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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
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Journal of Agricultural and Food Chemistry
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Heterologous expression and characterization of a new clade of
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Aspergillus α-L-rhamnosidase suitable for citrus juice processing
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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
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*Corresponding
15
Professor Hui Ni
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College of Food and Biology Engineering, Jimei University Xiamen, Fujian Province
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3616021, China
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Phone: 13015914929
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Fax: 865926180470
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E-mail:
[email protected] author:
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ABSTRACT:
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α-L-Rhamnosidase is a glycoside hydrolase capable of removing naringin from
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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
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quality of citrus juice.
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KEYWORD: α-L-rhamnosidase, naringin, aroma profile, Aspergillus tubingensis,
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citrus juice processing
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INTRODUCTION
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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,
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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.
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In the carbohydrate-active enzymes (CAZy) database (www.cazy.org),
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α-L-rhamnosidases have been classified in three glycoside hydrolase families (GH),
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namely, the GH28 family, the GH78 family, and the GH106 family14, 15. To date, 29
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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
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the catalytic domain. Moreover, domain D (a β-sheet domain) of the crystal structure
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of SaRha78A was identified as a noncatalytic carbohydrate binding module (CBM67)
59
that is involved in enzyme function against insoluble substrates18. Despite this, the
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catalytic properties of α-L-rhamnosidases have not been extensively reported, which
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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
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structurally characterize α-L-rhamnosidases with varied catalytic properties from
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natural or artificial resources.
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Citrus fruits are popular fruits that are extensively processed to juices that contain 3
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a wide range of flavonoid glycosides, such as naringin, rutin, narirutin, neohesperidin
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and hesperidin21. Among the glycosides, naringin is the main component causing the
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bitter taste of citrus juices13. A number of techniques have been reported to remove
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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)
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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
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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
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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
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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
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barely act on other substrates, suggesting that this enzyme might be able to debitter
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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
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homogeneity, enzymatic properties and its effects on naringin and aroma-related
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volatiles of citrus juices. The specific content included (1) strain identification; (2) 4
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cloning and heterologous expression of the α-L-rhamnosidase in P. pastoris GS115;
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(3) characterization of enzymatic properties; and (4) illustration of the effects on
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naringin and aroma-related volatiles of citrus juices.
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MATERIALS AND METHODS
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Isolation of the α-L-rhamnosidase producing strain JMU-TS529. Compost
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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.
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China. For the enrichment of α-L-rhamnosidase producing strains, 50 mL enrichment
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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
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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
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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
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g/L, ZnSO4·7H2O 0.01 g/L, agar powder 20.0 g/L) after 7 days of cultivation at 28 °C.
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The phase-contrast microscopic image of the conidial head was recorded using a
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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
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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%
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ethanol. The dehydrated samples were set in a critical-point dryer (Autosamdri-815B
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Series C, BBE, Germany) for 20 min. The samples were then mounted on aluminum 6
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stubs and coated with a layer of 30 nm gold-palladium alloy. The fungal morphology
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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.
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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.
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The PCR began with an initial denaturing step at 95 ºC for 5 min, followed by 30
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cycles of sequential incubation (94 ºC for 40 s, 57 ºC for 40 s and 72 ºC for 30 s), and
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ended with a final extension at 72 ºC for 10 min. The primer pair ITS1 (5'-TCC GTA
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GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3')
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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
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conducted according to Chen et al.22 with some modifications. One unit (U) of the
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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
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composed of 0.95 mL of 20 mM citrate buffer (pH 4.0), 1 mL of 300 µg/mL naringin
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aqueous solution and 0.05 mL of enzyme solution, was incubated at 60 °C for 15 min,
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followed by heating at 100 °C for 30 min to inactivate the enzyme. After being
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filtered through a 0.22 µm membrane (Biotech, Shanghai, China), the reaction
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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
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detector (Agilent Technologies Co. Ltd, California) under the same conditions used in
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our previous study22. The control experiment was carried out using inactivated
154
enzyme instead of active enzyme.
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Cloning of the α-L-rhamnosidase encoding gene from JMU-TS529. The
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JMU-TS529 culture mixture was washed twice with PBS (0.1 M, pH 7.0) and
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centrifuged at 30,000×g for 10 min. The precipitates were ground with liquid nitrogen.
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Total RNA was extracted using an EasyPureTM Plant RNA kit (TIANGEN, China)
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and stored at -70 °C before use.
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The RNA was reverse transcribed into first-strand cDNA using the 5' RACE
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System (Invitrogen, USA). The AT-Rha protein coding region was amplified using
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the designed primer (CGTCTGCCTGGACGGCSCNAARMGNGA), termed AT-Rha,
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and the 3'-end universal primer, RACE 3' (CTATAGTCGACGGCTTATT). The 5'-
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and 3'-cDNA ends of the AT-Rha gene were amplified using specific PCR primers
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designed based on the sequence of Aspergillus niger CBS 513.88 hypothetical protein
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(XM_001395598.2). The complete fragment was constructed by overlapping PCR.
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PCRs were carried out by denaturing at 94 °C for 4 min, followed by 30 cycles of
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sequential incubation (94 °C for 1 min, 54 °C for 1 min and 72 °C for 0.5 min) and a
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final extension at 72 °C for 10 min. PCR products were purified using a Universal 8
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DNA Purification Kit (TIANGEN, China) and ligated into the pMD18-T vector and
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sequenced (its accession number in GenBank is KX664478).
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Sequence Analysis and Molecular Docking. The sequences of GH78
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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
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Simple Modular Architecture Research Tool (SMART). Multiple sequence alignment
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of the amino acid sequences was performed using the ClustalW2 program.
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Phylogenetic trees were constructed using MEGA 7.0 with the neighbor-joining
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method. Bootstrap values were calculated based on 1000 replications of the data.
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Structure-based
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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
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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
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plasmid. The recombinant plasmid (pPIC9K-AT-Rha) was transformed into P.
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pastoris GS115 by electroporation using a Pichia expression kit (Invitrogen, USA).
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The positive transformants were isolated from geneticin (G418) (Trans, China)
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containing agar plates and verified by PCR and sequencing. The confirmed
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α-L-rhamnosidase-expressing transformants were cultured at 30 °C in buffered
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glycerol-complex (BMGY) medium containing yeast nitrogen base (YNB) (13.4 g/L), 9
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yeast extract powder (14.0 g/L), peptone (28.0 g/L), potassium phosphate buffer (0.1
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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
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by comparison with the 5-245 kDa markers (Solarbio, China) on a 10% SDS-PAGE
205
gel after silver staining30.
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Purification of the recombinant α-L-rhamnosidase (AT-rRha) from P.
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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.
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The protein sample was then applied to a DEAE SepharoseTM Fast Flow column
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(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
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citric acid-disodium hydrogen phosphate buffer (pH 7.0) containing 1.0 M NaCl. The
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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
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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
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The active eluents were pooled for analysis of mass weight using gel filtration
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chromatography and SDS-PAGE. The pooled enzyme fraction from HiTrap Blue HP
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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.
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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.
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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.
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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
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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
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(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
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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.
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RESULTS AND DISCUSSIONS
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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
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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
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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
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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
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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
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optimal
13.
The fungal
pH45.
Fungal
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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
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48.
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Journal of Agricultural and Food Chemistry
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
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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
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structure
(Fig.
S2);
Page 23 of 39
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 24 of 39
Page 25 of 39
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 26 of 39
Page 27 of 39
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Page 28 of 39
Page 29 of 39
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
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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.
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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.
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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).
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Fig. 1
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Fig. 2 A
140
Enzyme activity (U/mg)
120 100 80 60 40 20 0
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Fig. 3
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Fig. 4 Optimal pH
A
pH stability
Relative enzyme activity (%)
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 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
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20
30
40
50 60 70 Temperature (°C
Optimal temperature
40
50 60 70 Temperature (°C)
Thermal stabili
80
90
Journal of Agricultural and Food Chemistry
Fig. 5 A Naringin
Time (min)
B Prunin
Time (min)
C
Control juice
AT-rRha treated juice Naringin
Prunin
Time (min)
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GRAPHIC FOR TABLE OF CONTENTS
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