Heterologous Expression and Characterization of a New Clade of

Subscriber access provided by MIDWESTERN UNIVERSITY

Biotechnology and Biological Transformations

Heterologous expression and characterization of a new clade of Aspergillus #-L-rhamnosidase suitable for citrus juice processing Li-Jun Li, Jianye Gong, Song Wang, Guiling Li, Ting Gao, Zedong Jiang, Yi-Sheng Cheng, Hui Ni, and Qingbiao Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06932 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019

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

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

Page 1 of 39

Journal of Agricultural and Food Chemistry

1

Heterologous expression and characterization of a new clade of

2

Aspergillus α-L-rhamnosidase suitable for citrus juice processing

3

Lijun Li †,‡,§, Jianye Gong†, Song Wang†, Guiling Li†, Ting Gao†, Zedong Jiang†, Yi-Sheng Cheng

4

§, ⊥,

5 6 7 8 9 10 11 12 13

Hui Ni *,†,‡,§, Qingbiao Li†

†: College of Food and Biological Engineering, Jimei University, Xiamen, Fujian Province 361021, China ‡: Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, Fujian Province 361021, China §: Research Center of Food Biotechnology of Xiamen City, Xiamen, Fujian Province 361021, China §: Department of Life Science, National Taiwan University, Taipei 10617, Taiwan ⊥:

Institute of Plant Biology, National Taiwan University, Taipei 10617, Taiwan

14

*Corresponding

15

Professor Hui Ni

16

College of Food and Biology Engineering, Jimei University Xiamen, Fujian Province

17

3616021, China

18

Phone: 13015914929

19

Fax: 865926180470

20

E-mail: [email protected]

author:

1

ACS Paragon Plus Environment


22

ABSTRACT:

23

α-L-Rhamnosidase is a glycoside hydrolase capable of removing naringin from

24

citrus juice. However, α-L-rhamnosidases always have broad substrate spectra,

25

causing negative effects on citrus juice. In this study, a α-L-rhamnosidase-expressing

26

fungal strain, JMU-TS529 was identified, and its α-L-rhamnosidase was characterized.

27

As a result, JMU-TS529 was identified as Aspergillus tubingensis via morphological

28

and molecular characteristics. The predicted protein sequence shared an amino acid

29

identity of less than 30% with previously characterized α-L-rhamnosidases. The

30

optimal pH and temperature were 4.0 and 50-60 °C, respectively. Most importantly,

31

the α-L-rhamnosidase showed a strong ability to hydrolyze naringin but scarcely acted

32

on other substrates. Furthermore, the enzyme could efficiently remove naringin from

33

pomelo juice without changing its attractive aroma. These results indicate that the

34

present enzyme represents a new clade of Aspergillus α-L-rhamnosidase that is

35

desirable for debittering citrus juice, providing a better alternative for improving the

36

quality of citrus juice.

37 38

KEYWORD: α-L-rhamnosidase, naringin, aroma profile, Aspergillus tubingensis,

39

citrus juice processing

2


Page 2 of 39

Page 3 of 39


41

INTRODUCTION

42

α-L-Rhamnosidase [E. C. 3.2.1.40] is a glycoside hydrolase that specifically

43

cleaves the terminal α-L-rhamnose from natural glycosides such as the flavonoids and

44

the terpenyl glycosides1. This enzyme is important for debittering citrus juice2-5,

45

promoting the aroma of wine6-8, eliminating the hesperidin crystals in orange juice9,

46

and enhancing the antioxidant activities of various juices10. In addition, this enzyme

47

has been studied for the potential to prepare L-rhamnose and derhamnosylated

48

coproducts that are useful precursors for pharmaceuticals, cosmetics, and food

49

products11-13.

50

In the carbohydrate-active enzymes (CAZy) database (www.cazy.org),

51

α-L-rhamnosidases have been classified in three glycoside hydrolase families (GH),

52

namely, the GH28 family, the GH78 family, and the GH106 family14, 15. To date, 29

53

α-L-rhamnosidases have been biochemically characterized, and five of them, namely,

54

BsRhaB, BT1001, SaRha78A, KoRha, and AtRha, have been reported in crystal

55

structures16-20. All five crystal structures contain a typical GH78 core catalytic

56

(α/α)6-barrel domain and several β-sheet domains that lay between the N-terminal and

57

the catalytic domain. Moreover, domain D (a β-sheet domain) of the crystal structure

58

of SaRha78A was identified as a noncatalytic carbohydrate binding module (CBM67)

59

that is involved in enzyme function against insoluble substrates18. Despite this, the

60

catalytic properties of α-L-rhamnosidases have not been extensively reported, which

61

might hinder the development of α-L-rhamnosidases with excellent catalytic

62

properties for industrial bioprocesses. Thus, it is of great interest to biochemically and

63

structurally characterize α-L-rhamnosidases with varied catalytic properties from

64

natural or artificial resources.

65

Citrus fruits are popular fruits that are extensively processed to juices that contain 3



Page 4 of 39

66

a wide range of flavonoid glycosides, such as naringin, rutin, narirutin, neohesperidin

67

and hesperidin21. Among the glycosides, naringin is the main component causing the

68

bitter taste of citrus juices13. A number of techniques have been reported to remove

69

naringin from citrus products by the aid of α-L-rhamnosidase or naringinase (the

70

enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase)

71

plethora of data has proven that many juices contain nonvolatile glycosidic precursors

72

that can be transformed to free volatiles via enzymatic treatment and thus release

73

aromatic volatiles25. In addition, most α-L-rhamnosidases and naringinases have been

74

reported to hydrolyze various glycosides, including naringin, hesperidin and the

75

glycosidic precursors of aromatic volatiles in citrus fruits1, 11, 13, 26, which might result

76

in significant changes in the aroma of juices. For instance, Ni et al. reported that a

77

naringinase treatment caused a negative impact on the aroma of the pomelo juice, i.e.,

78

enhancing a cooked or overripened odor, due to greatly increasing the content of

79

nonanal, (Z)- and (E)-linalool oxides27. Therefore, in the case of debittering citrus

80

juice, it is important to screen or develop α-L-rhamnosidase or naringinase with high

81

efficiency to hydrolyze naringin and low affinity to other glycosides, such as

82

glycosidic aroma precursors.

11, 13, 22-24.

A

83

In our previous study, a fungal strain JMU-TS529 producing extracellular

84

α-L-rhamnosidase was isolated from rotten pomelo compost. Furthermore, primary

85

experiments showed that α-L-rhamnosidase could effectively hydrolyze naringin but

86

barely act on other substrates, suggesting that this enzyme might be able to debitter

87

citrus juice without affecting the volatile components. In the above context, the

88

present study aimed to characterize the α-L-rhamnosidase in terms of sequence

89

homogeneity, enzymatic properties and its effects on naringin and aroma-related

90

volatiles of citrus juices. The specific content included (1) strain identification; (2) 4


Page 5 of 39


91

cloning and heterologous expression of the α-L-rhamnosidase in P. pastoris GS115;

92

(3) characterization of enzymatic properties; and (4) illustration of the effects on

93

naringin and aroma-related volatiles of citrus juices.

5



95

MATERIALS AND METHODS

96

Isolation of the α-L-rhamnosidase producing strain JMU-TS529. Compost

97

samples were collected from sites where rotten pomelo (Citrus grandis) were

98

discarded and piled for at least 2 years in Ping-he County, Fujian Province, P. R.

99

China. For the enrichment of α-L-rhamnosidase producing strains, 50 mL enrichment

100

medium (naringin 2.0 g/L, (NH4)2SO4 1.0 g/L, MgSO4·7H2O 0.5 g/L, K2HPO4 1.0

101

g/L, CaCl2 0.2 g/L, yeast extract 0.3 g/L, beef extract 0.3 g/L, pH 6.0) was inoculated

102

with 3 g of compost sample and incubated at 28 °C for 5 days. The α-L-rhamnosidase

103

producing strain, named JMU-TS529, was isolated from the enriched mixture by the

104

naringin hydrolysis ring method of Ni et al.28.

105

Morphological characterization of JMU-TS529. The macroscopic morphology

106

of JMU-TS529 colonies was observed on CYA medium (Czapek concentrate 10

107

mL/L, sucrose 30.0 g/L, yeast extract 5.0 g/L, K2HPO4 1.0 g/L, CuSO4·5H2O 0.005

108

g/L, ZnSO4·7H2O 0.01 g/L, agar powder 20.0 g/L) after 7 days of cultivation at 28 °C.

109

The phase-contrast microscopic image of the conidial head was recorded using a

110

NIKON eclipse ts100 inverted microscope. The detailed conidial head and

111

conidiophore morphology were examined by scanning electron microscopy (SEM).

112

Briefly, the strain was grown on CYA medium at 28 °C for 5 days, and the agar plugs

113

were fixed in half-strength Karnovsky’s fixative for 4 h, rinsed twice in cacodylate

114

buffer (10 min each rinse), and incubated in 2% osmium tetroxide: 0.2 M cacodylate

115

buffer (1:1) for 2 h. The plugs were further rinsed twice in 0.1 M cacodylate buffer

116

over a period of 10 min and then dehydrated twice for a 30-min period by sequentially

117

soaking in a set of ethanol dehydration solutions that formed 35, 70, 95, and 100%

118

ethanol. The dehydrated samples were set in a critical-point dryer (Autosamdri-815B

119

Series C, BBE, Germany) for 20 min. The samples were then mounted on aluminum 6


Page 6 of 39

Page 7 of 39


120

stubs and coated with a layer of 30 nm gold-palladium alloy. The fungal morphology

121

was examined under an SEM (SU5000, Hitachi High-Technologies, Japan) at an

122

accelerating potential of 25 kV.

123

Phylogenetic analysis of JUM-TS529. JMU-TS529 was cultured in 25 mL of

124

PDA medium at 28 ºC for 48 h. The mycelium was recovered by centrifugation,

125

washed with distilled water, frozen in liquid nitrogen and ground to a fine powder.

126

Genomic DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN,

127

Beijing, China). PCRs were carried out to amplify the calmodulin gene sequence and

128

the fragment between the internal transcribed spacer (ITS) and the 5.8S rDNA region.

129

The PCR began with an initial denaturing step at 95 ºC for 5 min, followed by 30

130

cycles of sequential incubation (94 ºC for 40 s, 57 ºC for 40 s and 72 ºC for 30 s), and

131

ended with a final extension at 72 ºC for 10 min. The primer pair ITS1 (5'-TCC GTA

132

GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3')

133

were used for ITS-5.8S rDNA amplification, and the primer pair cmd5 (5'-CCG AGT

134

ACA AGG AGG CCT TC-3') and cmd6 (5'-CCG ATA GAG GTC ATA ACG

135

TGG-3') were used for calmodulin gene sequence amplification. The PCR products

136

were purified using a Universal DNA Purification Kit (TIANGEN, Beijing, China)

137

and sequenced by Bioray Biotechnology (Xiamen, China). The DNA sequences were

138

analyzed

139

(http://www.ncbi.nlm.nih.gov/BLAST/), edited and assembled using the MEGA 7.0

140

software (http://www.megasoftware.net/).

for

DNA

homology

using

the

BLASTN

Tool

141

Analysis of the α-L-rhamnosidase activity. The enzymatic analysis was

142

conducted according to Chen et al.22 with some modifications. One unit (U) of the

143

enzyme was defined as the amount of enzyme that hydrolyzed 1 µmole of naringin (4’,

144

5,7’-trihydroxyflavonone-7-rhamnoglucoside) per minute at 60 ºC. An assay mixture, 7



145

composed of 0.95 mL of 20 mM citrate buffer (pH 4.0), 1 mL of 300 µg/mL naringin

146

aqueous solution and 0.05 mL of enzyme solution, was incubated at 60 °C for 15 min,

147

followed by heating at 100 °C for 30 min to inactivate the enzyme. After being

148

filtered through a 0.22 µm membrane (Biotech, Shanghai, China), the reaction

149

mixture was then subjected to HPLC to determine the concentrations of the substrate

150

and the product. HPLC was performed on an Agilent 1260 HPLC equipment coupled

151

with a Symmetry C18 reversed-phase column (4.6×150 mm, 3.5 μm) and a UV

152

detector (Agilent Technologies Co. Ltd, California) under the same conditions used in

153

our previous study22. The control experiment was carried out using inactivated

154

enzyme instead of active enzyme.

155

Cloning of the α-L-rhamnosidase encoding gene from JMU-TS529. The

156

JMU-TS529 culture mixture was washed twice with PBS (0.1 M, pH 7.0) and

157

centrifuged at 30,000×g for 10 min. The precipitates were ground with liquid nitrogen.

158

Total RNA was extracted using an EasyPureTM Plant RNA kit (TIANGEN, China)

159

and stored at -70 °C before use.

160

The RNA was reverse transcribed into first-strand cDNA using the 5' RACE

161

System (Invitrogen, USA). The AT-Rha protein coding region was amplified using

162

the designed primer (CGTCTGCCTGGACGGCSCNAARMGNGA), termed AT-Rha,

163

and the 3'-end universal primer, RACE 3' (CTATAGTCGACGGCTTATT). The 5'-

164

and 3'-cDNA ends of the AT-Rha gene were amplified using specific PCR primers

165

designed based on the sequence of Aspergillus niger CBS 513.88 hypothetical protein

166

(XM_001395598.2). The complete fragment was constructed by overlapping PCR.

167

PCRs were carried out by denaturing at 94 °C for 4 min, followed by 30 cycles of

168

sequential incubation (94 °C for 1 min, 54 °C for 1 min and 72 °C for 0.5 min) and a

169

final extension at 72 °C for 10 min. PCR products were purified using a Universal 8


Page 8 of 39

Page 9 of 39


170

DNA Purification Kit (TIANGEN, China) and ligated into the pMD18-T vector and

171

sequenced (its accession number in GenBank is KX664478).

172

Sequence Analysis and Molecular Docking. The sequences of GH78

173

α-L-rhamnosidases

were

174

http://www.cazy.org/GH78_characterized.html. Amino acid sequences of the catalytic

175

domains were retrieved from the GenBank database. The conservative domain was

176

analyzed through Conserved Domain Search Service (CD Search) of NCBI and

177

Simple Modular Architecture Research Tool (SMART). Multiple sequence alignment

178

of the amino acid sequences was performed using the ClustalW2 program.

179

Phylogenetic trees were constructed using MEGA 7.0 with the neighbor-joining

180

method. Bootstrap values were calculated based on 1000 replications of the data.

181

Structure-based

182

three-dimensional structure was predicted through homology modeling using the

183

Modeler 9.15 program. The simulated three-dimensional structure was docked against

184

substrates using AutoDock 4.2. Visualization and analysis of the structural model

185

were performed using PyMOL software.

alignment

obtained

was

performed

from

using

the

ENDscript

website

2.0.

The

186

Expression of AT-Rha in P. pastoris GS115. The cDNA was amplified by PCR

187

using the primers 9kTF (GAGACCCTAGGATGGCAGCGTTGGAGGA) and 9kTR

188

(ATAGTTTAGCGGCCGCTCAACCTCTGACGGCA) and cloned into the pPIC9K

189

plasmid. The recombinant plasmid (pPIC9K-AT-Rha) was transformed into P.

190

pastoris GS115 by electroporation using a Pichia expression kit (Invitrogen, USA).

191

The positive transformants were isolated from geneticin (G418) (Trans, China)

192

containing agar plates and verified by PCR and sequencing. The confirmed

193

α-L-rhamnosidase-expressing transformants were cultured at 30 °C in buffered

194

glycerol-complex (BMGY) medium containing yeast nitrogen base (YNB) (13.4 g/L), 9



195

yeast extract powder (14.0 g/L), peptone (28.0 g/L), potassium phosphate buffer (0.1

196

M, pH 6.0), biotin (0.4‰, w/w), and glycerol (1%, w/w). Cells were harvested when

197

OD600 reached 2.0. The harvested cell pellet was resuspended in buffered

198

methanol-complex (BMMY) medium (similar to BMGY except for the substitution of

199

0.5% MeOH for glycerol) and cultured at 30 °C for the induction of the enzyme.

200

The supernatant was collected by centrifugation at 10,000×g for 5 min. The

201

protein content was monitored by UV spectrophotometry (A280), and the protein

202

concentration was determined by the Coomassie brilliant blue method with bovine

203

serum albumin (BSA) as the standard29. The MW of α-L-rhamnosidase was estimated

204

by comparison with the 5-245 kDa markers (Solarbio, China) on a 10% SDS-PAGE

205

gel after silver staining30.

206

Purification of the recombinant α-L-rhamnosidase (AT-rRha) from P.

207

pastoris. The crude enzyme solution was fractionated with (NH4)2SO4 solutions at

208

increasing concentrations from 50% to 90%. The pellets were dissolved in 20 mM

209

citric acid-disodium hydrogen phosphate buffer (pH 7.0) and dialyzed at 4 °C for 24 h.

210

The protein sample was then applied to a DEAE SepharoseTM Fast Flow column

211

(1.5×20 cm) pre-equilibrated with 20 mM citric acid-disodium hydrogen phosphate

212

buffer (pH 7.0). The unbound fractions were removed by washing with 10 bed

213

volumes of the equilibration buffer. The bound proteins were eluted with 20 mM

214

citric acid-disodium hydrogen phosphate buffer (pH 7.0) containing 1.0 M NaCl. The

215

fractions showing α-L-rhamnosidase activity were pooled and further purified using a

216

HiTrap Blue HP column (5 mL) that was pre-equilibrated with 20 mM citrate buffer

217

(pH 3.0). The protein samples were eluted with 20 mM citric acid-disodium hydrogen

218

phosphate buffer (pH 7.0) containing 1.0 M (NH4)2SO4. The enzyme was eluted with

219

20 mM citrate buffer (pH 7.0) containing 0.15 M NaCl. 10


Page 10 of 39

Page 11 of 39


220

The active eluents were pooled for analysis of mass weight using gel filtration

221

chromatography and SDS-PAGE. The pooled enzyme fraction from HiTrap Blue HP

222

column was concentrated using an ultrafilter membrane (30 kDa Millipore, Germany)

223

and further eluted with a Sephacryl S-200 HR column (1.6×100 cm) that was

224

pre-equilibrated with 20 mM citrate buffer (pH 7.0) containing 0.15 M NaCl.

225

Furthermore, it was analyzed using 10% SDS-PAGE. In addition, for evaluate mass

226

weight of the deglycosylated protein, it was deglycosylated by PNGase F (New

227

England BioLabs, China), according to the manufacturer’s instructions, followed by

228

analysis using 10% SDS-PAGE.

229

Substrate specificity of AT-rRha. Naringin, ginsenoside Rg2, myricitrin, rutin,

230

hersperidin, saikosaponin C, quercitrin and pNPR (Sigma, St. Louis, America) were

231

used as alternative substrates. Enzymatic activity was determined in 20 mM citric acid

232

buffer (pH 4.0) at 60 °C. The structures of these substrates are shown in the

233

supplementary data (Fig. S1).

234

Kinetic parameters of the enzyme towards naringin. The enzyme activities of

235

α-L-rhamnosidase were measured at various naringin concentrations (0.02, 0.04, 0.09,

236

0.17, 0.21, 0.25, 0.30, 0.34, 0.38, 0.43, 0.47 and 0.51 mM (incomplete solvent)) at 60

237

°C and pH 4.0. The kinetic parameters, including the Michaelis constant (Km), the

238

maximum velocity (Vmax), the turnover number (kcat), and the catalytic efficiency

239

(kcat/Km), were calculated according to the Michaelis-Menten equation by Origin

240

2016.

241 242

The effects of pH and temperature on the activity and stability of AT-rRha towards the substrate naringin.

243

To determine the optimal pH, the enzyme AT-rRha was incubated with naringin

244

in 50 mM citric acid-disodium hydrogen phosphate buffer (pH 3.0-8.0) at 50 °C. The 11



245

enzyme activities under different pH conditions were measured and normalized to the

246

highest value.

247

For pH stability, the enzyme was incubated with naringin at various pH values

248

ranging from 3.0 to 8.0 with an interval of 1.0 at 4 °C for 24 h, and the residual

249

enzyme activities were measured. The residual activities were normalized to the

250

highest enzyme activity value.

251

For enzyme thermal stability, the enzyme was incubated with substrate in 50 mM

252

citrate buffer (pH 4.0) at different temperatures varying from 30 to 90 °C for 60 min.

253

The residual enzymatic activities were measured and normalized to the highest

254

activity.

255

The effects of various reagents and metal ions on AT-rRha activity towards

256

naringin. AT-rRha was incubated with various reagents at different concentrations,

257

i.e., glucose (1 mM and 10 mM), L-rhamnose (1 mM and 10 mM), EDTA (1 mM and

258

10 mM), DTT (1 mM and 10 mM), β-ME (1.0% and 10% v/v), and SDS (1 mM and

259

10 mM), respectively. Enzyme activities were measured.

260

To detect the effects of metal ions, AT-rRha was first dialyzed in 20 mM

261

citrate-phosphate buffer (pH 4.0) in the presence of 10 mM EDTA at 4 °C for 12 h to

262

remove the residual metal ions. The dialysis buffer was switched to the EDTA-free

263

buffer to remove EDTA. AT-rRha was then treated with different salts, including

264

NaCl, KCl, AgNO3, MnCl2, CoCl2, MgSO4, CaCl2, FeSO4, Hg(NO3)2, BaCl2, ZnSO4,

265

CuCl2, Al(NO3)3, and FeCl3, at final concentrations of 1 mM and 10 mM, respectively.

266

After treatment at 30 °C for 1 h, the residual enzyme activity was measured as

267

described in the previous section. The activity of α-L-rhamnosidase without the

268

addition of any reagents or metal ions was defined as 100%.

269

Enzymatic hydrolysis of naringin in citrus juice. Eight milliliters of citrus 12


Page 12 of 39

Page 13 of 39


270

(pomelo) juice with 0.013 U AT-rRha was incubated at 60 ºC for 30 min. Thereafter,

271

the juice was centrifuged at 30,000×g for 10 min and then filtered through a 0.22 µm

272

membrane and subjected to HPLC analysis of the concentrations of naringin and its

273

hydrolysis product prunin at 280 nm as previously described22.

274

Analysis of volatile compounds in the enzyme-treated citrus juice using

275

mass spectrum-coupled gas chromatography (GC-MS). The juice was freshly

276

extracted from peeled pomelo fruits. To analyze effects of the enzyme on the aroma,

277

reaction solution was composed of 1 mL citrus juice, 9 mL pure water and 0.65 U of

278

AT-rRha. For the positive control, AT-rRha was replaced by 0.65 U of the

279

α-L-rhamnosidase r-Rha1 that was previously identified from A. niger JMU-TS528 to

280

hydrolyze a number of glycosidic substrates

281

closed system at 40 ºC for 0.5 h to ensure that delicate aroma volatiles were neither

282

lost nor damaged.

26.

All samples were incubated in a

283

The volatile desorption was conducted as described30. After 0.5 h of enzymatic

284

reaction, each sample was added in 10 µL/L of cyclohexanone (Sigma, St. Louis,

285

USA) as the internal standard, followed by volatile extraction using a

286

DVB/CAR/PDMS (50/30 µm) SPME fiber (Supelco, Bellefonte, USA) at 40 ºC for

287

20 min prior to chemical analysis by GC-MS with a gas chromatography coupled with

288

a QP 2010 plus mass spectrometer (Shimadzu Corporation, Kyoto, Japan) and the

289

Rtx-5 MS column (60 m×0.32 mm×0.25 µm, Restek Corporation, USA).

290

The volatiles were identified by matching individual MS spectra and Kovats

291

Retention Indices (RI) to the Mass Spectral Library (NIST08, NIST08s, FFNSC1.3)

292

and the standards (Sigma, St. Louis, USA). Volatiles were quantified according to

293

their respective calibration curves using the internal standard method (cyclohexanone)

294

by fixing the Selective Ion Monitoring (SIM) mode. To accurately detect the volatile 13



295

changes resulted from the enzymatic reaction, volatiles in 0.65 U of AT-rRha and

296

Rha1 were also analyzed and deducted from those of the juice samples respectively

297

treated with AT-rRha and Rha1.

298

To evaluate if the volatiles have a sensible effect on the aroma, the odor

299

characteristics were referred to the corresponding references31-35, and the odor activity

300

values (OAVs) were calculated by dividing the measured concentrations with the

301

respective odor threshold values in water27.

302

Statistical analysis. All of the experiments were performed at least in triplicate

303

independent experiments. SPSS 17.0 (SPSS Inc. H, IL) was used to analyze the

304

statistical significance through Duncan’s multiple range tests.

305

Accession numbers. The sequence of the ITS-5.8S rDNA sequence and the

306

calmodulin gene of A. tubingensis JMU-TS529 are available in GenBank under

307

accession numbers KX649892 and KX664477, respectively. The protein sequence of

308

AT-rRha has been deposited in the NCBI Protein database under accession number

309

ANZ93894.1.

14


Page 14 of 39

Page 15 of 39

311


RESULTS AND DISCUSSIONS

312

Identification of the strain JMU-TS529 by polyphasic taxonomy. The

313

JMU-TS529 strain grew slowly on CYA at 28 °C, attaining a colony of approximately

314

7.5 cm diameter in 7 days. The colony center was green, velvety, plane, more or less

315

funiculus, consisting of basal-like green aerial mycelium with white margins (Fig.

316

S2A). JMU-TS529 produced an Aspergillus-like upright conidial head that rooted in

317

the swollen vesicle, with conidiophores on top (Fig. S2B). The conidiophores were

318

branched with 34-38 µm long stipes, and the primary conidiophores were in phialide

319

shape, 70-75 µm long under SEM (Fig. S2C, S2D). In short, the morphology of

320

colony and conidiophores were identical to those of typical Aspergillus species (Fig.

321

S2).

322

The phylogenetic analysis of ITS-5.8S rDNA sequences (GenBank: KX649892)

323

showed that the sequence from JMU-TS529 matched that from A. tubingensis and A.

324

niger (Fig. S3A). Since the A. tubingensis and A. niger species are very similar, a

325

secondary sequence identification, such as a calmodulin gene sequence phylogenetic

326

analysis, is needed, as suggested by Samson et al.36. The calmodulin gene sequence

327

phylogenetic analysis (GenBank: KX664477) showed that the calmodulin gene in

328

JMU-TS529 matched exactly that from A. tubingensis, whereas the calmodulin gene

329

sequence in A. niger was slightly different (Fig. S3B). Based on the phylogenetic

330

analyses, it is reasonable to conclude that strain JMU-529 is a member of A.

331

tubingensis.

332

The Aspergillus section Nigri is the most complex section in taxonomic studies

333

due to the subtle differences among species and was shown to exert various

334

glycosidases. A polyphasic taxonomy on this section that accounts for all available

335

evidence, including morphologic, physiologic, metabolic, and genetic molecular 15



Page 16 of 39

336

phylogeny data, is often demanded35. In previous studies, α-L-rhamnosidase has been

337

characterized functionally and enzymatically in a number of Aspergillus species,

338

including A. nidulans37, A. kawachii38, A. flavus39 A. niger30,

339

aculeatus22, A. awamori5 and A. clavato-nanicus42. In this study, based on

340

morphological characteristics and phylogenetic analysis of the ITS-5.8S rDNA

341

sequence and calmodulin gene, the α-L-rhamnosidase synthesizing strain JMU-TS529

342

was identified as A. tubingensis, adding another piece of evidence to support that most

343

Aspergillus species could synthesize α-L-rhamnosidase.

40,

A. terreus41, A.

344

Cloning and sequence analysis of α-L-rhamnosidase (AT-Rha) from A.

345

tubingensis JMU-TS529. The amino acid sequence of the protein that was purified

346

from the fermented broth of A. tubingensis was determined using MALDI-TOF/TOF

347

mass spectrometry (Table S1). The result of mass spectrometry agreed with a

348

hypothetical glycoside hydrolase of A. kawachii IFO 4308 (GenBank: GAA86560).

349

Based on the amino acid sequence, a full-length 2,667 bp cDNA from JMU-TS529

350

was cloned and sequenced (GenBank: KX664478). The open reading frame (ORF) of

351

the cDNA putatively encoded a protein with 888 amino acids. One conserved domain,

352

named the Bac_rhamnosid domain, was discovered in AT-Rha by sequence analysis

353

in SMART and the Conserved Domain Search Service of NCBI (Fig. S4). The

354

Bac_rhamnosid domain is a (α/α)6 TIM-barrel catalytic domain that typically exists in

355

glycoside hydrolase family 78 (GH78) proteins. Furthermore, a few highly conserved

356

sites and regions in the (α/α)6 TIM-barrel catalytic domain were identified based on

357

the multiple sequence alignments (Fig. S5). These two findings indicated that AT-Rha

358

belongs to GH78.

359

Blast analysis was used to compare the amino acid sequence of AT-Rha to other

360

α-L-rhamnosidases. The closest characterized α-L-rhamnosidase sequence is from 16


Page 17 of 39


361

Alternaria sp. L1 (GenBank: AFA41506.1), which had a similarity of 29% to the

362

AT-Rha. In addition, the AT-Rha amino acid sequence had high sequence similarity

363

(approximately 97%) with two hypothetical glycoside hydrolases, one from A.

364

costaricaensis CBS 115574 (GenBank: XP_025540132.1) and the other from A. niger

365

(GenBank: GAQ44900.1) 43. Furthermore, a phylogenetic tree was established based

366

on alignment of the AT-Rha amino acid sequence with all of the characterized

367

α-L-rhamnosidases up to date and other typical α-L-rhamnosidases (Fig. 1). AT-Rha

368

and four putative α-L-rhamnosidases, i.e., the α-L-rhamnosidase of A. costaricaensis

369

CBS 115574 (GenBank: XP_025540132.1), the α-L-rhamnosidase of A. niger

370

(GenBank: GAQ44900.1), the α-L-rhamnosidase of A. kawachii IFO 4308 (GenBank:

371

GAA86560) and A. tubingensis CBS 134.48 (GenBank: OJI80484.1) were grouped

372

into the same cluster. Furthermore, this clade clearly separated from other

373

α-L-rhamnosidases,

374

α-L-rhamnosidases represents a new clade of Aspergillus α-L-rhamnosidases. To date,

375

none of the sequences in this clade have been experimentally verified in function. Our

376

work indicated the sequence from A. tubingensis embodying α-L-rhamnosidase,

377

which verified the annotation of this new clade.

indicating

that

AT-Rha

as

well

as

four

putative

378

Expression and purification of the recombinant α-L-rhamnosidase

379

(AT-rRha) in P. pastoris GS115. The P. pastoris GS115 hosting AT-Rha gene was

380

cultivated and induced by methanol for 7 days in flasks. Then the expressed

381

recombinant α-L-rhamnosidase (AT-rRha) was purified to appear single elution peak

382

using gel filtration chromatography (Fig. S6A), and was detected to have single

383

electrophoretic band on SDS gel (Fig. S6B). Both gel filtration and SDS-PAGE

384

indicated the molecular weight of the enzyme was approximately 110 kDa (Fig. S6A

385

and B). After the treatment with a glycosidase PNGase F to remove the 17



386

asparagine-linked oligosaccharides, the purified fragment of AT-rRha was analyzed to

387

have a molecular weight of approximately 96 kDa (Fig. S6C), which was identical to

388

the predicted molecular weight based on its amino acid sequence, indicating that the

389

expressed AT-rRha protein was secreted in the glycosylated form, which contained

390

approximately 14 kDa of glycosidic chain.

391

Substrate specificity and the kinetic constants for naringin hydrolysis.

392

AT-rRha showed negligible activities on rutin, hesperidin, quercitrin, ginsenoside Rg2,

393

myricitrin, saikosaponin C (Fig. 2A). Neither did AT-rRha show any activity on the

394

artificial substrate, pNPR (4-nitrophenyl-α-L-rhamnoside) (Fig. 2A). Interestingly,

395

AT-rRha exhibited high hydrolytic activity on naringin, of which the L-rhamnose

396

residue was linked to β-D-glucoside via an α-1,2-glycosidic bond (Fig. S1A).

397

Furthermore, AT-rRha was estimated to have a Km value of 0.47 mM and a Vmax value

398

of 4169.80 U/mg (Fig. 2B). The calculated turnover constant (kcat) of AT-rRha

399

towards naringin was 4.89×104 s-1, and the catalytic efficiency (kcat /Km) was 1.02×105

400

s-1 M-1, indicating that AT-rRha has high catalytic efficiency for naringin. The Km

401

values of previously reported α-L-rhamnosidases towards naringin are 0.021-7.0

402

mM11, 36. In comparison, the Km of AT-rRha was less than most of those of previously

403

reported α-L-rhamnosidases, indicating that it had a high affinity to hydrolyze

404

naringin.

405

Based on the structural model that was built using the Modeler 9.15

406

protein-modeling software, molecular docking studies were conducted to evaluate the

407

interaction and selectivity between AT-rRha and various substrates. Molecular

408

docking showed that substrates such as naringin are linked in the active site of

409

AT-rRha (Fig. 3A), with hydrogen bonds at Arg454 and Asp462 (Fig. 3B). In

410

addition, the binding energy with naringin (-6.69 kcal/mol) was significantly higher 18


Page 18 of 39

Page 19 of 39


411

than those of other substrates (Table 1), which explained why the affinity to naringin

412

was much higher than those of other substrates (Fig. 3A). Previous reports have

413

shown that most of the characterized α-L-rhamnosidases have catalytic activities

414

towards substrates containing α-1,2-, α-1,3-, α-1,4-, and α-1,6-glucosidic bonds11,

415

including naringin, ginsenoside, myricitrin, rutin, hesperidin, pNPR, etc11,

416

biochemical characterization revealed that AT-rRha hydrolyzed naringin but had no

417

activity towards the artificial substrate pNPR, which is distinct from the reported

418

α-L-rhamnosidases.

13.

The

419

Factors that affect the enzyme activities of AT-rRha. AT-rRha exhibited

420

optimal activity at pH 4.0 and maintained its enzyme activity over a broad pH range

421

between 3.0 and 8.0 (Fig. 4A). The optimal temperature was between 50-60 °C (Fig.

422

4B). At 60 °C, nearly 98% of the maximal activity of this enzyme was preserved after

423

1 h incubation. The stability decreased when incubated above 60 °C, and its activity

424

was completely lost when incubated at 70 °C for 1 h (Fig. 4B). The half-life of the

425

enzymatic activity of AT-rRha was approximately 33.3 min at 65 °C (Fig. 4C). The

426

optimal temperature and pH values for AT-rRha were 50-60 °C and pH 4.0,

427

respectively, which were in agreement with those of α-L-rhamnosidases from other

428

fungi11, 41, 44. Although α-L-rhamnosidases with varied properties have been isolated

429

from animal tissues, plants, yeasts, fungi, and bacteria, only fungal- and

430

bacterial-originating enzymes have been further explored11,

431

α-L-rhamnosidases showed more acidic pH optima compared to their bacterial

432

counterparts,

433

α-L-rhamnosidases are more suitable for biotechnological processes that require

434

catalytic efficiency in acidic conditions, such as citrus fruit juice debittering and

435

clarifying2-5. Stability and activity at high temperatures were also desirable properties

which

favor

neutral

and

alkaline

19


optimal

13.

The fungal

pH45.

Fungal


Page 20 of 39

436

for this type of enzyme because most industrial processes were carried out at high

437

temperatures.

438

Only Hg2+ at 10 mM showed inhibition on the enzyme activity (Table S3), while

439

other tested metal ions have no noticeable effect on AT-rRha activity at both 1 and 10

440

mM. This result indicated that an important cysteine residue might exist in or close to

441

the active site, due to researchers have shown that Hg2+ possibly combines with a

442

sulfydryl group46. In addition, in the presence of 10 mM DTT, 10 mM EDTA, 500

443

mM glucose and 500 mM L-rhamnose, the AT-rRha retained more than 85% of the

444

original activity (Table S2). The desirable characteristics of AT-rRha make this

445

enzyme applicable in a broad range of special industrial applications, especially in

446

citrus juice processing.

447

Effect of AT-rRha on naringin hydrolysis and the volatile compounds in

448

pomelo juice. Citrus juice is considered one of the most commonly consumed

449

beverages because of its health benefits and distinctive aroma and taste47,

450

Debittering citrus juice by transforming the bitter agent “naringin” to less/nonbitter

451

substances, is the main biotechnological application of α-L-rhamnosidase in the food

452

industry. As shown in Fig. 5, naringin in the pomelo juice was completely hydrolyzed

453

after treatment with AT-rRha at 60 ºC for 30 min. Furthermore, GC-MS analysis

454

showed that a total of 25 volatiles were identified from fresh and AT-rRha-treated

455

juices, whereas the Rha1-treated juice had 28 volatiles (Table S4, Table 2). The

456

quantitive analysis showed that the fresh and the AT-rRha-treated juice had similar

457

volatile components, except that five volatiles, β-cubebene, 1-nonanol, nonanal, ethyl

458

acetate and ethyl benzoate, showed slight concentration differences (Table 2, Table

459

S4), which might result from the catalysis of unknown enzymes in the AT-rRha

460

solution. However, the α-L-rhamnosidase Rha1 that has been shown could hydrolyze 20


48.

Page 21 of 39


26,

461

a variety of substrates

was detected to significantly increase the content of 10

462

volatiles (i.e, (E)-2-hexnal, nonanal, ethyl benzoate, haxanoic acid, 3-hexen-1-ol,

463

1-hexanol, linalool, 1-nonanol, β-cubebene, and E-2-pentenal), including the alcohols

464

such as 3-hexen-1-ol, 1-hexanol, linalool, and 1-nonanol that have been reported to be

465

released from glycosidic precursors via enzymatic hydrolysis49. These results

466

indicated that Rha1 released more volatiles from glycosidic precursors than AT-rRha.

467

The aromas of citrus juice come from a complex combination of several aromatic

468

compounds, including esters, aldehydes, alcohols, ketones, and hydrocarbons with

469

large amounts of limonene, linalool, γ-terpinene, β-myrcene, α-pinene, and octanal50.

470

Ni et al.27 showed that the aroma compounds of naringinase-treated pomelo juice was

471

different from those of fresh pomelo juice. Odor activity value (OAV) analysis

472

demonstrated that the fresh and AT-rRha-treated juices had similar OAVs of the

473

volatile compounds, whereas the decanal, nonanal, linanool, and geraniol were

474

analyzed to have greater OAVs in r-Rha1-treated juice than those in the fresh and

475

AT-rRha-treated juices (Table S5). This result suggested that AT-rRha had little effect

476

on the aroma profile of citrus juice; whereas Rha 1 might noticeably affect the overall

477

aroma of a citrus juice.

478

In conclusion, an α-L-rhamnosidase was identified and cloned from a newly

479

isolated A. tubingensis strain. The phylogenetic and substrate specificity analysis

480

suggested that AT-rRha represents a new clade of Aspergillus α-L-rhamnosidase with

481

specific hydrolytic ability towards naringin. The enzyme showed high activity and

482

desirable stability at acidic pH and high temperature. It effectively hydrolyzed

483

naringin but hardly acted on other substrates. Most interestingly, the enzyme

484

treatment decreased the naringin content of pomelo juice under the bitter taste

485

threshold and maintained the natural aroma. The comprehensive results indicate that 21



Page 22 of 39

486

AT-rRha is superior to those of previously reported α-L-rhamnosidase in the case of

487

debittering citrus juice, which could be ascribed to the very narrow substrate

488

specificity towards naringin, providing a better alternative for improving the quality

489

of citrus juice using enzymatic debittering processes.

490

ABBREVIATIONS USED

491

AT-Rha: native α-L-rhamnosidase of A. tubingensis, AT-rRha: recombinant

492

α-L-rhamnosidase of A. tubingensis, r-Rha1: α-L-rhamnosidase of A. niger, pNPR:

493

p-nitrophenyl-α-L-rhamnoside, GH: glycoside hydrolase families.

494

ACKNOWLEDGEMENTS

495

This work was supported by the Natural Science Foundation of China (No. U1805235)

496

and the Science and Technology Project of Xiamen City (No. 3502Z20183029).

497

SUPPLEMENTARY MATERIALS

498

The mass spectrometry result of AT-rRha (Table S1); Effects of inhibitors and

499

detergents on AT-rRha (Table S2); Effects of metal ions on AT-rRha (Table S3);

500

Identification and qualification of volatiles in the fresh and α-L-rhamnosidase pomelo

501

juice (Table S4); Odor Activity Values (OAVs) and characteristic aromas of pomelo

502

juice (Table S5); Hydrolysis of naringin to prunin by α-L-rhamnosidase and structure

503

of substrates used in the substrate specificity analysis (Fig. S1); Morphological

504

observation of the JMU-TS529 colonies. (A) Colony morphology; (B) conidia cross

505

section;

506

Neighbor-joining tree showing the phylogenetic relationship between JMU-TS529

507

and other Aspergillus species (Fig. S3). Domain architectures of AT-rRha containing

508

the Bac_rhamnosid domain; Conservative structural domain is predicted by SMART

(C)

conidiophore;

(D)

bilayer

sporogenous

22


structure

(Fig.

S2);

Page 23 of 39


509

and Conserved Domain Search Service of NCBI (Fig. S4); Structure-based sequence

510

alignment of GH78 α-L-rhamnosidase with known crystal structure (Fig. S4); Fig. S5

511

Structure-based sequence alignment of GH78 α-L-rhamnosidase with known crystal

512

structure (Fig. S5); Sephacryl S-200 HR column chromatographic purification of the

513

AT-rRha and SDS-PAGE analysis of the purified recombinant enzyme AT-rRha

514

expressed in P. pastoris GS115 (Fig. S6). This material is available free of charge via

515

the Internet at http://pubs.acs.org.

516

REFERENCES

517

(1) Izzo, V.; Tedesco, P.; Notomista, E.; Pagnotta, E.; Donato, A.; Trincone, A.;

518

Tramice, A.; α-Rhamnosidase activity in the marine isolate Novosphingobium sp.

519

PP1Y and its use in the bioconversion of flavonoids. J Mol Catal B-Enzym 2014, 105,

520

95-103.

521

(2) Zhang, T.; Yuan, W.; Li, M.; Miao, M.; Mu, W., Purification and characterization

522

of an intracellular α-L-rhamnosidase from a newly isolated strain, Alternaria alternata

523

SK37.001. Food Chem 2018, 269, 63-69.

524

(3) Chien, P. J.; Sheu, F.; Yuan, T. S., Monitoring Enzymatic Debittering in

525

Grapefruit Juice by High Performance Liquid Chromatography. J Food Drug Anal

526

2001, 9, 115-120.

527

(4) Li, L. J.; Wu, Z. Y.; Yu, Y.; Zhang, L. J.; Zhu, Y. B.; Ni, H.; Chen, F.,

528

Development and characterization of an α-L-rhamnosidase mutant with improved

529

thermostability and a higher efficiency for debittering orange juice. Food Chem 2018,

530

245, 1070-1078. 23



531

(5) Yadav, S.; Yadav, R. S. S.; Yadav, KDS., An α-L-rhamnosidase from Aspergillus

532

awamori MTCC-2879 and its role in debittering of orange juice. Int J Food Sci Tech

533

2013, 48, 927-933.

534

(6) Spagna, G.; Barbagallo, R. N.; Martino, A.; Pifferi, P. G., A simple method for

535

purifying glycosidases: α-L-rhamnosidase from Aspergillus niger to increase the

536

aroma of Moscato wine. Enzyme Microb Tech 2000, 27, 522-530.

537

(7) Gallego, M. V.; Pinaga, F.; Ramon, D.; Valles, S., Purification and

538

characterization of an α-L-rhamnosidase from Aspergillus terreus of interest in

539

Winemaking. J Food Sci 2001, 66, 204-209.

540

(8) Caldinia, C.; Bonomia, F.; Pifferib, P. G., Kinetic and immobilization studies on

541

fungal glycosidases for aroma enhancement in wine. Enzyme Microb Tech 1994, 16,

542

286-291.

543

(9) Terada, Y.; Kometani, T.; Nishimura, T.; Takii, H.; Okada, S., Prevention of

544

hesperidin crystal formation in canned mandarin orange syrup and clarified orange

545

juice by hesperidin glycosides. Food Sci Technol Int 1995, 1, 29-33.

546

(10) Sun, T.; Powers, J. R.; Tang J., Enzyme-catalyzed change of antioxidants content

547

and antioxidant activity of asparagus juice. J Agr Food Chem 2007, 55, 56-60.

548

(11) Yadav, V.; Yadav, P. K.; Yadav, S.; Yadav, KDS., α-L-Rhamnosidase: A review.

549

Process Biochemistry 2010, 45, 1226-1235.

550

(12) Yadav, S.; Yadava, S.; Yadav, KDS., Secretion of α-L-rhamnosidase by some

551

indigenous fungal strains belonging to penicillium genera. In Chemistry of 24


Page 24 of 39

Page 25 of 39


552

Phytopotentials: Health.; Energy and Environmental Perspectives. Edited by

553

Khemani, L. D.; Srivastava, M. M.; Srivastava, S. B.; Heidelberg: Springer Berlin

554

Heidelberg; 2012, 77-81.

555

(13) Puri, M., Updates on naringinase: structural and biotechnological aspects. Appl

556

Microbiol Biot, 2012, 93, 49-60.

557

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

558

Henrissat, B., The carbohydrate-active enzymes database (CAZy): an expert resource

559

for glycogenomics. Nucleic Acids Res 2009, 37, D233-238.

560

(15) Henrissat, B.; Davies, G., Structural and sequence-based classification of

561

glycoside hydrolases. Curr Opin Struc Biol 1997, 7, 637-644.

562

(16) Bonanno, J. B.; Almo, S. C.; Bresnick, A.; Chance, M. R.; Fiser, A.;

563

Swaminathan, S.; Jiang, J.; Studier, F. W.; Shapiro, L.; Lima C. D., New

564

York-Structural GenomiX Research Consortium (NYSGXRC): a large scale center

565

for the protein structure initiative. J Struct Funct Genomics 2005, 6, 225-232.

566

(17) Cui, Z.; Maruyama, Y.; Mikami, B.; Hashimoto, W.; Murata, K., Crystal

567

structure of glycoside hydrolase family 78 α-L-rhamnosidase from Bacillus sp. GL1. J

568

Mol Biol 2007, 374, 384-398.

569

(18) Fujimoto, Z.; Jackson, A.; Michikawa, M.; Maehara, T.; Momma, M.; Henrissat,

570

B.; Gilbert, H. J.; Kaneko, S., The structure of a Streptomyces avermitilis

571

α-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the

572

six-domain arrangement. J Biol Chem 2013, 288, 12376-12385. 25



573

(19) O'Neill, E. C.; Stevenson, C. E.; Paterson, M. J.; Rejzek, M.; Chauvin, A. L.;

574

Lawson, D. M.; Field, R. A: Crystal structure of a novel two domain GH78 family

575

α-L-rhamnosidase from Klebsiella oxytoca with rhamnose bound. Proteins 2015, 83,

576

1742-1749.

577

(20) Pachl, P.; Skerlova, J.; Simcikova, D.; Kotik, M.; Krenkova, A.; Mader, P.;

578

Brynda, J.; Kapesova, J.; Kren, V.; Otwinowski, Z.; Rezacova, P., Crystal structure of

579

native α-L-rhamnosidase from Aspergillus terreus. Acta Crystallogr D Struct Biol

580

2018, 74, 1078-1084.

581

(21) Ramful, D.; Tarnus, E.; Aruoma, OI.; Bourdon, E.; Bahorun, T., Polyphenol

582

composition.; vitamin C content and antioxidant capacity of Mauritian citrus fruit

583

pulps. Food Res Int 2011, 44, 2088-2099.

584

(22) Chen, Y. L.; Ni, H.; Chen, F.; Cai, H. N.; Li, L. J.; Wang, S., Purification and

585

characterization of a naringinase from Aspergillus aculeatus JMUdb058. J Agric

586

Food Chem 2013, 61, 931-938.

587

(23) Ribeiro, M. H., Naringinases: occurrence.; characteristics.; and applications. Appl

588

Microbiol Biot 2011, 90, 1883-1895.

589

(24) Bram, B.; Solomons, G. L., Production of the enzyme naringinase by Aspergillus

590

niger. J Appl Microbiol 1965, 13, 842.

591

(25) Ren, J. N.; Tai, Y. N.; Dong, M.; Shao, J. H, Yang, S. Z, Pan, S. Y, Fan, G.,

592

Characterisation of free and bound volatile compounds from six different varieties of

593

citrus fruits. Food Chem 2015, 185, 25-32. 26


Page 26 of 39

Page 27 of 39


594

(26) Li L. J.; Yu Y.; Zhang, X.; Jiang, Z. D.; Zhu, Y. B.; Xiao, A. F.; Ni, H.; Chen, F.,

595

Expression and biochemical characterization of recombinant α-L-rhamnosidase

596

r-Rha1 from Aspergillus niger JMU-TS528. Int J Biol Macromol 2016, 85, 391-399.

597

(27) Ni, H.; Hong, P.; Ji, H. F.; Sun, H.; Chen, Y. H.; Xiao, A. F.; Chen, F.,

598

Comparative analyses of aromas of fresh naringinase-treated and resin-absorbed

599

juices of pummelo by GC-MS and sensory evaluation. Flavour Frag J 2015, 30,

600

245-253.

601

(28) Ni, H.; Li, L. J.; Xiao, A. F.; Cao, Y.; Chen, Y. L.; Cai H. N., Identification and

602

characterization of a new naringinase-producing strain, Williopsis californica

603

Jmudeb007. World J Microb Biot 2011, 27, 2857-2862.

604

(29) Kruger, N. J., The Bradford method for protein quantitation. The protein

605

protocols handbook 1994, 32, 17-24.

606

(30) Gharahdaghi, F.; Weinberg, C. R.; Meagher, D. A.; Imai, B. S.; Mische, S. M.,

607

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel:

608

A method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999,

609

20, 601-605.

610

(31) Lee, S. J.; Noble, A. C., Characterization of odor-active compounds in

611

Californian Chardonnay wines using GC-olfactometry and GC-mass spectrometry. J

612

Agr Food Chem 2003, 51, 8036-8044.

613

(32) Qiao, Y.; Xie, B. J.; Zhang, Y.; Zhang, Y.; Fan, G.; Yao, X. L.; Pan, S. Y.,

614

Characterization of Aroma Active Compounds in Fruit Juice and Peel Oil of Jinchen 27



615

Sweet Orange Fruit (Citrus sinensis (L.) Osbeck) by GC-MS and GC-O. Molecules

616

2008, 13, 1333-1344.

617

(33) Selli, S.; Cabaroglu, T.; Canbas, A., Volatile flavour components of orange juice

618

obtained from the cv. Kozan of Turkey. J Food Compos Anal 2004, 17, 789-796.

619

(34) Castro-Vázquez, L.; Díaz-Maroto, M. C.; Pérez-Coello, M. S., Aroma

620

composition and new chemical markers of Spanish citrus honeys. Food Chem 2007,

621

103, 601-606.

622

(35) Liu, C. H.; Cheng, Y. J.; Zhang, H. Y.; Deng, X. X.; Chen, F.; Xu, J., Volatile

623

constituents of wild citrus Mangshanyegan (Citrus nobilis Lauriro) peel oil. J Agric

624

Food Chem 2012, 60, 2617-2628.

625

(36) Samson, R. A.; Houbraken, JAMP.; Kuijpers, AFA.; Frank, J. M.; Frisvad, J. C.,

626

New ochratoxin A or sclerotium producing species in Aspergillus section Nigri. Stud

627

Mycol 2004, 50, 45-61.

628

(37) Manzanares, P.; Orejas, M.; Ibañez, E.; Vallés, S.; Ramón, D., Purification and

629

characterization of an α-L-rhamnosidase from Aspergillus nidulans. Lett Appl

630

Microbiol 2000, 31, 198-202.

631

(38) Koseki, T.; Mese, Y.; Nishibori, N.; Masaki, K.; Fujii, T.; Handa, T.; Yamane,

632

Y.; Shiono, Y.; Murayama, T.; Iefuji, H., Characterization of an α-L-rhamnosidase

633

from Aspergillus kawachii and its gene. Appl Microbiol Biot 2008, 80, 1007.

634

(39) Yadav, V, Yadav, S, Yadava, S, Yadav, KDS., α-L-Rhamnosidase from

635

Aspergillus flavus MTCC-9606 isolated from lemon fruit peel. Int J Food Sci Tech 28


Page 28 of 39

Page 29 of 39


636

2011, 46, 350-357.

637

(40) Liu, T.; Yu, H.; Zhang, C.; Lu, M.; Piao, Y.; Ohba, M.; Tang, M.; Yuan, X.; Wei,

638

S.; Wang, K., Aspergillus niger DLFCC-90 rhamnoside hydrolase.; a new type of

639

flavonoid glycoside hydrolase. Appl Environ Microb 2012, 78, 4752-4754.

640

(41) Abbate, E.; Palmeri, R.; Todaro, A.; Blanco, RM.; Spagna, G., Production of a

641

a-L-rhamnosidase from Aspergillus terreus using citrus solid waste as inducer for

642

application in juice industry. Chemical Engineering Transactions 2012. 27, 253-258.

643

(42) Yadav, V.; Yadav, S.; Yadav, S.; Yadav, KDS., α-L-rhamnosidase from

644

Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH. APPL BIOCHEM

645

MICRO 2012, 48, 295-301.

646

(43) de, Vries. R. P.; Riley, R.; Wiebenga, A.; Aguilar-Osorio, G.; Amillis, S.;

647

Uchima, C. A.; Anderluh, G.; Asadollahi, M.; Askin, M.; Barry, K., Comparative

648

genomics reveals high biological diversity and specific adaptations in the industrially

649

and medically important fungal genus Aspergillus. Genome Biol 2017, 18, 1-28.

650

(44) Ni, H.; Xiao, A. F.; Cai, H. N.; Chen, F.; You, Q.; Lu, Y. Z., Purification and

651

characterization of Aspergillus niger α-L-rhamnosidase for the biotransformation of

652

naringin to prunin. Afr J Microbiol Res 2012, 6, 5276-5284.

653

(45) De, L. F.; Mensitieri, F.; Tarallo, V.; Ventimiglia, N.; Vinciguerra, R.; Tramice,

654

A.; Marchetti, R.; Pizzo, E.; Notomista, E.; Cafaro, V., RHA-P: Isolation, expression

655

and characterization of a bacterial α-L-rhamnosidase from Novosphingobium sp.

656

PP1Y. J Mol Catal B-Enzym 2016, 134, 136-147. 29



657

(46) Khandeparkar, R.; Bhosle, N. B.; Purification and characterization of

658

thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112. Res

659

Microbiol, 2006, 157. 315-325.

660

(47) Rouseff, R. L.; Ruiz, Perez-Cacho, P.; Jabalpurwala, F., Historical review of

661

citrus flavor research during the past 100 years. J Agric Food Chem 2009, 57,

662

8115-8124.

663

(48) Kelebek, H.; Selli, S., Determination of volatile, phenolic, organic acid and sugar

664

components in a Turkish cv. Dortyol (Citrus sinensis L. Osbeck) orange juice. J Sci

665

Food Agric 2011, 91, 1855-1862.

666

(49) Camilla, V.; Mogens, L.; Leif, Poll.; Volatile Monoterpenes in Black Currant

667

(Ribes nigrum L.)Juice: Effects of Heating and Enzymatic Treatment by

668

β-Glucosidase. J Sci Food Agric 2016, 54, 2298-2302.

669

(50) Qiao, Y.; Xie, B-j.; Zhang, Y.; Zhou, H-y.; Pan, S-y.; Study on aroma

670

components in fruit from three different satsuma mandarin varieties. Agr Sci China

671

2007, 6, 1487-1493

30


Page 30 of 39

Page 31 of 39


FIGURE CAPTIONS Figure No.

Caption Phylogenetic tree of AT-rRha from A. tubingensis JMU-TS529 and other α-L-rhamnosidases

of

Glycoside

Hydrolase

Family

78.

The

individual

α-L-rhamnosidases are represented by their producers, and the GenBank accession numbers. All characterized α-L-rhamnosidases are in bold type, and AT-rRha is indicated by a black circle. Sequence alignment was performed by ClustalW2 and Fig. 1

the tree was created MEGA 7.0 using the unrooted neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method.

Fig. 2

The enzyme activities (A) and the Michaelis-Menten equation of AT-rRha (B). Dominant conformation analysis of binding of naringin with AT-rRha (A) and

Fig. 3

hydrogen bond interaction (B). The hydrogen bond interaction is represented in red. Optimal pH and temperature and the stability range for AT-rRha. (A) optimal pH

Fig. 4

and pH stability for AT-rRha activity; (B) optimal temperature and thermal stability for AT-rRha activity; (C) thermostability of AT-rRha at 65 °C. Hydrolysis of pomelo juice by AT-rRha at 60 ºC (pH 4, 30 min). (A) standard of

Fig. 5

naringin; (B) standard of prunin; (C) juice samples treated with or without AT-rRha.

31



Page 32 of 39

Table 1 The binding energy values for AT-rRha with substrates based on AutoDock results. Intermolecular energy (kcal/mol)

Internal energy (kcal/mol)

Torsional energy (kcal/mol)

Unbound energy (kcal/mol)

Binding energya (kcal/mol)

Naringin

-10.87

-6.20

4.18

-6.20

-6.69

Rutin

-8.16

0.00

4.47

0.00

-3.69

Hesperidin

-6.83

-9.76

4.47

-9.76

-2.36

Quercetin

-6.52

-5.97

2.98

-5.97

-3.54

Myricitrin

-6.86

-6.76

3.28

-6.76

-3.58

Ginsenoside Rg2

-8.74

9.94

5.37

-9.97

-3.37

Saikosaponin C

-8.41

-10.42

5.37

-10.42

-3.04

a

Binding Energy = Intermolecular Energy + Internal Energy + Torsional energy − Unbound energy.

32


Page 33 of 39


Table 2 Concentration of volatile components in the fresh, r-Rha1-treated and AT-rRha-treated pomelo juice No.

Chemical identification

Concentration (μg/L) Fresh juice

rRha 1treatment

AT-rRhatreatment

No.

Alcohols

Chemical identification

Concentration (μg/L) Fresh juice

rRha 1treatment

AT-rRhatreatment

Aldehydes

1

1-Pentanol

33.2 a

28.2 a

30.9 a

1

(E)-2-Pentenal

7.5 a

0

6.8 a

2

(Z)-2-Penten-1-ol

21.6 a

23.9 a

23.9 a

2

Hexanal

150.8 a

140.8 a

142.2 a

3

3-Hexen-1-ol

111.2 a

121.4 a

104.2 a

3

Furfural

0

7.9 a

0

4

1-Hexanol

102.3 a

86.8 a

95.3 a

4

(E)-2-Hexenal

7.0 a

11.0 a

6.4 a

5

1-Heptanol

2.6 a

2.6 a

2.1 a

5

Heptanal

1.5 a

1.2 a

1.4 a

6

(E)-2-Octen-1-ol

7.2 a

6.2 a

7.1 a

6

Octanal

2.7 a

2.3 a

2.3 a

7

1-Octanol

2.1 a

2.2 a

2a

7

Nonanal

4.6 a

5.5 a

3.3 b

8

Linalool

8.7 a

12.3 a

9.5 a

8

Decanal

0

8.5 a

0

9

1-Nonanol

2.3b

5.1 a

1.8 c

10

Geraniol

0

7.1 a

0

74.3 a

74.3 a

68.6 b

2.3 a

2.6 a

2.4 a

10 a

14.4 a

5.4 b

Terpenes 1

Esters 1 2

7.9 a

9.5 a

8.1 a

10.1 a

11.0 a

10.2 a

3

Limonene Benzene acetaldehyde β-Cubebene

32.3 b

41.5 a

50.1 a

1

Hexanoic acid

6.2 a

12 a

6.5 a

4

Caryophyllene

43.4 a

37.2 a

45.5 a

2

(Z)-Linalool oxide

10.1 a

10.3 a

10.5 a

3

(E)-Linalool oxide

12.5 a

8.4 a

10.5 a

2

3

Ethyl Acetate Ethyl 2-methylbutanoate Ethyl benzoate Others

Note: Superscripts with different letters (i.e., a,b) indicate significant differences (p < 0.05).

33



Fig. 1

34


Page 34 of 39

Page 35 of 39


Fig. 2 A

140

Enzyme activity (U/mg)

120 100 80 60 40 20 0

35



Fig. 3

36


Page 36 of 39

Page 37 of 39


Fig. 4 Optimal pH

A

pH stability

Relative enzyme activity (%)


120 100 80 60 40 20

120 100 80 60 40 20

0

0

2

7

3

4

5

pH

6

7

8

8

9

9

65 °C 120 Optimal temperature Thermal stability C 120 B Optimal pH pH stability 120 100 120 B 100 A 10080 100 80 8060 80 60 6040 60 40 4020 40 20 20 0 20 0 0 10 20 30 40 50 60 70 0 20 30 40 50 Time 60 (min) 70 80 90 100 0 Temperature (°C) 2 3 4 5 6 7 8 9 20 30 pH Relative enzyme activity (%)


Relative enzymeenzyme activityactivity(%) (%) Relative

pH stability

Relative enzyme activity(%)

°C

0 )

50

60

70

Optimal temperature

B

120

65 °C

C

100 80 60 40 20 0 0

10

20

30 40 Time (min)

50

60

70

37


20

30

40

50 60 70 Temperature (°C

Optimal temperature

40

50 60 70 Temperature (°C)

Thermal stabili

80

90


Fig. 5 A Naringin

Time (min)

B Prunin

Time (min)

C

Control juice

AT-rRha treated juice Naringin

Prunin

Time (min)

38


Page 38 of 39

Page 39 of 39


GRAPHIC FOR TABLE OF CONTENTS

39


Heterologous Expression and Characterization of a New Clade of

Recommend Documents