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Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3

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Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3 Subscriber access provided by Imperial College London | Library Article 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 29, 2016 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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Page 1 of 39 Journal of Agricultural and Food Chemistry 1 Discovery of a bacterial glycoside hydrolase family 3 (GH3) β-glucosidase with myrosinase activity 2 from a Citrobacter strain isolated from soil 3 4 Abdulhadi Albaser 1,5 , Eleanna Kazana 1,3 , Mark H Bennett 1, Fatma Cebeci 2, Vijitra Luang-In 1,4 Pietro 5 D Spanu 1 and John T Rossiter 1* 6 7 1 Imperial College London, Faculty of Life Sciences, London SW7 2AZ, UK 8 2 Food and Health Programme, Institute of Food Research, Norwich, NR4 7UA, UK 9 3Current address: School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ 10 4Current Address: Mahasarakham University, Faculty of Technology, Department of Biotechnology, 11 Natural Antioxidant Research Unit, Mahasarakham 44150, Thailand 12 5Current address: Division of Microbiology, Faculty of Sciences, University of Sebha, Sebha Libya 13 *Corresponding author; John T Rossiter, Imperial College London, Faculty of Life Sciences, London 14 SW7 2AZ, UK; email: [email protected] 15 16 17 18 19 20 21 1 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 2 of 39 22 ABSTRACT 23 A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin 24 as a sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and 25 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 27 InterPro sequence analysis, belongs to the family GH3, contains a signal peptide and is a periplasmic 28 protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out 29 using protein extracts from cell free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 30 mmol dm -3 min -1 mg -1 respectively with sinigrin as substrate. The optimum temperature and pH for 31 enzyme activity were 25 oC and 6.0 respectively. The enzyme was marginally activated with ascorbate by 32 a factor of 1.67. 33 34 Keywords: myrosinase, bacteria, isothiocyanate, sequence 35 36 37 38 39 40 41 42 43 2 ACS Paragon Plus Environment Page 3 of 39 Journal of Agricultural and Food Chemistry 44 INTRODUCTION 45 Bacterial myrosinases are not well understood and there has been little work on this topic. Bacterial 46 myrosinases were first purified some years ago as an active enzyme but without any characterisation in 47 terms of sequence.1, 2 Since then there have been various reports examining the metabolism of 48 glucosinolates by bacteria and attempts have been made to characterize the enzyme but with limited 49 success. 3-7 We have set out to isolate a bacterium that can grow on sinigrin, purify the myrosinase and 50 obtain a sequence. β-Glucosidases (EC 3.2.1.21) catalyse the hydrolysis of the glucosidic linkages of a 51 variety of β-glucosides and, depending on sequence and structure, can be placed in the glycoside 52 hydrolase (GH) families GH1, GH3, GH9, GH 30 and GH116.8-10 With the exception of GH9, the 53 remainder utilize a two-step mechanism that involves a catalytic nucleophile that displaces the aglycone 54 and is followed by protonation of the nucleofuge and attack by a water molecule to give a glycosyl 55 residue. Plant myrosinases (thioglucoside glucohydrolase EC 3.2.3.147) belong to the family GH1 and 56 their structure and mechanisms have been well studied.11-14 Myrosinases hydrolyse glucosinolates, a 57 group of plant secondary metabolites, to give a variety of products (Figure 1) depending on the presence 58 of specifier proteins and type of glucosinolate, but the default product is an isothiocyanate (ITC).15-18 The 59 mechanism of plant myrosinase does not utilize the typical catalytic acid/base employed by β- 60 glucosidases but instead uses ascorbate as a catalytic base. The Sinapis alba myrosinase is a dimer linked 61 by a zinc atom and has a characteristic (β/α) 8-barrel structure in common with GH1 enzymes. The insect 62 Brevicoryne brassicae (cabbage aphid) contains a myrosinase that has been studied in some detail and 63 also belongs to the glucose hydrolase family GH1 19-21 and its structure has been determined.22 In contrast 64 to the S. alba myrosinase, the aphid myrosinase does not require ascorbate for activity and its mechanism 65 is the same as for β-glucosidases i.e . a nucleophile (glutamic acid residue) that forms a glycosyl-enzyme 66 intermediate together with a glutamic acid residue that acts as a catalytic base/acid typical of most GH1 67 glucosidases. 3 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 4 of 39 68 There is, however, increasing interest in bacteria that can metabolise glucosinolates, particularly those of 69 the human gut, where the chemoprotective nature of glucosinolate hydrolysis products is of dietary 70 importance.23 In addition, soil bacteria are also of importance in terms of soil ecology and in the 71 application of biofumigation, where plants belonging to the Brassicaceae, which are high in glucosinolate 72 content, are used to fumigate soil.24-27 In this work we describe the isolation of a soil bacterium that 73 possesses an inducible β-glucosidase that transforms glucosinolates into isothiocyanates. The properties 74 and sequence of the enzyme are described. 75 76 77 78 79 80 81 82 83 84 85 86 87 88 4 ACS Paragon Plus Environment Page 5 of 39 Journal of Agricultural and Food Chemistry 89 METHODS 90 Glucosinolates 91 Glucosinolates were isolated from seed sources as previously described.28 92 Isolation of a glucosinolate-metabolizing bacterial strain by enrichment 93 Soil was taken from a field in Wye (Kent, UK), which had previously been used for growing oilseed rape, 94 and 1 gram was placed into 10 ml of M9 medium with sinigrin (10 mg) as the sole carbon source and 95 placed in the dark at room temperature for 2 weeks. After gentle agitation, 1 ml of solution was removed, 96 placed into fresh M9 media with sinigrin as the sole carbon source and incubated for a further 2 weeks. 97 This process was repeated a further 5 times. Finally, dilutions were made and a small amount of the 98 enrichment culture was streaked onto a nutrient agar plate for each dilution. The plates were incubated 99 overnight at 30 oC. Colonies were added to fresh M9 media containing sinigrin as the sole carbon source o 100 and incubated at 30 C, and the OD 600nm (optical density at 600nm) was monitored. The culture was 101 centrifuged at 10000 g for 5 min and stored at -20 oC until analysis. Sinigrin-metabolizing colonies were 102 grown overnight in M9 media supplemented with sinigrin as the sole carbon source, and the following 103 day glycerol stocks (40%) were made and stored at -80 oC. 104 HPLC analysis of sinigrin fermentation 105 The fermentation of sinigrin was monitored by removing 1 mL of culture into an Eppendorf (1.5 mL) tube 106 and centrifuging at 10000 g for 10 min. The supernatant was removed and stored at -20 oC until analysis. 107 Samples were thawed out (1 mL), 100 µL of a barium/lead acetate solution (equimolar, 1M) were added, 108 and samples were thoroughly mixed and allowed to stand for a few minutes. The samples were 109 centrifuged at 10000 g for 5 min and the supernatant was removed and transferred to a fresh Eppendorf 110 tube.
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