion-methyl (b) after enzymolysis of parathion-methyl

34 Discussions Study on microbial degradation of pesti-cides began in the late 40s and it has always been a hotresearch spot The action modes of microbes degradingpesticides mostly belong to enzymatic reactions which aremainly triggered by a microbial intracellular enzyme Forenzymatic reactions pesticides are firstly adsorbed on thesurface of microbial cells and then the pesticides penetrate

cell membranes into the cell interior Inside the membranethe pesticides can combine with degrading enzyme andtrigger the enzyme catalytic reactions [13ndash15]

Many researches have been carried out on themechanismof parathion-methyl biodegradation all over the world Mostreported biodegradations of parathion-methyl were achievedby producing pNP and dimethyl thiophosphate (DMTP) but

6 BioMed Research International

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

0

50

100

150

200

250

300

20 30 40 50

Deg

rada

tion

rate

(120583g

min

)

Temperature (∘C)

Figure 6 Effect of temperature on the degradation rate ◻20∘C 99877130∘C40∘C and ◼50∘C

0

50

100

150

200

250

300

5 6 7 8pH

Deg

rada

tion

rate

(120583g

min

)

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

Figure 7 Effect of pH on the degradation rate ◼pH 5 998771pH6 epH7 and ◻pH8

there were also reports of different metabolic pathways Houstudied the parathion-methyl degradation effect of detoxifi-cation enzyme from metabolism of Pseudomonas alcaligenesand concluded that parathion-methyl was degraded by detox-ification enzyme via PndashO bond [16] Meanwhile they foundthat pNP and DMTP were produced during the degradationprocess but there was no excessive accumulation of pNPindicating that there might be further degradation pathwaysParathion-methyl degradation enzyme screened by Liu et alcould directly mineralize parathion-methyl and there wereno intermediate metabolites or final degradation products[6] Pseudomonas aeruginosa screened by Zheng et al coulddegrade the parathion-methyl into pNP meanwhile it coulddegrade the metabolic intermediate material pNP whichavoided the secondary pollution caused by accumulation ofintermediatematerials [17]Arthrobacter sp isolated by Liu etal could degrade both parathion-methyl and pNP and almostno pNP was detected during the degradation [18] Hydrolyticenzyme extracted by Liu et al from parathion-methyl degra-dation bacteria could degrade the parathion-methyl into pNP

[6] Pseudoxanthomonas japonensis isolated by Feng et alcould degrade both parathion-methyl and pNP [19] DM-1bacteria isolated by Dong could degrade parathion-methylinto amino parathion-methyl with low toxicity [20] EsteraseB1 secreted by strain C5 could degrade parathion-methyland HPLC and GC-TOF mass spectrometry analysis of theproducts confirmed that pNP was one of the degradationproducts As for the other product it might be DMTP butit still needed further identification

4 Conclusions

In this study a high activity esterase B1 was obtained bymarine bacterium C5 The strain was identified as Bacil-lus subtilis A single isozyme with a molecular weight of86 kDa was observed by SDS-PAGE and native-PAGE Themechanism of esterase B1 degrading parathion-methyl wasexplored and the effects of temperature and pH on thedegradation rate were investigated p-Nitrophenol was one of

BioMed Research International 7

the degradation products of B1 degrading parathion-methyland the best degradation effect could be achieved at thetemperature of 40∘C and the neutral pH value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National High Tech-nology Research and Development Program of China(2011AA090703) and the Qingdao Municipal Science andTechnology Program (11-2-4-20-hz)

References

[1] L Loperena V Soria H Varela et al ldquoExtracellular enzymesproduced by microorganisms isolated from maritime Antarc-ticardquo World Journal of Microbiology and Biotechnology vol 28no 5 pp 2249ndash2256 2012

[2] C Zhang and S-K Kim ldquoApplication of marine microbialenzymes in the food and pharmaceutical industriesrdquo Advancesin Food and Nutrition Research vol 65 pp 423ndash435 2012

[3] A Asoodeh and T Ghanbari ldquoCharacterization of an extra-cellular thermophilic alkaline esterase produced by BacillussubtilisDR8806rdquo Journal of Molecular Catalysis B Enzymaticvol 85-86 pp 49ndash55 2013

[4] K Mandal B Singh M Jariyal and V K Gupta ldquoMicrobialdegradation of fipronil by Bacillus thuringiensisrdquo Ecotoxicologyand Environmental Safety vol 93 pp 87ndash92 2013

[5] G-N Lu X-Q Tao Z Dang and X-Y Yi ldquoAdvances of studyon pesticide-degrading strains and their gene engineeringrdquoBulletin of Mineralogy Petrology and Geochemistry vol 24 no3 pp 258ndash263 2005

[6] F-Y Liu M-Z Hong D-M Liu et al ldquoBiodegradation ofmethyl parathion by Acinetobacter radioresistens USTB-04rdquoJournal of Environmental Sciences vol 19 no 10 pp 1257ndash12602007

[7] B Wang L Xiong Y Zheng et al ldquoCloning and expression ofthempd gene fromanewly isolatedmethylparathion-degradingstrain of bacteriardquo Acta Scientiae Circumstantiae vol 10 pp1969ndash1974 2008

[8] X-X Wang Z Chi S-G Ru and Z-M Chi ldquoGenetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica forremoval of methyl parathion in waterrdquo Biodegradation vol 23no 5 pp 763ndash774 2012

[9] A Ekkhunnatham B Jongsareejit W Yamkunthong and JWichitwechkarn ldquoPurification and characterization of methylparathion hydrolase from Burkholderia cepacia capable ofdegrading organophosphate insecticidesrdquo World Journal ofMicrobiology and Biotechnology vol 28 no 4 pp 1739ndash17462012

[10] K-D Kim J-H Ahn T Kim et al ldquoGenetic and phenotypicdiversity of fenitrothion-degrading bacteria isolated from soilsrdquoJournal of Microbiology and Biotechnology vol 19 no 2 pp 113ndash120 2009

[11] J Tian P Wang S Gao X Chu N Wu and Y Fan ldquoEnhancedthermostability of methyl parathion hydrolase from Ochrobac-trum sp M231 by rational engineering of a glycine to proline

mutationrdquo The FEBS Journal vol 277 no 23 pp 4901ndash49082010

[12] P Vandamme B Pot M Gillis P de Vos K Kersters andJ Swings ldquoPolyphasic taxonomy a consensus approach tobacterial systematicsrdquoMicrobiological Reviews vol 60 no 2 pp407ndash438 1996

[13] A W Boyle C J Silvin J P Hassett J P Nakas and S WTanenbaum ldquoBacterial PCB biodegradationrdquo Biodegradationvol 3 no 2-3 pp 285ndash298 1992

[14] K Sato ldquoEffect of nutrients on interaction between pesti-cide penta-chlorophenol and microorganisms in soil biore-mediation through rhizosphere technologyrdquo in Bioremediationthrough Rhizosphere Technology pp 43ndash55 AmericanChemicalSociety Washington DC USA 1994

[15] V T Gajbhiye N P Agnihotri and H K Jain ldquoCypermethrinresidues on teardquo Pesticide Research Journal vol 1 pp 83ndash871989

[16] Y X Hou J N Zhang L Han et al ldquoDegradation analysis ofmethyl parathion by detoxifying enzymerdquo Journal of Agricul-tural Science and Technology vol 13 pp 110ndash115 2011 (Chinese)

[17] Y L Zheng D L Liu S L Chen et al ldquoStudy on isolation andcharacteristic identification of a novel high-efficiency degradingstrain of methyl parathionrdquo Research of Environmental Sciencesvol 19 pp 100ndash104 2006

[18] P-P Liu Y-C Yan and X-P Xie ldquoThe mechanism of purifica-tion and degradation of methylparathion-degrading bacteriumYL8rdquo China Environmental Science vol 26 no 2 pp 206ndash2092006 (Chinese)

[19] Z Z Feng B Liang J Zhang et al ldquoDegradation characteristicsof methyl-parathion by Pseudoxanthomonas sp XY3rdquo JiangsuAgricultural Sciences vol 4 pp 375ndash378 2010 (Chinese)

[20] M Dong Y L Yuan and X M Guo ldquoIsolation and identifica-tion of methyl parathion degrading strain Pseudomonas spinosaand the characteristics of the degradationrdquo in Proceedings of thePesticide and environment safety International Conference pp148ndash153 2003 (Chinese)

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table 2 Summary of esterase B1 purification from Bacillus subtilis C5

Purification step Total activity (U) Total protein (ug) Specific activity (Umg) Yield () Purification foldCulture supernatant 755 22162 3407 100 10(NH4)2SO4 precipitation 212 116085 18262 5616 536Sephadex G-100 355 6375 55686 94 1634

Bacillus methylotrophicus strain Mo-Bm (HQ325853)Bacillus pumilus strain 1Re3 (EF178456)

Bacillus amyloliquefaciens strain sdg-31 (FJ436406)Bacillus mucilaginosus strain s-2 (HM849728)

Bacillus velezensis strain BCRC 17467 (EF433407)Bacillus atrophaeus strain LSSC3 (GU994860)Strain C5 (JN942155)

Bacillus subtilis subsp inaquosus strain NRRL B-23052 (EU138467)Bacillus cereus (AB523742)

Bacillus anthracis strain me-12 (EU652063)Bacillus flexus strain PL1 (GU001887)Bacillus levoglucosan-8 (EU661931)Bacillus circulans strain NBY8 (HQ851051)Staphylococcus saprophyticus strain YSY1-5 (GU197534)Bacillus licheniformis strain IV1 (EU366371)Lysinibacillus fusiformis (AB547327)Bacillus sphaericus (AJ311894)

Sporosarcina saromensis strain ML-42 (HQ848095)Lentibacillus halodurans (NR042837)Virgibacillus pantothenticus (AB617570)

89

99

99

100 99

73 88100

100

98

Figure 2 Phylogenetic tree of strain C5 based on the 16S rRNA gene sequence homology (in parentheses were the GenBank login numbers)

M(kDa) 1

116

662

450

350

250

184

144

(a)

2 3

(b)

Figure 3 SDS-PAGE and isozyme analysis of esterase purified from C5 (a) SDS-PAGE gel (b) Native-PAGE gel stained with Fast blue B saltLanes M molecular mass protein marker 1 purified esterase 2 esterase crude extract and 3 purified esterase

BioMed Research International 5

0

002

004

006

008

01

012

014

0 2 4 6 8 10

(AU

)

(min)

minus002

(a)

0

001

002

003

004

005

006

007

0 2 4 6 8 10

(AU

)

(min)

minus001

(b)

0001002003004005006007

0 2 4 6 8 10

(AU

)

(min)

minus001

(c)

Figure 4 HPLC of parathion-methyl enzymolysis (a) Standard sample of pNP (b) before enzymolysis of parathion-methyl (c) afterenzymolysis of parathion-methyl

OO

PS

O

O

N+

OminusCH3

CH3

Parathion-methyl

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

minus5

50 500

(a)

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

Parathion-methyl

p-Nitrophenol

50 500

minus5

OO

PS

O

O

N+

OminusCH3

CH3

O

HO N+

Ominus

(b)

Figure 5 TOF mass spectrogram (a) Before enzymolysis of parathion-methyl (b) after enzymolysis of parathion-methyl

34 Discussions Study on microbial degradation of pesti-cides began in the late 40s and it has always been a hotresearch spot The action modes of microbes degradingpesticides mostly belong to enzymatic reactions which aremainly triggered by a microbial intracellular enzyme Forenzymatic reactions pesticides are firstly adsorbed on thesurface of microbial cells and then the pesticides penetrate

cell membranes into the cell interior Inside the membranethe pesticides can combine with degrading enzyme andtrigger the enzyme catalytic reactions [13ndash15]

Many researches have been carried out on themechanismof parathion-methyl biodegradation all over the world Mostreported biodegradations of parathion-methyl were achievedby producing pNP and dimethyl thiophosphate (DMTP) but

6 BioMed Research International

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

0

50

100

150

200

250

300

20 30 40 50

Deg

rada

tion

rate

(120583g

min

)

Temperature (∘C)

Figure 6 Effect of temperature on the degradation rate ◻20∘C 99877130∘C40∘C and ◼50∘C

0

50

100

150

200

250

300

5 6 7 8pH

Deg

rada

tion

rate

(120583g

min

)

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

Figure 7 Effect of pH on the degradation rate ◼pH 5 998771pH6 epH7 and ◻pH8

there were also reports of different metabolic pathways Houstudied the parathion-methyl degradation effect of detoxifi-cation enzyme from metabolism of Pseudomonas alcaligenesand concluded that parathion-methyl was degraded by detox-ification enzyme via PndashO bond [16] Meanwhile they foundthat pNP and DMTP were produced during the degradationprocess but there was no excessive accumulation of pNPindicating that there might be further degradation pathwaysParathion-methyl degradation enzyme screened by Liu et alcould directly mineralize parathion-methyl and there wereno intermediate metabolites or final degradation products[6] Pseudomonas aeruginosa screened by Zheng et al coulddegrade the parathion-methyl into pNP meanwhile it coulddegrade the metabolic intermediate material pNP whichavoided the secondary pollution caused by accumulation ofintermediatematerials [17]Arthrobacter sp isolated by Liu etal could degrade both parathion-methyl and pNP and almostno pNP was detected during the degradation [18] Hydrolyticenzyme extracted by Liu et al from parathion-methyl degra-dation bacteria could degrade the parathion-methyl into pNP

[6] Pseudoxanthomonas japonensis isolated by Feng et alcould degrade both parathion-methyl and pNP [19] DM-1bacteria isolated by Dong could degrade parathion-methylinto amino parathion-methyl with low toxicity [20] EsteraseB1 secreted by strain C5 could degrade parathion-methyland HPLC and GC-TOF mass spectrometry analysis of theproducts confirmed that pNP was one of the degradationproducts As for the other product it might be DMTP butit still needed further identification

4 Conclusions

In this study a high activity esterase B1 was obtained bymarine bacterium C5 The strain was identified as Bacil-lus subtilis A single isozyme with a molecular weight of86 kDa was observed by SDS-PAGE and native-PAGE Themechanism of esterase B1 degrading parathion-methyl wasexplored and the effects of temperature and pH on thedegradation rate were investigated p-Nitrophenol was one of

BioMed Research International 7

the degradation products of B1 degrading parathion-methyland the best degradation effect could be achieved at thetemperature of 40∘C and the neutral pH value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National High Tech-nology Research and Development Program of China(2011AA090703) and the Qingdao Municipal Science andTechnology Program (11-2-4-20-hz)

References

[1] L Loperena V Soria H Varela et al ldquoExtracellular enzymesproduced by microorganisms isolated from maritime Antarc-ticardquo World Journal of Microbiology and Biotechnology vol 28no 5 pp 2249ndash2256 2012

[2] C Zhang and S-K Kim ldquoApplication of marine microbialenzymes in the food and pharmaceutical industriesrdquo Advancesin Food and Nutrition Research vol 65 pp 423ndash435 2012

[3] A Asoodeh and T Ghanbari ldquoCharacterization of an extra-cellular thermophilic alkaline esterase produced by BacillussubtilisDR8806rdquo Journal of Molecular Catalysis B Enzymaticvol 85-86 pp 49ndash55 2013

[4] K Mandal B Singh M Jariyal and V K Gupta ldquoMicrobialdegradation of fipronil by Bacillus thuringiensisrdquo Ecotoxicologyand Environmental Safety vol 93 pp 87ndash92 2013

[5] G-N Lu X-Q Tao Z Dang and X-Y Yi ldquoAdvances of studyon pesticide-degrading strains and their gene engineeringrdquoBulletin of Mineralogy Petrology and Geochemistry vol 24 no3 pp 258ndash263 2005

[6] F-Y Liu M-Z Hong D-M Liu et al ldquoBiodegradation ofmethyl parathion by Acinetobacter radioresistens USTB-04rdquoJournal of Environmental Sciences vol 19 no 10 pp 1257ndash12602007

[7] B Wang L Xiong Y Zheng et al ldquoCloning and expression ofthempd gene fromanewly isolatedmethylparathion-degradingstrain of bacteriardquo Acta Scientiae Circumstantiae vol 10 pp1969ndash1974 2008

[8] X-X Wang Z Chi S-G Ru and Z-M Chi ldquoGenetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica forremoval of methyl parathion in waterrdquo Biodegradation vol 23no 5 pp 763ndash774 2012

[9] A Ekkhunnatham B Jongsareejit W Yamkunthong and JWichitwechkarn ldquoPurification and characterization of methylparathion hydrolase from Burkholderia cepacia capable ofdegrading organophosphate insecticidesrdquo World Journal ofMicrobiology and Biotechnology vol 28 no 4 pp 1739ndash17462012

[10] K-D Kim J-H Ahn T Kim et al ldquoGenetic and phenotypicdiversity of fenitrothion-degrading bacteria isolated from soilsrdquoJournal of Microbiology and Biotechnology vol 19 no 2 pp 113ndash120 2009

[11] J Tian P Wang S Gao X Chu N Wu and Y Fan ldquoEnhancedthermostability of methyl parathion hydrolase from Ochrobac-trum sp M231 by rational engineering of a glycine to proline

mutationrdquo The FEBS Journal vol 277 no 23 pp 4901ndash49082010

[12] P Vandamme B Pot M Gillis P de Vos K Kersters andJ Swings ldquoPolyphasic taxonomy a consensus approach tobacterial systematicsrdquoMicrobiological Reviews vol 60 no 2 pp407ndash438 1996

[13] A W Boyle C J Silvin J P Hassett J P Nakas and S WTanenbaum ldquoBacterial PCB biodegradationrdquo Biodegradationvol 3 no 2-3 pp 285ndash298 1992

[14] K Sato ldquoEffect of nutrients on interaction between pesti-cide penta-chlorophenol and microorganisms in soil biore-mediation through rhizosphere technologyrdquo in Bioremediationthrough Rhizosphere Technology pp 43ndash55 AmericanChemicalSociety Washington DC USA 1994

[15] V T Gajbhiye N P Agnihotri and H K Jain ldquoCypermethrinresidues on teardquo Pesticide Research Journal vol 1 pp 83ndash871989

[16] Y X Hou J N Zhang L Han et al ldquoDegradation analysis ofmethyl parathion by detoxifying enzymerdquo Journal of Agricul-tural Science and Technology vol 13 pp 110ndash115 2011 (Chinese)

[17] Y L Zheng D L Liu S L Chen et al ldquoStudy on isolation andcharacteristic identification of a novel high-efficiency degradingstrain of methyl parathionrdquo Research of Environmental Sciencesvol 19 pp 100ndash104 2006

[18] P-P Liu Y-C Yan and X-P Xie ldquoThe mechanism of purifica-tion and degradation of methylparathion-degrading bacteriumYL8rdquo China Environmental Science vol 26 no 2 pp 206ndash2092006 (Chinese)

[19] Z Z Feng B Liang J Zhang et al ldquoDegradation characteristicsof methyl-parathion by Pseudoxanthomonas sp XY3rdquo JiangsuAgricultural Sciences vol 4 pp 375ndash378 2010 (Chinese)

[20] M Dong Y L Yuan and X M Guo ldquoIsolation and identifica-tion of methyl parathion degrading strain Pseudomonas spinosaand the characteristics of the degradationrdquo in Proceedings of thePesticide and environment safety International Conference pp148ndash153 2003 (Chinese)

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

0

002

004

006

008

01

012

014

0 2 4 6 8 10

(AU

)

(min)

minus002

(a)

0

001

002

003

004

005

006

007

0 2 4 6 8 10

(AU

)

(min)

minus001

(b)

0001002003004005006007

0 2 4 6 8 10

(AU

)

(min)

minus001

(c)

Figure 4 HPLC of parathion-methyl enzymolysis (a) Standard sample of pNP (b) before enzymolysis of parathion-methyl (c) afterenzymolysis of parathion-methyl

OO

PS

O

O

N+

OminusCH3

CH3

Parathion-methyl

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

minus5

50 500

(a)

05

101520253035404550

0 5 10 15 20 25 30 35 40(min)

(kco

unts)

Parathion-methyl

p-Nitrophenol

50 500

minus5

OO

PS

O

O

N+

OminusCH3

CH3

O

HO N+

Ominus

(b)

Figure 5 TOF mass spectrogram (a) Before enzymolysis of parathion-methyl (b) after enzymolysis of parathion-methyl

34 Discussions Study on microbial degradation of pesti-cides began in the late 40s and it has always been a hotresearch spot The action modes of microbes degradingpesticides mostly belong to enzymatic reactions which aremainly triggered by a microbial intracellular enzyme Forenzymatic reactions pesticides are firstly adsorbed on thesurface of microbial cells and then the pesticides penetrate

cell membranes into the cell interior Inside the membranethe pesticides can combine with degrading enzyme andtrigger the enzyme catalytic reactions [13ndash15]

Many researches have been carried out on themechanismof parathion-methyl biodegradation all over the world Mostreported biodegradations of parathion-methyl were achievedby producing pNP and dimethyl thiophosphate (DMTP) but

6 BioMed Research International

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

0

50

100

150

200

250

300

20 30 40 50

Deg

rada

tion

rate

(120583g

min

)

Temperature (∘C)

Figure 6 Effect of temperature on the degradation rate ◻20∘C 99877130∘C40∘C and ◼50∘C

0

50

100

150

200

250

300

5 6 7 8pH

Deg

rada

tion

rate

(120583g

min

)

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

Figure 7 Effect of pH on the degradation rate ◼pH 5 998771pH6 epH7 and ◻pH8

there were also reports of different metabolic pathways Houstudied the parathion-methyl degradation effect of detoxifi-cation enzyme from metabolism of Pseudomonas alcaligenesand concluded that parathion-methyl was degraded by detox-ification enzyme via PndashO bond [16] Meanwhile they foundthat pNP and DMTP were produced during the degradationprocess but there was no excessive accumulation of pNPindicating that there might be further degradation pathwaysParathion-methyl degradation enzyme screened by Liu et alcould directly mineralize parathion-methyl and there wereno intermediate metabolites or final degradation products[6] Pseudomonas aeruginosa screened by Zheng et al coulddegrade the parathion-methyl into pNP meanwhile it coulddegrade the metabolic intermediate material pNP whichavoided the secondary pollution caused by accumulation ofintermediatematerials [17]Arthrobacter sp isolated by Liu etal could degrade both parathion-methyl and pNP and almostno pNP was detected during the degradation [18] Hydrolyticenzyme extracted by Liu et al from parathion-methyl degra-dation bacteria could degrade the parathion-methyl into pNP

[6] Pseudoxanthomonas japonensis isolated by Feng et alcould degrade both parathion-methyl and pNP [19] DM-1bacteria isolated by Dong could degrade parathion-methylinto amino parathion-methyl with low toxicity [20] EsteraseB1 secreted by strain C5 could degrade parathion-methyland HPLC and GC-TOF mass spectrometry analysis of theproducts confirmed that pNP was one of the degradationproducts As for the other product it might be DMTP butit still needed further identification

4 Conclusions

In this study a high activity esterase B1 was obtained bymarine bacterium C5 The strain was identified as Bacil-lus subtilis A single isozyme with a molecular weight of86 kDa was observed by SDS-PAGE and native-PAGE Themechanism of esterase B1 degrading parathion-methyl wasexplored and the effects of temperature and pH on thedegradation rate were investigated p-Nitrophenol was one of

BioMed Research International 7

the degradation products of B1 degrading parathion-methyland the best degradation effect could be achieved at thetemperature of 40∘C and the neutral pH value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National High Tech-nology Research and Development Program of China(2011AA090703) and the Qingdao Municipal Science andTechnology Program (11-2-4-20-hz)

References

[1] L Loperena V Soria H Varela et al ldquoExtracellular enzymesproduced by microorganisms isolated from maritime Antarc-ticardquo World Journal of Microbiology and Biotechnology vol 28no 5 pp 2249ndash2256 2012

[2] C Zhang and S-K Kim ldquoApplication of marine microbialenzymes in the food and pharmaceutical industriesrdquo Advancesin Food and Nutrition Research vol 65 pp 423ndash435 2012

[3] A Asoodeh and T Ghanbari ldquoCharacterization of an extra-cellular thermophilic alkaline esterase produced by BacillussubtilisDR8806rdquo Journal of Molecular Catalysis B Enzymaticvol 85-86 pp 49ndash55 2013

[4] K Mandal B Singh M Jariyal and V K Gupta ldquoMicrobialdegradation of fipronil by Bacillus thuringiensisrdquo Ecotoxicologyand Environmental Safety vol 93 pp 87ndash92 2013

[5] G-N Lu X-Q Tao Z Dang and X-Y Yi ldquoAdvances of studyon pesticide-degrading strains and their gene engineeringrdquoBulletin of Mineralogy Petrology and Geochemistry vol 24 no3 pp 258ndash263 2005

[6] F-Y Liu M-Z Hong D-M Liu et al ldquoBiodegradation ofmethyl parathion by Acinetobacter radioresistens USTB-04rdquoJournal of Environmental Sciences vol 19 no 10 pp 1257ndash12602007

[7] B Wang L Xiong Y Zheng et al ldquoCloning and expression ofthempd gene fromanewly isolatedmethylparathion-degradingstrain of bacteriardquo Acta Scientiae Circumstantiae vol 10 pp1969ndash1974 2008

[8] X-X Wang Z Chi S-G Ru and Z-M Chi ldquoGenetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica forremoval of methyl parathion in waterrdquo Biodegradation vol 23no 5 pp 763ndash774 2012

[9] A Ekkhunnatham B Jongsareejit W Yamkunthong and JWichitwechkarn ldquoPurification and characterization of methylparathion hydrolase from Burkholderia cepacia capable ofdegrading organophosphate insecticidesrdquo World Journal ofMicrobiology and Biotechnology vol 28 no 4 pp 1739ndash17462012

[10] K-D Kim J-H Ahn T Kim et al ldquoGenetic and phenotypicdiversity of fenitrothion-degrading bacteria isolated from soilsrdquoJournal of Microbiology and Biotechnology vol 19 no 2 pp 113ndash120 2009

[11] J Tian P Wang S Gao X Chu N Wu and Y Fan ldquoEnhancedthermostability of methyl parathion hydrolase from Ochrobac-trum sp M231 by rational engineering of a glycine to proline

mutationrdquo The FEBS Journal vol 277 no 23 pp 4901ndash49082010

[12] P Vandamme B Pot M Gillis P de Vos K Kersters andJ Swings ldquoPolyphasic taxonomy a consensus approach tobacterial systematicsrdquoMicrobiological Reviews vol 60 no 2 pp407ndash438 1996

[13] A W Boyle C J Silvin J P Hassett J P Nakas and S WTanenbaum ldquoBacterial PCB biodegradationrdquo Biodegradationvol 3 no 2-3 pp 285ndash298 1992

[14] K Sato ldquoEffect of nutrients on interaction between pesti-cide penta-chlorophenol and microorganisms in soil biore-mediation through rhizosphere technologyrdquo in Bioremediationthrough Rhizosphere Technology pp 43ndash55 AmericanChemicalSociety Washington DC USA 1994

[15] V T Gajbhiye N P Agnihotri and H K Jain ldquoCypermethrinresidues on teardquo Pesticide Research Journal vol 1 pp 83ndash871989

[16] Y X Hou J N Zhang L Han et al ldquoDegradation analysis ofmethyl parathion by detoxifying enzymerdquo Journal of Agricul-tural Science and Technology vol 13 pp 110ndash115 2011 (Chinese)

[17] Y L Zheng D L Liu S L Chen et al ldquoStudy on isolation andcharacteristic identification of a novel high-efficiency degradingstrain of methyl parathionrdquo Research of Environmental Sciencesvol 19 pp 100ndash104 2006

[18] P-P Liu Y-C Yan and X-P Xie ldquoThe mechanism of purifica-tion and degradation of methylparathion-degrading bacteriumYL8rdquo China Environmental Science vol 26 no 2 pp 206ndash2092006 (Chinese)

[19] Z Z Feng B Liang J Zhang et al ldquoDegradation characteristicsof methyl-parathion by Pseudoxanthomonas sp XY3rdquo JiangsuAgricultural Sciences vol 4 pp 375ndash378 2010 (Chinese)

[20] M Dong Y L Yuan and X M Guo ldquoIsolation and identifica-tion of methyl parathion degrading strain Pseudomonas spinosaand the characteristics of the degradationrdquo in Proceedings of thePesticide and environment safety International Conference pp148ndash153 2003 (Chinese)

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

0

50

100

150

200

250

300

20 30 40 50

Deg

rada

tion

rate

(120583g

min

)

Temperature (∘C)

Figure 6 Effect of temperature on the degradation rate ◻20∘C 99877130∘C40∘C and ◼50∘C

0

50

100

150

200

250

300

5 6 7 8pH

Deg

rada

tion

rate

(120583g

min

)

0

20

40

60

80

100

0 5 10 15 20 25Time (min)

Resid

ual q

uant

ity (

)

Figure 7 Effect of pH on the degradation rate ◼pH 5 998771pH6 epH7 and ◻pH8

there were also reports of different metabolic pathways Houstudied the parathion-methyl degradation effect of detoxifi-cation enzyme from metabolism of Pseudomonas alcaligenesand concluded that parathion-methyl was degraded by detox-ification enzyme via PndashO bond [16] Meanwhile they foundthat pNP and DMTP were produced during the degradationprocess but there was no excessive accumulation of pNPindicating that there might be further degradation pathwaysParathion-methyl degradation enzyme screened by Liu et alcould directly mineralize parathion-methyl and there wereno intermediate metabolites or final degradation products[6] Pseudomonas aeruginosa screened by Zheng et al coulddegrade the parathion-methyl into pNP meanwhile it coulddegrade the metabolic intermediate material pNP whichavoided the secondary pollution caused by accumulation ofintermediatematerials [17]Arthrobacter sp isolated by Liu etal could degrade both parathion-methyl and pNP and almostno pNP was detected during the degradation [18] Hydrolyticenzyme extracted by Liu et al from parathion-methyl degra-dation bacteria could degrade the parathion-methyl into pNP

[6] Pseudoxanthomonas japonensis isolated by Feng et alcould degrade both parathion-methyl and pNP [19] DM-1bacteria isolated by Dong could degrade parathion-methylinto amino parathion-methyl with low toxicity [20] EsteraseB1 secreted by strain C5 could degrade parathion-methyland HPLC and GC-TOF mass spectrometry analysis of theproducts confirmed that pNP was one of the degradationproducts As for the other product it might be DMTP butit still needed further identification

4 Conclusions

In this study a high activity esterase B1 was obtained bymarine bacterium C5 The strain was identified as Bacil-lus subtilis A single isozyme with a molecular weight of86 kDa was observed by SDS-PAGE and native-PAGE Themechanism of esterase B1 degrading parathion-methyl wasexplored and the effects of temperature and pH on thedegradation rate were investigated p-Nitrophenol was one of

BioMed Research International 7

the degradation products of B1 degrading parathion-methyland the best degradation effect could be achieved at thetemperature of 40∘C and the neutral pH value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National High Tech-nology Research and Development Program of China(2011AA090703) and the Qingdao Municipal Science andTechnology Program (11-2-4-20-hz)

References

[1] L Loperena V Soria H Varela et al ldquoExtracellular enzymesproduced by microorganisms isolated from maritime Antarc-ticardquo World Journal of Microbiology and Biotechnology vol 28no 5 pp 2249ndash2256 2012

[2] C Zhang and S-K Kim ldquoApplication of marine microbialenzymes in the food and pharmaceutical industriesrdquo Advancesin Food and Nutrition Research vol 65 pp 423ndash435 2012

[3] A Asoodeh and T Ghanbari ldquoCharacterization of an extra-cellular thermophilic alkaline esterase produced by BacillussubtilisDR8806rdquo Journal of Molecular Catalysis B Enzymaticvol 85-86 pp 49ndash55 2013

[4] K Mandal B Singh M Jariyal and V K Gupta ldquoMicrobialdegradation of fipronil by Bacillus thuringiensisrdquo Ecotoxicologyand Environmental Safety vol 93 pp 87ndash92 2013

[5] G-N Lu X-Q Tao Z Dang and X-Y Yi ldquoAdvances of studyon pesticide-degrading strains and their gene engineeringrdquoBulletin of Mineralogy Petrology and Geochemistry vol 24 no3 pp 258ndash263 2005

[6] F-Y Liu M-Z Hong D-M Liu et al ldquoBiodegradation ofmethyl parathion by Acinetobacter radioresistens USTB-04rdquoJournal of Environmental Sciences vol 19 no 10 pp 1257ndash12602007

[7] B Wang L Xiong Y Zheng et al ldquoCloning and expression ofthempd gene fromanewly isolatedmethylparathion-degradingstrain of bacteriardquo Acta Scientiae Circumstantiae vol 10 pp1969ndash1974 2008

[8] X-X Wang Z Chi S-G Ru and Z-M Chi ldquoGenetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica forremoval of methyl parathion in waterrdquo Biodegradation vol 23no 5 pp 763ndash774 2012

[9] A Ekkhunnatham B Jongsareejit W Yamkunthong and JWichitwechkarn ldquoPurification and characterization of methylparathion hydrolase from Burkholderia cepacia capable ofdegrading organophosphate insecticidesrdquo World Journal ofMicrobiology and Biotechnology vol 28 no 4 pp 1739ndash17462012

[10] K-D Kim J-H Ahn T Kim et al ldquoGenetic and phenotypicdiversity of fenitrothion-degrading bacteria isolated from soilsrdquoJournal of Microbiology and Biotechnology vol 19 no 2 pp 113ndash120 2009

[11] J Tian P Wang S Gao X Chu N Wu and Y Fan ldquoEnhancedthermostability of methyl parathion hydrolase from Ochrobac-trum sp M231 by rational engineering of a glycine to proline

mutationrdquo The FEBS Journal vol 277 no 23 pp 4901ndash49082010

[12] P Vandamme B Pot M Gillis P de Vos K Kersters andJ Swings ldquoPolyphasic taxonomy a consensus approach tobacterial systematicsrdquoMicrobiological Reviews vol 60 no 2 pp407ndash438 1996

[13] A W Boyle C J Silvin J P Hassett J P Nakas and S WTanenbaum ldquoBacterial PCB biodegradationrdquo Biodegradationvol 3 no 2-3 pp 285ndash298 1992

[14] K Sato ldquoEffect of nutrients on interaction between pesti-cide penta-chlorophenol and microorganisms in soil biore-mediation through rhizosphere technologyrdquo in Bioremediationthrough Rhizosphere Technology pp 43ndash55 AmericanChemicalSociety Washington DC USA 1994

[15] V T Gajbhiye N P Agnihotri and H K Jain ldquoCypermethrinresidues on teardquo Pesticide Research Journal vol 1 pp 83ndash871989

[16] Y X Hou J N Zhang L Han et al ldquoDegradation analysis ofmethyl parathion by detoxifying enzymerdquo Journal of Agricul-tural Science and Technology vol 13 pp 110ndash115 2011 (Chinese)

[17] Y L Zheng D L Liu S L Chen et al ldquoStudy on isolation andcharacteristic identification of a novel high-efficiency degradingstrain of methyl parathionrdquo Research of Environmental Sciencesvol 19 pp 100ndash104 2006

[18] P-P Liu Y-C Yan and X-P Xie ldquoThe mechanism of purifica-tion and degradation of methylparathion-degrading bacteriumYL8rdquo China Environmental Science vol 26 no 2 pp 206ndash2092006 (Chinese)

[19] Z Z Feng B Liang J Zhang et al ldquoDegradation characteristicsof methyl-parathion by Pseudoxanthomonas sp XY3rdquo JiangsuAgricultural Sciences vol 4 pp 375ndash378 2010 (Chinese)

[20] M Dong Y L Yuan and X M Guo ldquoIsolation and identifica-tion of methyl parathion degrading strain Pseudomonas spinosaand the characteristics of the degradationrdquo in Proceedings of thePesticide and environment safety International Conference pp148ndash153 2003 (Chinese)

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

the degradation products of B1 degrading parathion-methyland the best degradation effect could be achieved at thetemperature of 40∘C and the neutral pH value

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National High Tech-nology Research and Development Program of China(2011AA090703) and the Qingdao Municipal Science andTechnology Program (11-2-4-20-hz)

References

[1] L Loperena V Soria H Varela et al ldquoExtracellular enzymesproduced by microorganisms isolated from maritime Antarc-ticardquo World Journal of Microbiology and Biotechnology vol 28no 5 pp 2249ndash2256 2012

[2] C Zhang and S-K Kim ldquoApplication of marine microbialenzymes in the food and pharmaceutical industriesrdquo Advancesin Food and Nutrition Research vol 65 pp 423ndash435 2012

[3] A Asoodeh and T Ghanbari ldquoCharacterization of an extra-cellular thermophilic alkaline esterase produced by BacillussubtilisDR8806rdquo Journal of Molecular Catalysis B Enzymaticvol 85-86 pp 49ndash55 2013

[4] K Mandal B Singh M Jariyal and V K Gupta ldquoMicrobialdegradation of fipronil by Bacillus thuringiensisrdquo Ecotoxicologyand Environmental Safety vol 93 pp 87ndash92 2013

[5] G-N Lu X-Q Tao Z Dang and X-Y Yi ldquoAdvances of studyon pesticide-degrading strains and their gene engineeringrdquoBulletin of Mineralogy Petrology and Geochemistry vol 24 no3 pp 258ndash263 2005

[6] F-Y Liu M-Z Hong D-M Liu et al ldquoBiodegradation ofmethyl parathion by Acinetobacter radioresistens USTB-04rdquoJournal of Environmental Sciences vol 19 no 10 pp 1257ndash12602007

[7] B Wang L Xiong Y Zheng et al ldquoCloning and expression ofthempd gene fromanewly isolatedmethylparathion-degradingstrain of bacteriardquo Acta Scientiae Circumstantiae vol 10 pp1969ndash1974 2008

[8] X-X Wang Z Chi S-G Ru and Z-M Chi ldquoGenetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica forremoval of methyl parathion in waterrdquo Biodegradation vol 23no 5 pp 763ndash774 2012

[9] A Ekkhunnatham B Jongsareejit W Yamkunthong and JWichitwechkarn ldquoPurification and characterization of methylparathion hydrolase from Burkholderia cepacia capable ofdegrading organophosphate insecticidesrdquo World Journal ofMicrobiology and Biotechnology vol 28 no 4 pp 1739ndash17462012

[10] K-D Kim J-H Ahn T Kim et al ldquoGenetic and phenotypicdiversity of fenitrothion-degrading bacteria isolated from soilsrdquoJournal of Microbiology and Biotechnology vol 19 no 2 pp 113ndash120 2009

[11] J Tian P Wang S Gao X Chu N Wu and Y Fan ldquoEnhancedthermostability of methyl parathion hydrolase from Ochrobac-trum sp M231 by rational engineering of a glycine to proline

mutationrdquo The FEBS Journal vol 277 no 23 pp 4901ndash49082010

[12] P Vandamme B Pot M Gillis P de Vos K Kersters andJ Swings ldquoPolyphasic taxonomy a consensus approach tobacterial systematicsrdquoMicrobiological Reviews vol 60 no 2 pp407ndash438 1996

[13] A W Boyle C J Silvin J P Hassett J P Nakas and S WTanenbaum ldquoBacterial PCB biodegradationrdquo Biodegradationvol 3 no 2-3 pp 285ndash298 1992

[14] K Sato ldquoEffect of nutrients on interaction between pesti-cide penta-chlorophenol and microorganisms in soil biore-mediation through rhizosphere technologyrdquo in Bioremediationthrough Rhizosphere Technology pp 43ndash55 AmericanChemicalSociety Washington DC USA 1994

[15] V T Gajbhiye N P Agnihotri and H K Jain ldquoCypermethrinresidues on teardquo Pesticide Research Journal vol 1 pp 83ndash871989

[16] Y X Hou J N Zhang L Han et al ldquoDegradation analysis ofmethyl parathion by detoxifying enzymerdquo Journal of Agricul-tural Science and Technology vol 13 pp 110ndash115 2011 (Chinese)

[17] Y L Zheng D L Liu S L Chen et al ldquoStudy on isolation andcharacteristic identification of a novel high-efficiency degradingstrain of methyl parathionrdquo Research of Environmental Sciencesvol 19 pp 100ndash104 2006

[18] P-P Liu Y-C Yan and X-P Xie ldquoThe mechanism of purifica-tion and degradation of methylparathion-degrading bacteriumYL8rdquo China Environmental Science vol 26 no 2 pp 206ndash2092006 (Chinese)

[19] Z Z Feng B Liang J Zhang et al ldquoDegradation characteristicsof methyl-parathion by Pseudoxanthomonas sp XY3rdquo JiangsuAgricultural Sciences vol 4 pp 375ndash378 2010 (Chinese)

[20] M Dong Y L Yuan and X M Guo ldquoIsolation and identifica-tion of methyl parathion degrading strain Pseudomonas spinosaand the characteristics of the degradationrdquo in Proceedings of thePesticide and environment safety International Conference pp148ndash153 2003 (Chinese)

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology