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Efficient degradation of malathion in the presence of detergents using an engineered organophosphorus hydrolase highly expressed by Pichia pastoris without methanol induction Yun-Peng Bai, Xiao-Jing Luo, Yu-Lian Zhao, Chun-Xiu Li, Dian-sheng Xu, and Jian-He Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03405 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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Journal of Agricultural and Food Chemistry

1


2

Efficient degradation of malathion in the presence of detergents

3

using an engineered organophosphorus hydrolase highly expressed

4

by Pichia pastoris without methanol induction

5

Yun-Peng Bai, 1,* Xiao-Jing Luo,1 Yu-Lian Zhao,2 Chun-Xiu Li,1 Dian-Sheng Xu 2

6

and Jian-He Xu1,*

7

1

8

East China University of Science and Technology, Shanghai 200237, P. R. China.

9

*Corresponding authors. Tel.: +86-21-6425-2498; Fax: +86-21-6425-0840; E-mails:

10

State Key Laboratory of Bioreactor Engineering, and

2

School of Biotechnology,

[email protected] (Y.P. Bai); [email protected] (J.H. Xu).

1

ACS Paragon Plus Environment


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Abstract

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The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an

13

efficient enzyme production technology in industry. Herein, a Pichia pastoris strain

14

was constructed for the extracellular expression of PoOPHM9, an engineered

15

malathion-degrading enzyme. After optimization, the maximum titer and yield of

16

fermentation reached 50.8 kU/L and 4.1 gprotein/L after 3 days, with the highest

17

space-time yield (STY) reported so far, 640 U·L-1·h-1. PoOPHM9 displayed its high

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activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04

19

mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution

20

within 20 min. Furthermore, 12 µmol malathion on apples and cucumbers surfaces

21

was completely removed by 0.05 mg/mL PoOPHM9 in tap water after 35 min washing.

22

The efficient production of the highly active PoOPHM9 has cleared a major barrier to

23

biodegradation of pesticide residues in food industry.

24

Keywords:

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extracellular expression; malathion

biodegradation;

Pichia

pastoris;

organophosphorus

2


hydrolase;

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Introduction

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Organophosphates (OPs) are a class of synthetic chemicals that are widely used as the

28

effective components in pesticides, plasticizers, flame retardants and chemical warfare

29

agents. Malathion was first registered for use as an OP insecticide in the US in 1956

30

by the U.S. Department of Agriculture, and is currently widely used for agricultural

31

production, residential landscaping and mosquito eradication.1 It was estimated by the

32

U.S. Environmental Protection Agency that over 30 million pounds of malathion were

33

used in 1998.2 Some metabolites degraded or metabolized from malathion are toxic,

34

e.g. malaoxon, and the accumulation of malathion and its metabolites on agricultural

35

products has raised serious concerns regarding food safety and public health.3 Novel

36

bacteria capable of degrading OP pesticides have been discovered for removal of

37

toxic compounds from contaminated soil and water.4-6 However, enzyme-catalyzed

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biodegradation has been regarded as a safe, clean and efficient technology for

39

removing residual OPs for decontamination in end-use applications in the food

40

industry, for example in fruit and vegetable washing.7

41

In

recent

years,

several

organophosphorus

hydrolases

such

as

42

phosphotriesterase,8-13 OpdA,14 MPH15-16, and SsoPox17, and so on, have been

43

engineered by directed evolution to improve their activity and thermal stability for

44

biodegradation. In previous studies, we proposed a hierarchical iteration mutagenesis

45

strategy to perform combinatorial evolution of a newly-discovered phosphotriesterase

46

(PoOPH),10 resulting in a mutant named PoOPHM9 (V55I, L57G, K83G, T114A,

47

L137I, M188F, H250I, V262A, I263W) that possesses good thermal stability and high 3



48

activity toward malathion.18 However, the application of these evolved enzymes is

49

still hindered by the lack of highly-productive host strains that can express enzymes

50

with a cost low enough for large-scale industrial fermentation. For instance, the

51

expression level of PoOPHM9 in Escherichia coli was very low, and autolysis

52

occurred during high-density fermentation in our previous study. More importantly,

53

the biodegradation effect of the enzyme developed in lab should be demonstrated

54

under conditions where commercially available detergents are used together with the

55

enzyme during a washing process. Recently, Manco and co-workers evolved an

56

efficient OPH, SsoPox, and tested its performance together with two detergents for the

57

cleaning of pesticide-contaminated surfaces,17 although large-scale production of this

58

enzyme was not reported.

59

Yeasts are single-celled eukaryotes used widely for expressing proteins that cannot

60

be properly folded or correctly glycosylated in E. coli. Pichia pastoris is an attractive

61

choice of host species because it allows simple genetic manipulation and strict

62

transcription regulated by the promoter alcohol oxidase (AOX). P. pastoris can also

63

constitutively express proteins using the glyceraldehyde-3-phosphate dehydrogenase

64

(GAP) promoter. In the GAP system, cell growth and protein expression are

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performed simultaneously without induction.19 Although P. pastoris has been

66

successfully employed for expression of OPHs,20-21 the expression levels were not

67

satisfactory for industrial requirements. Recently, Yan et al. reported a

68

high-OPH-producing P. pastoris strain,22 but the productivity relied on multiple

69

copies of plasmids assembled in the strain; plasmids may be lost during multi-batch 4


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fermentation. In addition, the induction of protein expression required methanol,

71

which increases the fermentation cost and potential safety risks in industrial

72

production.

73

In this work, several recombinant P. pastoris strains were constructed bearing AOX

74

or GAP promoters and their productivities were compared in high-density

75

fermentation for the extracellular expression of PoOPHM9. The secretory protein

76

PoOPHM9 was further used to degrade malathion in aqueous solution together with

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various commercially available detergents, and its degradation effects were

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systematically investigated in different conditions to mimic real application situations

79

in decontamination processes. The high expression level and high tolerance of

80

detergents demonstrate that PoOPHM9 is a promising candidate for the effective

81

removal of residual organophosphates in end-use applications.

82

Materials and methods

83

Materials

84

All chemicals of reagent grade were purchased from Sigma (St. Louis, MO).

85

Malathion (99% purity) was purchased from Shanghai Pesticide Research Center.

86

Restriction enzymes, DNaseI, T4 DNA ligase, and Taq DNA polymerase were

87

purchased from New England Biolabs (Beverley, MA). Exnase II ligase was

88

purchased from Vazyme Biotech (Nanjing, China). P. pastoris strains X33 (Mut+His+)

89

and GS115 (Mut+His-) were stored in our lab. Methanol of analytical grade was

90

purchased from Titan (Shanghai, China). Glucose and glycerol of analytical grade

91

were purchased from Lingfeng Chemicals (Shanghai, China). The expression vectors 5



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pPIC9k, pPICZαA and pGAPZαA were purchased from Invitrogen (Carlsbad, CA).

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The gene encoding the mutated phosphotriesterase (poophm9) was described

94

previously.18 Recipes of all media used are described in the supporting information.

95

Detergents containing coconut oil derivatives (COD, Komi, Lot 6907974981509),

96

Sodium dodecyl sulphate (SDS, Mama Lemon, Lot 6903624600158) and alkyl

97

polyglycoside (APG, Diaopai, Lot 6910019001841), apples, cucumbers and cotton

98

tissue were purchased from a local supermarket without further treatment.

99

Construction of plasmids

100

pPIC9k-poophm9,

pPICZαA-poophm9

and

pGAPZαA-poophm9:

The

101

poophm9 gene was inserted into plasmids pPIC9k, pPICZαA and pGAPZαA

102

following the normal protocols for the molecular kits used.

103

pPIC9k-∆poophm9: The ∆poophm9 gene (which lacks the nucleotides encoding

104

a predicted N-terminal signal sequence of PoOPHM9) was directly linked after the

105

α-signal peptide reading frame, replacing the poophm9 gene in pPIC9k-poophm9 via

106

seamless cloning. First, the pPIC9k-poophm9 plasmid was linearized and amplified

107

by PCR. The PCR mixture contained 1 µL pPIC9k-poophm9, 3.0 µL primers (Table

108

S1), 1 µL KOD-plus-Neo polymerase, 5 µL 10× PCR buffer, 5 µL 2 mM dNTPs, 3 µL

109

25 mM MgSO4 and 32 µL deionized (DI) water. The PCR procedure was: 94°C for 2

110

min; followed by 30 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 5 min; and a

111

final extension at 68°C for 5 min. After PCR, 2 µL of DpnI was added to digest the

112

template plasmid at 37°C for 2 h. After agarose gel electrophoresis, the linearized

113

plasmid was recovered. Ligation was performed in 10 µL CE II buffer containing 90 6


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ng linearized plasmid, 20 ng ∆poophm9 fragment and Exnase II ligase at 37°C for 30

115

min. After reaction, the mixture was cooled for 5 min in an ice bath. Then, the

116

recombinant plasmid was transformed into E. coli Top 10, which were subsequently

117

cultured at 37°C in 900 µL Luria-Bertani (LB) medium for 1 h. The culture was then

118

streaked onto an agar plate containing 50 mg/L kanamycin and incubated at 37°C

119

overnight. The plasmid pPIC9k-∆poophm9 was extracted from positive clones.

120

pPICZαA-∆poophm9 and pGAPZαA-∆poophm9: The ∆poophm9 gene was

121

directly linked after the α-signal peptide reading frame. The pPICZαA, pGAPZαA

122

plasmids and the ∆poophm9 gene fragment were double digested with restriction

123

enzymes at 37°C for 3 h as follows: 48 µL gene or empty plasmid, 6 µL 10× buffer,

124

and 3 µL XhoI, 3 µL NotI. The fragments were recovered and ligated at 16°C for 2h as

125

follows: 2 µL digested plasmid, 6.5 µL digested gene fragment, 1 µL buffer, 0.5 µL T4

126

ligase. Recombinant plasmids were recovered as described above.

127

Construction of the recombinant P. pastoris cells

128

Preparation of linearized recombinant plasmids: pPIC9k-∆poophm9 and

129

pPICZαA-∆poophm9 were digested by SalI, and pGAPZαA-∆poophm9 was digested

130

by AvrII. The digestions were performed in a mixture of 6 µL 10× buffer, 1–10 µg

131

plasmid, 3 µL restriction enzyme, and 51 µL DI water, at 37°C for 3 h. The digested

132

products were collected, purified and dissolved in 30 µL DI water.

133

Transformation of P. pastoris: Linearized plasmids (10 µL) were added to an

134

80-µL suspension of competent P. pastoris cells and mixed in a precooled cup for 5

135

min. After electroporation, the mixture was immediately transferred to 500 µL 7



136

precooled 1 M sorbitol and 500 µL YPD medium. The suspension was placed at 30°C

137

for 1 h, and then cultured in a shaker at 30°C and 220 rpm for 2 h. Cell culture (200

138

µL) was streaked onto MD solid medium or YPD solid medium containing 300–2000

139

mg/L zeocin and cultured at 30°C for 3 days to obtain positive clones.

140

Screening of recombinant P. pastoris strains for high expression of PoOPHM9

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Screening of GS115/pPIC9k-∆poophm9 and X33/pPICZαA-∆poophm9:

142

Single clones were picked up from solid culture medium, inoculated into 25 mL

143

BMGY medium and cultured at 30°C with shaking at 300 rpm for approximately 18 h

144

until the OD600 reached 2–6. Afterwards, the cells were suspended in BMMY medium

145

to OD600 = 1.0. The cell suspension was cultured at 30°C and 300 rpm for the

146

induction of PoOPHM9, and methanol was added every 24 h to a final concentration of

147

1% (v/v). Aliquots of the broth supernatant were withdrawn every 24 h to measure the

148

enzyme activity.

149

Screening of X33/pGAPZαA-∆poophm9: Single clones were picked up from

150

solid culture medium, inoculated into 5 mL YPD medium, and cultured at 30°C and

151

300 rpm overnight. Culture (50 µL) was taken and inoculated again into 25 mL YPD

152

medium at 30°C and 300 rpm. The supernatant of the culture broth was sampled every

153

24 h to measure the enzyme activity.

154

Fermentation

155

Strain X33/pPICZαA-∆poophm9 was employed for a three-step fermentation and

156

enzyme expression was induced by the addition of methanol. In contrast, strain

157

X33/pGAPZαA-∆poophm9 was exploited to express enzymes via a two-step 8


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fermentation in a constitutive manner.

159

In a typical three-step fermentation, seed culture was inoculated into the

160

fermentation medium (BSM) added with 8.7 mL trace-element solution PTM1 (see SI

161

for details), and the dissolved oxygen (DO) was maintained at >20% by adjusting the

162

agitation speed. After fermentation was conducted for 18–24 h, glycerol was depleted

163

and the DO level increased rapidly up to 100%. Then, a glycerol solution (50% v/v)

164

containing 12 mL/L PTM1 was fed at a rate of 18 mL/h until the OD600 reached

165

approximately 200 when the feeding was stopped. After 45 min, methanol containing

166

12 mL/L of trace-element solution PTM1 was fed in at a rate increasing gradually

167

from 3 to 12 mL/h. Aliquots of supernatant were taken at fixed time intervals to

168

measure the enzyme titers of the culture broth. The fermentation was terminated when

169

the enzyme production did not increase further.

170

In a typical two-step fermentation process, seed culture was inoculated into BSM

171

or FM22 medium, and the DO was kept above 20% by adjusting the agitation speed.

172

After the fermentation was performed for 18–24 h, glycerol or glucose was depleted

173

and the DO increased rapidly up to 100%. Then, a glycerol solution (50%, v/v)

174

containing 12 mL/L PTM1 was fed at a rate of 18 mL/h until the enzyme titer of the

175

broth supernatant did not increase further.

176

Measurement of PoOPHM9 activity

177

The production of PoOPHM9 in cell culture broth was monitored by determining

178

the enzyme activity, i.e., the increase in absorbance at 412 nm caused by the reaction

179

between

the

hydrolysis

product,

mercaptan,

and

9


the

Ellman

reagent,


180

5,5'-dithiobis-(2-nitrobenzoic acid) (DNTB).18 The assay was performed in 96-well

181

half-area plates. Each reaction contained 1 mL of 45 mM Tris-HCl buffer (pH 9.0)

182

containing final concentrations of 1.8 mM DNTB, 0.5 mM malathion and 10% (v/v)

183

cell culture. Absorbance readings were taken every min at 30°C using a PowerWave

184

XS2 spectrophotometer (BioTek, USA). One unit of enzyme activity was defined as

185

the amount of enzyme that catalyzed the hydrolysis of 1 µmol malathion per min in

186

the above conditions.

187

Degradation of malathion and malaoxon by PoOPHM9 in the presence of

188

detergents

189

The degradation of malathion and malaoxon in aqueous solution was monitored

190

by measuring absorbance of the reaction mixture at 412 nm. Typically, 980 µL of 50

191

mM Tris-HCl buffer (pH 9.0) containing 0.3 mM DNTB and 0.1% (w/w) of a

192

detergent was mixed with 10 µL of 15 mM malathion or malaoxon and 10 µL of 4

193

mg/mL PoOPHM9. The mixture was kept at 25°C for 20 min and absorbance readings

194

were taken every minute. The absorbance of completely hydrolyzed product was used

195

as the positive control. The hydrolysis percentage was calculated by comparing the

196

absorbance at different times with the positive control. To confirm the degradation

197

effect, the reaction mixture was immediately extracted with 1 mL ethyl acetate after

198

the reaction, and the organic supernatant was subsequently subjected to GC-MS

199

analysis. To determine the enzyme stability in detergent-containing solutions, the

200

residual activity was measured at different times during a 60-min incubation in 50

201

mM Tris-HCl buffer (pH 9.0) containing 0.5 mM malathion, 0.01 mg/mL pure 10


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enzyme, and 1 mg/mL DNTB.

203

Decontamination of malathion and malaoxon on apples, cucumbers and cotton

204

tissues in tap water by PoOPHM9

205

Apples and cucumbers: Fresh apples (477 g) or cucumbers (300 g) were

206

contaminated with 12 µmol malathion. The analytical method for evaluating the

207

removal effect of malathion was modified and applied in a 2-L reaction system

208

according to the previous study.17 After 30 min, apples or cucumbers were submerged

209

in a 2-L solution of tap water (pH 7.5) containing 5 mM Tris-HCl, 50 µg/mL

210

PoOPHM9 and 0.02 g DNTB. The solution was stirred for 30 min at 25°C, and

211

aliquots of the washing solution were withdrawn at different times and analyzed at

212

412 nm. For malaoxon, apples and cucumbers were contaminated with 3 µmol

213

malaoxon and subsequently washed with 42 µg/mL PoOPHM9 in 250 mL tap water

214

under the same condition described above.

215

Cotton tissues: Cotton clothes were cut into squares (25×25 cm), and each

216

surface of the tissues was contaminated with 7.5 µmol malathion. The tissues were

217

stood for 30 min and then submerged in 2 L tap water (pH 7.5) containing 0.02 g

218

DNTB, 5 mM Tris-HCl and 20 µg/mL of PoOPHM9. The solution was stirred for 30

219

min at 25°C, and aliquots of the washing solution were withdrawn at different times

220

and analyzed at 412 nm. The hydrolysis percentage was calculated as described above.

221

For malaoxon, cotton clothes were contaminated with 3 µmol malaoxon and

222

subsequently washed with 42 µg/mL PoOPHM9 in 250 mL tap water under the same

223

condition described above. 11



224

GC-MS analysis: Before washing, one apple (300 g), one cucumber (197 g),

225

and one piece of cotton tissue (25 × 25 cm) after contamination with malathion were

226

selected and extracted with 6.0 mL ethyl acetate, and 1.0 mL of the extract solution

227

was subjected to GC-MS analysis. After washing, the same apple, cucumber and

228

tissue were dried and extracted with 6.0 mL ethyl acetate, and 1.0 mL of the solution

229

was analyzed by GC-MS. Washing solution (1 mL) was also taken at 35 min and

230

extracted with 1.0 mL ethyl acetate, which was then analyzed by GC-MS. GC-MS

231

analysis was conducted using a Shimadzu GCMS-QP2010 SE equipped with a

232

DB-5MS quartz capillary column (0.25 µm × 0.25 mm × 30m). High-purity helium of

233

was used as the carrier gas (1.0 mL/min). The temperature of the inlet was set to

234

270°C, and the column temperature started at 50°C, was held for 1 min, and then

235

increased to 180°C at 20°C/min. 180°C was maintained for 2 min, and then increased

236

to 240°C at the rate of 10°C/min. Electron ionization was used with an electron

237

energy of 70 eV and temperature of 230°C. The temperatures at the quadrupole and

238

the transmission line were set to 150°C and 280°C, respectively. Selected ion monitor

239

was used as the detection mode and the characteristic peaks of malathion were

240

compared and confirmed with the database in the software.

241

Results and Discussion

242

Strain construction and optimization of fermentation conditions. The codon

243

usage of the PoOPHM9 coding gene was optimized. As Figure S1 shows, the gene

244

contains no rare codon and has a codon adaptation index of 0.97 after optimization.

245

The poophm9 gene encodes a putative signal peptide of 24 amino acids at the protein 12


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246

N-terminus. To avoid interference with enzyme secretion, this signal peptide was

247

removed to yield a gene named ∆poophm9, which was linked directly after the

248

reading frame of the α-signal peptide in the expression plasmids.

249

Subsequently, three plasmids (pPIC9k, pPICZαA and pGAPZαA) were

250

constructed for transformation of P. pastoris (Figure S2; see Table S1 for the details

251

of primers). After ligation, pPIC9k-∆poophm9 was transformed into P. pastoris strain

252

GS115, whereas pPICZαA-∆poophm9 and pGAPZαA-∆poophm9 were transformed

253

into strain X33 as suggested by the protocol of plasmids. Since the location and copy

254

number of the recombinant gene may influence protein expression, transformed cells

255

were screened for strains with the highest expression level before fermentation.

256

The average malathion-degrading activity of X33/pPICZαA-∆poophm9 was

257

higher than that of GS115/pPIC9k-∆poophm9 (Figure 1A). Since both strains have

258

AOX promoters, a clone of X33/pPICZαA-∆poophm9 that displayed the highest

259

activity, 2.39 kU/L, was selected for fermentation. Meanwhile, the activity of

260

X33/pGAPZαA-∆poophm9, which has the GAP promoter, was screened (Figure 1B),

261

and a clone with the highest activity (353 U/L) was selected.

262

The selected strains were cultured in shaken-flasks for optimization of

263

fermentation conditions (Figure 2). For X33/pPICZαA-∆poophm9 strain, the highest

264

fermentation activity was found at pH 7.0 with 1.5% methanol as the inducer. The

265

enzyme production in the culture increased with induction time and the maximum

266

activity was observed after 4 days. For strain X33/pGAPZαA-∆poophm9, the

13



Page 14 of 36

267

optimum pH was 7.0, and the best carbon source was glucose, followed by glycerol,

268

sorbitol and methanol.

269

Fermentation for high extracellular expression of PoOPHM9. Firstly, we used

270

recombinant E. coli BL21 (DE3) as the control for fermentation in a 5-L tank. The

271

fermentation was short (18 h), (Figure S3A), and protein expression was quite low

272

because most of the protein was in inclusion bodies (Figure S3B). The final enzyme

273

titer was 3.6 kU/L with a protein concentration of only 0.1 g/L, indicating that E. coli

274

is not suitable for PoOPHM9 production on a large scale.

275

Then we selected the most productive clone of P. pastoris strain

276

X33/pPICZαA-∆poophm9 for a three-step fermentation. The fermentation was

277

performed in BSM at pH 5.0 and 6.0 with DO maintained above 20% during the

278

process. As Figure 3 shows, the maximum titer was 38.7 kU/L at pH 5.0 and 47.6

279

kU/L at pH 6.0. Higher activity at pH 6.0 than at pH 5.0 was consistent with the

280

results of shaken-flask fermentations. At pH 7.0, protease was produced in

281

high-cell-density fermentation and salts in the medium precipitated; thus, pH 6.0 was

282

used in the following experiments. After purification, the specific activity of

283

PoOPHM9 was 12.5 U/mg, and the protein yield was 3.8 g/L.

284

Strain X33/pGAPZαA-∆poophm9 was also employed for fermentation in FM22

285

medium containing glucose and BSM containing glycerol (Figure 4). Fermentation

286

with glycerol resulted in a slightly higher titer (50.8 kU/L) and an enzyme yield of 4.1

287

g/L. Therefore, BSM containing glycerol is more suitable for the constitutive

288

expression

of

∆poophm9.

Sodium

dodecyl

sulphate-polyacrylamide

14


gel

Page 15 of 36


289

electrophoresis (SDS-PAGE) analysis showed that protein degradation did not occur

290

in the constitutive expression, but it was observed at 72 h in induced-fermentation

291

using strain X33/pPICZαA-∆poophm9 (Figure S4). PoOPHM9 was readily recovered

292

from the fermentation broth and freeze-dried to give enzyme powder for convenient

293

storage and further study.

294

The fermentation productivities of PoOPHM9 in different recombinant strains are

295

listed in Table 1. The space-time yields (STYs) of strains X33/pPICZαA and

296

X33/pGAPZαA reached 850 and 640 U·L-1·h-1, respectively, which were much higher

297

than the value 200 U·L-1·h-1 for E. coli. Because of the high fermentation titer and the

298

short fermentation time, the STYs of the two strains investigated in detail in this work

299

are also significantly better than that of 70 U·L-1·h-1 for recombinant P. pastoris for

300

production of another OPH (OPHC2)16. Yp/x and Yp/s, the ratios of the protein mass

301

divided by the dry cell weight and by the weight of consumed carbon source, were

302

45.0 and 18.0 mg/g for strain X33/pGAPZαA, respectively. Of the two recombinant P.

303

pastoris strains, X33/pGAPZαA showed higher titer and protein yield without enzyme

304

degradation or the need for methanol induction. Therefore, it is a better candidate

305

strain for PoOPHM9 production. The material cost of 100 kU enzyme produced by

306

fermentation of X33/pGAPZαA with glucose as the carbon source was only 11.0

307

RMB (approximately 1.63 US dollars) (Table S2), which seems cheap enough for

308

large-scale production of PoOPHM9 in industry.

309

Effect of detergents on PoOPHM9 activity. It has been reported that SDS can

310

activate phosphotriesterase (PTE) and enhance its activity,17, 23 but other types of 15



311

surfactants have not yet been tested. To clarify the effect of detergents on PoOPHM9

312

activity, its kinetic parameters were measured in the presence of three different

313

detergents, including a natural surfactant containing coconut oil derivatives (COD)

314

extracted from plants, an anionic detergent containing SDS, and a non-ionic detergent

315

containing alkyl polyglycoside (APG). In the absence of detergent, PoOPHM9

316

displayed specific activity of 35.0 U/mg, but this decreased to 8.7, 3.8 and 3.0 U/mg

317

in the presence of 0.1% (w/w) COD, APG and SDS, respectively (Table 2). The kcat

318

decreased in all three cases (24.2 ± 0.32 s−1 for PoOPHM9), with the highest and the

319

lowest values of 6.5 ± 0.17 s−1 and 1.8 ± 0.03 s−1 in the presence of COD and SDS,

320

respectively. Interestingly, the Km value for PoOPHM9 (91.2 ± 5.3 µM) also decreased,

321

to 40.2 µM, in the presence of SDS, consistent with a previous report.17 Although the

322

Km value increased for PoOPHM9 in the presence of COD (197.1 µM) and APG

323

(139.1 µM), these values were still lower than the Km values of ArPTES308L/Y309A (410

324

µM)24 and PTEM7 (290 µM)25 reported previously, which is favored because

325

hydrolysis of pesticides is usually conducted at low substrate concentration (0.1–10

326

ppm). The catalytic efficiency (kcat/Km) of PoOPHM9 in the presence of the three

327

detergents decreased compared to that of free enzyme. Therefore, PoOPHM9 is not

328

activated by detergents.

329

Degradation of malathion and malaoxon by PoOPHM9 in the presence of

330

detergents. In daily life or in the food industry, detergents are often used in washing

331

fruits and vegetables. To remove the residual malathion in food that detergents cannot

332

degrade, it is always expected that enzymes can be used together with detergents. 16


Page 16 of 36

Page 17 of 36


333

Therefore, it is necessary to evaluate PoOPHM9 in the presence of commercially

334

available detergents for degrading malathion in aqueous solution. First, the time

335

course of enzymatic hydrolysis of malathion was investigated in the presence of the

336

above detergents. As Figure 5A shows, about 80% of 0.15 mM malathion was

337

degraded by 0.04 mg/mL PoOPHM9 in only 5 min at room temperature without any

338

detergents, and 100% of malathion was removed within 20 min. PoOPHM9 was able

339

to remove 100% of malathion in the presence of COD in 20 min, but the initial

340

degradation rate was lower than that of the free enzyme. PoOPHM9 was less effective

341

in the presence of APG because its initial degradation rate was lower, and the final

342

hydrolysis percentage reached only 95%. Notably, the hydrolysis rate in the presence

343

of SDS was the lowest, with a final hydrolysis percentage of 89% at 20 min. Although

344

PoOPHM9 was specially engineered to improve its activity toward malathion, it shows

345

a moderate specific activity of 6.7 U/mg toward malaoxon which is 22 times more

346

toxic than malathion (Malathion: Human Health Draft Risk Assessment for

347

Registration Review, US-EPA, 2016). Thus, we also tested the degradation effect of

348

malaoxon with 0.04 mg/mL PoOPHM9 (Figure 5B). Again, 0.15 mM malaoxon in 1

349

mL solution was completely degraded with free enzyme or in the presence of 0.1%

350

COD, but the degradation rate decreased significantly in the presence of 0.1% APG

351

and 0.1% SDS with 80% and 60% hydrolysis percentage of malaoxon at 20 min,

352

respectively. Interestingly, PoOPHM9 showed higher activity in COD than in APG

353

and SDS in degradation of malathion and malaoxon, indicating that COD is more

354

suitable to use together with the enzyme. To confirm the degrading effect, the reaction 17



355

solutions of degrading malathion and malaoxon in the absence or in the presence of

356

0.1% COD were analysed by GC-MS after reaction (Figure S5). Clearly, no residual

357

malathion and malaoxon were detected in the samples, demonstrating the

358

effectiveness of PoOPHM9 in removing malathion and malaoxon.

359

Subsequently, we increased the concentration of detergent in the solution to 1%

360

and 10% (w/w) to investigate the influence of detergent concentration on degradation

361

(Figure 5C). In the presence of 1.0% APG and SDS, the hydrolysis percentage

362

decreased to 24% and 17% respectively after 20 min, and it dropped further to 6% and

363

0% in the presence of 10% detergent. Notably, the hydrolysis of malathion by

364

PoOPHM9 was less affected by the presence of COD, since it degraded 73% and 31%

365

malathion in the presence of 1% and 10% COD, respectively. The stability of

366

PoOPHM9 in detergents was tested by measuring the residual activity of the enzyme in

367

solutions containing 0.1% detergent at different times during 1-h incubation of the

368

aqueous solution (Figure 5D). PoOPHM9 maintained approximately 70% residual

369

activity in 0.1% COD after 1 h, which was close to the activity of free enzyme in

370

solution without added detergent. However, the activity of the enzyme in solutions

371

containing 0.1% SDS and 0.1% APG decreased dramatically, to 25% after 1 h. These

372

results indicate that the plant-derived detergent COD has the best biocompatibility

373

with PoOPHM9. The stability of OPH in the surfactant-containing solution is

374

influenced by the interaction of the enzyme with the surfactant interface.26 The

375

structural parameters and the phase behaviour of the micelles formed in the solution

376

can change the kinetic properties of OPH. Polymers that can associate with OPH to 18


Page 18 of 36

Page 19 of 36


377

form different structures were used to enhance the activity and stability of OPH.27-28

378

However, due to the complex composites in the natural extract of coconut oil

379

derivatives and the various micelle structures, the mechanism of the interaction

380

between OPH and COD is unclear to date, which will need more effort to reveal the

381

influence of COD on the stability of OPH.

382

Degradation of malathion and malaoxon from contaminated apples,

383

cucumbers and cotton tissue by PoOPHM9. To further demonstrate the

384

decontamination efficiency of PoOPHM9, fresh apples, cucumbers and clean cotton

385

tissues were contaminated with 12 or 7.5 µmol malathion and then washed in 2 L tap

386

water (pH 7.5) with a dosage of 50 or 20 µg/mL enzyme (Figure 6A–C). With

387

malathion-contaminated apples and cucumbers, tap water containing PoOPHM9

388

hydrolyzed 80% and 100% of the malathion after 10 and 35 min, respectively (Figure

389

6D). The hydrolysis rate of contaminated cotton tissues was slower, and ~80% of the

390

malathion was removed after 20 min. Extending the washing time helped with

391

decontamination of malathion from tissues as 96.7% of the malathion was removed

392

after 35 min. To confirm if the residual malathion was completely removed, samples

393

from the surfaces and the washing solutions before and after decontamination were

394

analyzed by GC-MS (Figure S5). At the beginning (t = 0 min), malathion was

395

detected in all the samples extracted by ethyl acetate from the surfaces of apples,

396

cucumbers and cotton tissues. At 35 min, the corresponding malathion peak

397

disappeared from samples from the surfaces and the washing solutions, demonstrating

398

malathion was completely hydrolyzed by PoOPHM9. Again, these results indicate that 19



399

PoOPHM9 secreted from P. pastoris has high activity in detoxifying malathion from

400

contaminated fruits and vegetables in conditions similar to everyday washing.

401

Furthermore, we also used 42 µg/mL PoOPHM9 to decontaminating 3 µmol malaxon

402

on apples, cucumbers and cotton tissues in 250 mL tap water (Figure 6E). 100%, 93%

403

and 87% malaoxon on cucumbers, cotton tissues and apples had been removed after

404

35 min, respectively. Compared with the degradation of malathion, malaoxon is more

405

difficult to degrade because PoOPHM9 is intrinsically engineered to improve its

406

activity toward malathion. Although the catalytic efficiency (kcat/Km) of degrading

407

malaoxon is one magnitude lower than that of malathion (1.3×104 vs. 2.6×105 M-1S-1),

408

the application of PoOPHM9 in a washing process could still effectively remove the

409

majority of residual malaoxon on fruits and clothes.

410

In this study, P. pastoris strain X33/pGAPZαA-∆poophm9 was constructed for

411

constitutive secretory expression of the phosphotriesterase PoOPHM9. Titer of 50.8

412

kU/L was achieved, higher than reported for other constructed recombinant strains.

413

This strain also shows the highest space-time yield for the constitutive expression of

414

an OPH in the literature, 640 U·L-1·h-1. Moreover, the fermentation requires no

415

addition of inducers such as methanol, which facilitates the operation process in

416

industrial production. The secretory enzyme shows good activity and stability in

417

degrading malathion in aqueous solution. In the presence of 0.1% (w/w) of the

418

plant-derived detergent COD, 0.04 mg/mL PoOPHM9 could hydrolyze 100% of 0.15

419

mM malathion within 20 min, and the enzyme could hydrolyze 31% of the malathion

420

in the presence of 10% (w/w) COD. Notably, PoOPHM9 dissolved in tap water could 20


Page 20 of 36

Page 21 of 36


421

also effectively degrade the residual malathion on the surfaces of apples, cucumbers

422

and cotton tissues (depending on the concentrations of malathion and enzyme used).

423

Herein,

424

biodegradation clears a major hurdle on the way to real application of phosphorous

425

hydrolases in detoxification/decontamination, e.g. for fruit and vegetable washing in

426

the food industry.

427

Supporting information

the

combination

of

highly-productive

fermentation

and

efficient

428

Additional experimental results including the construction of plasmids,

429

SDS-PAGE analysis of the protein production in fermentation and other data are

430

available free of charge via the Internet at http://pubs.acs.org.

431

Acknowledgements

432

This work was financially sponsored by the National Key Research and

433

Development Program of China (2016YFA0204300), National Natural Science

434

Foundation of China (Nos. 21505044, 21506055 & 21536004) and Shanghai Pujiang

435

Program (15PJ1401200).

436

References

437

(1) U.S. EPA. Malathion. URL (https://www.epa.gov/mosquitocontrol/malathion.)

438

(2) Donaldson, D. K.; Kiely A. G. Pesticides industry sales and usage: 1998 and 1999

439

market estimates. U.S.EPA. Office of Pesticide Programs. 2002, 14-15.

440

(3) Singh, B. K. Organophosphorus-degrading bacteria: ecology and industrial

441

applications. Nat. Rev. Microbiol. 2009, 7 (2), 156-164.

442

(4) Ishag, A. E.; Abdelbagi, A. O.; Hammad, A. M.; Elsheikh, E. A.; Elsaid, O. E.; 21



443

Hur, J. H.; Laing, M. D. Biodegradation of chlorpyrifos, malathion, and dimethoate

444

by three strains of bacteria isolated from pesticide-polluted soils in Sudan. J. Agric.

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Food Chem. 2016, 64 (45), 8491-8498.

446

(5) Tian, J.; Dong, Q.; Yu, C.; Zhao, R.; Wang, J.; Chen, L. Biodegradation of the

447

organophosphate trichlorfon and its major degradation products by a novel

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Aspergillus sydowii PA F-2. J. Agric. Food Chem. 2016, 64 (21), 4280-4287.

449

(6) Wu, X.; Wang, W.; Liu, J.; Pan, D.; Tu, X.; Lv, P.; Wang, Y.; Cao, H.; Wang, Y.;

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Hua, R. Rapid biodegradation of the herbicide 2,4-dichlorophenoxyacetic acid by

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Cupriavidus gilardii T-1. J. Agric. Food Chem. 2017, 65 (18), 3711-3720.

452

(7) Theriot, C. M.; Grunden, A. M. Hydrolysis of organophosphorus compounds by

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microbial enzymes. Appl. Microbiol. Biotechnol. 2011, 89 (1), 35-43.

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(8) Cherny, I.; Greisen, P. J.; Ashani, Y.; Khare, S. D.; Oberdorfer, G.; Leader, H.;

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Baker, D.; Tawfik, D. S. Engineering V-type nerve agents detoxifying enzymes using

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computationally focused libraries. ACS Chem. Biol. 2013, 8 (11), 2394-2403.

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(9) Khare, S. D.; Kipnis, Y.; Greisen, P. J.; Takeuchi, R.; Ashani, Y.; Goldsmith, M.;

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Song, Y. F.; Gallaher, J. L.; Silman, I.; Leader, H.; Sussman, J. L.; Stoddard, B. L.;

459

Tawfik, D. S.; Baker, D. Computational redesign of a mononuclear zinc

460

metalloenzyme for organophosphate hydrolysis. Nat. Chem. Biol. 2012, 8 (3),

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294-300.

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(10) Luo, X. J.; Kong, X. D.; Zhao, J.; Chen, Q.; Zhou, J. H.; Xu, J. H. Switching a

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newly discovered lactonase into an efficient and thermostable phosphotriesterase by

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simple double mutations His250Ile/Ile263Trp. Biotechnol. Bioeng. 2014, 111 (10), 22


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465

1920-1930.

466

(11) Abe, K.; Yoshida, S.; Suzuki, Y.; Mori, J.; Doi, Y.; Takahashi, S.; Kera, Y.

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Haloalkylphosphorus hydrolases purified from Sphingomonas sp strain TDK1 and

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Sphingobium sp strain TCM1. Appl. Environ. Microbiol. 2014, 80 (18), 5866-5873.

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(12) Xiang, D. F.; Bigley, A. N.; Ren, Z. J.; Xue, H. R.; Hull, K. G.; Romo, D.;

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Raushel, F. M. Interrogation of the Substrate Profile and Catalytic Properties of the

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phosphotriesterase from Sphingobium sp strain TCM1: an enzyme capable of

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hydrolyzing organophosphate flame retardants and plasticizers. Biochemistry-US

473

2015, 54 (51), 7539-7549.

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(13) Bigley, A. N.; Xiang, D. F.; Ren, Z. J.; Xue, H. R.; Hull, K. G.; Romo, D.;

475

Raushel, F. M. Chemical mechanism of the phosphotriesterase from Sphingobium sp

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strain TCM1, an enzyme capable of hydrolyzing organophosphate flame retardants. J.

477

Am. Chem. Soc. 2016, 138 (9), 2921-2924.

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(14) Jackson, C. J.; Weir, K.; Herlt, A.; Khurana, J.; Sutherland, T. D.; Horne, I.;

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Easton, C.; Russell, R. J.; Scott, C.; Oakeshott, J. G. Structure-based rational design

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of a phosphotriesterase. Appl. Environ. Microbiol. 2009, 75 (15), 5153-5156.

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(15) Chen, J.; Luo, X. J.; Chen, Q.; Pan, J.; Zhou, J. H.; Xu, J. H. Marked

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enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single

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residue substitution Ile211Ala. Bioresour. Bioprocess. 2015, 2 (1), 39-46.

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(16) Liu, X. Y.; Chen, F. F.; Li, C. X.; Luo, X. J.; Chen, Q.; Bai, Y. P.; Xu, J. H.

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Improved efficiency of a novel methyl parathion hydrolase using consensus approach.

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Enzyme Microbiol. Technol. 2016, 93-94, 11-17. 23



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(17) Del Giudice, I.; Coppolecchia, R.; Merone, L.; Porzio, E.; Carusone, T. M.;

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Mandrich, L.; Worek, F.; Manco, G. An efficient thermostable organophosphate

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hydrolase and its application in pesticide decontamination. Biotechnol. Bioeng. 2016,

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113 (4), 724-734.

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(18) Luo, X. J.; Zhao, J.; Li, C. X.; Bai, Y. P.; Reetz, M. T.; Yu, H. L.; Xu, J. H.

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Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by

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hierarchical iteration mutagenesis. Biotechnol. Bioeng. 2016, 113 (11), 2350-2357.

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(19) Potvin, G.; Ahmad, A.; Zhang, Z. S. Bioprocess engineering aspects of

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heterologous protein production in Pichia pastoris: a review. Biochem. Eng. J. 2012,

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64, 91-105.

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(20) Chu, X. Y.; Wu, N. F.; Deng, M. J.; Tian, J.; Yao, B.; Fan, Y. L. Expression of

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organophosphorus

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characterization. Protein Expres. Purif. 2006, 49 (1), 9-14.

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(21) Wang, P.; Huang, L.; Jiang, H.; Tian, J.; Chu, X. Y.; Wu, N. F. Improving the

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secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its

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N-terminal sequence. Plos One 2014, 9 (5).

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(22) Shen, W.; Shu, M.; Ma, L. X.; Ni, H.; Yan, H. High level expression of

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organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.

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Protein Expres. Purif. 2016, 119, 110-116.

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(23) Merone,

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phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression

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and properties. Extremophiles 2005, 9 (4), 297-305.

L.;

hydrolase

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OPHC2

L.;

in

Rossi,

Pichia

M.;

pastoris:

Manco,

24


G.

purification

and

A thermostable

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(24)Naqvi, T.; Warden, A. C.; French, N.; Sugrue, E.; Carr, P. D.; Jackson, C. J.; Scott,

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C. A 5000-fold increase in the specificity of a bacterial phosphotriesterase for

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malathion through combinatorial active site mutagenesis. Plos One 2014, 9 (4),

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e94177.

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(25) Schofield, D. A.; Dinovo, A. A. Generation of a mutagenized organophosphorus

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hydrolase for the biodegradation of the organophosphate pesticides malathion and

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demeton-S. J. Appl. Microbiol. 2010, 109 (2), 548-57.

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(26) Komives, C. F.; Lilley, E.; Russell, A. J. Biodegradation of pesticides in nonionic

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water-in-oil microemulsions of Tween 85: relationship between micelle structure and

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activity. Biotechnol. Bioeng. 1994, 43 (10), 946-959.

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(27) Mills, C. E.; Obermeyer, A; Dong, X. H.; Walker, J.; Olsen, B. D. Complex

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coacervate core micelles for the dispersion and stabilization of organophosphate

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hydrolase in organic solvents. Langmuir 2016, 32, 13367-13376.

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(28) Kim, M.; Gkikas, M.; Huang, A.; Kang, J. W.; Suthiwangcharoen, N.; Nagarajan,

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R.; Olsen, B. D. Enhanced activity and stability of organophosphorus hydrolase via

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interaction with an amphiphilic polymer. Chem. Commun. 2014, 50, 5345-5348.

25



Page 26 of 36

Scheme and figure legends Figure 1. Screening of recombinant P. pastoris strains containing plasmids pPIC9K or pPICZαA (A) and pGAPZαA (B) for high protein expression. Figure 2. Optimization of fermentation conditions of recombinant P. pastoris in shaken flasks. The pH (A), methanol concentration (B) and induction time (C) were optimized for strain X33/pPICZαA-∆poophm9 (red), while the pH (A) and carbon source (D) were optimized for strain X33/pGAPZαA-∆poophm9 (blue). Figure

3.

Time

course

of

high-cell-density

fermentation

using

strain

using

strain

X33/pPICZαA-∆poophm9 in BSM at pH 5.0 (A) or pH 6.0 (B). Figure

4.

Time

course

of

high-cell-density

fermentation

X33/pGAPZαA-∆poophm9 in BSM + glycerol (A) and FM22 medium + glucose (B). Figure 5. Malathion (A) or malaoxon (B) was degraded by PoOPHM9 in the presence of 0.1% (w/w) coconut oil derivatives (COD), Sodium dodecyl sulphate (SDS), and alkyl polyglycoside (APG). (C) The hydrolysis of malathion in the aqueous phase with different concentrations of detergents was measured after 20-min incubation. (D) The residual activity of PoOPHM9 was measured at different time intervals during a 1-h incubation of the aqueous solution containing PoOPHM9 and different detergents. Figure 6. Degradation of 12 µmol malathion from contaminated cucumbers (A, 300 g), apples (B, 477 g), and 7.5 µmol malathion on cotton tissue (C, 25 × 25 cm) in 2 L tap water using 50 (A, B) or 20 (C) µg/mL PoOPHM9. DNTB (0.02 g) was added to measure the hydrolysis percentage of malathion or malaoxon during the washing process at room temperature. (D) Malathion hydrolysis was measured during the 26


Page 27 of 36


washing process. (E) Degradation of 3 µmol malaoxon from contaminated cucumbers, apples and cotton tissue was measured during the washing processs.

27



Page 28 of 36

Table 1. Comparison of fermentation profiles of different recombinant strains. Yield (g/L)

Yp/x

Yp/s

(mg/g)

(mg/g)

0.1

11.0

3.0

This work

850

3.8

53.0

24.0

This work

50.8

640

4.1

45.0

18.0

This work

10.1

70

5.5

n.d.

n.d.

16

Strains

Time (h)

Titer (kU/L)

STY

BL21/pET28a

18

3.6

(U·L ·h ) 200

X33/pPICZαA

56

47.6

X33/pGAPZαA

72

GS115/pPIC9k

150

-1

-1

28


References

Page 29 of 36


Table 2. Kinetic parameters of PoOPHM9 in the absence or presence of detergents. Enzyme+detergents PoOPHM9 PoOPHM9+0.1% COD PoOPHM9+0.1% APG PoOPHM9+0.1% SDS

Specific activity (U/mg)

kcat (S-1)

Km (µM)

kcat/Km (M-1S-1) 5

35.0

24.2 ± 0.32

91.2 ± 5.3

(2.6 ± 0.20) × 10

8.7 ± 0.28 3.8 ± 0.06 3.0 ± 0.09

6.5 ± 0.17 2.5 ± 0.04 1.8 ± 0.03

197.1 ± 5.0 139.1 ± 1.0 40.2 ± 0.6

(2.3 ± 0.32) × 104 (1.8 ± 0.03) × 104 (4.4 ± 0.10) × 104

29


References 18 This work This work This work


Figure 1. Screening of recombinant P. pastoris strains containing plasmids pPIC9K or pPICZαA (A) and pGAPZαA (B) for high protein expression.

30


Page 30 of 36

Page 31 of 36


Figure 2. Optimization of fermentation conditions of recombinant P. pastoris in shaken flasks. The pH (A), methanol concentration (B) and induction time (C) were optimized for strain X33/pPICZαA-∆poophm9 (red), while the pH (A) and carbon source (D) were optimized for strain X33/pGAPZαA-∆poophm9 (blue).

31



Figure

3.

Time

course

of

high-cell-density

fermentation

X33/pPICZαA-∆poophm9 in BSM at pH 5.0 (A) or pH 6.0 (B).

32


Page 32 of 36

using

strain

Page 33 of 36


Figure

4.

Time

course

of

high-cell-density

fermentation

using

strain

X33/pGAPZαA-∆poophm9 in BSM + glycerol (A) and FM22 medium + glucose (B).

33



Figure 5. Malathion (A) or malaoxon (B) was degraded by PoOPHM9 in the presence of 0.1% (w/w) coconut oil derivatives (COD), Sodium dodecyl sulphate (SDS), and alkyl polyglycoside (APG). (C) The hydrolysis of malathion in the aqueous phase with different concentrations of detergents was measured after 20-min incubation. (D) The residual activity of PoOPHM9 was measured at different time intervals during a 1-h incubation of the aqueous solution containing PoOPHM9 and different detergents.

34


Page 34 of 36

Page 35 of 36


Figure 6. Degradation of 12 µmol malathion from contaminated cucumbers (A, 300 g), apples (B, 477 g), and 7.5 µmol malathion on cotton tissue (C, 25 × 25 cm) in 2 L tap water using 50 (A, B) or 20 (C) µg/mL PoOPHM9. DNTB (0.02 g) was added to measure the hydrolysis percentage of malathion or malaoxon during the washing process at room temperature. (D) Malathion hydrolysis was measured during the washing process. (E) Degradation of 3 µmol malaoxon from contaminated cucumbers, apples and cotton tissue was measured during the washing processs.

35



TOC graphic

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Efficient degradation of malathion in the presence ... - ACS Publications

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