Identification, purification and characterization of a novel glycosidase (BgLm1) from Leuconostoc mesenteroides

Journal Pre-proof Identification, purification and characterization of a novel glycosidase (BgLm1) from Leuconostoc mesenteroides Raquel del Pino-García, Annalisa Porrelli, Patricia Rus-Fernández, Antonio SeguraCarretero, José Antonio Curiel PII:

S0023-6438(19)31171-5

DOI:

https://doi.org/10.1016/j.lwt.2019.108829

Reference:

YFSTL 108829

To appear in:

LWT - Food Science and Technology

Received Date: 12 September 2019 Revised Date:

23 October 2019

Accepted Date: 8 November 2019

Please cite this article as: Pino-García, R.d., Porrelli, A., Rus-Fernández, P., Segura-Carretero, A., Curiel, José.Antonio., Identification, purification and characterization of a novel glycosidase (BgLm1) from Leuconostoc mesenteroides, LWT - Food Science and Technology (2019), doi: https:// doi.org/10.1016/j.lwt.2019.108829. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Identification, purification and characterization of a novel glycosidase (BgLm1)

2

from Leuconostoc mesenteroides

3

Raquel del Pino-García1, Annalisa Porrelli1, Patricia Rus-Fernández1, Antonio Segura-

4

Carretero1,2, and José Antonio Curiel1,3,*

5 6

Author information:

7

1

8

Park, 18016 Granada, Spain

9

2

Department of Analytical Chemistry, University of Granada, 18071 Granada, Spain

10

3

Torres Morente S.A.U., Bussines Park Metropolitano, 18130 Escúzar, Granada, Spain

11

*

12

Correspondence: [email protected]; Tel.: +34-958-637-206.

Functional Food Research and Development Center, Health Science Technological

Author to whom correspondence should be addressed

13 14 15 16 17 18 19 20 21 22 23 24 25

1

26

ABSTRACT

27

This study describes the identification and characterization of a novel recombinant

28

Leuconostoc mesenteroides glycosidase (BgLm1). Since the protein encoded by

29

LEUM_0847 gene was annotated as putative β-glucosidase, characterization procedures

30

were done using p-nitrophenyl-β-D-glucopyranoside as substrate. A high yield of

31

purified recombinant BgLm1 was obtained (12 mg/L). The enzyme showed an optimal

32

activity at pH 6.0 and 40ºC and preserved 65% of residual activity after 48 h of

33

incubation at 25ºC. Ca2+ and Mn2+ ions greatly increased the β-glucosidase activity.

34

Moreover, BgLm1 demonstrated β-galactosidase and β-fucosidase activities. Kinetic

35

parameters of BgLm1 revealed its low affinity to p-nitrophenyl-β-D-glucopyranoside

36

(Km of 9.93 mmol/L).

37

Then, although LEUM_0847 gene was annotated as a β-glucosidase, our results suggest

38

that BgLm1can be indeed considered as a β-galactosidase since high hydrolysis using

39

skimmed milk (lactose) as natural substrate and high affinity (Km of 0.56 mmol/L) and

40

specific constant (2254 mmol/L-1 s-1) to p-nitrophenyl-β-D-galactopyranoside were

41

observed. In conclusion, the enzymatic properties observed in this study, support the

42

interest of BgLm1 for food industrial applications.

43 44

Keywords

45

Galactosidase, Glucosidase, Leuconostoc mesenteroides, LEUM_0847, Lactose

46

Abbreviations

47

Glycoside hydrolase (GH), glucose oxidase/peroxidase enzyme (GOPOD), lactic acid

48

bacteria (LAB), p-nitrophenyl (pNP), p-nitrophenyl-β-D-glucopyranoside (pNPG), p-

49

nitrophenyl -β-D-fucopyranoside (pNPFU), p-nitrophenyl -β-D-galactopyranoside

50

(pNPGAL).

2

51

1. INTRODUCTION

52

Glycosidases (E.C.3.2.1) catalyze the hydrolysis of glycosidic bonds in complex sugars

53

(Speciale, Thompson, Davies, & Williams, 2014). Among the glycosidases, β-

54

glucosidases are the enzymes that catalyze the hydrolysis of the glycosidic bonds of a

55

part of carbohydrates to release nonreducing terminal glycosyl residues, glycosides and

56

oligosaccharides (Li et al., 2013). Because of their enzymatic properties, the food

57

industry uses β-glucosidases to decrease bitterness by the hydrolysis of specific

58

compounds and to release flavored compounds from glycosylated precursors present in

59

fruit juices, wine tea and fermented products (Acebrón, Curiel, de las Rivas, Muñoz, &

60

Mancheño, 2009; Olguín, Oriol-Alegret, Bordons, & Reguant, 2011). Besides, β-

61

glucosidases are used as cheap biocatalysts in the synthesis of oligosaccharides and

62

alkyl-glycosides synthesis (Bruins, Strubela, van Lieshoutb, Janssena, & Boom, 2003).

63

In addition to applications in the of food industry, β-glucosidases are used for the

64

production of biofuel and ethanol from biomass (Li et al., 2013), in the pharmaceutical

65

sector, for the synthesis of phytoestrogen precursors from daizin and genistin (Otieno,

66

Ashton, & Shah, 2005), and also in chemical, cosmetic, and detergent industries

67

(Bankova, Bakalova, Petrova, & Kolev, D. (2006).

68

β-glucosidase activity is widespread among Lactic Acid Bacteria (LAB) mainly

69

involved in carbohydrate metabolism. Considering the industrial intereset of β-

70

glucosidases and the fact that LAB are classified as Generally Regarded as Safe

71

(GRAS), several β-glucosidases from LAB have been identified and their biochemical

72

properties characterized, as for example those isolated from Lactobacillus brevis

73

(Michlmayr, Schumann, Barreira Braz Da Silva, Kulbe, & Del Hierro, 2010a),

74

Oenococcus oeni (Dong et al., 2014; Michlmayr et al., 2010b), Lactobacillus plantarum

75

(Sestelo, Poza, & Villa, 2004), Lactobacillus casei (Coulon, Chemardin, Gueguen,

3

76

Arnaud, & Galzy, 1998), Weissella cibaria (Lee, Han, & Kim, 2012), and Lactococcus

77

sp.( Fang et al., 2014).

78

Leuconostoc mesenteroides is a LAB species widely used in food industry as starter

79

leading kimchi and sauerkraut fermentations (Zabat, Sano, Wurster, Cabral, & Belenky,

80

2018). Moreover, L. mesenteroides is mainly characterized for the synthesis of

81

exopolysaccharides such as dextran, with broad medical and chemical applications

82

(Lule, Singh, Pophaly, Pooan, & Tomar, 2016; Han et al, 2014) and bacteriocins as well

83

(Okuda, Tulini, Winkëlstroter, & De Martinis, 2017). As for others LAB, β-glucosidase

84

activity in L. mesenteroides has been previously described (Eom, Hwang, Kim, Kim, &

85

Paik, 2018; Lee et al., 2016; Zhu, Wang, & Zhang, 2019), and an enzyme has been

86

partially-purified and characterized (Gueguen, Chemardin, Labrot, Arnaud, & Galzy,

87

1997). However, to our knowledge no β-glucosidase from L. mesenteroides has been

88

previously genetically identified and characterized for its physicochemical properties.

89

Thus, because of i) the potential industrial applications of β-glucosidases from LAB;

90

and ii) although β-glucosidase activity has been described in L. mesenteroides, the

91

enzyme identification and physicochemical properties still remain unknown; this study

92

describes for the first time the genetic identification and biochemical characterization of

93

a recombinant glycosidase from L. mesenteroides.

94 95

2. MATERIAL AND METHODS

96

2.1. Bacterial strains and Materials

97

Sixteen Leuconostoc mesenteroides strains belonging to the PJ collection from CIDAF,

98

isolated from different raw vegetable matrices, and propagated in MRS broth medium at

99

30ºC without shaking were screened for their extracellular β-glucosidase activity

100

following the procedure described previously (Landete et al., 2014) using p-nitrophenyl

4

101

β-D-glucopyranoside (pNPG) as substrate. Escherichia coli DH5α was used for all

102

DNA manipulations. E. coli BL21 (DE3) was used for expression in pLATE52 vector

103

(ThermoFisher). E. coli strains were cultured in Luria-Bertani (LB) medium at 37ºC and

104

180 rpm. Amplicilin and IPTG were added to the medium at a concentration of 100

105

µg/mL and 0.4 mmol/L respectively when required.

106

The assayed substrates in this study were 4-Nitrophenyl β-D-glucopyranoside (pNPG)

107

(Sigma N7006), 4-Nitrophenyl α-D-glucopyranoside (Sigma N1377), 4-Nitrophenyl β-

108

D-galactopyranoside (pNPGAL) (Sigma N1252), 4-Nitrophenyl α-D-galactopyranoside

109

(Sigma N0877), 4-Nitrophenyl β-D-fucopyranoside (pNPFU) (Sigma N3378),

110

Trehalose (Sigma T9531), D-(+)-Cellobiose (Sigma C7252), Cellulose (Sigma C6288),

111

and Lactose (skimmed milk).

112 113

2.2. Identification of glycosidase BgLm1 from L. mesenteroides

114

The search in silico for β-glucosidases was performed in NCBI database (GenBank)

115

concerning to the representative genome of L. mesenteroides subsp. mesenteroides

116

ATCC 8293. A search for similar sequences was addressed in the NCBI Database

117

(GenBank) using the BLASTp algorithm. A distance-matrix (neighbour-joining) tree

118

was constructed with the Blast Tree View Widget.

119 120

2.3. Expression and purification of the glycosidase BgLm1

121

The gene coding for the Leuconostoc mesenteroides PJ128 glycosidase ΒgLm1 (named

122

LEUM_0847 in the L. mesenteroides subsp. mesenteroides ATCC 8293), was cloned

123

and overexpressed using the pLATE52 vector (ALICator Ligation Scientific Cloning

124

and Expression System, ThermoFisher, USA) according to the manufacturer’s

125

instructions. Briefly, the gene was PCR-amplified with Phusion Flash High-Fidelity 5

126

DNA polymerase (ThermoFisher, USA) using the primers ΒgLm1F (5’-

127

GCGTCCGGTTGGGAATTGCAAatgattaaaggtgttaatttaggtgg) and ΒgLm1R (5’-

128

GGAGATGGGAAGTCAttaaatcttggcccattttttg) (in italics the nucleotides pairing the

129

expression vector sequence, in lower case the nucleotides pairing the βgLm1 gene

130

sequence). The corresponding 1.1-kb purified PCR product was treated with T4 DNA

131

polymerase to generate the necessary 5’ and 3’ overhands for inserting the gene by LIC

132

cloning. E. coli DH5α cells were transformed, recombinant plasmids were isolated, and

133

those containing the correct insert were identified and verified by DNA sequencing, and

134

then transformed into E. coli BL21 (DE3) cells. Expression vector pLATE52 is

135

designed for expressing a protein attached to a N-terminal target sequence for WELQut

136

protease followed by a six-histidine affinity tag.

137

Recombinant E. coli BL21-pLATE52-βgLm1cells have grown in 1 L of Luria-Bertani

138

media kept in 2 L flask at 37ºC, 180 rpm, containing ampicillin (100 µg/mL) until they

139

reached an optical density (OD) of 0.4 at 600 nm, and induced by the addition of IPTG

140

(0.4 mmol/L final concentration). After induction, the cells were cultured at 37ºC, with

141

shaking for 3 h and collected by centrifugation. Cells were resuspended in 50 mmol/L

142

sodium phosphate buffer, pH 7.0, 100 mmol/L NaCl, 0.1% Triton X-100. Crude

143

extracts were prepared by vortexing the cellular suspension with quartz sand (150 µm)

144

for 10 min with occasional cooling in the ice bath. The insoluble fraction of the lysate

145

was removed by centrifugation at 38000g for 30 min at 4ºC.

146

The supernatant was filtered through a 0.22 µm PTFE filter and applied to a His

147

GraviTrap affinity column (GE Healthcare, Sweden) equilibrated with binding buffer

148

(50 mmol/L sodium phosphate buffer, pH 7.0, 100 mmol/L NaCl, and 20 mmol/L

149

imidazole). The bound enzyme was released by applying the elution buffer (50 mmol/L

150

sodium phosphate buffer, pH 7.0, 100 mmol/L NaCl, and 500 mmol/L imidazole). The

6

151

eluted protein was then dialyzed (3500 cutoff membrane) against 50 mmol/L sodium

152

phosphate buffer, pH 7.0 at 4ºC overnight, prior to further analysis. The grade of the

153

enzyme purification was determined by 12% sodium-dodecyl sulfate polyacrylamide-

154

gel electrophoresis (SDS-PAGE) in Tris-glycine buffer. Protein concentration was

155

measured according to the Bradford method using a protein assay kit (Bio-Rad) with

156

bovine serum albumin (BSA) as standard.

157 158

2.4. Enzyme Activity Assay

159

The β-glucosidase activity of purified ΒgLm1 was determined using as substrate pNPG.

160

Ten micrograms of ΒgLm1 was mixed with 1  mmol/L pNPG in 50  mmol/L sodium

161

phosphate buffer, pH 7.0, in a final volume of 500 µL and incubated at 40ºC for 5 min.

162

After this incubation, the reaction was stopped by adding a volume of 0.5 M Na2CO3.

163

The absorbance was detected using a microplate reader (Synergy Mx, Biotek) at

164

400 nm. A standard curve was prepared using p-nitrophenol (pNP) concentrations

165

ranging from 0.125 to 1 mmol/L. One unit (U) of enzyme activity is defined as the

166

amount of enzyme that catalyzed the formation of 1 µmol pNP per minute under the

167

conditions of the assay. Enzyme assays were performed at least in triplicates for each

168

analysis and the average activities were quantified. The results are shown as means ±

169

standard deviations.

170 171

2.5. pH-Dependence and Optimal Temperature

172

The optimal pH value for β-glucosidase activity of ΒgLm1 was determined by

173

analyzing its pH-dependence within the pH range 3 and 10 at 40ºC. Acetic acid-sodium

174

acetate buffer was used for pH 3, 4 and 5, citric acid-sodium citrate buffer for pH 6,

175

sodium phosphate buffer for pH 7, Tris-HCl for pH 8 and 9, and sodium carbonate-

7

176

bicarbonate for pH 10. The concentration used for all buffers was 100 mmol/L. To

177

determinate the optimal temperature, the recombinant L. mesenteroides ΒgLm1 β-

178

glucosidase activity was measured at 25, 30, 37, 40, 50 and 60 ºC in 50 mmol/L sodium

179

phosphate buffer, pH 7.0. Finally, to evaluate the temperature stability of recombinant

180

ΒgLm1, the enzyme was incubated in 50  mmol/L sodium phosphate buffer, pH 7.0 at

181

25, 30, 40, 50, and 60ºC for 5, 15, 30 min and 1, 2, 3, 4, 22, 24 and 48 h to determine

182

the temperature stability measurements. After incubations, the residual activity was

183

measured as described above.

184 185

2.6. Effect of additives on glycosidase activity

186

To assay the effects of different additives on the activity of recombinant glycosidase,

187

the enzymatic activity was measured in presence of several metals and inhibitors at 1

188

mmol/L of final concentration. The additives analyzed were β-mercaptoethanol, CaCl2,

189

DMSO, EDTA, FeCl3, Glucose, HgCl2, MgCl2, MnCl2, and Tween 80. The activity was

190

calculated relatively to the control sample containing no additive.

191 192

2.7. Substrate specificity analysis of L. mesenteroides glycosidase ΒgLm1 activity

193

ΒgLm1was incubated in 50 mmol/L sodium phosphate buffer, pH 6.0 in presence of

194

CaCl2 (1 mmol/L final concentration) and each of the p-nitrophenyl derivatives used in

195

this study (1 mmol/L final concentration) for1 h at 40 °C and measured following the

196

same procedure described above.

197

Besides, the hydrolysis activity of recombinant BgLm1 was assayed on natural

198

substrates such as cellulose, cellobiose, threalose and lactose (skimmed milk). The

199

glucose released by BgLm1 activity was monitored following the Glucose Assay kit

200

procedure (Megazyme, Ireland). Mixtures of glucose oxidase/peroxidase enzymes

8

201

(GOPOD) and products of BgLm1 reaction were incubated at 40ºC for 20 min. After

202

this incubation, the absorbance at 510 nm was measured on a spectrophotometer. A

203

standard curve was prepared using glucose concentrations ranging from 0.12 to 3 g/L.

204

Previous tests showed that GOPOD reacts with glucose but not with disaccharides or

205

polysaccharides. Blank reactions without enzyme were carried out for each substrate,

206

data were collected in triplicate, and the average activities were quantified. The results

207

are shown as means ± standard deviations.

208

Kinetic analysis was performed under conditions of pH 7.0 at 40°C for 5 min in 50

209

mmol/L sodium phosphate buffer containing substrate pNPG, pNPGAL or pNPFU at

210

different concentrations ranging from 0.1 to 5 mmol/L. The value of Km and Vmax were

211

calculated by fitting the initial rates as a function of the substrate concentration

212

according to the Michaelis-Menten equation.

213 214 215

3. RESULTS AND DISCUSSION

216

3.1. Identification of Leuconostoc mesenteroides BgLm1 gene

217

Leuconostoc mesenteroides PJ128 strain isolated from carrots was selected among the

218

other L. mesenteroides PJ strains for the subsequence studies in this work due to its

219

highest extracellular β-glucosidase activity (Figure S1). Since L. mesenteroides is

220

widely used as starter leading kimchi fermentations (Zabat et al., 2018) and its β-

221

glucosidase ability could improve the sensorial properties of this and other fermented

222

products by releasing aromatic compounds from glycoside precursors, our interest was

223

directed to identify and characterize its β-glucosidase since none glycosidase had been

224

genetically identified in this microorganism before.

9

225

Glucosidases were searched in NCBI database (GenBank) concerning to the

226

representative genome of L. mesenteroides subsp. mesenteroides ATCC 8293 and using

227

as topic “glucosidase”. In that search, it was found out that three α-glucosidases

228

(LEUM_0828; LEUM_0897; LEUM_0899) were annotated in the genome by

229

automated computational protein homology analysis (Makarova et al., 2006), but also

230

one β-glucosidase (LEUM_0847).

231

Often in literature there are situations in which genes that actually encode β-

232

galactosidases, were initially annotated as putative β-glucosidases (Acebron et al.,

233

2009). In this context, our efforts were addressed to elucidate the physicochemical

234

properties and confirm the nature of that enzyme (accession no. WP_011679619).

235

According to the predicted protein sequence, the putative β-glucosidase encoded by

236

LEUM_0847 gene from L. mesenteroides (hereinafter referred to as BgLm1) belongs to

237

the glycoside hydrolase family 1. Based on topology prediction software (ExPASy

238

Bioinformatics Resource Portal, Swiss Institute of Bioinformatics) the protein can be

239

considered cytoplasmic, since no transmembrane region or target sequence has been

240

predicted.

241

Figure 1 shows a phylogenetic tree constructed from related protein sequences obtained

242

by a BLASTp search in the NCBI database. High occurrence of LEUM_0847 gene in

243

genera Leuconostoc and Lactobacillus was found. As expected, the closest similarities

244

were found in the genomes of L. mesenteroides.

245

Previously, Michlmayr et al. (2010b) identified and characterized a β-glucosidase from

246

Oenococcus oeni. In L. mesenteroides, these authors also predicted the presence of a

247

different β-glucosidase enzyme (accession no. YP_818356 and annotated as

248

LEUM_0875) based on its high similarity to the O. oeni β-glucosidase sequence.

249

Nevertheless, when searching for glucosidase enzymes in the NCBI database, the

10

250

information provided from these authors was not updated in the L. mesenteroides ATCC

251

8293 genome. Thus, considering that both BgLm1 and LEUM_0875 amino acid

252

sequences exhibit low similarities (26%), the presence of more β-glucosidases in L.

253

mesenteroides is suggested, which is supported by previous results (Gueguen et al.,

254

1997). In this sense, further studies should be performed to elucidate the activity of the

255

enzyme encoded by LEUM_0875.

256 257

3.2. Production and Enzymatic Activity of Recombinant BgLm1

258

To confirm that BgLm1 gene encodes for a functional β-glucosidase, this gene was

259

amplified from Leuconostoc mesenteroides PJ128 strain and expressed in E. coli under

260

the control of the T7 RNA polymerase-inducible promoter.

261

Hyperproduced BgLm1 was detected in cell extracts from recombinant E. coli, whereas

262

control cells harboring pLATE52 expression vector alone did not showed expression

263

over the time course analyzed (Figure 2). The molecular mass of the recombinant

264

protein corresponded to that inferred from the nucleotide sequence (45 KDa). As our

265

gene was cloned containing an affinity poly-His tag, BgLm1 was purified on a His

266

GraviTrap crude chelating column and eluted with a stepwise gradient of imidazole

267

(Figure 2). Purified BgLm1 was dialyzed to eliminate the imidazole and then checked

268

for its β-glucosidase activity with pNPG to confirm the activity assigned by the

269

automated computational protein homology analysis (Makarova et al., 2006). The

270

presence of the His tag had no apparent effect on the catalytic activity.

271

A total of 12 mg of recombinant enzyme with specific activity of 6.02 x 103 U/mg were

272

obtained per 1 L of cultures following the protocol herein described.

273

Up to now, only the biochemical characterization of a partially purified β-glucosidase

274

from L. mesenteroides has been reported (Gueguen et al., 1997). However, since that

11

275

partially purified β-glucosidase (88 KDa) showed different molecular mass on the SDS-

276

PAGE analysis respect to BgLm1 (45 KDa), we can affirm that a novel glycosidase

277

from L. mesenteroides was identified and characterized in this work.

278

Recombinant L. mesenteroides PJ128 BgLm1 showed an optimal pH around 6, being

279

also highly active at pH 5 (Figure 3A). This pH value is similar to that previously

280

described for the partially purified β-glucosidase from L. mesenteroides (Gueguen et al.,

281

1997), as well as for most β-glucosidases described from lactic acid bacteria and fungi,

282

with all of them showing an optimal pH value around 5-6 (Coulon et al., 1998; Gueguen

283

et al., 1997; Liu et al., 2012; Michlmayr et al., 2010a,b; Moreira Souza et al., 2010).

284

Concerning to the temperature, recombinant BgLm1 showed ~40ºC as optimum (Figure

285

3B) in agreement with the optimal temperature described for other β-glucosidase

286

isolated from Lactobacillus plantarum (Sestelo et al., 2004), Lactobacillus brevis

287

(Michlmayr et al., 2010b), and Oenococcus oeni 31MBR (Dong et al., 2014).

288

Nevertheless, β-glucosidases isolated from Leuconostoc mesenteroides and Oenococcus

289

oeni ATCC BAA-1163 showed the highest activity at 50ºC (Gueguen et al., 1997;

290

Michlmayr et al., 2010b). Furthermore, BgLm1 represents a technological advantage

291

over fungal β-glucosidases which show an average optimal temperature in the range of

292

60-70ºC (Chen, Fu, Ng, & Ye, 2011; Kalyani et al., 2012; Liu et al., 2012; Moreira

293

Souza et al., 2010; Zahoor, Javed, Aftab, Latif, & ul-Haq, 2011).

294

Regarding to the thermal resistance, recombinant BgLm1 showed a markedly decreased

295

residual β-glucosidase activity after incubation at 40ºC or higher temperatures (Figure

296

3C), maintaining more than 65% of residual activity after 48h at 20ºC (Figure 3C).

297

The effects of some additives (1 mmol/L of final concentration) on L. mesenteroides

298

PJ128 recombinant BgLm1 were assayed (Table 1). Control was carried out following

299

the standard conditions (50 mmol/L of phosphate buffer pH 7.0, at 40ºC during 5 min).

12

300

The enzymatic activity of BgLm1 was increased by most of the additives assayed, being

301

CaCl2 and MnCl2 those in which BgLm1 showed the highest activity (137-133%

302

respectively). Our data are in line with those reported for the β-glucosidase isolated

303

from O. oeni 31MNR, as this enzyme showed an activity increment of 30% and 20% in

304

presence of both Ca2+ and Mn2+ ions respectively (Dong et al., 2014). Among all

305

additives assayed, FeCl3, HgCl2, DMSO, and glucose inhibited the BgLm1 activity to

306

66, 64, 58 and 53% respectively.

307

As Ca2+ ions greatly increased the BgLm1 activity, the substrate specificity assays were

308

carried out using the optimal conditions for the enzyme (phosphate buffer pH 6.0, in the

309

presence of CaCl2 and at 40ºC).

310 311

3.3. Substrate specificity of recombinant L. mesenteroides BgLm1

312

Since BgLm1 was annotated as β-glucosidase by automated computational protein

313

homology analysis (Makarova et al., 2006), in order to clarify the nature of recombinant

314

L. mesenteroides PJ128 BgLm1, substrate specificity analysis were carried out using

315

different p-nitrophenyl (pNP) derivatives and natural substrates such as cellulose,

316

cellobiose, threalose and lactose (skimmed milk) (Table 2).

317

According to the predicted protein sequence, we observed that BgLm1 belongs to the

318

glycoside hydrolase family 1 (GH1). GH1 enzymes have β-glucosidase activity (EC

319

3.2.1.21) but also a wide range of specificities, including β-galactosidase (EC 3.2.1.23),

320

6-phospho-β-galactosidase (EC 3.2.1.85), 6-phospho-β-glucosidase (EC 3.2.1.86),

321

myrosinase or sinigrinase (EC 3.2.1.147), and lactase-phlorizin hydrolase (EC

322

3.2.1.62/108) activities (Henrissat, 1991).

323

To demonstrate biochemically that BgLm1 encodes a functional β-glucosidase,

324

characterization assays were performed with p-nitrophenyl β-D-glucopyranoside

13

325

(pNPG) as substrate. Serendipity, the protein also showed 100% and 79% of relative

326

activity when pNP-β-D-fucopyranoside (pNPFU) and pNP-β-D-galactopyranoside

327

(pNPGAL) were respectively used as substrates in the reaction (Table 2). Despite the

328

recombinant BgLm1 activity was observed with pNPG as substrate, the efficiency to

329

hydrolyze cellobiose was quite low (20%). On the other hand, substrate specificity

330

assay demonstrated that BgLm1 greatly hydrolyzed lactose (Table 2).

331

Kinetic properties of BgLm1 were also investigated using pNPG, pNPGAL, and

332

pNPFU as substrates (Table 3). According to the substrate specificity assay results,

333

BgLm1 revealed higher affinity to the substrate pNPGAL (Km = 0.56 mmol/L) than

334

pNPG and pNPFU substrates (Km = 9.93 and 6.55 mmol/L, respectively). Vmax were

335

calculated in order to elucidate the specificity constants (Kcat/Km) of BgLm1 against

336

assayed substrates, showing the highest specificity for pNPGAL substrate (Table 3).

337

To our knowledge, taking into account the substrate specificity assay and kinetic results,

338

the BgLm1 from L. mesenteroides should be considered as a β-galactosidase enzyme.

339

Similarly, Acebrón et al. (2009) confirmed that Lactobacillus plantarum lp_3629 gene

340

encodes a functional β-galactosidase enzyme, while it was first annotated as a putative

341

β-glucosidase.

342

Both β-glucosidase and β-galactosidase activities were previously described in L.

343

mesenteroides (Lee et al., 2016). However, recombinant L. mesenteroides BgLm1

344

showed great β-galactosidase, and residual β-glucosidase and β-fucosidase activities.

345

This wide range of activities may seem unusual but we have found out in literature other

346

characterized glycosidases with the same activities, as those identified by metagenomic

347

analysis of samples from the Baltic Sea water (Wierzbicka-Woś, Bartasun, Cieśliński,

348

& Kur, 2013), from bovine liver (Rodriguez, Cabezas, & Calvo, 1982) and another

349

glycosidase from Helicella ericetorum (Calvo, Santamaria, Melgar, & Cabezas, 1983).

14

350

The wide spectrum of BgLm1 glycosidase activities could be interesting for the dairy

351

industry in the manufacture of lactose-free products, the synthesis of galacto-

352

oligosaccharides (GOS), the valorization of waste water from cheese factories, and the

353

improvement of the rheological properties of several dairy products, among many other

354

applications in food industry (Saqib, Akram, Halim, & Tassaduq, 2017).

355 356

4. CONCLUSIONS

357

In this study, putative protein encoded by LEUM_0847 in Leuconostoc mesenteroides

358

(BgLm1) has been identified as a novel functional β-glycosidase for the first time.

359

Recombinant L. mesenteroides BgLm1 was heterologously expressed in E. coli and

360

biochemically characterized successfully. BgLm1 exhibited great β-glucosidase, β-

361

galactosidase, and β-fucosidase activities using artificial pNP derivatives. The wide

362

spectrum of specificity and the tolerance under different types of additives make

363

BgLm1 potentially interesting for industry. Despite LEUM_0847 gene was annotated

364

for encoding a β-glucosidase, the substrate specificity assays indicated that BgLm1 can

365

be considered as a β-galactosidase. Since GH1 β-glycosidases are involved in the

366

synthesis of oligosaccharides, further studies should be performed to elucidate the

367

transglycosylation ability of BgLm1.

368 369

ACKNOWLEDGMENT

370

R. del Pino-García has a postdoctoral contract with the research program “Juan de la

371

Cierva-Formación” funded by MICINN (FJCI-2016-29091). P. Rus-Fernández and A.

372

Porrelli were scholarships from TFG (UGR) and TUCEP (Erasmus) respectively. J.A.

373

Curiel was recipient of postdoctoral contracts from research program “Torres Quevedo”

374

co-funded by MICINN and Torres Morente S.A.U. (PTQ-16-08434). The authors

15

375

appreciate the technical support and equipment availability of AGR-274 “Bioactive

376

Ingredients” team.

377 378

BIBLIOGRAPHY

379

Acebrón, I., Curiel, J. A., de las Rivas, B., Muñoz, R., & Mancheño, J. M. (2009).

380

Cloning, production, purification and preliminary crystallographic analysis of a

381

glycosidase from the lactic acid bacterium Lactobacillus plantarum CECT 748T.

382

Protein Expression and Purification, 68, 177–182.

383

Bankova, E., Bakalova, N., Petrova, S., & Kolev, D. (2006). Enzymatic synthesis of

384

oligosaccharides and alkylglycosides in waterorganic media via

385

transglycosylation of lactose. Biotechnology & Biotechnological Equipment, 20,

386

114–119.

387

Bruins, M. E., Strubela, M., van Lieshoutb, J. F. T., Janssena, A. E. M., & Boom R. M.

388

(2003). Oligosaccharide synthesis by the hyperthermostable β-glucosidase

389

from Pyrococcus furiosus: kinetics and modeling. Enzyme and Microbial

390

Technology, 33, 3-11.

391

Calvo, P., Santamaria, M. G., Melgar, M. J., & Cabezas, J. A. (1983). Kinetic evidence

392

for two active sites in β-d-fucosidase of Helicella ericetorum. International

393

Journal of Biochemistry, 15, 685-693.

394

Chen, P., Fu, X. Y., Ng, T. B., & Ye, X. Y. (2011). Expression of a secretory beta-

395

glucosidase from Trichoderma reesei in Pichia pastoris and its characterization.

396

Biotechnology Letters, 33, 2475–2479.

397

Coulon, S., Chemardin, P., Gueguen, Y., Arnaud, A., & Galzy, P. (1998). Purification

398

and characterization of an intracellular b-glucosidase from Lactobacillus casei

399

ATCC 393. Applied Biochemistry and Biotechnology, 74, 105–114.

400

Dong, M., Fan, M., Zhang, Z., Xu, Y., Li, A., Wang, P., & Yang, K. (2014).

401

Purification and characterization of β-glucosidase from Oenococcus

402

oeni 31MBR. European Food Research and Technology, 239, 995–1001.

403

Eom, S. J., Hwang, J. E., Kim, H. S., Kim, K. T., & Paik, H. D. (2018). Anti-

404

inflammatory and cytotoxic effects of ginseng extract bioconverted by

405

Leuconostoc mesenteroides KCCM 12010P isolated from kimchi. International

406

Journal of Food Science Technology, 53, 1331-1337. 16

407

Fang, S., Chang, J., Lee, Y., Guo, W., Choi, Y., & Zhou, Y. (2014). Cloning and

408

characterization of a new broadspecific β-glucosidase from Lactococcus sp.

409

FSJ4. World Journal of Microbiology & Biotechnology, 30, 213-223.

410

Gueguen, Y., Chemardin, P., Labrot, P., Arnaud, A., & Galzy, P. (1997). Purification

411

and characterization of an intracellular β-glucosidase from a new strain of

412

Leuconostoc mesenteroides isolated from cassava. Journal of Applied

413

Microbiology, 82, 469–476.

414

Han, J., Hang, F., Guo, B., Liu, Z., You, C., & Wu, Z. (2014). Dextran synthesized by

415

Leuconostoc mesenteroides BD1710 in tomato juice supplemented with sucrose.

416

Carbohydrate Polymers, 112, 556-562.

417 418 419

Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities, Biochemical Journal, 280, 309–316. Kalyani, D., Lee, K., Tiwari, M. K., Ramachandran, P., Kim, H., Kim, I., Jeya, M., &

420

Lee, J. (2012). Characterization of a recombinant aryl beta-glucosidase from

421

Neosartorya fischeri NRRL181. Applied Microbiology and Biotechnology, 94,

422

413–423.

423

Landete, J. M., Curiel, J. A., Rodríguez, H., de las Rivas, B., & Muñoz, R. (2014). Aryl

424

glycosidases from Lactobacillus plantarum increase antioxidant activity of

425

phenolic compounds. Journal of Functional Foods, 7, 322–329.

426

Lee, K. W., Han, N. S., & Kim, J. H. (2012). Purification and characterization of beta-

427

glucosidase from Weissella cibaria 37. The Journal of Microbiology and

428

Biotechnology, 22, 1705–1713.

429

Lee, K. W., Shim, J. M., Park, S. K., Heo, H. J., Kim, H. J., Ham, K. S., & Kim, J. H.

430

(2016). Isolation of lactic acid bacteria with probiotic potentials from kimchi,

431

traditional Korean fermented vegetable. LWT - Food Science and Technology,

432

71, 130-137.

433

Li, D., Li, X., Dang, W., Tran, P. L., Park, S., Oh, B., Hong, W., Lee, J., & Park, K.

434

(2013). Characterization and application of an acidophilic and thermostable b-

435

glucosidase from Thermofilum pendens. Journal of Bioscience and

436

Bioengineering, 115, 490–496.

437

Liu, D., Zhang, R., Yang, X., Zhang, Z., Song, S., Miao, Y., & Shen, Q. (2012).

438

Characterization of a thermostable beta-glucosidase from Aspergillus fumigatus

439

Z5, and its functional expression in Pichia pastoris X33. Microbial Cell

440

Factories, 11, 25. 17

441

Lule, V. K., Singh, R., Pophaly, S. D., Pooan, & Tomar, S. K. (2016). Production and

442

structural characterisation of dextran from an indigenous strain of Leuconostoc

443

mesenteroides BA08 in Whey. International Journal of Dairy Technology, 69,

444

520–531.

445

Makarova, K., Slesarev, A., Wolf, Y., Sorokin, A., Mirkin, B., Koonin, E., Pavlov, A.,

446

Pavlova, N., Karamychev, V., Polouchine, N., Shakhova, V., Grigoriev, I., Lou,

447

Y., Rohksar, D., Lucas, S., Huang, K., Goodstein, D. M., Hawkins, T.,

448

Plengvidhya, V., Welker, D., Hughes, J., Goh, Y., Benson, A., Baldwin, K., Lee,

449

J. H., Díaz-Muñiz, I., Dosti, B., Smeianov, V., Wechter, W., Barabote, R.,

450

Lorca, G., Altermann, E., Barrangou, R., Ganesan, B., Xie, Y., Rawsthorne, H.,

451

Tamir, D., Parker, C., Breidt, F., Broadbent, J., Hutkins, R., O'Sullivan, D.,

452

Steele, J., Unlu, G., Saier, M., Klaenhammer, T., Richardson, P., Kozyavkin, S.,

453

Weimer, B., & Mills, D. (2006). Comparative genomics of the lactic acid

454

bacteria. Proceedings of the National Academy of Sciences, 103, 15611–15616.

455

Michlmayr, H., Schumann, C., Barreira Braz Da Silva, N. M., Kulbe, K. D., & Del

456

Hierro, A. M. (2010a). Isolation and basic characterization of a b-glucosidase

457

from a strain of Lactobacillus brevis isolated from a malolactic starter culture.

458

Journal of Applied Microbiology, 108, 550–559.

459

Michlmayr, H., Schumann, C., Wurbs, P., da Silva, N. M. B. B., Rogl, V., Kulbe, K. D.,

460

& del Hierro, A. M. (2010b). A b-glucosidase from Oenococcus oeni ATCC

461

BAA-1163 with potential for aroma release in wine: cloning and expression in

462

E. coli. World Journal of Microbiology & Biotechnology, 26, 1281–1289.

463

Moreira Souza, F. H., Nascimento, C. V., Rosa, J. C., Masui, D. C., Leone, F. A., Jorge,

464

J. A., & Furriel, R. P. M. (2010). Purification and biochemical characterization

465

of a mycelial glucose- and xylosestimulated beta-glucosidase from the

466

thermophilic fungus Humicola insolens. Process Biochemistry, 45, 272–278.

467

Okuda, N. K., Tulini, F. L., Winkëlstroter, L. K. & De Martinis, E. C. (2017). Partial

468

characterisation of a bacteriocin produced by Leuconostoc mesenteroides A11

469

and evaluation of bacteriocin production using whey as culture medium.

470

International Journal of Dairy Technology, 71, 240–244.

471

Olguín, N., Oriol-Alegret, J., Bordons, A., & Reguant, C. (2011). β-Glucosidase activity

472

and bgl gene expression of Oenococcus oeni strains in model media and

473

Cabernet Sauvignon wine. American Journal of Enology and Viticulture, 62, 99-

474

105. 18

475

Otieno, D. O., Ashton, F. J., & Shah, N. P. (2005). Stability of β-glucosidase activity

476

produced by Bifidobacterium and Lactobacillus spp. in fermented soymilk

477

during processing and storage. Journal of Food Science, 70, 236-241.

478

Rodriguez, J.A., Cabezas, J. A., & Calvo, P. (1982). β-Fucosidase, β-glucosidase and β-

479

galactosldase activities associated in bovine liver. International Journal of

480

Biochemistry, 14, 695–698.

481 482 483

Saqib, S., Akram, A., Halim, S. A., & Tassaduq, R. (2017). Sources of b-galactosidase and its applications in food industry. 3 Biotech, 7, 79. Sestelo, A. B. F., Poza, M., & Villa, T. G. (2004). β-Glucosidase activity in a

484

Lactobacillus plantarum wine strain. World Journal of Microbiology &

485

Biotechnology, 20, 633–637.

486

Speciale, G., Thompson, A. J., Davies, G. J., & Williams, S. J. (2014). Dissecting

487

conformational contributions to glycosidase catalysis and inhibition. Current

488

Opinion in Structural Biology, 28, 1–13.

489

Wierzbicka-Woś, A., Bartasun, P., Cieśliński, H., & Kur, J. (2013). Cloning and

490

characterization of a novel coldactive glycoside hydrolase family 1 enzyme with

491

β-glucosidase, β-fucosidase and β-galactosidase activities. BMC Biotechnology,

492

13, 22.

493

Zabat, M. A., Sano, W. H., Wurster, J. I., Cabral, D. J., & Belenky, P. (2018). Microbial

494

community analysis of sauerkraut fermentation reveals a stable and rapidly

495

established community. Foods, 7, 77.

496

Zahoor, S., Javed, M. M., Aftab, S., Latif, F., & ul-Haq, I. (2011). Metabolic

497

engineering and thermodynamic characterization of an extracellular beta-

498

glucosidase produced by Aspergillus niger. African Journal of Biotechnology,

499

10, 8107–8116.

500

Zhu, Y., Wang, Z., & Zhang, L. (2019). Optimization of lactic acid fermentation

501

conditions for fermented tofu whey beverage with high-isoflavone aglycones.

502

LWT - Food Science and Technology, 111, 211–217.

503 504 505 506

19

507

FIGURE AND LEGENDS

508

Figure 1. Distance matrix-tree of β-glucosidase LEUM_0847. Protein sequences were

509

aligned with Blast tree view.

510

Figure 2. Analysis of the expression and purification of the BgLm1 enzyme from L.

511

mesenteroides PJ128. Cell extracts of the IPTG induced E. coli BL21 (DE3) pLATE52,

512

line 1; cell extracts of the IPTG induced E. coli BL21 (DE3) pLATE52-BgLm1, line 2;

513

fraction of BgLm1 eluted after His chelating affinity column, line 3. SDS-PAGE (12%)

514

stained with Coomassie blue.

515

Figure 3. Biochemical properties of recombinant L. mesenteroides PJ128 BgLm1. (A)

516

Relative activity of BgLm1 under different pH. Enzyme activity was assayed at 40ºC

517

during 5 min. (B) Relative activity of BgLm1 under different temperatures. Enzyme

518

activity was assayed at pH 7.0. (C) Residual activities of the recombinant BgLm1 after

519

preincubation at 25℃ (♦), 30℃ (■), 40℃ (▲), 50℃ (○) and 60℃ (□). The maximum

520

activity was defined as the 100% in all cases.

521

Figure S1. Leuconostoc mesenteroides strains screening for their extracellular β-

522

glucosidase activity. Reactions containing cell free protein extracts (1 mg), and pNPG

523

(1 mmol/L) in sodium phosphate buffer (50 mmol/L, pH 7.0) were incubated at 37 ºC

524

for 1 h, stopped by the addition of 800 µl of 0.5 mol/L Na2CO3 and clarified by

525

centrifugation (10000 g, 3 min). Supernatants (200 µl) were transferred to a 96-well

526

plate and the absorbance at 400 nm was determined using a microplate reader. One unit

527

(U) of enzyme activity is defined as the amount of enzyme that catalyzed the formation

528

of 1 µmol pNP per minute.

529

530 20

531

TABLES

532

Table 1. Effects of different additives on recombinant L. mesenteroides PJ128 BgLm1. Additives (1 mmol/L) β-mercaptoethanol Control CaCl2 DMSO EDTA FeCl3 Glucose HgCl2 MgCl2 MnCl2 Tween 80

Relative activity (%) 124 ±2 100 ±2 137 ±2 58 ±3 119 ±1 66 ±1 53 ±3 64 ±5 125 ±3 133 ±6 121 ±2

533

534 535 536 537 538 539 540 541 542 543 544 545 546 547 548

21

549

Table 2. Substrate specificity of recombinant L. mesenteroides PJ128 BgLm1. Substrate (1 mmol/L final concentration) p-nitrophenyl β-D-glucopyranoside p-nitrophenyl α-D-glucopyranoside

100 ± 4a 0 ± 0a

p-nitrophenyl β-D-galactopyranoside

79 ± 3a

p-nitrophenyl α-D-galactopyranoside

0 ± 0a

p-nitrophenyl β-D-fucopyranoside

100 ± 6a

Cellulose

0 ± 0b

Cellobiose

20 ± 1b

Lactose (skimmed milk)

91 ± 5b

Threalose 550 551 552 553 554

Relative activity (%)

0 ± 0b

a

The activity was calculated as relative to the p-nitrophenyl β-D-glucopyranoside substrate reaction. b The activity was calculated as relative to the released glucose against the theoretical.

555

556

557

558

559

560

561

562

563

564

22

565 566

Table 3. Kinetic parameters of BgLm1 from L. mesenteroides against different pNP β-D substrates. Km (mmol/L)

Vmax (µmol min-1)

Kcat (s-1)

Kcat /Km (mmol/L-1 s-1)

p-nitrophenyl β-D-glucopyranoside

9.93±1.11

232±25

2320±251

257±1

p-nitrophenyl β-D-galactopyranoside

0.56±0.08

126±11

1262±106

2254±134

p-nitrophenyl β-D-fucopyranoside 567

6.55±0.05

74±1

739±12

112±2

568

569

570

571

572

573

574

575

576

577

578

579

580

581

23

Highlights

•

LEUM_0847 gene is identified as a functional β-glycosidase enzyme

•

Recombinant L. mesenteroides BgLm1 has been high-yield purified and characterized

•

BgLm1 showed β-galactosidase, β-glucosidase and β-fucosidase activities

•

This study describes BgLm1 as a β-galactosidase since greatly hydrolyzed lactose.

the authors declare no conflict of interests