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Article

Discovery of a bacterial glycoside hydrolase family 3 (GH3) #-glucosidasewith myrosinase activity from a Citrobacter strain isolated from soil

Abdulhadi Albaser, Eleanna Kazana, Mark Bennett, FatmaCebeci, Vijitra Luang-In, Pietro D. Spanu, and John T. Rossiter

J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05381 • Publication Date (Web): 28 Jan 2016

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1

Discovery of a bacterial glycoside hydrolase family 3 (GH3) β-glucosidase with myrosinase activity 1

from a Citrobacter strain isolated from soil 2

3

Abdulhadi Albaser 1,5

, Eleanna Kazana 1,3

, Mark H Bennett 1, Fatma Cebeci

2, Vijitra Luang-In

1,4 Pietro 4

D Spanu 1 and John T Rossiter

1* 5

6

1 Imperial College London, Faculty of Life Sciences, London SW7 2AZ, UK 7

2 Food and Health Programme, Institute of Food Research, Norwich, NR4 7UA, UK 8

3Current address: School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ 9

4Current Address: Mahasarakham University, Faculty of Technology, Department of Biotechnology, 10

Natural Antioxidant Research Unit, Mahasarakham 44150, Thailand 11

5Current address: Division of Microbiology, Faculty of Sciences, University of Sebha, Sebha Libya 12

*Corresponding author; John T Rossiter, Imperial College London, Faculty of Life Sciences, London 13

SW7 2AZ, UK; email: [email protected]

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Journal of Agricultural and Food Chemistry

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ABSTRACT 22

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin 23

as a sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and 24

gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal 25

peptide sequence of the purified protein were determined and used to identify the gene which, based on 26

InterPro sequence analysis, belongs to the family GH3, contains a signal peptide and is a periplasmic 27

protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out 28

using protein extracts from cell free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 29

mmol dm-3

min-1

mg-1

respectively with sinigrin as substrate. The optimum temperature and pH for 30

enzyme activity were 25 oC and 6.0 respectively. The enzyme was marginally activated with ascorbate by 31

a factor of 1.67. 32

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Keywords: myrosinase, bacteria, isothiocyanate, sequence 34

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INTRODUCTION 44

Bacterial myrosinases are not well understood and there has been little work on this topic. Bacterial 45

myrosinases were first purified some years ago as an active enzyme but without any characterisation in 46

terms of sequence.1, 2

Since then there have been various reports examining the metabolism of 47

glucosinolates by bacteria and attempts have been made to characterize the enzyme but with limited 48

success.3-7

We have set out to isolate a bacterium that can grow on sinigrin, purify the myrosinase and 49

obtain a sequence. β-Glucosidases (EC 3.2.1.21) catalyse the hydrolysis of the glucosidic linkages of a 50

variety of β-glucosides and, depending on sequence and structure, can be placed in the glycoside 51

hydrolase (GH) families GH1, GH3, GH9, GH 30 and GH116.8-10

With the exception of GH9, the 52

remainder utilize a two-step mechanism that involves a catalytic nucleophile that displaces the aglycone 53

and is followed by protonation of the nucleofuge and attack by a water molecule to give a glycosyl 54

residue. Plant myrosinases (thioglucoside glucohydrolase EC 3.2.3.147) belong to the family GH1 and 55

their structure and mechanisms have been well studied.11-14

Myrosinases hydrolyse glucosinolates, a 56

group of plant secondary metabolites, to give a variety of products (Figure 1) depending on the presence 57

of specifier proteins and type of glucosinolate, but the default product is an isothiocyanate (ITC).15-18

The 58

mechanism of plant myrosinase does not utilize the typical catalytic acid/base employed by β-59

glucosidases but instead uses ascorbate as a catalytic base. The Sinapis alba myrosinase is a dimer linked 60

by a zinc atom and has a characteristic (β/α)8-barrel structure in common with GH1 enzymes. The insect 61

Brevicoryne brassicae (cabbage aphid) contains a myrosinase that has been studied in some detail and 62

also belongs to the glucose hydrolase family GH1 19-21

and its structure has been determined.22

In contrast 63

to the S. alba myrosinase, the aphid myrosinase does not require ascorbate for activity and its mechanism 64

is the same as for β-glucosidases i.e. a nucleophile (glutamic acid residue) that forms a glycosyl-enzyme 65

intermediate together with a glutamic acid residue that acts as a catalytic base/acid typical of most GH1 66

glucosidases. 67

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There is, however, increasing interest in bacteria that can metabolise glucosinolates, particularly those of 68

the human gut, where the chemoprotective nature of glucosinolate hydrolysis products is of dietary 69

importance.23

In addition, soil bacteria are also of importance in terms of soil ecology and in the 70

application of biofumigation, where plants belonging to the Brassicaceae, which are high in glucosinolate 71

content, are used to fumigate soil.24-27

In this work we describe the isolation of a soil bacterium that 72

possesses an inducible β-glucosidase that transforms glucosinolates into isothiocyanates. The properties 73

and sequence of the enzyme are described. 74

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METHODS 89

Glucosinolates 90

Glucosinolates were isolated from seed sources as previously described.28

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Isolation of a glucosinolate-metabolizing bacterial strain by enrichment 92

Soil was taken from a field in Wye (Kent, UK), which had previously been used for growing oilseed rape, 93

and 1 gram was placed into 10 ml of M9 medium with sinigrin (10 mg) as the sole carbon source and 94

placed in the dark at room temperature for 2 weeks. After gentle agitation, 1 ml of solution was removed, 95

placed into fresh M9 media with sinigrin as the sole carbon source and incubated for a further 2 weeks. 96

This process was repeated a further 5 times. Finally, dilutions were made and a small amount of the 97

enrichment culture was streaked onto a nutrient agar plate for each dilution. The plates were incubated 98

overnight at 30 oC. Colonies were added to fresh M9 media containing sinigrin as the sole carbon source 99

and incubated at 30 oC, and the OD600nm (optical density at 600nm) was monitored. The culture was 100

centrifuged at 10000g for 5 min and stored at -20 oC until analysis. Sinigrin-metabolizing colonies were 101

grown overnight in M9 media supplemented with sinigrin as the sole carbon source, and the following 102

day glycerol stocks (40%) were made and stored at -80 oC. 103

HPLC analysis of sinigrin fermentation 104

The fermentation of sinigrin was monitored by removing 1 mL of culture into an Eppendorf (1.5 mL) tube 105

and centrifuging at 10000g for 10 min. The supernatant was removed and stored at -20 oC until analysis. 106

Samples were thawed out (1 mL), 100 µL of a barium/lead acetate solution (equimolar, 1M) were added, 107

and samples were thoroughly mixed and allowed to stand for a few minutes. The samples were 108

centrifuged at 10000g for 5 min and the supernatant was removed and transferred to a fresh Eppendorf 109

tube. DEAE Sephadex-A25 (Sigma, UK) was mixed with an excess of NaOAc buffer (1M, pH 5), which 110

was changed several times over 6 h and finally left overnight to equilibriate. The excess buffer was 111

decanted and the ion exchanger was mixed with deionized water, decanted (x 2) and finally stored in 20 112

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% EtOH to give an approximately 50% settled gel. One mL of suspended DEAE Sephadex-A25 slurry 113

was pipetted into Poly-Prep Chromatography Columns (BioRad, UK) and allowed to drain. The ion 114

exchange material was washed twice with 1 mL de-ionised water, and the sample was applied and 115

allowed to drain. The column was washed with 2 x 1 mL de-ionised water, followed by two washes of 0.5 116

mL sodium acetate buffer (0.05 M, pH 5.0). Sulfatase (Sigma, UK) was added (75 µL, 0.3U/mL) 117

carefully to the top of the gel and the column was left overnight at room temperature. The desulfo-118

glucosinolate (DS-GSL) was eluted with 3 x 0.5 mL of deionized water and stored at -20 oC until HPLC 119

analysis. The DS-GSLs were analysed by HPLC on a Synergi Hydro-RP column (4 µm, 15 x 0.2 cm, 120

Phenomenex Inc. Torrance, CA). The following conditions were used for the HPLC analysis: Water 121

(solvent A), ACN (solvent B); 2% B (15 min), 2-25% B (2 min), 25-70% B (2 min), 70% (2 min), 70-2% 122

B (2 min), 70-2%B (15 min); flow rate 0.2 mL/min, column temperature 35 oC. HPLC was carried out 123

using an Agilent HP1100 system with a Waters UV detector (model 486) at a wavelength of 229 nm. 124

GC-MS analysis of glucosinolate metabolites 125

One mL of culture or cell free extract was removed and centrifuged at 10000g for 10 min. The 126

supernatant was removed and transferred to a 2 mL Eppendorf tube, 0.5 mL DCM was added and the tube 127

was vortexed for 30 s. The tubes were centrifuged at 10000g for 5 min and the DCM was carefully 128

removed with a glass syringe. The sample was re-extracted with a further 0.5 mL DCM and combined 129

with the first extraction. A small amount of dried MgSO4 was added to the DCM extract to remove any 130

water and briefly vortexed. The extract was centrifuged and the DCM was carefully removed, transferred 131

to an Agilent 2 mL screw cap vial and stored at 4 oC until analysis. 132

An Agilent HP 6890 series system connected to an Agilent HP 5973 mass selective detector was used for 133

the GC-MS analysis. A capillary column, Agilent HP-5MS (5% Phenylmethylsiloxane, 30 m × 0.25 mm 134

i.d.; film thickness, 0.25 µm), was used with helium as the carrier gas (split mode, 25:1; splitter inlet 135

pressure, 40 kPa). The temperature was kept at 40 °C for 10 min and ramped up to 150 °C at the rate of 136

4°C/min for 25 min and then to 250°C at the rate of 4°C/min for 15 min, with the flow at 1 mL/min, 137

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average velocity of 36 cm/s, pressure at 7.56 kPa and injection volume of 1 µL. Mass spectra were 138

obtained by electron ionization (EI) over a range of 50−550 atomic mass units. Ion source temperature 139

was 230 °C and the electron multiplier voltage was 70.1 eV. The analysis of allyl amine, allyl nitrile and 140

2-propenoic acid (Sigma UK) were carried out on a Machery-Nagel (UK) Optima Delta-6 column (30 m 141

× 0.25 mm i.d.; film thickness, 0.25 µm) using the same programme as previously described, with the 142

exception that the final oven temperature was 200 °C. The limit of detection for pure ITC/NIT standards 143

by GC-MS analysis was 10 ng/mL. 144

16S rRNA Sequence determination, genomic DNA assembly and preparation of phylogenetic tree 145

An overnight culture was centrifuged at 13000g for 5 min, and the pellet was washed with milliQ water 146

(x1), resuspended in 15 µL milliQ water and boiled at 95 oC for 5 min. The sample was cooled on ice, 147

spun for 5 min at 13,000g and the supernatant was collected. The gene was amplified by PCR using the 148

primers AMP_F: 5': GAG AGT TTG ATY CTG GCT CAG, Tm: 60.5 and AMP_R: 5': AAG GAG GTG 149

ATC CAR CCG CA, Tm: 68.9, GoTaq DNA polymerase, PCR buffer (Promega) and dNTPs (200 µM 150

each). The PCR conditions were the following: initial denaturation 95 oC, 2 min (1 cycle); denaturation 151

95oC, 30 s (20 cycles); annealing 55

oC, 30 s (20 cycles); extension 72 °C, 1 min 30 s (20 cycles); final 152

extension 72 °C, 1 min (1 cycle). The PCR product (1500 bp) was purified by QIAquick PCR 153

purification kit (Qiagen) and quantified by a Nanodrop 2000 spectrometer (Thermo scientific). The 154

product was sequenced with both primers (AMP_F and AMP_R; Eurofins Sequencing). The sequences 155

were assembled using Seqman to create a contig and this contig was searched on the RDP database 156

(https://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp). The genomic DNA of the Citrobacter WYE1 157

was sequenced at The Genome Analysis Centre, Norwich, UK, using the Illumina MiSeq platform as 158

previously reported (29). The 251bp length of DNA reads from Genomic DNA sequencing was analysed 159

and quality-trimmed extended contigs were produced by using SPAdes program version 3.6.1.29

These 160

contigs of Citrobacter WYE1 were used in construction of a phylogenetic tree. Draft genomes of different 161

Citrobacter species were obtained from Genbank whole genome shutgun projects. Core genome single 162

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nucleotide polymorphisms (SNPs) in draft genomes and Citrobacter WYE1 were used to prepare the 163

phylogenetic tree using parSNP program Version 1.2 30

. 164

Myrosinase assay 165

Myrosinase activity determinations were based on the glucose oxidase/peroxidase enzyme assay of 166

glucose.31

Preparation of the GOD-PERID (glucose oxidase-peroxidase) reagent (all reagents purchased 167

from Sigma, UK): 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) 250 mg, glucose oxidase 12.7 168

mg (157.5 U/mg) and peroxidase 4.7 mg (148U/mg) were dissolved in Tris buffer (3 g Tris, final 169

concentration 0.1M, pH 7.2) to a volume of 250 mL. The reagent was mixed well, wrapped with 170

aluminium foil and kept at 4°C. A calibration curve was obtained using glucose. Samples were assayed in 171

a total volume of 300 µL containing the myrosinase extract (maximum volume 20 µL), 2 mM sinigrin, 172

CPB (citrate phosphate buffer, 20 mM, pH 6.0), and incubated at 25 oC for 2 h or less depending on the 173

activity. The reaction was stopped by placing in a heating block (95 oC) for 5 min and cooled on ice. 1 174

mL of GOD-PERID reagent was added to the sample and incubated for 15 min at 37 °C. The absorbance 175

at 420 nm was measured and the glucose concentration was determined from a calibration graph. 176

General assay conditions for myrosinase activity 177

As there was insufficient pure protein for characterization studies, preliminary studies were carried out 178

using cell-free protein extracts. An overnight culture of Citrobacter WYE1 was grown on nutrient broth 179

(NB), inoculated into M9 medium (50 mL) with 50 mg of sinigrin and incubated aerobically overnight at 180

30 °C in a shaking incubator at 180 rpm. The culture was centrifuged, the pellet was washed as described 181

in the purification methods and the cells re-suspended in 5 ml of extraction buffer (CPB, 50 µL 100 xs 182

protease inhibitor cocktail (Melford Ltd) and 1 mM DTT) and lysed using a one shot cell disruptor 183

(CONSTANT SYSTEMS Ltd, Northants, UK). The lysate was centrifuged at 10,000 g for 15 min at 4oC. 184

The cell-free protein extract was desalted against CPB using Econo-Pac 10DG Desalting Columns 185

(BIORAD, UK) and stored at 4oC. The protein concentration was determined using Bradford reagent 186

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(BIORAD, UK). The activity of the myrosinase was initially determined to ensure linearity and an 187

appropriate amount of protein used for each assay. As the activity of the myrosinase markedly diminished 188

over time, the cell-free protein extracts were used within 72 h of preparation. 189

pH Optimisation 190

Citrate phosphate buffer (pH range of 3.6-7.6, 20 mM) was used to determine the optimum pH for 191

Citrobacter WYE1myrosinase. Crude protein extract (5 µg) was added to a mixture of sinigrin (2 mM) in 192

CPB to a final volume of 300 µL at specific pH values over the range 3.6 -7.6 and incubated at 37 °C for 193

2 h. The reaction was stopped by immersing the tubes in boiling water for 5 min. Glucose release 194

measurements were then carried out using the GOD-PERID assay. 195

Temperature optimization 196

The effect of temperature on the activity of the Citrobacter WYE1 myrosinase was measured over a range 197

of temperatures (5-70 ºC). A total volume (300 µL) with protein (5µg), sinigrin (2 mM) and CPB was 198

incubated at the specified temperature for 30 min and glucose release was assayed by the GOD Perid 199

assay. 200

Substrate specificity 201

Myrosinase assays were carried out using CPB, 2 mM glucosinolate substrate, 60 min incubation and 20 202

µg protein. Samples for qualitative GC-MS analysis were incubated overnight and extracted with DCM as 203

previously described for glucosinolate metabolites. 204

Activation by ascorbate 205

A mixture of the protein (16 µg) and CPB was incubated (30 ºC, 1h) with ascorbate concentrations in the 206

range 0.025-20 mM and gluconasturtiin (0.5 mM) to a volume of 300 µL. The reaction was stopped by 207

boiling (5 min). ITCs were extracted with dichloromethane and quantitated by GC-MS using an Agilent 208

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HP-5MS column as previously described. Quantitation was carried out using a calibration graph of known 209

phenethylisothiocyanate concentrations versus total ion current. 210

Determination of apparent Km & Vmax for sinigrin 211

A range of sinigrin concentrations (0.1-5 mM) was incubated (25 °C, 10 min) with protein (2 µg) and 212

CPB in a total volume of 300 µL. The reaction was stopped by boiling (5 min) and cooled before the 213

GOD Perid reagent (1 mL) was added and incubated (15 min, 37 °C), followed by absorbance 214

measurement (420 nm). Assays were carried out in triplicate and the kinetic parameters evaluated using 215

GraphPad version 6. 216

Estimation of pI 217

A range of buffers at different pH (N-methyl piperazine pH 4.5 and 5; L- histidine pH 5.5 and 6.0; bis-218

Tris pH 6.6; bis-Tris propane pH 7; triethanolamine pH 7.6; Tris pH 8) was used to determine the 219

approximate pI of the Citrobacter myrosinase. Mini anion Q columns (Thermo scientific) were 220

equilibrated with the appropriate buffer (400 µL) and centrifuged on an Eppendorf 5425 centrifuge 221

(2000g, 4 ºC, 5 min). The protein extract was desalted using Zebra spin desalting columns 0.5 mL 222

(Thermo scientific) against the appropriate pH buffer and applied to the mini anion Q column and then 223

centrifuged (2000 x g, 4 ºC, 5 min). The column was washed twice with the appropriate buffer (400µL). 224

Finally the protein was eluted with buffer that contained NaCl (1 M) and desalted against CPB before 225

being assayed for activity. 226

Inducibility of myrosinase 227

Citrobacter WYE1 was grown overnight in M9 media and separately supplemented with sinigrin (10 228

mM) or glucose (10 mM). Cell protein extracts were prepared as previously described (general assay 229

conditions) and assayed for myrosinase activity using 5 µg of crude desalted protein extract/assay. 230

Purification of Citrobacter WYE1 myrosinase 231

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An overnight culture of Citrobacter WYE1 was grown on nutrient broth (NB), inoculated into M9 232

medium (500 mL) with 0.5 g of sinigrin and incubated aerobically overnight at 30 °C in a shaking 233

incubator at 180 rpm. The bacteria were harvested by centrifugation (Avanti J26 XP, Beckman coulter, 234

USA) (8000g, 15 min, 4 °C) and washed twice with CPB. The cell pellet was re-suspended in extraction 235

buffer (CPB, 250 µL 100 Xs protease inhibitor cocktail (Melford Ltd), 1 mM DTT) and lysed using a one 236

shot cell disruption system. The cell debris was pelleted by centrifugation (20,000 x g, 20 min, 4°C) and 237

the supernatant was desalted against CPB using a desalting column (Econo-Pac 10DG) and concentrated 238

to 11 mL using an ultrafiltration membrane tube (molecular weight cut-off 10 KDa, 4 mL UFC801024, 239

Amicon ultra, Millipore, USA). The protein concentration was measured using Bradford’s reagent 240

(BioRad, UK) and the activity of the extract using the GOD-PERID reagent. 241

Proteins were separated on a Mono Q HR 515 column (50 x 5 mm I.D., Pharmacia, Uppsala, Sweden). 242

The column was attached to a WATERS HPLC purification system (650 E, Millipore, USA) equipped 243

with a UV detector (ABI 757 absorbance detector, Applied Biosystems) and protein elution monitored at 244

A280nm. Both the initial starting buffer (Tris 20 mM, pH 8.0) and elution buffer (Tris 20 mM, plus NaCl 245

1M pH 8.0) were filtered using a Millipore membrane filter. The column was pre-equilibrated with 246

starting buffer. The sample (40 mg of protein) was desalted against the initial starting buffer using Econo-247

Pac 10DG desalting columns then filtered through a 0.2 µm mini-Sart filter prior to injection. Fractions 248

(1.5 mL) were collected every 2 min at flow rate of 0.75 mL/min. The bound protein was eluted by using 249

a linear buffer concentration gradient from 0-1M NaCl (Supplementary information S1). 250

Fractions with myrosinase activity were combined and desalted against starting buffer using Econo-Pac 251

10DG desalting columns and concentrated using an ultrafiltration membrane tube. The sample (0.77 mg) 252

was applied to an ion exchange column (Mono Q column) equilibrated with the same buffer. The 253

separation was carried out exactly as previously outlined except that 0.75mL fractions were collected. 254

The active fractions following the ion exchange purification second step were combined, desalted against 255

CPB (50 mM, pH 6.0) using Econo-Pac 10DG desalting columns and concentrated by ultrafiltration 256

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(molecular weight cut-off 10 KDa, 4 mL UFC801024, Amicon ultra, Millipore, USA). The sample (50 257

µg) was loaded onto a Superdex 75 gel filtration column (Superdex 75, 75, 10/300 GL, GE Healthcare 258

Life Sciences, Sweden) equilibrated with CPB buffer containing NaCl (150 mM) and sodium azide 259

(0.02%). Fractions (0.35 mL) were collected every 1 min at flow rate of 0.35 mL/min and assayed for 260

myrosinase activity. Molecular weight markers 12 - 79 kDa (GE Healthcare Ltd) were used to calibrate 261

the column: cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (45 kDa), albumin bovine 262

serum (66 kDa) and transferrin (79 kDa). 263

SDS PAGE 264

Gels (13%) were prepared as previously described32

and stained with SimplyBlue™ SafeStain (Life 265

Technologies). 266

N-Terminal sequencing and internal peptide sequencing 267

Pure protein was used for N-terminal sequencing (Edman degradation) which was carried out at the 268

Protein Sequence Analysis Facility, Department of Biochemistry, Cambridge (UK). Internal peptide 269

analysis was carried out by slicing out the pure protein from an SDS-PAGE gel stained with 270

SimplyBlue™ SafeStain. The gel bands were processed and analysed by LC-MS as previously 271

described.33

272

The full-length DNA coding sequence was obtained using Geneious Pro 5.6.6. Software. 273

GenBank accession numbers 274

Citrobacter_WYE1_Myr_KT821094; Citrobacter_WYE1_16sRNA_KT825141 275

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RESULTS 283

Strain isolation and identification 284

Serial dilutions were made from the enrichment culture and 100 µL spread onto nutrient agar plates 285

followed by incubation at 30 oC. A single colony was picked and cultured in M9 medium with sinigrin as 286

the sole carbon source overnight. The pure isolate was identified by 16S rRNA sequence analysis. S_ab 287

scores of > 0.95 for twenty matches with known Citrobacter species were obtained and it was deduced 288

that the isolated organism belongs to the family Enterobacteriaceae and the genus Citrobacter and was 289

named Citrobacter WYE1 to indicate the origin of the isolate. It was not possible to assign Citrobacter 290

WYE1 to a specific species based on the 16S rRNA sequence. A phylogenetic tree was constructed to 291

show its relationship among selected Citrobacter strains (Figure 2). It is likely that Citrobacter WYE1 is 292

a new species. 293

Fermentation of sinigrin 294

The metabolism of sinigrin was monitored by HPLC and OD values were recorded over a 24 h period 295

(Figure 3). Sinigrin was completely utilized although no product could be identified. The potential 296

products allyisothiocyanate, or corresponding nitrile, carboxylic acid or amine could not be detected by 297

GC-MS. 298

Properties of the myrosinase activity 299

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Fresh cell-free protein extracts were used for all assays and were tested for activity prior to use. The 300

testing of activity was necessary as there were differences between preparations. An experiment was 301

carried out to determine whether the myrosinase was constitutive or inducible. Growth of Citrobacter 302

WYE1 in M9 media supplemented with glucose gave only low levels of myrosinase activity as did 303

nutrient broth. It was clear, however, that in the presence of sinigrin, myrosinase activity was 304

substantially induced (Figure 4). Sinigrin was stable over the 24 h period in M9 medium alone. The 305

approximate pI of the protein was between pH 6-7, based on binding of the enzyme to miniQ ion 306

exchange columns. Thus, for purification purposes, ion exchange buffers of pH 8 were used. The 307

temperature optimum was 25 oC for the myrosinase (Figure 5), which is unlike most plant and aphid 308

myrosinases where temperature optima are generally higher.17, 18

The pH optimum of the enzyme was 309

found to be 6, while at pH 7.6 the activity was markedly diminished (Figure 6). With this in mind, it was 310

necessary to desalt all purified fractions against CPB in order to obtain the full myrosinase activity. The 311

myrosinase was active towards three other glucosinolates tested: glucoerucin had similar specific activity 312

to sinigrin, while glucotropaeolin and glucoraphanin had lower values, suggesting that the nature of the 313

side chain is important (Figure 7). In addition to the GOD-PERID assays, the ITCs were identified by 314

GC-MS. All of the substrates gave the expected ITCs and no other products such as nitriles were observed 315

(Supplementary information S2). GC-MS analysis of the hydrolysis products was used to determine the 316

activation of myrosinase by ascorbate, as this compound interferes with the GOD-PERID assay. In 317

addition, gluconasturtiin, which results in phenethylisothiocyanate on hydrolysis, was used as the 318

substrate, since allylisothiocyanate produced by sinigrin is very volatile and can result in significant 319

losses during work up. The ascorbate-treated myrosinase assays resulted in an activation of 1.67 (Figure 320

8) which is very low in comparison to the plant enzymes. The apparent Km and Vmax were 0.46 mM and 321

4.91 mmol dm-3

min-1

mg-1

respectively (Figure 9). The Km value of the Citrobacter WYE1 myrosinase 322

was similar to those obtained for the aphid myrosinase19, 20, 22

and some plant myrosinases.18

323

Purification of Citrobacter WYE1 myrosinase 324

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The enzyme was purified by ion exchange chromatography (Supplementary information S1) on a high 325

resolution monoQ ion exchange column, followed by gel filtration The procedure gave a pure single band 326

with an approximate molecular mass of 66 kDa on analysis by SDS-PAGE (Figure 10). The gel filtration 327

step using a high resolution Superdex 75 column gave a native molecular size of approximately 66 kDa, 328

based on GE-Health Care molecular weight markers. Thus it would appear that the bacterial myrosinase is 329

a monomeric protein. The myrosinase is however of low abundance, with a yield of 4.5 µg pure protein 330

from a fermentation of 500 ml (Table 1). 331

Sequence analysis 332

The N-terminal sequence (SIQSAQQPELGYDTV) of the purified protein was determined (Figure 11a) as 333

well as peptides from tryptic digestion. The N-terminal peptide was used as a query to identify the 334

respective gene by tBLASTn against the genomic sequence reads obtained by Illumina. The DNA 335

sequences found were then assembled; the resulting consensus sequence was then used in iterative 336

searches of the non-assembled genome reads to extend the locus encoding the myrosinase gene, until a 337

full-length coding sequence corresponding to a plausible ORF for myrosinase was obtained. No other 338

genes could be identified using the sequence data obtained, suggesting that there is only one myrosinase 339

gene. The validity of the DNA sequence was confirmed by carrying out PCR of the genomic DNA 340

(unpublished data). 341

BLAST analysis of the translated sequence of Citrobacter WYE1 myrosinase showed a high degree of 342

homology with a number of bacterial β-O-glucosidases (Figure 11b), the greatest being with a 343

hypothetical protein sequence from Citrobacter sp. 30_2. This bacterium was made available (Dr. Emma 344

Allen-Vercoe, University of Guelph) to us but showed no activity towards glucosinolates. There was no 345

significant homology with either plant or aphid myrosinases. Using InterProt34

, the protein was shown to 346

belong to the GH 3 family of glucosidases. This is in contrast to known myrosinases which so far all 347

belong to GH 1. 348

Page 15 of 39



16

The full length gene appears to code for an N-terminal signal peptide which presumably targets the 349

enzyme to the periplasm. This observation fits with the purified protein which appears to have lost a 350

peptide as evident from the actual N-terminal sequencing of the protein (Figure 11a). 351

352

353

354

355

356

357

DISCUSSION 358

The bacterial myrosinase is of low abundance in comparison with plant and aphid myrosinases18, 20, 21

but 359

nonetheless is an active inducible enzyme (Figure 4). In contrast to both plant and insect myrosinases, 360

which appear to have a function in defence, the bacterial myrosinases are more likely to be involved in 361

scavenging glucose from glucosides. Citrobacter WYE1 is a soil microorganism which was isolated from 362

a field where oilseed rape had previously grown. The stubble and roots left in the field were ploughed 363

back into the soil which would consequently contain a significant amount of glucosinolate that would be 364

available for bacterial growth. It would seem therefore that bacteria can adapt35

to changes in the 365

environment, and it is likely that other soil bacteria will also have the ability of the Citrobacter WYE1 to 366

metabolise glucosinolates. This is of importance in the field of biofumigation with Brassicaceae crops 367

where glucosinolate metabolism can yield the bioactive isothiocyanates, and is key to the success of this 368

technology. Thus bacteria that degrade glucosinolates can potentially counter the effects of biofumigation 369

by decreasing the available glucosinolate, although at this stage more work is required to establish the 370

Page 16 of 39



17

products of hydrolysis in the soil.25, 36, 37

On the other hand, bacteria that can metabolise glucosinolates in 371

the human gut play a key role in the generation of the chemoprotective isothiocyanates.3, 28, 38

372

The Citrobacter WYE1 myrosinase belongs to the GH3 family of the glucosidases 9 and INTERPRO 373

analysis shows that the full length gene codes for a signal peptide, which presumably targets it to the 374

periplasm. In terms of sequence homology there is no resemblance to either plant or aphid myrosinases, 375

although there is high homology with other bacterial β-O-glucosidases (Figure 11b). A feature of the GH3 376

glucosidases is the conserved motif ‘SDW’ which contains aspartate as the nucleophile, as part of the 377

mechanism for hydrolysis.8, 39

The SWISS-MODEL webpage tools40

were used to generate models based 378

on available templates. The scores, however, were too low to obtain a meaningful model. With the GH3 379

glucosidases, the general acid/base catalytic component of the mechanism is much more variable and will 380

require structure determination to fully elucidate the mechanism, work that is currently underway. Recent 381

reports41

have suggested that E. coli 0157:H7 possesses 6-phospho-β-glucosidases (bglA and ascbB) and, 382

following gene disruption, the sinigrin degradation capacity of the bacteria is substantially diminished. 383

This result41

is supported by previous work7 where it was not possible to isolate myrosinase activity in 384

cell free extracts of bacteria, despite the production of isothiocyanates and nitriles during fermentation. A 385

differential proteomic study of E. coli VL8 with or without sinigrin did not reveal any glucosidase 386

associated with glucosinolate hydrolysis.33

A glucose-specific phosphotransferase system was however 387

induced, which suggests that glucosinolates may undergo phosphorylation at the 6-hydroxyl group of the 388

sugar residue. If this is the case, then it may well be that 6-phospho-β-glucosidases in cell free extracts 389

cannot recognize glucosinolates without prior phosphorylation or that they are membrane-bound and loss 390

of integrity results in loss of activity. This might explain why it has proven so difficult to show 391

myrosinase activity in cell free extracts of bacteria4, 28

that can metabolise glucosinolates, although 392

enzyme instability cannot be ruled out. Thus a synthesis, either chemical or chemoenzymatic42

, of a 6-393

phospho-glucosinolate would go some way to probing the mechanism of glucosinolate metabolism if 6-394

phospho-β-glucosidases are involved. Clearly at this time there are several potential distinct mechanisms 395

Page 17 of 39



18

for glucosinolate hydrolysis by bacteria that may involve phosphorylation, membrane-bound enzyme 396

systems and a more typical hydrolysis by a glucosidase as shown in our work. 397

It was not possible to identify the fermentation products at this time, although they were not any of the 398

usual glucosinolate hydrolysis products, i.e. nitrile, carboxylic acid, amine or thiocyanate (unpublished 399

data) that have been previously described.17, 18

Previous work43

has shown isothiocyanates to be unstable 400

over a period of 24 h in phosphate buffers, although cell free extracts of the Citrobacter WYE1 produce 401

isothiocyanates in citrate phosphate buffer. Of course, it may also be possible that Citrobacter WYE1 402

possesses a detoxification pathway. M9 media contain not just phosphate but also ammonium ions which 403

likely add to the possible interactions with ITCs. Thus at this stage it is not clear what the actual 404

degradation metabolites are in vivo and more work is required on this topic. In conclusion, this is the first 405

report to describe a fully characterized bacterial myrosinase in terms of its properties and sequence. 406

407

ACKNOWLEDGEMENTS 408

Abdul Albaser was funded by the Libyan Government and Vijitra Luang-In by the Royal Thai 409

Government. We thank Dr Arnoud Van Vliet of the Institute of Food Research, Norwich, UK for help 410

with the bioinformatics. 411

SUPPORTING INFORMATION 412

A table describing the FPLC programme for the ion exchange chromatography is shown in S1. 413

Figures for the qualitative GC-MS analysis of cell free extracts with sinigrin, glucoerucin, glucoraphanin, 414

glucotropaeolin, glucoraphanin and gluconasturtiin are shown in S2. This material is available free of 415

charge via the internet at http://pubs.acs.org. 416

417

418

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419

420

421

422

423

424

425

426

427

428

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Their Breakdown Products in Foods. Angew. Chem., Int. Ed. 2014, 53, 11430-11450. 470

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from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore. Eur. J. Biochem. 2001, 268, 1041-476

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close relationship to beta-glucosidases. Insect Biochem. Mol. Biol. 2005, 35, 1311-1320. 486

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cancer risk: epidemiologic evidence and mechanistic basis. Pharmacol. Res. 2007, 55, 224-236. 488

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and Their Degradation Products against Brassica-Pathogenic Bacteria and Fungi. Appl. Environ. 495

Microbiol. 2015, 81, 432-440. 496

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540

541

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542

543

544

545

546

547

548

549

550

551

Figure Legends. 552

Figure 1. Generalised scheme of the hydrolysis of glucosinolates by myrosinase. RNCS, isothiocyanate; 553

RCN, nitrile; RSCN, thiocyanate; ETN, epithionitriles. 554

Figure 2. Phylogenetic tree of Citrobacter WYE1 compared with related Citrobacter species. Draft 555

genomes of Citrobacter species were obtained from Genbank WGS projects (accession prefix 556

supplied). Core genome single nucleotide polymorphisms (SNPs) in draft genomes were used to 557

prepare the phylogenetic tree using the parSNP program. 558

Figure 3. The metabolism of sinigrin (10 mM) in M9 media by Citrobacter WYE1 over 24 h. The 559

metabolism of sinigrin was monitored by HPLC and OD600nm recorded. 560

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25

Figure 4. The induction of myrosinase activity in cultures grown with sinigrin. Cell free protein extracts 561

were prepared as described in the methods and assayed for myrosinase activity with 5 µg of crude 562

desalted protein extract/assay. 563

Figure 5. The effect of temperature on the activity of the Citrobacter WYE1 myrosinase was measured 564

over a range of temperatures (5-70 ºC). Assays were carried out as described in the methods with 5µg 565

crude cell free protein. 566

Figure 6. The pH optimisation of the enzymatic degradation of sinigrin. Crude protein extract (5 µg) was 567

added to a mixture of sinigrin (2 mM) in CPB to a final volume of 300 µL at specific pH values over the 568

range 3.6 -7.6 and incubated at 37 °C for 2 h. Activity was measured by determining the release of 569

glucose using the GOD-PERID assay. 570

Figure 7. The enzymatic degradation of a range of glucosinolates (10 mM) in CPB. Activity was 571

measured by determining the release of glucose using the GOD-PERID assay. 572

Figure 8. The effect of differing concentrations of ascorbate on the enzymatic activity of myrosinase. 573

Activity was determined by monitoring the production of phenethylisothiocyanate using GC-MS. ● = 574

ascorbate, ■ = control. 575

Figure 9. The determination of the apparent Km and Vmax of the myrosinase. 576

Figure 10. SDS PAGE analysis of the purification steps of Citrobacter WYE1 myrosinase; M= marker; 577

IE1= Ion exchange first run; IE2= Ion exchange second run; GF= gel filtration fraction of pure 578

myrosinase at 66 kDa. Gels were washed three times with MilliQ H2O (100 mL) and stained with 579

SimplyBlue SafeStain (Invitrogen LC6060) for an hour and destained in MilliQ H2O. 580

Figure 11a. Full length protein based on the first ORF derived from the annotated gene. Putative signal 581

peptide is underlined, N-terminal sequence in bold and italics and internal peptide sequence in bold type. 582

Figure 11b. BLAST search results of the Citrobacter WYE1 showing the homology with a range of 583

bacterial β-O-glucosidases. The signature ‘SDW’ of the GH3 glucosidases is highlighted. 584

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585

586

587

588

589

590

591

592

593

594

595

596

597

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27

Purification step

Volume

(mL)

Protein

(mg)

Specific activity

(nmole/mg

protein/min)

Total

activity

Crude extract 10 40 611 24,444

Ion exchange 1st (Mono Q

column ) 2.5 0.775 1111 861

Ion exchange 2nd (Mono Q

column ) 0.5 0.05 593 29.6

Gel filtration (Superdex 75

column) 0.35 0.0045 2686 12.1

Table 1 Summary of the purification steps of Citrobacter myrosinase. Activity assays were carried out

with sinigrin (10 mM) at 25°C (pH 6.0, CPB 20 mM) and are based on glucose release (GOD-Perid assay).

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Figure 1

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Figure 2

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Figure 3

h

0 4 8 12 16 20 24 28

0

20

40

60

80

100

0.0

0.2

0.4

0.6

0.8

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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MLTAFKMNTVSLAVLVCLPLSVSASIQSAQQPELGYDTVPLLHLSGLSFKDLNRDGKLNPYEDWRLSPQTRAADLVKRMSVAEKAGVM

MHGTAPAEGSTFGNGSVYDSEATRKMVVDAHVNSLITRLNGEEPARLAEQNNMIQKTAETTRLGIPVTISTDPRNSYQALVGISNPAGK

FTQWPEAIGLGAAGSEALAQEYADHVRREYRAVGITEALSPQADITTEPRWARISGTFGEDPELAKKLVRGYITGMQKGGQGLNPQSV

AAVVKHWVGYGAAEDGWDGHNAYGKHTVLSNESLQKHIIPFRGAFEANVAAVMPTYSVMKGVTWNGRETEQVAAGFSHFLLTDLL

RKQNNFSGVIISDWLITNDCDDECVNGSAPGKKPVAGGMPWGVESLSKERRFVKAVNAGIDQFGGVTDSAVLVTAVEKGLITQARLDA

SVERILQQKFELGLFEQPYVDAKLAEKIVGAPDTKKAADDAQFRTLVLLQNKNILPLKPGTKVWLYGADKSAAEKAGLEVVSEPEDAD

VALMRTSAPFEQPHYNYFFGRRHHEGSLEYREDNKDFAVLKRVSKHTPVIMTMYMERPAVLTNVTDKTSGFIANFGLSDEVFFSRLTSD

TPYTARLPFALPSSMASVLKQKSDEPDDLDTPLFQRGFGLTR

Figure 11a

Figure 11b

361 420Citrobacter WYE1 VIISDWLITNDCDDECVNGSAPGKKPVAGGMPWGVESLSKERRFVKAVNAGIDQFGGVTD Enterobacter cloacae β-glucosidase(70%) LIISDWLITADCDEECVNGTTGDKKPVPGGMPWGVEHLSKEQRFVKAVNAGIDQFGGVTD

Citrobacter sp.30_2 hypothetical protein(71%) VIISDWLITNDCDDECIHGAPGGKKPVTGGMPWGVESLTQEQRFVKAVQAGIDQFGGVTD Escherichia vulneris β-glucosidase (72%) VIISDWLITNDCDNECLNGAPEGKKPVTGGMPWGVESLTKEQRFVKAIHAGIDQFGGVTD

Rahnella aquatilis β-glucosidase VILSDWLITSNCEGECLNGSPEGKEPVPGGMPWGVENLTPQQRFVKAVLAGVDQFGGVTN Pectobacterium carotovorum β-glucosidase(60%) VILSDWLITNDCKDDCVTGAKPNEKPVPRGMPWGVENLTVEQRFIKAVEAGVDQFGGVTN

Serratia sp 1,4- β-xylosidase (61%) VILSDWLITQDCKADCINGFKPGEKPVPRGMPWGVEHMTVDQRFIQAVKAGIDQFGGVTD Dickeya solani β-xylosidase periplasmic (61%) VILSDWLITNDCKGDCLTGVKPGEKPVPRGMPWGVENLTPAERFIKAVNAGVDQFGGVTD

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Table of Contents Graphic

334x202mm (96 x 96 DPI)

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with myrosinase activity from a citrobacter strain ... · note that technical editing may introduce...

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