Biodegradation of the insecticide flonicamid by Alcaligenes faecalis

Subscriber access provided by Nottingham Trent University

Agricultural and Environmental Chemistry

Biodegradation of the insecticide flonicamid by Alcaligenes faecalis CGMCC 17553 via hydrolysis and hydration pathways mediated by nitrilase Wen-Long Yang, Lei-Lei Guo, Zhi-Ling Dai, Ruo-Chen Qin, Yun-Xiu Zhao, and Yijun Dai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04245 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

Journal of Agricultural and Food Chemistry

1

Biodegradation of the insecticide flonicamid by Alcaligenes

2

faecalis CGMCC 17553 via hydrolysis and hydration pathways

3

mediated by nitrilase

4

5

Wen-Long Yang, Lei-Lei Guo, Zhi-Ling Dai, Ruo-Chen Qin, Yun-Xiu Zhao, Yi-

6

Jun Dai*

7

8

Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and

9

Technology Research Center for Industrialization of Microbial Resources, College of Life

10

Science, Nanjing Normal University, Nanjing 210023, People’s Republic of China

11

12

*Address correspondence to: Yi-Jun Dai, tel: 86-25-85891731, fax: 86-25-85891067, e-mail:

13

[email protected]

14

1 / 43

ACS Paragon Plus Environment


16

Abstract: Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO), a novel selective

17

systemic pyridinecarboxamide insecticide, effectively controls hemipterous pests. However,

18

microbial degradation of flonicamid, along with the enzymatic mechanism, has not been studied.

19

Here, bacterial isolate PG13, which converts flonicamid into 4-(trifluoromethyl) nicotinol glycine

20

(TFNG) and N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM), was isolated and

21

identified as Alcaligenes faecalis CGMCC 17553. The genome of CGMCC 17553 contained five

22

nitrilases but no nitrile hydratase, and recombinant Escherichia coli strains harboring CGMCC

23

17553 nitrilase genes nitA or nitD acquired the ability to degrade flonicamid. Purified NitA

24

catalyzed flonicamid into both TFNG and TFNG-AM, indicating dual functionality, while NitD

25

could only produce TFNG-AM. Three-dimensional homology modeling revealed that aromatic

26

amino acid residues in the catalytic pocket affected nitrilase activity. These findings further our

27

understanding of the enzymatic mechanism of flonicamid metabolism in the environment and may

28

help develop a potential bioremediation agent for the elimination of flonicamid contamination.

29

Keywords: insecticide; biodegradation; Alcaligenes faecalis; flonicamid; nitrilase

2 / 43


Page 2 of 43

Page 3 of 43

30


Introduction

31

Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO), a pyridinecarboxamide

32

insecticide, demonstrates highly selective activity against pests belonging to the order Hemiptera,

33

including cotton leafhopper, aphids, and mosquitoes.1-3 As such, flonicamid is commonly used to

34

control pests on a variety of crops, such as hops, cabbage, cotton, wheat, and potatoes.4-7

35

Mechanistically, flonicamid has been shown to block A-type potassium channels,8 causing

36

toxicity by inhibiting inward rectifier K+ (Kir) channels of insect pests and disrupting salivary and

37

renal excretion.9 Biological effects include loss of directed movement and suppression of feeding.

38

Globally, neonicotinoids are the most commonly used class of insecticides; however, they pose

39

a significant risk to wild bees and honeybees.10-12 Therefore, several countries have banned the

40

use of neonicotinoid insecticides,13 necessitating the development of alternative pest control

41

products. Compared with neonicotinoid insecticides, studies have shown that flonicamid is less

42

toxic to bees, honeybees, and natural predators of insect pests; hence, it was recommended for

43

widespread pest control.2, 7, 14, 15

44

However, despite having decreased toxicity and being responsible for lower levels of residue

45

build-up in the environment compared with neonicotinoid insecticides, flonicamid toxicity against

46

predators of insect pests has been reported. For example, flonicamid was found to be toxic to

47

coccinellid and chrysoperla, predators of pomegranate aphids,16 while a 17.39% mortality rate

48

was observed in adult Coccophagus japonicus wasps following exposure to flonicamid.17

49

flonicamid also had a sublethal impact on Nesidiocoris tenuis, a generalist predator of the

50

Mediterranean Basin that is commonly used in biological control programs.18 Additionally, 3 / 43



Page 4 of 43

51

flonicamid reportedly reduced egg hatch numbers in Episyrphus balteatus19 and decreased the

52

parasitism rates of Microplitis mediator and the parasitoid beetle Aleochara bilineata.20

53

Flonicamid residue has also been detected in harvested food crops, including cabbages, bell

54

peppers, and cucumbers,5, 21, 22 as well as in several watersheds around the Great Lakes Basin in

55

the United States.23 Flonicamid and its metabolites, 5-trifluoromethylnicotinic acid (TFNA), 4-

56

(trifluoromethyl) nicotinol glycine (TFNG), and N-(4-trifluoromethylnicotinoyl) glycinamide

57

(TFNG-AM), were detected following application of the insecticide in orange groves in field

58

studies,26 while flonicamid, TFNG, TFNA, and 4-trifluoromethylnicotinamide (TFNA-AM) were

59

detected in dried hops.4 Even more worryingly, flonicamid residue has even detected in human

60

serum and urine samples.24 This is of particular concern given that high doses of flonicamid

61

caused DNA degradation and severe genome damage in mice.25

62

With the increasing usage of flonicamid worldwide, environmental build-up of flonicamid

63

residues seems more and more likely, which is a significant environmental concern. To remove

64

flonicamid residues from the environment, photooxidation-based remediation methods have been

65

trialed by several research groups. However, results were suboptimal when persulfate was added

66

to the tested wastewater. The addition of ZnO- and TiO2-coated magnetic particles to the systems

67

greatly enhanced the flonicamid degradation rate but was not economically viable.27,

68

Microorganisms have been used as the basis of many insecticide degradation protocols that are

69

considered fairly economical.29 However, there are currently no reports on the microbial

70

degradation of flonicamid. Thus, in the current study, we intend to isolate flonicamid-degrading

71

microorganisms from soil and characterize the underlying metabolic pathways and enzymes 4 / 43


28

Page 5 of 43


72

required for insecticide degradation. Our findings significantly enhance our understanding

73

microbial flonicamid degradation in the environment and may help in the development of a novel

74

bacterial-based method for flonicamid bioremediation.

75

MATERIALS AND METHODS

76

Chemicals and Media. Flonicamid (95% purity) was purchased from Hubei

77

Zhengxingyuan Fine Chemical Co. (Wuhan, China) and TFNG was purchased from Sigma-

78

Aldrich (St. Louis, MI). High-performance liquid chromatography (HPLC)-grade acetonitrile was

79

purchased from Tedia Co. (Fairfield, OH). All other reagents were of analytical grade and were

80

purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Mineral salt medium

81

(MSM) used for screening and isolation of flonicamid-degrading microbes was prepared as

82

described previously.30 Lysogeny broth (LB) contained 5 g of yeast extract, 10 g of tryptone, and

83

10 g of NaCl in 1 L of deionized water (pH 7.2). LB agar medium was prepared by the addition

84

of 20 g of agar to 1 L of liquid medium. The preparation of TFNG-AM is described in the

85

Supporting Information.

86

Isolation and Identification of Flonicamid-Degrading Bacterial Strain PG13.

87

The method used to isolate flonicamid-degrading bacterial strains is outlined in the Supporting

88

Information. Flonicamid-degrading isolates were identified by morphological analysis and 16S

89

rRNA gene sequencing. Cell morphology was observed using an optical microscope after Gram

90

staining. For all isolates, the 16S rRNA gene was amplified by colony polymerase chain reaction

91

(PCR) using primers K1 (5ʹ-AACTGAAGAGTTTGATCCTGGCTC-3ʹ) and K2 (5ʹ-

92

TACGGTTACCTTGTTACGACTT-3ʹ). The resulting amplicons were sequenced by Springern 5 / 43



93

Co. (Nanjing, China). Nucleotide sequence data were deposited in the GenBank database. The

94

16S rRNA gene sequence of PG13 was then compared against those already present in the

95

GenBank database using the Basic Local Alignment Search Tool-nucleotide (BLASTn). A

96

neighbor-joining phylogenetic tree for PG13 was then generated based on the 16S rRNA gene

97

sequences using MEGA 6.

98

Liquid Chromatography-Mass Spectrometry (LC-MS) Analyses. To identify

99

the metabolites of flonicamid, flonicamid-transformed samples by PG13 were characterized by

100

LC-MS analysis using an Agilent LC-MS system (model 1290 Infinity LC/6460 Triple Quad MS;

101

Agilent, Santa Clara, CA) equipped with an electrospray ion source operated in both the positive

102

ion mode and the negative ion mode. The detailed High-performance Liquid Chromatography

103

(HPLC) Analysis condition was described in the Supporting Information.

104

Biodegradation of Flonicamid in Soil. For soil biodegradation experiments, soil

105

samples which was no pesticide application were collected from Nanjing, China. The

106

physicochemical properties were measured in the Institute of Soil Science of the Chinese

107

Academy of Sciences. The measurements were: pH 6.0, organic matter 17.24 g/kg, cation

108

exchange capacity 21.61 cmol/kg. The soil sample was smashed and sieved by 0.55 mm sifter,

109

then dry heat sterilization was practiced at 150℃ for 5 h. 10 g of sterilized dry soil samples were

110

spread uniformly in 100-mL flasks. Bacteria pre-cultured in LB broth (30℃, 220 rpm for 18 h)

111

were then collected and re-suspended in 200 mg/L sterilized FLO solution (50 mmol/L FLO in

112

phosphate-buffered saline (PBS) [pH 7.0]) to an optical density at 600 nm (OD600) = 5 and aliquots

113

(4 mL) of the bacterial suspension were then added to each soil sample immediately , resulting in 6 / 43


Page 6 of 43

Page 7 of 43


114

a final FLO concentration of 80 mg/kg soil. For control, 4 mL of the bacterial suspension was

115

instead by 4 mL sterilized FLO solution (50 mmol/L FLO in phosphate-buffered saline (PBS) [pH

116

7.0]) added to each soil sample. The flasks were then incubated at 30°C and 35% humidity in the

117

dark. Following incubation for the indicated time (0, 3, 6, 9 days), 12 mL of acetonitrile were

118

added to each flask to examine FLO degradation. The flasks were then sealed and placed in a

119

rotary shaker at 220 rpm for 2 h. Supernatants were collected following centrifugation at 10,000

120

× g for 10 min and filtered prior to HPLC analysis described in the Supporting Information.

121

Genomic DNA Sequencing and Cloning and Over-Expression of Nitrilase

122

Genes in E. coli Rosetta (DE3). Total Genomic DNA was extracted from the flonicamid-

123

degrading isolate using a MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, Dalian,

124

China). Whole-genome sequencing and annotation was conducted by Beijing Genomics Institute

125

Co. (Beijing, China). The primers used for amplification of nitrilase genes contained EcoRI and

126

XhoI restriction enzyme sites are listed in Table 1. Primers were synthesized by Sangon Biotech

127

Co. (Shanghai, China). Reaction mixtures for PCR analysis contained 1× PrimeSTAR Max

128

Premix (TaKaRa), forward and reverse primers (1 mmol/L), 1 ng of DNA template, and ultrapure

129

water to a final volume of 20 μL. Amplification was performed using a Bio-Rad Thermal Cycler

130

(Bio-Rad, Hercules, CA) with the following program: hot start at 95°C for 5 min, followed by 31

131

cycles of 95°C for 50 s, 54°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10

132

min. The resulting amplicons were verified by electrophoresis on 1% agarose gels in 1× TAE

133

buffer followed by staining with ethidium bromide.

134

Following verification, a ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., 7 / 43



135

Nanjing, China) was used to recombine the EcoRI/XhoI-digested PCR products into expression

136

vector pET28a as per the manufacturer’s protocols. The resulting recombinant plasmids

137

containing nitrilase genes were individually transformed into competent E. coli Rosetta cells as

138

described by Sun et al.31

139

Enzymatic Activity Assay. Nitrilase gene expression was induced in the recombinant E.

140

coli Rosetta (DE3) strains by the addition of 0.2 mmol/L isopropyl β-D-thiogalactoside (IPTG),

141

0.2 mmol/L CoCl2 was added. Following incubation for 6 h at 30°C, sodium dodecyl sulphate

142

polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess protein expression.

143

Gels were stained with Coomassie Brilliant Blue. The activity of the expressed proteins against

144

flonicamid was then examined as described in the resting cells biodegradation assays, with

145

incubation at 30°C, 220 rpm, for 20 min. The culture supernatants were then collected and

146

subjected to HPLC analysis described in the Supporting Information.

147

Enzyme Purification and Biochemical Characterization. N-terminal 6× His-

148

tagged NitA and NitD overexpressed in E. coli Rosetta (DE3) were purified by affinity

149

chromatography as per the manufacturer’s instructions (Novagen, Madison, WI). SDS-PAGE was

150

then performed to assess protein expression. Gels were stained with Coomassie Brilliant Blue.

151

Nitrilase activity was determined by HPLC analysis condition was described in the Supporting

152

Information. One unit (U) of enzyme activity was defined as the amount of enzyme required to

153

catalyze the generation of 1 μmol of TFNG or TFNG-AM in 1 min. The optimal temperature for

154

the degradation of flonicamid by NitA and NitD was determined by adding purified protein

155

(concentration) to PBS (50 mmol/L, pH 7.0) containing 200 mg/L flonicamid and incubating the 8 / 43


Page 8 of 43

Page 9 of 43


156

mixtures at temperatures ranging from 20–50°C (NitA) or 20–60°C (NitD) for 2 h. The optimal

157

pH for the degradation of flonicamid by NitA and NitD was determined using the same system at

158

30°C and 800 rpm for 2 h in PBS, citric acid/sodium citrate (CA/SC) or Tis/HCl, pH values

159

ranging from 5.0–9.0. The same system was also used to examine the influence of various metal

160

ions on flonicamid degradation by NitA and NitD. For these assays, PBS was supplemented with

161

0.1 mmol/L EDTA, MgSO4, CoCl2, CaCl2, CuCl2, FeCl2, MnCl2, MoCl5, or ZnCl2.

162

The kinetic parameters of NitA and NitD were examined in a 1-mL reaction mixture containing

163

5 μL of purified NitA (2.75 mg/mL) and 995 μL of flonicamid (50–1000 mg/L) dissolved in PBS

164

(50 mmol/L, pH 7.0). Mixtures were incubated at 40°C and 800 rpm for 30 min. The kinetic

165

parameters of NitD were examined in a 1-mL reaction mixture containing 5 μL of purified NitD

166

(1.09 mg/mL) and 995 μL of flonicamid (400–3000 mg/L) dissolved in PBS (50 mmol/L, pH 8.0).

167

Mixtures were incubated at 40°C and 800 rpm for 10 h. The kinetic parameters of NitA and NitD

168

were analyzed by Origin 8.5 nonlinear curve fit (MichaelisMenten).

169

The substrate specificity of NitA and NitD was examined in a 1-mL reaction mixture containing

170

20 μL of purified protein (NitA, 7.28 mg/mL; NitD, 11.73 mg/mL) and 980 μL of 200 mg/L

171

flonicamid, acetamiprid (ACE), thiacloprid (THI), indolyl-3-acetonitrile (IAN), or 3-

172

cyanopyridine (3-CP) dissolved in PBS (50 mmol/L, pH 7.0). Mixtures were incubated at 30°C

173

and 800 rpm for 1 h (NitA) or 3 h (NitD). All the reactions were analyzed by HPLC analysis

174

described in the Supporting Information.

175

NitA Activity Against TFNG-AM. The enzymatic activity of NitA against TFNG-AM

176

was examined in a 1-mL reaction mixture containing 30 μL of NitA (7.49 mg/mL) and 970 μL of 9 / 43



Page 10 of 43

177

TFNG-AM (250 mg/L) dissolved in PBS (50 mmol/L, pH 7.0). Mixtures were incubated at 30°C

178

and 800 rpm for 30 min. The reaction was analyzed by HPLC analysis described in the Supporting

179

Information.

180

Homology Modeling of the Nitrilases. Three-dimensional homology models of the five

181

nitrilases

182

(https://swissmodel.expasy.org/interactive). The crystal structure of Nit6803 (PDB accession

183

code 3WUY, x-ray diffraction resolution 3.10 Å) from Synechocystis sp. PCC6803 was used as

184

template for NitA model construction. The crystal structure of N-carbamoylputrescine

185

amidohydrolase from Medicago truncatula (PDB accession code 5H8I, x-ray diffraction

186

resolution 1.97 Å) was used as a template for NitB model construction. The crystal structure of

187

Nit2 from Saccharomyces cerevisiae strain ATCC 204508/S288c (PDB accession code 4H5U, x-

188

ray diffraction resolution 1.92 Å) was used as template for NitC and NitE model construction.

189

The crystal structure of PH0642 from Pyrococcus horikoshii (PDB accession code 1J31, x-ray

190

diffraction resolution 1.60 Å) was used as template for NitD model construction. The qualities of

191

the constructed nitrilase structures were then evaluated using GMQE32 and QMEAN33. The

192

structures of the modeled nitrilases were analyzed by Chimera (www.cgl.ucsf.edu/ chimera),

193

while active sites and catalytic triads within the nitrilases were predicted by BLAST sequence

194

analysis against other nitrilase sequences in the Reference proteins (refseq_protein) database

195

(Figures S7, S8, S9, S10, and S11 of the Supporting Information).

196

RESULTS AND DISCUSSION

197

were

developed

using

the

SWISS-MODEL

workspace

Isolation and Identification of Flonicamid-Degrading Microbes. A total of 15 10 / 43


Page 11 of 43


198

colonies showing obvious morphological differences on LB agar plates following culture in MSM

199

broth containing 500 mg/L flonicamid were selected and tested for their ability to degrade

200

flonicamid. Among these, one isolate, designated PG13, was confirmed to degrade flonicamid.

201

Assays showed that PG13 could transform flonicamid into two products, referred to as P1 and P2

202

(Figure 1A). PG13 formed yellow, wettish, roundish, convex colonies on LB agar, with

203

morphological analysis showing that the isolate was a Gram negative, rod-shaped, non-spore-

204

forming bacterium. Nucleotide BLAST analysis of the 16S rRNA gene (GenBank accession

205

number MK888682) revealed that PG13 showed 96.83% nucleotide sequence identity to the

206

corresponding gene from A. faecalis strain PNP6. The neighbor-joining phylogenetic tree

207

generated by MEGA 6 based on 16S rRNA gene sequences showed that PG13 clustered with A.

208

faecalis strain PNP6 but had more distant evolutionary relationships with other Alcaligenes

209

species (Figure S1 in Supplementary Information). Therefore, PG13 was identified as A. faecalis

210

and was deposited at the China General Microbiological Culture Collection Center (CGMCC)

211

(Beijing, China) under accession number CGMCC 17553.

212

Many other A. faecalis strains have demonstrated the ability to degrade organic pollutants,

213

including phenanthrene, endosulfan, and nicosulfuron.34-36 Endosulfan is a chlorinated cyclodiene

214

insecticide, while nicosulfuron is a sulfonylurea herbicide. However, this is the first report of the

215

degradation of flonicamid, a cyano-group-containing insecticide, by an A. faecalis strain.

216

Identification of the Metabolites. HPLC analysis showed that the retention times of

217

products P1 and P2 were 3.5 and 4.5 min, respectively, which correspond to the retention times

218

of TFNG and TFNG-AM, respectively. LC-MS analysis revealed that metabolite P1 exhibited 11 / 43



219

peaks at 249, 271, 287, 519, 535, 247, and 495 m/z, corresponding to [M+H]+, [M+Na]+, [M+K]+,

220

[2M+Na]+, [2M+K]+, [M−H]−, and [2M−H]−, respectively (Figure 1B and 1C). Metabolite P2 had

221

peaks at 248, 270, 286, and 246 m/z, corresponding to [M+H]+, [M+Na]+, [M+K]+, and [M−H]−,

222

respectively (Figure 1D and 1E). Therefore, the relative molecular weights of P1 and P2 were

223

calculated as 248 and 247, respectively. Previous reports have shown that metabolic products of

224

flonicamid, including TFNG, TFNG-AM, TFNA, and TFNA-AM, were present in crops

225

following flonicamid application. The relative molecular weights of TFNG and TFNG-AM are

226

248 and 247, respectively, which is consistent with the calculated molecular weights of P1 and

227

P2, respectively (Figure 2A). These results therefore suggest that A. faecalis CGMCC 17553

228

degrades flonicamid via hydrolysis and hydration pathways, two general chemical reaction

229

pathways of cyano-group insecticides.

230

Time Course of Flonicamid Degradation by Resting Cells and Degradation

231

in Soil by A. faecalis CGMCC 17553. As shown in Figure 3A, resting A. faecalis CGMCC

232

17553 cells reduced the initial concentration of flonicamid from 915 μmol/L to 11 μmol/L over

233

96 h, corresponding to an overall degradation rate of 98.8%. During this period, 799 μmol/L

234

TFNG and 101 μmol/L TFNG-AM were produced, resulting in a molar conversion rate of 99.5%.

235

Overall, 88.3% and 11.2% of the substrate was transformed into TFNG and TFNG-AM,

236

respectively. The half-life of flonicamid in the presence of A. faecalis CGMCC 17553 was 15 h.

237

These results indicated that A. faecalis CGMCC 17553 efficiently degraded flonicamid to TFNG

238

via hydrolysis, and that this is the main metabolic pathway of flonicamid degradation.

239

Inoculation of A. faecalis CGMCC 17553 into soil resulted in a flonicamid degradation rate of 12 / 43


Page 12 of 43

Page 13 of 43


240

85.0% within 9 days, with the half-life of flonicamid calculated as 3.3 days. Specifically, soil

241

concentrations of flonicamid were decreased from 0.40 μmol/g of soil to 0.06 μmol/g of soil over

242

the 9-day period. In the absence of A. faecalis CGMCC 17553 inoculation, 42.0% of the

243

flonicamid was degraded in the same time period, corresponding to a half-life of 9.8 days (Figure

244

4B). Therefore, inoculation of A. faecalis CGMCC 17553 into soil accelerated flonicamid

245

degradation, indicating that microorganisms may be one of the major factors affecting flonicamid

246

degradation in soil.

247

A. faecalis CGMCC 17553 Genome Sequencing and Cloning and

248

Overexpression of Nitrilase Genes in E. coli Rosetta (DE3). TFNG and TFNG-AM

249

are the products of flonicamid hydrolysis and hydration pathways, respectively, which require the

250

activity of nitrile hydratase/amidases or nitrilases.37 Analysis of the complete genome sequence

251

of A. faecalis CGMCC 17553 revealed only five nitrilase-encoding genes: nitA (GenBank

252

accession no. MK888683), nitB (GenBank accession no. MK926977), nitC (GenBank accession

253

no. MK926978), nitD (GenBank accession no. MK926979), and nitE (GenBank accession no.

254

MK926980). Interestingly, no nitrile hydratase-encoding genes were identified in the A. faecalis

255

CGMCC 17553 genome. Nitrilases (EC 3.5.5.1) either hydrolyze nitriles into their corresponding

256

carboxylic acids or hydrate them into their corresponding carboxylic amides.38 The five nitrilase-

257

encoding genes from A. faecalis CGMCC 17553 were amplified by PCR and ligated into

258

expression vector pET28a. The recombinant plasmids were then transformed into E. coli Rosetta

259

(DE3) and protein expression was induced. SDS-PAGE analysis showed that all of the target

260

proteins were successfully expressed with the major soluble fractions (Figure 4A). E. coli Rosetta 13 / 43



261

(DE3) overexpressing NitA degraded most of the available flonicamid into TFNG and TFNG-

262

AM, while the NitD over-expressing strain only degraded 9.4% of the initial flonicamid, with

263

only the TFNG-AM degradation product detected. Interestingly, E. coli Rosetta strains

264

overexpressing NitB, NitC, or NitE showed no catalytic activity against flonicamid (Table 2).

265

Results of SDS-PAGE analysis of the purified NitA and NitD proteins are shown in Figure 4B

266

(lane 2) and Figure 4C (lane 2). Purified NitA degraded 77.9% of the starting concentration of

267

flonicamid (200 mg/L) within 180 min, with 60.9% of the product identified as TFNG and the

268

remaining 30.1% identified as TFNG-AM (Figure 3C), suggesting that TFNG is the major

269

degradation product. Purified NitD degraded 44.1% of the starting flonicamid concentration (200

270

mg/L) within the allotted 180 min (Figure 3D), but only produced TFNG-AM. Overall, the

271

enzymatic behavior of the purified proteins was consistent with that of the A. faecalis CGMCC

272

17553 cells.

273

Enzymatic Characterization of NitA. The optimal pH for flonicamid degradation

274

by NitA was 7.0, with 100% enzyme activity observed at this pH. However, significant differences

275

in the activity of NitA against flonicamid were observed as the pH was adjusted between pH 5.0

276

and pH 9.0 (Figure 5A). At pH 5.0, enzyme activity was only 22.87% of that at the optimal pH,

277

but increased slightly when the pH was increased to 6.0. Activity again decreased when the pH

278

was adjust from pH 7.0 to pH 8.0, with another major drop in activity observed following an

279

increase to pH 9.0. Thus, NitA only exhibited activity within a narrow pH range. When NitA was

280

preincubated at pH values ranging from 6.0–9.0, there was only a slight change in enzymatic

281

activity against flonicamid, with 100% activity observed at pH 8.0. In comparison, preincubation 14 / 43


Page 14 of 43

Page 15 of 43


282

at pH 5.0 resulted in activity levels only 44.95% of that of the highest enzyme activity (Figure

283

5B). Thus, NitA remained stable across a broad pH range.

284

Maximum NitA activity (100%) was observed at 40°C. However, at 50°C, only 4.1% of the

285

maximum activity remained, indicating that NitA cannot tolerate high temperatures (Figure 5C)

286

and only exhibits activity within a narrow temperature range. When preincubated at temperatures

287

ranging from 30–50°C, the highest NitA activity against flonicamid was observed at 30°C, which

288

was defined as 100% activity. Activity levels decreased when temperatures were increased

289

between 30°C and 50°C, confirming that the enzyme was not stable at high temperatures (Figure

290

5D).

291

Co2+ had a positive effect on flonicamid degradation by NitA, with an observed 18.4%

292

increase in enzyme activity compared with the unsupplemented control. In comparison, Ca2+,

293

EDTA, and Fe2+ inhibited NitA enzyme activity by 7.2%, 14.3%, and 34.0%, respectively, while

294

Mg2+ and Mn2+ exhibited no effect on NitA enzyme activity. Importantly, Cu2+ and Zn2+

295

completely inhibited the ability of NitA to degrade flonicamid (Figure 5E). All of the tested

296

organic solvents inhibited NitA activity against flonicamid to some degree, with the observed

297

activity of NitA in the presence of ethyl alcohol, methyl alcohol, methylene dichloride, ethyl

298

acetate, and n-butyl alcohol decreased by 10.3%, 19.4%, 23.9%, 48.1%, and 24.8%, respectively

299

(Figure 5F).

300

Analysis of the kinetic parameters of the reaction revealed that NitA degradation of

301

flonicamid showed classic Michaelis-Menten kinetics. For the formation of TFNG, the Vmax was

302

1.18 U/mg and the Km was 1.62 mmol/L (Figure 6A), with corresponding values of 0.58 U/mg 15 / 43



303

and 0.59 mmol/L, respectively, for the formation of TFNG-AM (Figure 6B). Substrate specificity

304

assays showed that NitA had no activity against ACE, THI, IAN, or 3-CP, indicating that NitA

305

has a narrow substrate specificity (Figure 2B).

306

Enzymatic Characterization of NitD. The optimal pH for flonicamid degradation by

307

NitD was 8.0, with enzyme activity at pH 8.0 defined as 100%. As with NitA, the activity of NitD

308

against flonicamid decreased with alterations in the pH away from the optimum, with only 9.34%

309

and 57.10% of the highest enzyme activity remaining at pH 5.0 and pH 9.0, respectively (Figure

310

7A). Therefore, both NitA and NitB could be used to degrade flonicamid under neutral or slightly

311

alkaline pH conditions, although the activity of NitA is slightly higher. When NitD was

312

preincubated at pH values ranging from 5.0–9.0, there was little alteration in enzyme activity

313

against flonicamid, and the highest enzyme activity was observed at pH 8.0 (defined as 100%

314

enzyme activity) (Figure 7B). Thus, although decreased compared with optimal conditions, NitD

315

demonstrated activity across a broad range of pH conditions.

316

Maximum (100%) NitD enzyme activity against flonicamid was observed at 40°C, and large

317

alterations in activity were observed across the tested temperature range (20–70°C). At 70°C, only

318

7.5% of the maximum activity remained, indicating that NitD cannot tolerate high temperatures

319

(Figure 7C). When preincubated at temperatures ranging from 30–60°C, the highest (100%) NitD

320

activity against flonicamid was observed at 20°C. However, activity decreased as temperatures

321

increased from 20°C to 60°C, with only 1.6% of the highest activity remaining at 60°C. These

322

results confirmed that NitD does not tolerate high temperatures (Figure 7D).

323

EDTA, Ca2+, Co2+, Fe2+, Mg2+, and Mn2+ exhibited no effect on the activity of NitD, while Cu2+ 16 / 43


Page 16 of 43

Page 17 of 43


324

and Zn2+ inhibited NitD activity by 64.9% and 49.6%, respectively, compared with the control

325

(Figure 7E). While Cu2+ and Zn2+ strongly inhibited NitD enzyme activity, the inhibition was less

326

pronounced than that observed for NitA. Methylene dichloride enhanced NitD activity by 7.6%,

327

while ethyl alcohol, methyl alcohol, ethyl acetate, and n-butyl alcohol decreased the activity of

328

NitD by 3.5%, 5.8%, 12.7%, and 8.7%, respectively, compared with the control (Figure 7F).

329

Except for methylene dichloride, the levels of NitD inhibition by the organic solvents were similar

330

to results obtained for NitA.

331

Analysis of the kinetic parameters indicated that NitD degradation of flonicamid demonstrated

332

classic Michaelis-Menten kinetics. The Vmax of the NitD degradation reaction was 0.18 mU/mg,

333

while the Km was 145.87 mmol/L (Figure 6C). NitD demonstrated a reduced ability to form

334

TFNG-AM compared with NitA. Substrate specificity assays showed that NitD was not active

335

against ACE, THI, IAN, or 3-CP, displaying a similarly narrow substrate specificity to NitA.

336

NitA Activity Against TFNG-AM. Activity assays showed that NitA had no activity

337

against TFNG-AM. This indicated that TFNG and TFNG-AM were formed independently.

338

Therefore, we propose that flonicamid degradation by A. faecalis CGMCC 17553 is achieved

339

through the hydration of flonicamid into TFNG-AM by both NitA and NitD, and the hydrolysis

340

of flonicamid into TFNG by NitA only. NitA is thus a bifunctional enzyme.

341

Analysis of the Nitrilase Homology Models. Homology modeling of the nitrilases

342

from A. faecalis CGMCC 17553 was conducted using the SWISS-MODEL workspace (Figure

343

8). The homology model of NitA, which was generated based on the model crystal structure of

344

Nit6803, had a GMQE value of 0.62 and a QMEANA value of −2.71 (Figure 8A and Figure S2 17 / 43



345

of the Supporting Information). The amino acid sequence identity between NitA and Nit6803 was

346

35.2%. Based on alignments against selected nitrilase sequences, Glu-48, Lys-133, and Cys-167

347

were predicted to constitute the catalytic triad, while Glu-48, Lys-133, Cys-167, Thr-137, Glu-

348

140, Gly-168, Asn-170, Thr-171, and Tyr-191 were predicted to make up the putative active site

349

(Figure 9A and Figure S7 of the Supporting Information).

350

The homology model of NitB was generated based on the model crystal structure of M.

351

truncatula MtCPA and had a GMQE value of 0.51 and QMEANA value of −4.77 (Figure 8B and

352

Figure S3 of the Supporting Information). The sequence identity between NitB and Nit6803 was

353

19.58%. Alignment against selected nitrilase sequences revealed that Glu-47, Lys-129, and Cys-

354

163 made up the catalytic triad, while Glu-47, Lys-129, Thr-133, Cys-163, Trp-164, His-166,

355

Leu-167, and Trp-187 were predicted to form the putative active site (Figure 9B and Figure S8 of

356

the Supporting Information).

357

The homology model of NitC, generated based on the model crystal structure of S. cerevisiae

358

Nit2, had a GMQE value of 0.72 and a QMEANA value of −1.58 (Figure 8C and Figure S4 of the

359

Supporting Information). The sequence identity between NitB and Nit6803 was 35.97%.

360

Alignment against selected nitrilase sequences showed that the catalytic triad comprised Glu-42,

361

Lys-112, and Cys-149, while Glu-42, Lys-112, Tyr-116, Glu-123, Cys-149, Tyr-150, Leu-152,

362

Arg-153, and Ala-174 were predicted to form the putative active site (Figure 9C and Figure S9 of

363

the Supporting Information).

Page 18 of 43

364

The homology model of NitD was generated by SWISS-MODEL based on the model crystal

365

structure of hypothetical protein PH0642 (Figure 8D and Figure S5 of the Supporting Information), 18 / 43


Page 19 of 43


366

to which NitD showed 25.97% similarity. The GMQE value was 0.57 and the QMEANA value

367

was −3.38. Alignment against selected nitrilase sequences revealed that Glu-42, Lys-129, and

368

Cys-163 constituted the catalytic triad, while Glu-42, Lys-129, Thr-133, Glu-136, Cys-163, Trp-

369

164, Asn-166, Tyr-167, and Thr-187 were predicted to form the putative active site (Figure 9D

370

and Figure S10 of the Supporting Information).

371

The homology model of NitE was generated based on the model crystal structure of S.

372

cerevisiae Nit2, with a resulting GMQE value of 0.70 and a QMEANA value of −2.06 (Figure 8E

373

and Figure S6 of the Supporting Information). The sequence identity between NitB and Nit6803

374

was 33.08%. Alignment against selected nitrilase sequences showed that Glu-43, Lys-114, and

375

Cys-154 formed the predicted catalytic triad, while Glu-43, Asn-97, Lys-114, Tyr-118, Glu-129,

376

Cys-154, Tyr-155, Met-157, Arg-158, and Ala-179 made up the putative active site (Figure 9D

377

and Figure S11 of the Supporting Information).

378

While all five nitrilases showed a similar three-dimensional structure and a catalytic triad

379

comprising Glu, Lys, and Cys residues, their active sites were more varied. In NitA, the active

380

site amino acid residue adjacent to catalytic triad amino acid residue Cys-167 was Gly-168, which

381

has a hydrogen side chain, is non-polar, and occupies little space. In comparison, aromatic amino

382

acid residues Trp-164 (NitB), Tyr-116 and Tyr-150 (NitC), Trp-164 (NitD), and Tyr-118 and Tyr-

383

155 (NitE) were located in the corresponding positions in the catalytic pockets of the other

384

nitrilases. These residues occupy more space and may inhibit the binding of flonicamid within the

385

catalytic pocket, thereby inhibiting enzymatic activity against flonicamid. In addition, active site

386

residues Tyr-116 of NitC and Tyr-118 of NitE also occupied more space within the catalytic 19 / 43



387

pocket than the Thr-137 residue located at the corresponding position of NitA, any may therefore

388

also block the binding of flonicamid. The random coil of NitE at the catalytic pocket may also

389

take up space, further blocking the binding of flonicamid.

390

In conclusion, this is the first report of microbial degradation of flonicamid. A. faecalis strain

391

CGMCC 17553 was shown to efficiently transform flonicamid into TFNG and TFNG-AM. The

392

bifunctional nitrilase, NitA, of A. faecalis CGMCC 17553 was responsible for the transformation

393

of flonicamid into both TFNG and TFNG-AM, while nitrilase NitD only converted flonicamid

394

into TFNG. Aromatic amino acids within the catalytic pockets of several of the A. faecalis

395

CGMCC 17553 nitrilases may inhibit the flonicamid binding and therefore reduce nitrilase

396

activity. This study enhances our understanding of flonicamid degradation by microbes in the

397

environment and describes a novel bioremediation bacterium for the degradation of flonicamid

398

residues.

399

ASSOCIATED CONTENT

400

Supporting Information

401

The Supporting Information is available free of charge on the ACS Publications website.

402

Methods for the preparation of TFNG-AM and the isolation of bacteria from soil are provided

403

here. High-performance liquid chromatography (HPLC) analysis method used in this study. A

404

neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences of A. faecalis CGMCC

405

17553 and closely related species is shown in Figure S1. Alignments of nitrilase sequences with

406

template sequences are provided in Figures S2, S3, S4, S5, and S6. Alignments of A. faecalis

20 / 43


Page 20 of 43

Page 21 of 43


407

CGMCC 17553 nitrilase active sites against selected nitrilase sequences are shown in Figures S7,

408

S8, S9, S10, and S11.

409

Funding

410

This research was financed by the National Science Foundation of China (grant no. 31570104)

411

and the Program for Jiangsu Excellent Scientific and Technological Innovation Team

412

(17CXTD00014).

413

Notes

414

The authors declare no competing financial interest.

21 / 43



1

REFERENCES

2

(1) Morita, M.; Ueda, T.; Yoneda, T.; Koyanagi, T.; Haga, T. Flonicamid, a novel insecticide

3

with a rapid inhibitory effect on aphid feeding. Pest Manag. Sci. 2007, 63 (10), 969-973.

4

(2) Abbas, N.; Abbas, N.; Ejaz, M.; Shad, S. A.; Asghar, I.; Irum, A.; Binyameen, M. Resistance

5

in field populations of Amrasca devastans (Hemiptera: Cicadellidae) to new insecticides in

6

Southern Punjab, Pakistan. Phytoparasitica 2018, 46 (4), 533-539.

7

(3) Taylor-Wells, J.; Gross, A. D.; Jiang, S.; Demares, F.; Clements, J. S.; Carlier, P. R.;

8

Bloomquist, J. R. Toxicity, mode of action, and synergist potential of flonicamid against

9

mosquitoes. Pestic. Biochem. Physiol. 2018, 151, 3-9.

10

(4) Hengel, M. J.; Miller, M. Analysis of flonicamid and its metabolites in dried hops by liquid

11

chromatography-tandem mass spectrometry. J. Agric. Food Chem. 2007, 55 (20), 8033-8039.

12

(5) Wang, S. S.; Jin, F.; Cao, X. L.; Shao, Y.; Wang, J.; She, Y. X.; Qi, Y.; Zhang, C.; Li, H.;

13

Jin, M. J.; Wang, J.; Shao, H.; Zheng, L. F. Residue behaviors and risk assessment of flonicamid

14

and its metabolites in the cabbage field ecosystem. Ecotox. Environ. Safe. 2018, 161, 420-429.

15

(6) Korr, V. Teppeki (R) - a new insecticide for tillage (wheat and potatoes). Gesunde Pflanzen

16

2007, 59 (3), 95-99.

17

(7) Chawla, S.; Gor, H. N.; Patel, H. K.; Parmar, K. D.; Patel, A. R.; Shukla, V.; Ilyas, M.;

18

Parsai, S. K.; Somashekar; Meena, R. S.; Shah, P. G. Validation, residue analysis, and risk

19

assessment of fipronil and flonicamid in cotton (Gossypium sp.) samples and soil. Environ. Sci.

20

Pollut. R. 2018, 25 (19), 19167-19178. 22 / 43


Page 22 of 43

Page 23 of 43


21

(8) Tariq, K.; Noor, M.; Backus, E. A.; Hussain, A.; Ali, A.; Peng, W.; Zhang, H. The toxicity

22

of flonicamid to cotton leafhopper, Amrasca biguttula (Ishida), is by disruption of ingestion: an

23

electropenetrography study. Pest Manag. Sci. 2017, 73 (8), 1661-1669.

24

(9) Ren, M. M.; Niu, J. G.; Hu, B.; Wei, Q.; Zheng, C.; Tian, X. R.; Gao, C. F.; He, B. J.; Dong,

25

K.; Su, J. Y. Block of Kir channels by flonicamid disrupts salivary and renal excretion of insect

26

pests. Insect Biochem. Molec. 2018, 99, 17-26.

27

(10) Tosi, S.; Burgio, G.; Nieh, J. C. A common neonicotinoid pesticide, thiamethoxam, impairs

28

honey bee flight ability. Sci. Rep. 2017, 7.

29

(11) Whitehorn, P. R.; O'Connor, S.; Wackers, F. L.; Goulson, D. Neonicotinoid pesticide

30

reduces bumble bee colony growth and queen production. Science 2012, 336 (6079), 351-352.

31

(12) Raine, N. E. Pesticide affects social behavior of bees Neonicotinoid exposure impairs the

32

social dynamics of bumblebees inside the nest. Science 2018, 362 (6415), 643-644.

33

(13) Dupraz-Dobias, P. France bans all uses of neonicotinoid pesticides. Chem. Eng. News 2018,

34

96 (36), 15-15.

35

(14) Kodandaram, M. H.; Kumar, Y. B.; Banerjee, K.; Hingmire, S.; Rai, A. B.; Singh, B. Field

36

bioefficacy, phytotoxicity and residue dynamics of the insecticide flonicamid (50 WG) in okra

37

Abelmoschus esculenta (L) Moench. Crop Protection 2017, 94, 13-19.

38

(15) Zhao, M.-A.; Yu, A.; Zhu, Y.-Z.; Kim, J.-H. Potential dermal exposure to flonicamid and

39

risk assessment of applicators during treatment in apple orchards. J. Occup. Environ. Hyg. 2015,

40

12 (8), D147-D152. 23 / 43



41

(16) Pathania, M.; Arora, P. K.; Pathania, S.; Kumar, A. Studies on population dynamics and

42

management of pomegranate aphid, Aphis punicae Passerini (Hemiptera: Aphididae) on

43

pomegranate under semi-arid conditions of South-western Punjab. Sci. Hortic. 2019, 243, 300-

44

306.

45

(17) Shen, S.; Niu, L.; Zhang, F.; Fu, W.; Zhang, Y.; Fu, Y.; Zhu, J. The toxicities of several

46

common insecticides on Coccophagus japonicus compere. Chinese J. Trop. Crop. 2018, 39 (3),

47

553-558.

48

(18) Wanumen, A. C.; Sanchez-Ramos, I.; Vinuela, E.; Medina, P.; Adan, A. Impact of feeding

49

on contaminated prey on the life parameters of Nesidiocoris Tenuis (Hemiptera: Miridae)

50

Adults. J. Insect Sci. 2016, 16(1), 1-7.

51

(19) Moens, J.; De Clercq, P.; Tirry, L. Side effects of pesticides on the larvae of the hoverfly

52

Episyrphus balteatus in the laboratory. Phytoparasitica 2011, 39 (1), 1-9.

53

(20) Jansen, J. P.; Defrance, T.; Warnier, A. M. Side effects of flonicamide and pymetrozine

54

on five aphid natural enemy species. Biocontrol 2011, 56 (5), 759-770.

55

(21) Lopez-Ruiz, R.; Romero-Gonzalez, R.; Martinez Vidal, J. L.; Garrido Frenich, A.

56

Determination of flonicamid and its metabolites in bell pepper using ultra-high-performance

57

liquid chromatography coupled to high-resolution mass spectrometry (Orbitrap). Food Addit.

58

Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2016, 33 (11), 1685-1692.

59

(22) Abdel-Ghany, M. E.; Hussein, L. A.; El Azab, N. E. Multiresidue analysis of five

60

neonicotinoid insecticides and their primary metabolite in cucumbers and soil using high24 / 43


Page 24 of 43

Page 25 of 43


61

performance liquid chromatography with diode-array detection. J. AOAC Int. 2017, 100 (1),

62

176-188.

63

(23) Metcalfe, C. D.; Helm, P.; Paterson, G.; Kaltenecker, G.; Murray, C.; Nowierski, M.;

64

Sultana, T. Pesticides related to land use in watersheds of the Great Lakes basin. Sci. Total

65

Environ. 2019, 648, 681-692.

66

(24) Lopez-Ruiz, R.; Ruiz-Muelle, A. B.; Romero-Gonzalez, R.; Fernandez, I.; Vidal, J. L. M.;

67

Frenich, A. G. The metabolic pathway of flonicamid in oranges using an orthogonal approach

68

based on high-resolution mass spectrometry and nuclear magnetic resonance. Analytical

69

Methods 2017, 9 (11), 1718-1726.

70

(25) Yamamuro, T.; Ohta, H.; Aoyama, M.; Watanabe, D. Simultaneous determination of

71

neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted

72

extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr. B 2014, 969,

73

85-94.

74

(26) Sabry, A. K. H.; Salem, L. M.; Ali, N. I.; Ahmed, S. S. E. Genotoxic effect of flonicamid

75

and etofenprox on mice. Biosci. Res. 2018, 15 (3), 2295-2303.

76

(27) Garrido, I.; Pastor-Belda, M.; Campillo, N.; Vinas, P.; Yanez, M. J.; Vela, N.; Navarro, S.;

77

Fenoll, J. Photooxidation of insecticide residues by ZnO and TiO2 coated magnetic

78

nanoparticles under natural sunlight. J. Photoch. Photobio. A 2019, 372, 245-253.

25 / 43



79

(28) Vela, N.; Fenoll, J.; Garrido, I.; Perez-Lucas, G.; Flores, P.; Hellin, P.; Navarro, S.

80

Reclamation of agro-wastewater polluted with pesticide residues using sunlight activated

81

persulfate for agricultural reuse. Sci. Total Environ. 2019, 660, 923-930.

82

(29) Liu, Z.; Dai, Y.; Huang, G.; Gu, Y.; Ni, J.; Wei, H.; Yuan, S. Soil microbial degradation

83

of neonicotinoid insecticides imidacloprid, acetamiprid, thiacloprid and imidaclothiz and its

84

effect on the persistence of bioefficacy against horsebean aphid Aphis craccivora Koch after

85

soil application. Pest Manag. Sci. 2011, 67 (10), 1245-52.

86

(30) Ge, F.; Zhou, L. Y.; Wang, Y.; Ma, Y.; Zhai, S.; Liu, Z. H.; Dai, Y. J.; Yuan, S. Hydrolysis

87

of the neonicotinoid insecticide thiacloprid by the N 2 -fixing bacterium Ensifer meliloti

88

CGMCC 7333. Int. Biodeterior. Biodegradation 2014, 93, 10-17.

89

(31) Sun, S.; Lu, T.; Yang, W.; Rui, X.; Mao, S.; Zhou, L. Y.; Dai, Y. Characterization of a

90

versatile nitrile hydratase of the neonicotinoid thiacloprid-degrading bacterium Ensifer meliloti

91

CGMCC 7333. RSC Adv. 2016, 6 (19), 15501-15508.

92

(32) Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer,

93

F.T., De Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., Schwede, T. SWISS-MODEL:

94

Homology modelling of protein structures and complexes. Nucleic Acids Research. 2018, 46

95

(Web Server issue), W296-W303.

96

(33) Benkert, P., Biasini, M., Schwede, T. Toward the estimation of the absolute quality of

97

individual protein structure models. Bioinformatics. 2011, 27, 343-35

98

(34) Lakshmi, M. B.; Velan, M., Biodegradation of the toxic polycyclic aromatic hydrocarbon,

26 / 43


Page 26 of 43

Page 27 of 43

99


phenanthrene by an indigenously isolated. Alcaligenes faecalis MVMB1strain. Int. Proc. Chem.

100

Biol. Environ. Eng. 6, 440-444.

101

(35) Zhao, W.; Wang, C.; Xu, L.; Zhao, C.; Liang, H.; Qiu, L. Biodegradation of nicosulfuron

102

by a novel Alcaligenes faecalis strain ZWS11. J. Environ. Sci. 2015, 35 (9), 151-162.

103

(36) Kong, L.; Zhu, S.; Zhu, L.; Hui, X.; Kai, W.; Yan, T.; Wang, J.; Wang, J.; Wang, F.; Sun,

104

F. Colonization of Alcaligenes faecalis strain JBW4 in natural soils and its detoxification of

105

endosulfan. APPL. MICROBIOL. BIOT. 2014, 98 (3), 1407-1416.

106

(37) Angelini, L. M. L.; da Silva, A. R. M.; Rocco, L. D. C.; Milagre, C. D. D. A high-

107

throughput screening assay for distinguishing nitrile hydratases from nitrilases. Braz. J.

108

Microbiol. 2015, 46 (1), 113-116.

109

(38) Fernandes, B. C. M.; Mateo, C.; Kiziak, C.; Chmura, A.; Wacker, J.; van Rantwijk, F.;

110

Stolz, A.; Sheldon, R. A. Nitrile hydratase activity of a recombinant nitrilase. Adv. Synth. Catal.

111

2006, 348 (18), 2597-2603.

27 / 43



1

Table 1. Primers used for the amplification of nitrilase genes from A. faecalis

2

CGMCC 17553. nitA-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGATGCTGGAATTGC GTCAG

nitA-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTACACTTCCTTGTC GTCCAGAAC

nitB-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGCAGACAAGAAAA ATCGTCC

nitB-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAGGACGGTTCCT GCACC

nitC-F

ACAGCAAATGGGTCGCGGATCCGAATTCGTGGTCATGAAAGTTG CATTGG

nitC-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAACCCAGTTCGG GCG

nitD-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGAGCAAAGTTGCTG TTATACAAG

nitD-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTCGCCCGAGT CTTCG

nitE-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGTCTACGACACGTG TTGCC

nitE-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAAGAAGAAATAG GATACAGATCAC

28 / 43


Page 28 of 43

Page 29 of 43


1

Table 2. Degradation of flonicamid (initial concentration was 976.0 μmol/L) by

2

recombinant E. coli Rosetta (DE3) strains expressing nitrilase genes from A. faecalis CGMCC 17553.

3

Concentration (μmol/L) TFNG nitA

4 5 6

TFNG-AM

556.0±12.4 285.6±1.9

Reduced flonicamid 891.5±17.8a

nitB

ND

ND

12.9±10.1c

nitC

ND

ND

22.9±10.8c

nitD

ND

92.2±1.3

82.8±7.2b

nitE

ND

ND

21.9±9.7c

control

ND

ND

21.0±9.4c

Data represent the mean values of triplicate samples. Superscript letters indicate that the mean values (± SD) within that column are significantly different at p ≤ 0.05 based on analysis by Duncan’s test.

29 / 43



Page 30 of 43

1

Figure legends

2

Figure

3

chromatography-mass spectrometry (LC-MS) analysis of metabolites produced during

4

the degradation of flonicamid by A. faecalis CGMCC 17553. (A) HPLC analysis of the

5

transformation of flonicamid by A. faecalis CGMCC 17553. (B) Positive and (C) negative

6

ion mode mass spectrometry analysis of degradation product 1 (P1). (D) Positive and (E)

7

negative ion mode spectrometry analysis of degradation product 2 (P2).

8

Figure 2. Metabolic pathways of flonicamid and molecular structures used for substrate

9

specificity testing. (A) Various pathways for the metabolism of flonicamid. (B) Structure

1.

High-performance

liquid

chromatography

(HPLC)

and

liquid

10

of molecular used for substrate specificity testing.

11

Figure 3. Time course analysis of flonicamid degradation and kinetic parameters of the

12

reactions catalyzed by NitA and NitD. (A) Time course of flonicamid degradation by

13

resting A. faecalis CGMCC 17553 cells. (B) Degradation of flonicamid in soil by

14

faecalis CGMCC 17553. (C) Degradation of flonicamid by E. coli Rosetta (DE3)

15

expressing nitA from A. faecalis CGMCC 17553. (D) Degradation of flonicamid by E.

16

coli Rosetta (DE3) expressing nitD from A. faecalis CGMCC 17553.

17

Figure 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

18

analysis of NitA, NitB, NitC, NitD, and NitE overexpressed in E. coli Rosetta (DE3)

19

along with purified NitA and NitD. (A) SDS-PAGE analysis of NitA, NitB, NitC, NitD,

20

and NitE overexpressed in E. coli Rosetta (DE3). Lanes 1, 3, 5, 7, 9, and 11, total protein

30 / 43


A.

Page 31 of 43


21

extracts from E. coli Rosetta (DE3) strains containing pET28a (control), pET28a-nitC,

22

pET28a-nitB, pET28a-nitA, pET28a-nitD, and pET28a-nitE, respectively. Lanes 2, 4, 6,

23

8, 10, and 12, soluble protein fractions from E. coli Rosetta (DE3) strains containing

24

pET28a (control), pET28a-nitC, pET28a-nitB, pET28a-nitA, pET28a-nitD, and pET28a-

25

nitE, respectively. Lane M, standard protein markers (116.0, 66.2, 45.0, and 35.0 kDa).

26

(B) SDS-PAGE analysis of NitA expression in recombinant E. coli Rosetta (DE3) and

27

purified NitA. Lane 1, soluble protein fraction from E. coli Rosetta (DE3) containing

28

pET28a-nitA; lane 2, purified NitA with an N-terminal His-tag; lane M, standard protein

29

markers (116.0, 66.2, 45.0, and 35.0 kDa). (C) SDS-PAGE analysis of NitD expression

30

in recombinant E. coli Rosetta (DE3) and purified NitD, Lane 1, soluble protein fraction

31

from E. coli Rosetta (DE3) containing pET28a-nitD; lane 2, purified NitD with an N-

32

terminal His-tag; lane M, standard protein markers (116.0, 66.2, 45.0, and 35.0 kDa) .

33

Figure 5. Enzymatic characterization of NitA. (A) Effects of pH on NitA activity. (B)

34

Effects of pH on NitA stability. (C) Effects of temperature on NitA activity. (D) Effects

35

of temperature on NitA stability. (E) Effects of metalo ions on NitA activity. (F) Effects

36

of organic solvents on NitA activity. Superscript letters indicate that mean values (± SD)

37

within a column are significantly different at p ≤ 0.05 according to analysis by Duncan’s

38

test.

39

Figure 6. Kinetic parameters of flonicamid degradation reactions catalyzed by NitA

40

and NitD. (A) Kinetic parameters of TFNG formation by NitA. (B) Kinetic parameters of

41

TFNG-AM formation by NitA. (C) Kinetic parameters of TFNG-AM formation by NitD. 31 / 43



42

Figure 7. Enzymatic characterization of NitD. (A) Effects of pH on NitD activity. (B)

43

Effects of pH on NitD stability. (C) Effects of temperature on NitD activity. (D) Effects

44

of temperature on NitD stability. (E) Effects of metalo ions on NitD activity. (F) Effects

45

of organic solvents on NitD activity. Superscript letters indicate that mean values (± SD)

46

within a column are significantly different at p ≤ 0.05 according to analysis by Duncan’s

47

test.

48

Figure 8. Homology models of nitrilases from A. faecalis CGMCC 17553. (A) Three-

49

dimensional homology model of NitA. (B) Three-dimensional homology model of NitB.

50

(C) Three-dimensional homology model of NitC. (D) Three-dimensional homology

51

model of NitD. (E) Three-dimensional homology model of NitE.

52

Figure 9. Identification of actives sites within nitrilases from A. faecalis CGMCC

53

17553. (A–E) Active sites in NitA, NitB, NitC, NitD, and NitE, respectively. Amino acid

54

residues marked in green constitute the putative catalytic triad of each nitrilase. Amino

55

acid residues marked in yellow make up the active site of each nitrilase.

32 / 43


Page 32 of 43

Page 33 of 43


1

Figure 1

2 33 / 43



1

Figure 2

2

34 / 43


Page 34 of 43

Page 35 of 43


1

Figure 3

2

35 / 43



1

Figure 4

2

36 / 43


Page 36 of 43

Page 37 of 43


1

Fig 5

2

37 / 43



1

Figure 6

38 / 43


Page 38 of 43

Page 39 of 43


2 39 / 43



1

Figure 7

2

40 / 43


Page 40 of 43

Page 41 of 43


1

Figure 8

2

41 / 43



1

Figure 9

2

42 / 43


Page 42 of 43

Page 43 of 43


1

TOC GRAPHICS

2 3

43 / 43


Biodegradation of the insecticide flonicamid by Alcaligenes faecalis

Recommend Documents