Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) Î²

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Discovery of a bacterial glycoside hydrolase family 3 (GH3) #-glucosidase with myrosinase activity from a Citrobacter strain isolated from soil Abdulhadi Albaser, Eleanna Kazana, Mark Bennett, Fatma Cebeci, 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 Downloaded from http://pubs.acs.org on January 28, 2016

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Journal of Agricultural and Food Chemistry

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

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from a Citrobacter strain isolated from soil

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Abdulhadi Albaser 1,5, Eleanna Kazana 1,3, Mark H Bennett 1, Fatma Cebeci 2, Vijitra Luang-In 1,4 Pietro

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D Spanu 1 and John T Rossiter 1*

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

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

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

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

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Natural Antioxidant Research Unit, Mahasarakham 44150, Thailand

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

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

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

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ABSTRACT

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

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

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

26

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

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

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

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

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mmol dm-3 min-1 mg-1 respectively with sinigrin as substrate. The optimum temperature and pH for

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

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a factor of 1.67.

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Keywords: myrosinase, bacteria, isothiocyanate, sequence

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INTRODUCTION

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

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

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terms of sequence.1, 2 Since then there have been various reports examining the metabolism of

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

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success.3-7 We have set out to isolate a bacterium that can grow on sinigrin, purify the myrosinase and

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

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

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

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

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

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

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their structure and mechanisms have been well studied.11-14 Myrosinases hydrolyse glucosinolates, a

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

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

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

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

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

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

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also belongs to the glucose hydrolase family GH1 19-21 and its structure has been determined.22 In contrast

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

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

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

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glucosidases.

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

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

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importance.23 In addition, soil bacteria are also of importance in terms of soil ecology and in the

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

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content, are used to fumigate soil.24-27 In this work we describe the isolation of a soil bacterium that

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

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and sequence of the enzyme are described.

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METHODS

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Glucosinolates

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Glucosinolates were isolated from seed sources as previously described.28

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Isolation of a glucosinolate-metabolizing bacterial strain by enrichment

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

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

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

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

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

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

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

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

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

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

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

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HPLC analysis of sinigrin fermentation

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

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

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

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

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

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

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

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decanted and the ion exchanger was mixed with deionized water, decanted (x 2) and finally stored in 20 5 ACS Paragon Plus Environment


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

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

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

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

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

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

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

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

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

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

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

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

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GC-MS analysis of glucosinolate metabolites

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

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

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

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

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

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

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

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

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

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

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

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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, 6 ACS Paragon Plus Environment

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

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

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

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

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

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

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by GC-MS analysis was 10 ng/mL.

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

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

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

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

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

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

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

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95oC, 30 s (20 cycles); annealing 55 oC, 30 s (20 cycles); extension 72 °C, 1 min 30 s (20 cycles); final

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extension

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

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

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

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

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

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

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

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

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

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



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

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phylogenetic tree using parSNP program Version 1.2 30.

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Myrosinase assay

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

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glucose.31 Preparation of the GOD-PERID (glucose oxidase-peroxidase) reagent (all reagents purchased

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

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

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

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

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

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

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

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

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

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General assay conditions for myrosinase activity

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

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

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

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

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

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

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

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

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


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

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

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

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pH Optimisation

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

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

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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

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

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

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Temperature optimization

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

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

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

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assay.

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Substrate specificity

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

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

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previously described for glucosinolate metabolites.

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Activation by ascorbate

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

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

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



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

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phenethylisothiocyanate concentrations versus total ion current.

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Determination of apparent Km & Vmax for sinigrin

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

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

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

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

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GraphPad version 6.

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Estimation of pI

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

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

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

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

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

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

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

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

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being assayed for activity.

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Inducibility of myrosinase

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

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

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

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Purification of Citrobacter WYE1 myrosinase


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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CPB (50 mM, pH 6.0) using Econo-Pac 10DG desalting columns and concentrated by ultrafiltration 11 ACS Paragon Plus Environment


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

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

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

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

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

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

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

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SDS PAGE

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Gels (13%) were prepared as previously described32 and stained with SimplyBlue™ SafeStain (Life

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Technologies).

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N-Terminal sequencing and internal peptide sequencing

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

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

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

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

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described.33

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

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GenBank accession numbers

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Citrobacter_WYE1_Myr_KT821094; Citrobacter_WYE1_16sRNA_KT825141

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RESULTS

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Strain isolation and identification

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

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

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

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

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

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

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

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

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a new species.

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Fermentation of sinigrin

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

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

297

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

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GC-MS.

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Properties of the myrosinase activity



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

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

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

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

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

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

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

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

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

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myrosinases where temperature optima are generally higher.17, 18 The pH optimum of the enzyme was

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

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

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

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

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

315

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

316

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

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

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

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

320

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

321

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

322

4.91 mmol dm-3 min-1 mg-1 respectively (Figure 9). The Km value of the Citrobacter WYE1 myrosinase

323

was similar to those obtained for the aphid myrosinase19, 20, 22 and some plant myrosinases.18

324

Purification of Citrobacter WYE1 myrosinase


Page 15 of 39


325

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

326

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

327

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

328

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

329

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

330

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

331

from a fermentation of 500 ml (Table 1).

332

Sequence analysis

333

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

334

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

335

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

336

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

337

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

338

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

339

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

340

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

341

(unpublished data).

342

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

343

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

344

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

345

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

346

significant homology with either plant or aphid myrosinases. Using InterProt34, the protein was shown to

347

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

348

belong to GH 1.



Page 16 of 39

349

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

350

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

351

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

352 353 354 355 356 357 358

DISCUSSION

359

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

360

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

361

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

362

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

363

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

364

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

365

available for bacterial growth. It would seem therefore that bacteria can adapt35 to changes in the

366

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

367

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

368

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

369

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

370

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


Page 17 of 39


371

products of hydrolysis in the soil.25, 36, 37 On the other hand, bacteria that can metabolise glucosinolates in

372

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

373

The Citrobacter WYE1 myrosinase belongs to the GH3 family of the glucosidases

374

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

375

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

376

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

377

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

378

mechanism for hydrolysis.8, 39 The SWISS-MODEL webpage tools40 were used to generate models based

379

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

380

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

381

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

382

reports41 have suggested that E. coli 0157:H7 possesses 6-phospho-β-glucosidases (bglA and ascbB) and,

383

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

384

This result41 is supported by previous work7 where it was not possible to isolate myrosinase activity in

385

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

386

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

387

associated with glucosinolate hydrolysis.33 A glucose-specific phosphotransferase system was however

388

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

389

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

390

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

391

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

392

myrosinase activity in cell free extracts of bacteria4,

393

enzyme instability cannot be ruled out. Thus a synthesis, either chemical or chemoenzymatic42, of a 6-

394

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

395

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

28

9

and INTERPRO

that can metabolise glucosinolates, although



Page 18 of 39

396

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

397

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

398

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

399

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

400

data) that have been previously described.17, 18 Previous work43 has shown isothiocyanates to be unstable

401

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

402

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

403

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

404

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

405

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

406

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

407 408

ACKNOWLEDGEMENTS

409

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

410

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

411

with the bioinformatics.

412

SUPPORTING INFORMATION

413

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

414

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

415

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

416

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

417 418 18 ACS Paragon Plus Environment

Page 19 of 39


419 420 421 422 423 424 425 426 427 428 429

REFERENCES

430

(1) Tani, N.; Ohtsuru, M.; Hata, T., Studies on bacterial myrosinase .1. Isolation of myrosinase producing

431

microorganism. Agric. Biol. Chem. 1974, 38, 1617-1622.

432

(2) Tani, N.; Ohtsuru, M.; Hata, T., Studies on bacterial myrosinase .2. Purification and general

433

characteristics of bacterial myrosinase produced by enterobacter-cloacae. Agric. Biol. Chem. 1974, 38,

434

1623-1630.

435

(3) Saha, S.; Hollands, W.; Teucher, B.; Needs, P. W.; Narbad, A.; Ortori, C. A.; Barrett, D. A.; Rossiter,

436

J. T.; Mithen, R. F.; Kroon, P. A., Isothiocyanate concentrations and interconversion of sulforaphane to

437

erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli.

438

Mol. Nutr. Food Res. 2012, 56, 1906-1916.

439

(4) Palop, M. L.; Smiths, J. P.; Tenbrink, B., Degradation of sinigrin by lactobacillus-agilis strain r16. Int.

440

J. Food Microbiol. 1995, 26, 219-229.



441

(5) Rabot, S.; Nugonbaudon, L.; Raibaud, P.; Szylit, O., Rapeseed meal toxicity in gnotobiotic-rats -

442

influence of a whole human fecal flora or single human strains of escherichia-coli and bacteroides-

443

vulgatus. Br. J. Nutr. 1993, 70, 323-331.

444

(6) Nugonbaudon, L.; Rabot, S.; Wal, J. M.; Szylit, O., Interactions of the intestinal microflora with

445

glucosinolates in rapeseed meal toxicity - 1st evidence of an intestinal lactobacillus possessing a

446

myrosinase-like activity invivo. J. Sci. Food Agric. 1990, 52, 547-559.

447

(7) Luang-In, V., Influence of Human Gut Microbiota on the Metabolic Fate of Glucosinolates. PhD

448

Thesis, Imperial College London 2013.

449

(8) Ketudat Cairns, J. R.; Esen, A., beta-Glucosidases. Cell. Mol. Life. Sci. 2010, 67, 3389-405.

450

(9) Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B., The

451

Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids

452

Res. 2009, 37, D233-D238.

453

(10) Sansenya, S.; Opassiri, R.; Kuaprasert, B.; Chen, C.-J.; Cairns, J. R. K., The crystal structure of rice

454

(Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing beta-

455

glucosidase with significant thioglucohydrolase activity. Arch. Biochem. Biophys. 2011, 510, 62-72.

456

(11) Mahn, A.; Angulo, A.; Cabanas, F., Purification and Characterization of Broccoli (Brassica oleracea

457

var. italica) Myrosinase (beta-Thioglucosidase Glucohydrolase). J. Agric. Food Chem. 2014, 62, 11666-

458

11671.

459

(12) Burmeister, W. P.; Cottaz, S.; Driguez, H.; Iori, R.; Palmieri, S.; Henrissat, B., The crystal structures

460

of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the

461

substrate recognition and active-site machinery of an S-glycosidase. Structure. 1997, 5, 663-675.

462

(13) Burmeister, W. P.; Cottaz, S.; Rollin, P.; Vasella, A.; Henrissat, B., High resolution x-ray

463

crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the

464

catalytic base. J. Biol. Chem. 2000, 275, 39385-39393.

465

(14) Cottaz, S.; Henrissat, B.; Driguez, H., Mechanism-based inhibition and stereochemistry of

466

glucosinolate hydrolysis by myrosinase. Biochemistry. 1996, 35, 15256-15259. 20 ACS Paragon Plus Environment

Page 20 of 39

Page 21 of 39


467

(15) Dosz, E. B.; Ku, K.-M.; Juvik, J. A.; Jeffery, E. H., Total Myrosinase Activity Estimates in Brassica

468

Vegetable Produce. J. Agric. Food Chem. 2014, 62, 8094-8100.

469

(16) Hanschen, F. S.; Lamy, E.; Schreiner, M.; Rohn, S., Reactivity and Stability of Glucosinolates and

470

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

471

(17) Bones, A. M.; Rossiter, J. T., The enzymic and chemically induced decomposition of glucosinolates.

472

Phytochemistry. 2006, 67, 1053-1067.

473

(18) Bones, A. M.; Rossiter, J. T., The myrosinase-glucosinolate system, its organisation and

474

biochemistry. Physiol. Plant. 1996, 97, 194-208.

475

(19) Pontoppidan, B.; Ekbom, B.; Eriksson, S.; Meijer, J., Purification and characterization of myrosinase

476

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

477

1048.

478

(20) Jones, A. M. E.; Bridges, M.; Bones, A. M.; Cole, R.; Rossiter, J. T., Purification and

479

characterisation of a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.). Insect

480

Biochem. Mol. Biol. 2001, 31, 1-5.

481

(21) Jones, A. M. E.; Winge, P.; Bones, A. M.; Cole, R.; Rossiter, J. T., Characterization and evolution of

482

a myrosinase from the cabbage aphid Brevicoryne brassicae. Insect Biochem. Mol. Biol. 2002, 32, 275-

483

284.

484

(22) Husebye, H.; Arzt, S.; Burmeister, W. P.; Hartel, F. V.; Brandt, A.; Rossiter, J. T.; Bones, A. M.,

485

Crystal structure at 1.1 angstrom resolution of an insect myrosinase from Brevicoryne brassicae shows its

486

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

487

(23) Higdon, J. V.; Delage, B.; Williams, D. E.; Dashwood, R. H., Cruciferous vegetables and human

488

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

489

(24) Neubauer, C.; Heitmann, B.; Mueller, C., Biofumigation potential of Brassicaceae cultivars to

490

Verticillium dahliae. Eur. J. Plant Pathol. 2014, 140, 341-352.



491

(25) Ngala, B. M.; Haydock, P. P. J.; Woods, S.; Back, M. A., Biofumigation with brassica juncea,

492

raphanus sativus and eruca sativa for the management of field populations of globodera pallida. J.

493

Nematol. 2014, 46, 210-211.

494

(26) Sotelo, T.; Lema, M.; Soengas, P.; Cartea, M. E.; Velasco, P., In Vitro Activity of Glucosinolates

495

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

496

Microbiol. 2015, 81, 432-440.

497

(27) Taylor, F. I.; Kenyon, D.; Rosser, S., Isothiocyanates inhibit fungal pathogens of potato in in vitro

498

assays. Plant Soil. 2014, 382, 281-289.

499

(28) Luang-In, V.; Narbad, A.; Nueno-Palop, C.; Mithen, R.; Bennett, M.; Rossiter, J. T., The metabolism

500

of methylsulfinylalkyl- and methylthioalkyl- glucosinolates by a selection of human gut bacteria. Mol.

501

Nutr. Food Res. 2014, 58, 875-883.

502

(29) Nurk, S.; Bankevich, A.; Antipov, D.; Gurevich, A. A.; Korobeynikov, A.; Lapidus, A.; Prjibelski,

503

A. D.; Pyshkin, A.; Sirotkin, A.; Sirotkin, Y.; Stepanauskas, R.; Clingenpeel, S. R.; Woyke, T.; McLean,

504

J. S.; Lasken, R.; Tesler, G.; Alekseyev, M. A.; Pevzner, P. A., Assembling Single-Cell Genomes and

505

Mini-Metagenomes From Chimeric MDA Products. J. Comput. Biol. 2013, 20, 714-737.

506

(30) Treangen, T. J.; Ondov, B. D.; Koren, S.; Phillippy, A. M., The Harvest suite for rapid core-genome

507

alignment and visualization of thousands of intraspecific microbial genomes. Genome biology 2014, 15,

508

524.

509

(31) Huggett, A. S.; Nixon, D. A., Use of glucose oxidase, peroxidase, and O-dianisidine in determination

510

of blood and urinary glucose. Lancet (London, England). 1957, 273, 368-70.

511

(32) James, D. C.; Rossiter, J. T., Development and characteristics of myrosinase in brassica-napus during

512

early seedling growth. Physiol. Plant. 1991, 82, 163-170.

513

(33) Luang-In, V.; Narbad, A.; Cebeci, F.; Bennett, M.; Rossiter, J. T., Identification of Proteins Possibly

514

Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8. Protein J. 2015, 34, 135-46.

515

(34) Jones, P.; Binns, D.; Chang, H. Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.;

516

Mitchell, A.; Nuka, G.; Pesseat, S.; Quinn, A. F.; Sangrador-Vegas, A.; Scheremetjew, M.; Yong, S. Y.; 22 ACS Paragon Plus Environment

Page 22 of 39

Page 23 of 39


517

Lopez, R.; Hunter, S., InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014,

518

30, 1236-40.

519

(35) Diaz, E., Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility. Int.

520

Microbiol. 2004, 7, 173-180.

521

(36) Gimsing, A. L.; Kirkegaard, J. A., Glucosinolate and isothiocyanate concentration in soil following

522

incorporation of Brassica biofumigants. Soil Biol. Biochem. 2006, 38, 2255-2264.

523

(37) Gimsing, A. L.; Kirkegaard, J. A., Glucosinolates and biofumigation: fate of glucosinolates and their

524

hydrolysis products in soil. Phytochem. Rev. 2009, 8, 299-310.

525

(38) Mullaney, J. A.; Kelly, W. J.; McGhie, T. K.; Ansell, J.; Heyes, J. A., Lactic Acid Bacteria Convert

526

Glucosinolates to Nitriles Efficiently Yet Differently from Enterobacteriaceae. J. Agric. Food Chem.

527

2013, 61, 3039-3046.

528

(39) Withers, S. G., Mechanisms of glycosyl transferases and hydrolases. Carbohydrate Polymers 2001,

529

44, 325-337.

530

(40) Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Cassarino,

531

T. G.; Bertoni, M.; Bordoli, L.; Schwede, T., SWISS-MODEL: modelling protein tertiary and quaternary

532

structure using evolutionary information. Nucleic Acids Res. 2014, 42, W252-W258.

533

(41) Cordeiro, R. P.; Doria, J. H.; Zhanel, G. G.; Sparling, R.; Holley, R. A., Role of glycoside hydrolase

534

genes in sinigrin degradation by E. coli O157:H7. Int. J. Food Microbiol. 2015, 205, 105-111.

535

(42) Thompson, J.; Lichtenthaler, F. W.; Peters, S.; Pikis, A., beta-glucoside kinase (BglK) from

536

Klebsiella pneumoniae - Purification, properties, and preparative synthesis of 6-phospho-beta-D-

537

glucosides. J. Biol. Chem. 2002, 277, 34310-34321.

538

(43) Mays, J. R.; Roska, R. L. W.; Sarfaraz, S.; Mukhtar, H.; Rajski, S. R., Identification, synthesis, and

539

enzymology of non-natural glucosinolate chemopreventive candidates. ChemBioChem. 2008, 9, 729-747.

540 541 23 ACS Paragon Plus Environment


542 543 544 545 546 547 548 549 550 551 552

Figure Legends.

553

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

554

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

555

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

556

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

557

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

558

prepare the phylogenetic tree using the parSNP program.

559

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

560

metabolism of sinigrin was monitored by HPLC and OD600nm recorded.


Page 24 of 39

Page 25 of 39


561

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

562

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

563

desalted protein extract/assay.

564

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

565

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

566

crude cell free protein.

567

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

568

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

569

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

570

glucose using the GOD-PERID assay.

571

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

572

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

573

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

574

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

575

ascorbate, ■ = control.

576

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

577

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

578

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

579

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

580

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

581

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

582

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

583

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

584

bacterial β-O-glucosidases. The signature ‘SDW’ of the GH3 glucosidases is highlighted. 25 ACS Paragon Plus Environment


585 586 587 588 589 590 591 592 593 594 595 596 597


Page 26 of 39

Page 27 of 39


Specific activity

Purification step Crude extract

Volume

Protein

(nmole/mg

Total

(mL)

(mg)

protein/min)

activity

10

40

611

24,444

2.5

0.775

1111

861

0.5

0.05

593

29.6

0.0045

2686

12.1

Ion exchange 1st (Mono Q column )

Ion exchange 2nd (Mono Q column )

Gel filtration (Superdex 75 column)

0.35

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).



Figure 1


Page 28 of 39

Page 29 of 39


Figure 2



100

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0.8

80

0.6

60 0.4 40 0.2

20 0

0.0 0

4

8

12

16

20

24

h

Figure 3


28

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Figure 4



Figure 5


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Page 33 of 39


Figure 6



Figure 7


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Page 35 of 39


Figure 8



Figure 9


Page 36 of 39

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Figure 10



Page 38 of 39

MLTAFKMNTVSLAVLVCLPLSVSASIQSAQQPELGYDTVPLLHLSGLSFKDLNRDGKLNPYEDWRLSPQTRAADLVKRMSVAEKAGVM MHGTAPAEGSTFGNGSVYDSEATRKMVVDAHVNSLITRLNGEEPARLAEQNNMIQKTAETTRLGIPVTISTDPRNSYQALVGISNPAGK FTQWPEAIGLGAAGSEALAQEYADHVRREYRAVGITEALSPQADITTEPRWARISGTFGEDPELAKKLVRGYITGMQKGGQGLNPQSV AAVVKHWVGYGAAEDGWDGHNAYGKHTVLSNESLQKHIIPFRGAFEANVAAVMPTYSVMKGVTWNGRETEQVAAGFSHFLLTDLL RKQNNFSGVIISDWLITNDCDDECVNGSAPGKKPVAGGMPWGVESLSKERRFVKAVNAGIDQFGGVTDSAVLVTAVEKGLITQARLDA SVERILQQKFELGLFEQPYVDAKLAEKIVGAPDTKKAADDAQFRTLVLLQNKNILPLKPGTKVWLYGADKSAAEKAGLEVVSEPEDAD VALMRTSAPFEQPHYNYFFGRRHHEGSLEYREDNKDFAVLKRVSKHTPVIMTMYMERPAVLTNVTDKTSGFIANFGLSDEVFFSRLTSD TPYTARLPFALPSSMASVLKQKSDEPDDLDTPLFQRGFGLTR Figure 11a

Citrobacter WYE1 Enterobacter cloacae β-glucosidase(70%) Citrobacter sp.30_2 hypothetical protein(71%) Escherichia vulneris β-glucosidase (72%) Rahnella aquatilis β-glucosidase Pectobacterium carotovorum β-glucosidase(60%) Serratia sp 1,4- β-xylosidase (61%) Dickeya solani β-xylosidase periplasmic (61%)

361 420 VIISDWLITNDCDDECVNGSAPGKKPVAGGMPWGVESLSKERRFVKAVNAGIDQFGGVTD LIISDWLITADCDEECVNGTTGDKKPVPGGMPWGVEHLSKEQRFVKAVNAGIDQFGGVTD VIISDWLITNDCDDECIHGAPGGKKPVTGGMPWGVESLTQEQRFVKAVQAGIDQFGGVTD VIISDWLITNDCDNECLNGAPEGKKPVTGGMPWGVESLTKEQRFVKAIHAGIDQFGGVTD VILSDWLITSNCEGECLNGSPEGKEPVPGGMPWGVENLTPQQRFVKAVLAGVDQFGGVTN VILSDWLITNDCKDDCVTGAKPNEKPVPRGMPWGVENLTVEQRFIKAVEAGVDQFGGVTN VILSDWLITQDCKADCINGFKPGEKPVPRGMPWGVEHMTVDQRFIQAVKAGIDQFGGVTD VILSDWLITNDCKGDCLTGVKPGEKPVPRGMPWGVENLTPAERFIKAVNAGVDQFGGVTD

Figure 11b


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