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Purification and characterization of a novel #-cypermethrindegrading aminopeptidase from Pseudomonas aeruginosa GF31 Ai-Xing Tang, Hu Liu, You-Yan Liu, Qing-Yun Li, and Yi-Ming Qing J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03288 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Page 1 of 37

Journal of Agricultural and Food Chemistry

1 Purification and characterization of a novel β-cypermethrin-degrading aminopeptidase from Pseudomonas aeruginosa GF31

Ai-Xing Tanga,b, Hu Liua, You-Yan Liua,b*, Qing-Yun Lia,b, Yi-Ming Qinga

a

School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004,

Guangxi, P. R. China b

Key Laboratory of Guangxi Biorefinery, Nanning 530003, Guangxi, P. R. China

*Corresponding author: School of Chemistry and Chemical Engineering, Guangxi University, 100 Daxue Road, Nanning, Guangxi, 530004, P.R. China Tel.: +86 771 323 3583 Fax: +86 771 323 3718 E-mail address: [email protected] (YY. Liu) ORCID You-Yan Liu：0000-0003-4459-7841

ACS Paragon Plus Environment


Page 2 of 37

2 Abstract 1

In this study, a novel β-cypermethrin-degrading enzyme was isolated and purified by 32.8 fold

2

from the extracellular cell-free filtrate of Pseudomonas aeruginosa GF31with the protein

3

recovery of 26.6%. The molecular mass of the enzyme was determined to be 53 kDa. The

4

optimum temperature for the activity was surprisingly 60 °C and moreover, the purified enzyme

5

showed a good pH- stability, maintaining over 85% of its initial activity in the pH 5.0-9.0 range.

6

Most of the common metal ions exhibited little influence on the activity except for Hg2+, Ag+ and

7

Cu2+. After the complete gene sequence of the degrading enzyme was obtained by subcloning,

8

sequence analyses as well as enzymatic properties demonstrated that the islolated enzyme should

9

be an aminopeptidase. This is the first reported aminopeptidase for pyrethroid hydrolase,

10

providing new potential enzyme resources for the degradation of this type of pesticide.

11 12

Keywords: β-cypermethrin, pyrethroid hydrolase, aminopeptidase, purified enzyme,

13

Pseudomonas aeruginosa GF31


Page 3 of 37


3 14

Introduction

15

β-cypermethrin (β-CP) is a wide spectrum synthetic pyrethroid insecticide that was

16

previously thought to have a relatively low toxicity and is thus widely used to control insects in

17

agriculture, landscaping and household hygiene. However, increasing studies are revealing that

18

β-CP exhibits not only reproductive and developmental toxicities in humans1 but also acute

19

toxicities in off-target organisms such as bees, silkworms, fish and aquatic invertebrates, even at

20

concentrations of below 0.5g/kg.2 To reduce the environmental and public health risks

21

associated with pyrethroid use, it is necessary to develop rapid and effective methods to remove

22

or minimize the concentrations of insecticides in the environment. Of the various methods that

23

are used for remediation of contaminated environments, the biological approach is the most

24

promising and effective strategy. To date, numerous pyrethroid-degrading microorganisms (i.e.,

25

bacteria, fungi and actinomycetes) have been isolated and studied. Several species, including

26

Bacillus sp. AKD1,3 Pseudomonas aeruginosa JQ-41,4 Ochrobactrum anthropi JCm1,5

27

Aspergillus niger YAT6 and Actinomycetes HU-S-01,7 have been reported to have the ability to

28

degrade β-CP.

29

It is necessary to have a thorough knowledge of the metabolic pathways and key enzymes

30

that are involved in insecticide removal to develop efficient bioremediation technologies.8

31

Generally, the elimination of multiple lipophilic xenobiotics depends on their conversion to

32

water-soluble compounds.9 For pyrethroid, a hydrophobic ester, the most effective way to

33

increase the water solubility of the compound is through hydrolysis to the alcohol and carboxylic

34

acid. Carboxylesterase (CES, EC 3.1.1) plays a key role in the ester bond cleavage of



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4 35

pyrethroid.10 To date, several pyrethroid-degrading enzymes have been purified and

36

characterized, including permethrinase from Bacillus cereus SM3,11 pyrethroid hydrolase from

37

Aspergillus niger ZD11,12 EStP from Klebsiella sp. ZD112,13 PytH from Sphingobium sp. JZ-2,14

38

and PytY and PytZ from Ochrobactrum anthropi YZ-1.15,16 Additionally, Pye317 and Sys41018

39

were screened from the metagenome of soil that was contaminated by pyrethroid. These

40

degrading enzymes, whose molecular masses range from 31 to 73 kDa, can hydrolyze the

41

pyrethroids over a range of optimal temperatures (35–55 °C) and pH values (6.5–7.5). Although

42

the characteristics of these enzymes are different, they are generally considered to be CESs or

43

esterases (EC 3.1). To our knowledge, no other types of hydrolase have been reported.

44

In this study, we aimed to purify a β-CP-degrading enzyme from Pseudomonas

45

aeruginosa strain GF31. Based on a previous report,19 the target enzyme was extracellular, unlike

46

previously reported purified enzymes, indicating that GF31 may possess a distinct substrate

47

utilization pattern. Moreover, the characteristics of the target enzyme were unlike those of a

48

typical carboxylesterase, in contrast to previous reports. It is highly possible that the

49

β-CP-hydrolyzing enzyme from GF31 may be a new type of pyrethroid hydrolase.

50 51

Materials and Methods

52

Chemicals and Reagents

53

β-Cypermethrin (98%), Fenpropathrin (91.5%), Fenvalerate (90.8%), and deltamethrin

54

(98.2%) were obtained from Plant Protection Station of Guangxi, 3-phenoxybenzaldehyde

55

(3-PBH) and 3-phenoxybenzoic acid (3-PBA) were purchased from Sigma-Aldrich Chemical Co.


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5 56

(Shanghai, China). DEAE-Sepharose CL-6B (GE, USA), Sephadex G75 (Pharmacia, USA), the

57

low-molecular-weight protein standard and the SDS-PAGE reagent (Shanghai Sangon

58

Biotechnology Engineering Co., Ltd., China) were also used in this study. Restriction enzymes

59

and DNA polymerase were purchased from Takara (Dalian, China). All other chemicals were of

60

analytical grade and purchased from commercial sources. The beef extract-peptone medium

61

contained beef extract (5.0 g/L), peptone (10.0 g/L), and NaCl (5.0 g/L) (pH 7.0). The 20-mM

62

Phosphate-buffered solution (PBS) comprised Na2HPO4·12H2O (7.169 g/L) and KH2PO4 (2.72

63

g/L).

64 65 66 67

Bacterial Strains and Culture Conditions Pseudomonas aeruginosa GF31 was isolated from pesticide-contaminated soil. Culturing of the bacteria was performed as described previously.19

68 69 70

Purification of the Hydrolyzing Enzyme All experiments described below were carried out between 0 and 4 °C unless otherwise

71

specified.

72

(a)Preparation of the Extracellular Crude Enzyme

73

To prepare the crude enzyme, GF31 was cultured in a beef extract-peptone medium, the

74

culture was centrifuged at 12000 × g for 10 min at 4 °C, and the supernatant was filtered through

75

a 0.22-µm filter. The resulting liquid was collected and concentrated by ultrafiltration (10K,

76

Sartorius vivaflow 200) and then used as an enzyme source for subsequent enzyme purification.



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6 77

(b) Ammonium Sulfate Precipitation

78

The extracellular crude enzyme was brought to 40% ammonium sulfate saturation and

79

stirred for 30 min at 4°C. After standing for 12 h, the mixture was centrifuged at 12000 × g for

80

30 min at 4 °C, and the supernatant was removed and precipitated with 50% ammonium sulfate

81

saturation, stirred for 30 min, and allowed to stand for 12 h. The precipitate was collected by

82

centrifugation at 12000 × g for 30 min at 4 °C and subsequently suspended in a minimal volume

83

of 20 mM Bis-Tris buffer at pH 7.0.

84

(c) Ion-Exchange Chromatography

85

The enzyme solution from the ammonium sulfate fractions was loaded onto a

86

DEAE-Sepharose CL-6B column (1.6 cm × 60 cm) and equilibrated with 20 mM Bis-Tris buffer

87

at pH 7.0. The enzymes were eluted from the column with a linear gradient using the same buffer

88

containing 0 to 1 M NaCl at a flow rate of 1 mL/min.

89

(d) Gel Filtration through Sephadex G-75

90

Two milliliters of the enzyme solution from the ion-exchange chromatography was

91

loaded onto a Sephadex G-75 column (1.0 cm × 50 cm) that had been equilibrated with 20 mM

92

PBS at pH 7.0. The column was washed at a flow rate of 4 mL/h with 50 mL of the same buffer,

93

and 3-mL fractions were collected. The high-activity fractions were pooled, concentrated, and

94

stored at -20 °C for the various analyses, which included assessment of enzymatic properties,

95

metal dependence and purity.

96 97

Protein Determination


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7 98 99

The protein concentration was quantified using the Bradford method.20 Bovine serum albumin (BSA, Sigma) was used as the standard for calibration.

100 101

Enzyme Assay

102

The enzyme activity was measured by determining the decrease of β-cypermethrin. The

103

pyrethroid hydrolase activity was assayed with high-performance liquid chromatography using a

104

reaction mixture at a final volume of 3 mL phosphate buffer (50 mM, pH 7.0) that contained

105

substrate (50 mg/L) and an appropriate amount of protein. These assays were started by addition

106

of the substrate and incubating the reaction at 60 °C on a shaker at 120 rpm for 2 h, finally

107

stopped by adding 0.2 mL of 1 M HCl. Residual β-cypermethrin was extracted with ethyl acetate

108

and detected by an ULTIMATE 3000 high-performance liquid chromatography system (Idstein,

109

Germany) equipped with an ultraviolet detector. Assays were conducted at room temperature

110

using a Lichrospher 5-µm C18 column (250 × 4.6 mm) at a wavelength of 235 nm, and the

111

mobile phase was 85:15 (v/v) acetonitrile and water.19

112

The kinetic constants of the enzyme for β-cypermethrin, namely, the maximum reaction

113

rate (Vmax) and Michaelis constant (Km), were determined by measuring the enzyme activity

114

when initial β-cypermethrin concentrations were 20–200 mg L−1, and initial reaction velocities

115

measured at various substrate concentrations were fitted to the Lineweaver-Burk transformation

116

of the Michaelis-Menten equation. Catalytic constant (Kcat) was equal to the quotient of the Vmax

117

and enzyme concentration.

118

All experiments were performed in triplicate, and enzyme-free controls were included.



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8 119

One unit of enzyme activity was defined as the amount of enzyme required to catalyze the

120

consumption of 1 µmol of the substrate per min at 60 °C and pH 7.0 on a shaker at 120 rpm.

121 122

Determination of Enzymatic Properties

123

To determine the optimal pH and temperature, the enzyme activity was measured by

124

incubating the purified enzyme (0.2 µg/mL) with β-cypermethrin as the substrate at 60 °C for 2 h

125

in 50 mM buffered solution at a pH ranging from 3-9(pH3.0-6.0, Citric acid-disodium hydrogen

126

phosphate buffer; pH6.0-8.0, PBS; pH8.0-9.0, Tris-HCl buffer)and at pH 7 for 2 h in 50 mM

127

PBS at a temperature ranging from 20-70 °C. The relative activity was measured as described

128

above.

129

To investigate the thermal stability and pH stability of the enzyme, the enzyme was kept

130

at different temperatures in 50 mM PBS at pH 7.0 or at different pH values at 60 °C for 2 h, and

131

then the β-cypermethrin was put into the reaction system as a substrate to measure the relative

132

activity. As for the effects of metal ions and chemicals on the enzyme activity, the enzyme was

133

pre-incubated with chemicals in 50 mM PBS at pH 7.0 and 60 °C for 2 h and then measured for

134

activity using β-cypermethrin as the substrate.

135

The degradation products of β-CP hydrolyzed by purified enzyme were detected by

136

HPLC, and 3PBA and 3PBH standards were used as controls. The products’ extraction and

137

detection conditions were the same as those mentioned above.

138

The enzyme activity and kinetic constants of the enzyme for other pyrethroids including

139

fenpropathrin, fenvalerate, and deltamethrin were determined as well. The reaction conditions


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9 140

and the detection methods were similar to those for cypermethrin except that fenpropathrin was

141

detected at a wavelength of 226 nm and the concentrations of substrates, due to their low or high

142

specific activities, were appropriately adjusted when the kinetic parameters were measured.

143 144

All experiments were performed in triplicate and enzyme-free controls were included. Standard deviations of the mean were determined and reported.

145 146

Aminopeptidase Assay

147

Aminopeptidase activity was determined in 50 mM Tris-HCl buffer (pH 8.0) at 60 °C

148

using 2 mM Leu-pNA as the substrate. The reaction was initiated by adding the enzyme to the

149

preincubated substrate solution. The increase in absorbance at 405 nm was monitored at 10-s

150

intervals for 5 min on a spectrophotometer (Beckman Model 600), and the initial rate of

151

hydrolysis was calculated.21 One unit of the aminopeptidase activity was defined as the amount

152

of enzyme that produced 1 µmol of p-nitroaniline/min under standard conditions.

153

The values for Kcat and Km were calculated from two individual experiments. Each

154

experiment was carried out at five different substrate concentrations. The substrates, namely,

155

Leu-pNA, Ala-pNA, and Arg-pNA, were examined from 0.2 to 4 mM, from 0.2 to 4 mM, and

156

from 0.07 to 1.4 mM, respectively, due to their low solubility or high Km values. The kinetic

157

constants of the enzyme for p-nitroanilide, namely, the Vmax and Km, were estimated from a

158

Lineweaver-Burk plot, and a Kcat equal to the quotient of the Vmax and enzyme concentration.

159 160

Determination of the Molecular Mass and pI



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10 161

The molecular mass of the purified enzyme was determined by sodium dodecyl sulfate

162

polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed using the Laemmli

163

method22 on a 12% (w/v) polyacrylamide separation gel and 4% (w/v) polyacrylamide stacking

164

gel. After SDS-PAGE, proteins were stained with Coomassie brilliant blue R-250. The isoelectric

165

point (pI) was assayed according to the method by Liang et al.12

166 167

Protein Identification and NH2-terminal Amino Acid Sequencing

168

The purified pyrethroid-degrading enzyme was resolved by SDS-PAGE and used as a

169

sample for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass

170

spectrometry to identify the protein. The MALDI-TOF data collected during the LC-MS run

171

were submitted to the Mascot search software. Protein identification was carried out by

172

comparing the experimental data to the National Center for Biotechnology Information (NCBI)

173

nr databases (www.matrixseienee.com). Homology searches were performed using the NCBI

174

BLAST server (http://www.ncbi.nlm.nih.gov/BLAST). N-terminal amino acid sequence was

175

determined by the Edman degradation method as described by Borgo and Havranek.23

176 177

Sub-cloning of the Pyrethroid-degrading Enzyme Gene

178

Primers for PCR

179

Database searching for the peptide mass fingerprint of the purified enzyme showed that

180

the enzyme had a high sequence homology with the deduced aminopeptidase encoded by pepB

181

that was derived from Pseudomonas aeruginosa PAO1. The primer sequences were obtained


Page 11 of 37


11 182

according to the open reading frame of pepB from P. aeruginosa PAO1. To amplify the complete

183

ORF of the aminopeptidase gene of P. aeruginosa GF31, forward and reverse primers were

184

designed based on the 5′ and 3′ regions of the cDNA sequence using the Primer 5 and Oligo 7

185

software (synthesized by Invitrogen, Shanghai, China). The primers used were as follows:

186

forward,

187

5′-CCCAAGCTTTTACTTGATGAAGTCGT -3′(HindⅢ).

5′-GCCGAATTCATGAGCAACAAGAACAAT-3′(EcoRI)

and

reverse,

188 189

DNA Manipulation

190

Routine DNA manipulation was carried out as described by Sambrook et al.24 The

191

genomic DNA from P. aeruginosa GF31 was obtained using a bacterial genome DNA extraction

192

kit (DP302, TIANGEN) following the manufacturer’s instructions. Electrophoresis was carried

193

out with a 0.8% agarose gel in Tris-acetic acid-EDTA buffer. The PCR-amplified product was

194

resolved on a 1% (m/v) agarose gel and gel-extracted using the Universal DNA Purification Kit

195

(TIANGEN). The purified fragment was ligated into the pMD-18-T plasmid (Takara, Dalian,

196

China). This ligation mixture was used to transform DH5α E. coli. The white bacterial colonies

197

that contained the recombinant plasmids were selected on LB agar medium containing 0.1 mM

198

X-gal, 0.2 mM IPTG, and 50µg/mL ampicillin.

199 200

DNA Sequencing and Protein Sequence Comparison

201

DNA sequencing of the isolated plasmid was carried out using standard primers (T7

202

forward and T7 reverse) and an automated DNA sequencing system, namely, the ABI Prism 3700

203

DNA Analyzer (Waltham, MA, Thermo Fisher Scientific). The sequences obtained were



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12 204

assembled using the Sequencher DNA sequence analysis software (version 4.0.5; Gene Codes,

205

USA). The amino acid sequence was deduced using ExPasy [http://expasy.org/tools]. This

206

full-length amino acid sequence for the pyrethroid-degrading enzyme served as a template to

207

screen structurally similar proteins using BLASTP from NCBI.

208 209

Results

210

Enzyme Production and Purification

211

The extracellular crude enzyme was purified by ultrafiltration, ammonium sulfate

212

precipitation, ion exchange on DEAE-Sepharose CL-6B and gel filtration on Sephadex G-75. A

213

summary of the data for the purification is presented in Table 1.

214

The purified enzyme was examined by SDS-PAGE and showed a single band (Figure 1

215

and S1). The molecular mass of the purified enzyme was determined to be approximately 53.0

216

kDa, which was different from the molecular masses of the pyrethroid-hydrolyzing enzymes

217

from Sphingobium sp. JZ-2 (31 kDa), A. niger ZD11 (56 kDa), Nephotettix cincticeps Uhler

218

(58.6 kDa), mouse liver microsomes (60 kDa), B. cereus SM3 (61 kDa), C. pipiens (65 kDa) and

219

Klebsiella sp. ZD112 (67 kDa).11–14,25–27 The pI value of the purified enzyme was 7.67, which

220

was higher than that of Sphingobium sp. JZ-2 (4.85) and A. niger ZD11 (5.4).

221 222

Effect of pH and pH Stability on Enzyme Activity

223

The effects of pH and pH stability on the activity of the purified enzyme are shown in

224

Figure 2. The optimal pH of the purified enzyme was 7.0. At a pH between 5.0 and 9.0, the


Page 13 of 37


13 225

enzyme displayed a high relative activity that was ＞ 85%; however, the relative activity

226

significantly decreased at low pH, with 26% of the relative activity at pH 3.0. The stability

227

assessment of the enzyme at different pH values showed that the enzyme was stable at pH values

228

between 5.0 and 9.0, but a low pH effectively promoted an increase in the deactivation rate of the

229

enzyme. This is also why enzymatic reactions can be terminated by acidification.

230 231

Effect of Temperature and Thermal Stability on Enzyme Activity

232

The optimal temperature of the purified enzyme was 60 °C (Figure 3). At 50 °C and

233

70 °C, the enzyme showed high relative activity with 83% and 96% of the highest activity,

234

respectively. The relative activity significantly decreased outside of the range from 50-70 °C.

235

The pure enzyme was stable below 40 °C, with ＞ 92% relative activity. However, the stability

236

of the enzyme decreased as the temperature increased, particularly when it exceeded 70 °C,

237

where only 10% of the initial activity was retained.

238 239

Effect of Various Metallic Ions and Inhibitors on Enzyme Activity

240

The effects of various compounds on the purified enzyme activity are shown in Tables 2

241

and 3. The enzymatic activity was strongly inhibited by surfactants such as SDS (approximately

242

69% inhibition) and Triton-100 (approximately 52% inhibition), whereas chelating agents such

243

as EDTA and 1,10-phenanthroline displayed no inhibitory effect. Specific esterase inhibitor

244

malathion and nonspecific inhibitor PMSF showed no significant effect (less than 10%) on the

245

enzyme activity. From the reaction, Hg2+, Ag+ and Cu2+ (all at 1 mM) caused 71%, 43% and 34%



Page 14 of 37

14 246

reduction in enzymatic activity, respectively. The other metallic ions had little effect (less than

247

18%) on the enzyme activity.

248 249

Substrate Specificity

250

The substrate specificity toward various p-nitroanilides and pyrethroids is shown in Table

251

4. Of the nitroaniline derivatives examined, the purified enzyme showed the highest activity with

252

Leu-pNA (22312.6 U/mg), followed by Arg-pNA (2540 U/mg) and Ala-pNA (1276.9U/mg),

253

which indicates that the purified enzyme is an aminopeptidase. A range of pesticides including

254

β-cypermethrin, fenpropathrin, fenvalerate, and deltamethrin were used to test the substrate

255

specificity of the purified enzyme. Although the purified enzyme hydrolyzed all pesticides tested

256

at different rates, the hydrolysis rates of all pesticides were much lower than the hydrolysis rates

257

of several p-nitroanilides. Based on the data, the purified enzyme has a higher activity to

258

hydrolyze β-cypermethrin than other pyrethroids tested, which is consistent with the

259

phenomenon in the cell and the crude enzyme. On the other hand, the purified enzyme showed

260

the strongest affinity for deltamethrin (Km=11.8µM) and weakest affinity for β-cypermethrin

261

(Km=47.7 µM), thus leading to a relatively low catalytic efficacy (Kcat/Km) for β-cypermethrin.

262

This is why β-cypermethrin is always chosen as a pyrethroid model and needs to be focused on.

263 264

Product Analysis of the Purified Enzyme after Degradation of Cypermethrin

265

We used the purified enzyme to study the metabolic pathway of β-cypermethrin by

266

metabolite identification and enzymatic analysis. The HPLC-based metabolite identification of


Page 15 of 37


15 267

the purified enzyme after degradation of β-cypermethrin is shown in Figure S2. β-Cypermethrin

268

was converted to 3-phenoxybenzaldehyde (3-PBH) and 3-phenoxybenzoic acid (3-PBA)

269

compared to the control group, and the amount of 3-PBH was greater than the amount of 3-PBA.

270

According to the previously described pyrethroid-degrading pathway,28-30 we deduced that

271

β-cypermethrin was first degraded by hydrolysis of the carboxyl ester linkage to yield

272

3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate

273

cyano-3-phenoxybenzylalcohol, which indicated that the purified pyrethroid-degrading enzyme

274

is a hydrolase. Cyano-3-phenoxybenzylalcohol was then quickly converted to 3-PBH and slowly

275

converted to 3-PBA, spontaneously. The degradation pathway of cypermethrin by the purified

276

enzyme is shown in Figure 4.

(DCVA)

and

277 278

Protein Identification and NH2-terminal Amino Acid Sequencing

279

The degrading enzyme was identified and characterized by mass spectrometry. In

280

combination with the results from the SDS-PAGE gel electrophoresis and high performance gel

281

permeation chromatography (Figure S3), it appears that the purified enzyme we obtained is most

282

likely a single-subunit protein. Database searches for the peptide mass fingerprint of the purified

283

enzyme showed that it had a high sequence homology with putative aminopeptidases, such as

284

those from Pseudomonas aeruginosa M18 (57.8 kDa, gi|347304395), Pseudomonas aeruginosa

285

strain PAO1 (57.8 kDa, gi|15598135) and Pseudomonas aeruginosa PA7 (57.7 kDa,

286

gi|152984126).The N-terminal amino acid sequence of the purified enzyme was determined to be

287

NH2-Thr-Pro-Gly-Lys-Pro-Asn-Pro-Ser-Ile-Cys.



Page 16 of 37

16 288 289

Sub-cloning and Analysis of the Pyrethroid-degrading Enzyme Gene

290

The gene for the pyrethroid-degrading enzyme (GenBank accession number KT735188)

291

that was derived from Pseudomonas aeruginosa strain GF31 was obtained by PCR with primers

292

designed according to the sequence of the pepB gene of Pseudomonas aeruginosa PAO1; the

293

1611-bp DNA fragment was gel purified and subcloned into the pMD-18-T plasmid. The

294

pyrethroid-degrading enzyme showed an amino acid sequence identity of approximately 99.1%

295

with the deduced aminopeptidase from Pseudomonas aeruginosa PAO1. The bioinformatics

296

analysis of this pyrethroid-degrading enzyme gene showed that the enzyme consisted of 536

297

amino acids, with the first 24 amino acids representing a signal peptide, amino acids 25-36

298

representing a leading peptide, and the followed 500 amino acid residues representing a mature

299

protein (Figure S4); the molecular weight was 53.7 KDa, which is consistent with the molecular

300

weight of the purified protein.

301 302

Multiple Sequence Alignment and Metalloprotein Analysis

303

Multiple sequences were compared with the pyrethroid-degrading enzyme by selecting

304

homologous proteins from the NCBI database. These selected strains included Pseudomonas

305

aeruginosa PAO1,31 Arthrobacter crystallopoietes (A_cry),32 Spirillospora albida (S_alb),

306

Thermobispora bispora (T_bis),33 Bacillus subtilis (B_sub)34 and Saccharomyces cerevisiae

307

(S_cer)35 (Figure S5). The sequence comparison results indicate that the pyrethroid-degrading

308

enzyme had a typical aminopeptidase catalytic ternary-component (Glu341, Ser423 and His296)


Page 17 of 37


17 309

and five amino acid residues to be coordinated with Zn, including His296, Asp308, Glu341, and

310

His467. Analysis of the conserved domain (Figure S6) and amino acid sequence showed that the

311

pyrethroid-degrading enzyme could be classified as an aminopeptidase from the superfamily of

312

zinc peptidases (M28).

313 314

Discussion

315

Pseudomonas aeruginosa, which is one of the most potential pyrethroid-degrading

316

microbes,31 is a ubiquitous environmental bacterium. In addition to the strain GF31 of this study,

317

there are three strains of Pseudomonas aeruginosa: CH7,36 JCm85 and JQ-41.4 All three have

318

been isolated and tested for degradation of cypermethrin, but the key metabolic enzymes of these

319

strains have not been studied in depth. Even after the metabolites of CH7 and JQ-41 were

320

detected as DCVA and 3-PBA, the authors of these studies deduced that the key metabolic

321

enzyme was a carboxylesterase, which was similar to that of other pyrethroid-degrading bacteria.

322

This is different from our conclusion. Although we have detected the same metabolites, we

323

confirmed that these metabolites are the result of the hydrolysis by aminopeptidase from

324

Pseudomonas aeruginosa GF31. Furthermore, the degrading aminopeptidase was an

325

extracellular enzyme,19 in contrast to the other known pyrethroid-degrading enzymes, which are

326

intracellular. This means that strain GF31 can overcome the limitation of substrate uptake and

327

eliminate the barriers to the substrate through the cell membrane,

328

hydrolysis pathway.

329

37

indicating a new pyrethroid

There’re some reports on the structure and catalytic mechanism of aminopeptidase



Page 18 of 37

18 330

previously. It is generally believed that the active center of the aminopeptidase contains a

331

catalytic ternary structure consisting of Glu, Ser and His.38,39 Similarly, carboxylesterase has a

332

typical "Ser-His-Asp/Glu" catalytic triad.

333

carboxylate esterase, the nucleophilic serine is located at a prominent position in the active center,

334

and the oxygen on the serine residue engages in a nucleophilic attack on the carbonyl carbon of

335

the amido bond or ester bond in the catalytic process. We have confirmed that the

336

aminopeptidase from GF31 has a similar catalytic center (Figure S5), it is more possible that the

337

enzyme was hydrolyzing pyrethroids and peptides in the same active center. However, two

338

potent carboxylesterase inhibitors, malathion and PMSF, didn’t exert significant effects on

339

hydrolyzing pyrethroids. It seems that the catalytic mechanism in hydrolyzing was somewhat

340

different between two substrates. So far, we are not able to completely rule out the possibility

341

that the enzyme hydrolyzes pyrethroids and peptides in two different active centers until there is

342

conclusive evidence. Additionally, what role the PA domain (Figure S7), which provides a

343

scaffold for binding larger protein in the aminopeptidase, would play in the degradation of

344

pyrethroids is also unclear and requires a more in-depth investigation.

40

And moreover, whether it is aminopeptidase or

345

The investigation of the effects of various chemicals is the basis for future practical

346

applications of enzymes and may provide information on the structure and properties of enzymes

347

from another aspect. The purified enzyme was significantly inhibited by sulfhydryl oxidant

348

metals (Hg2+, Ag+), whereas other metal ions were not strongly inhibitory, suggesting that thiol

349

may be involved in the catalytic site of the enzyme.12 EDTA and 1,10-phenanthroline did not

350

have inhibitory effects on the enzyme activity, indicating that divalent cations are not required


Page 19 of 37


19 351

for enzyme activity.12 The strong inhibitory effects of SDS (10 mM) on the enzyme illustrated

352

that the enzyme should be sensitive to the denaturant and enzyme activity depend on the spatial

353

conformation. 14

354

The catalytic characteristics of an enzyme are the most important factors that influence its

355

practical application. The purified enzyme from Pseudomonas aeruginosa GF31 has a high

356

optimal temperature, good thermal stability and a wide pH tolerance level. These characteristics

357

benefit the actual bioremediation of the pure enzyme from GF31. Optimal purified enzyme

358

activity was obtained at 60 °C and pH 7.0. The temperature optimum of the purified enzyme

359

activity was much higher than that reported for A. niger ZD11 (45 °C), Sphingobium sp. JZ-2

360

(40 °C), Klebsiella sp. ZD112 (40 °C), and Bacillus cereus (37 °C). The temperature elevation,

361

particularly above 70 °C, resulted in only a 10% retention of the initial activity. The thermostable

362

enzymes are often associated with stability in solvents and detergents, enabling numerous

363

potential applications of these enzymes in multiple industries. The optimal pH of the purified

364

enzyme activity was similar to those recorded for Sphingobium sp. JZ-2 (pH 7.5), B. cereus SM3

365

(pH 7.5), A. niger ZD11 (pH 6.5) and Klebsiella sp. ZD112 (pH 7.0). The purified enzyme

366

exhibited a relatively wide pH range between pH 5.0 and 9.0, where it displayed more than 85%

367

of the relative activity. Additionally, the purified pyrethroid-degrading enzyme had high

368

aminopeptidase activity. Thus, this enzyme has potential for applications in the food industry.

369

In conclusion, we have purified an extracellular pyrethroid hydrolase from an important

370

environmental microbe, Pseudomonas aeruginosa, the hydrolase is an aminopeptidase. This

371

hydrolase is different from the pyrethroid hydrolases that have been widely reported, which are



Page 20 of 37

20 372

intracellular carboxylate esterases. This indicates that Pseudomonas aeruginosa may have a new

373

pyrethroid hydrolysis pathway and substrate utilization mechanism that is worth further studies.

374

This study also provides a new perspective for studying the degradation of other hydrophobic

375

organic compounds. Additionally, the purified enzyme from Pseudomonas aeruginosa GF31 has

376

a high optimal temperature, good thermal stability and wide pH tolerance level, which enables its

377

strong potential for various applications.

378 379

Funding Source

380

This study was support in part by the National Natural Science Foundation of China (No.

381

21276053) and Natural Science Foundation of Guangxi Province (No. 2016GXNSFAA380302)

382 383

Conflict of Interest

384

The authors declare that they have no conflicts of interest.

385 Supporting Information Metabolite identification and analysis of the Pyrethroid-degrading Enzyme Gene 386


Page 21 of 37


21 387

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Page 28 of 37

28 Figure Captions Figure 1. SDS-polyacrylamide gel electrophoresis of the purified enzyme from Pseudomonas aeruginosa GF31 (lane 1) and protein markers (lane 2) stained with Coomassie brilliant blue. Markers from top to bottom include phosphorylase b (97.4 kDa), BSA (66.2 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa). Figure 2. Effect of pH on activity (■) and stability (●) of the enzyme. Figure 3. Effect of temperature on activity (■) and stability (●) of the enzyme. Figure 4. Product analysis of the purified enzyme after degradation of cypermethrin.


Page 29 of 37


29 Tables Table 1. Summary of the purification process of cypermethrin hydrolase from Pseudomonas aeruginosa GF31.

Purification step

Protein

Total

Specific

concentration

activity

activity

(U)

(U/mg)

Volume (mL) (mg/mL)

Yield

Purification

(%)

(Fold)

Fermentation 2170

0.334

56854

78.4

100

1

Ultrafiltration

260

1.225

34567

108.4

60.8

1.4

(NH4)2SO4

23

4.387

29734

294.7

52.3

3.8

DEAE-Sepharose CL-6B

28

0.543

22230

1462.1

39.1

18.6

G75

24

0.245

15123

2571.9

26.6

32.8

liquor



Page 30 of 37

30 Table 2. Effect of various metallic ions on the relative activity of the enzyme. Substance

Relative activity (%)

Substance


None

100

1 mM NiSO4

85±0.8

1 mM LiCl

94±2.5

1 mM ZnSO4

82±0.8

1 mM KCl

84±0.7

1 mM Al(NO3)3

87±2.2

1 mM CaCl2

85±3.6

1 mM Co(NO3)2

94±1.5

1 mM MgSO4

85±1.3

1 mM CrCl3

90±0.7

1 mM MnSO4

89±1.0

1 mM CuSO4

66±2.5

1 mM FeCl3

102±1.7

1 mM AgNO3

56.7±1.2

1 mM BaCl2

83±3.9

1 mM Hg(NO3)2

29±0.5

1 mM Pb(NO3)2

90±1.6

_

_


Page 31 of 37


31 Table 3. Effect of various inhibitors on the relative activity of the enzyme. Substance

Relative Activity (%)

None

100

10 mM 1,10-phenanthroline

98±2.5

10 mM EDTA

101.5±4.2

10 mM Triton-100

48.6±3.1

10 mM SDS

31.1±0.8

0.5 mM Malathion

92±1.6

0.5 mM PMSF

93±1.4



Page 32 of 37

32 Table 4. Kinetic constants for various pesticides and p-nitroanilide derivatives. Specific activity (U/mg)

Km (µM)

Vmax (µM/s/mg protein)

β-cypermethrin

8.5±0.1

47.7

0.0017

0.008

0.17

Fenpropathrin

3.1±0.2

42.1

0.0014

0.0066

0.16

Fenvalerate

1.8±0.3

26.5

0.0012

0.0056

0.21

deltamethrin

2.8±0.1

11.8

0.0011

0.0051

0.43

Leu-pNA

22312.6±658.2

2668.8

0.99

183.3

68.7

Ala-pNA

1276.9±79.0

6507.9

0.47

17.4

2.7

Arg-pNA

2540.0±15.8

2986.9

2.05

23.6

7.9

Substrate

Kcat (s-1)


Kcat/Km (mM-1s-1)

Page 33 of 37


33 Figure graphics

Figure 1.



Page 34 of 37

34

100


80

60

40

20

0 3

4

5

6

7

8

9

PH

Figure 2.


Page 35 of 37


35

100


80

60

40

20

0 20

30

40

50

Temperature (

60

70

80

)

Figure 3.



Page 36 of 37

36

Figure 4.


Page 37 of 37


37 Table of Contents Graphics


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