Switching glycosyltransferase UGTBL1 regioselectivity toward polydatin synthesis using a semi-rational design

Article abstract of DOI:10.1039/c8ob00376a

The 62nd residue of glycosyltransferase UGT_BL1 was identified as a "hotspot" for glycosylation at 3-OH of resveratrol. Via semi-rational design including structure-guided alanine scanning and saturation mutations, the mutation I62G significantl

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Switching glycosyltransferase UGTBL1 regioselectivity toward
polydatin synthesis using semi-rational design
Bo Fana, Wenxin Donga, Tianyi Chena, Jianlin Chu*b,c and Bingfang He*a,b,c
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The 62nd residue of glycosyltransferase UGTBL1 was identified as a “hot spot” for glycosylation at 3-OH of resveratrol. Via
semi-rational design including structure-guided alanine scanning and saturation mutations, the mutation I62G significantly
switched the regioselectivity from 4′-OH to 3-OH of resveratrol and mainly produced polydatin (87.7%), a therapeutic
natural product.
www.rsc.org/
Introduction
Resveratrol (trans-3,5,4
′
-trihydroxystilbene) is
a natural
polyphenol with wide range of physiological and
a
pharmacological activities1, such as antioxidative2, anti-
obesity3, 4, anti-inflammatory5, and antitumor6. Despite the
importance of resveratrol, its application is limited because of
its poor solubility and bioavailability, which is dramatically
improved through the glycosylation of resveratrol7-9. Polydatin
(3-O-β-glucoside of resveratrol or piceid), with about 18-fold
water solubility of resveratrol, possesses improved
bioavailability and pharmaceutical properties8, 10, 11. Polydatin
presents various kinds of biological activities, such as anti-
Fig.
1 Switch glycosyltransferase UGTBL1 regioselectivity towards polydatin
synthesis.
economy”. For example, four tert-butyldimethylsilyl (TBS)
protected resveratrols were prepared in one pot and
aging10 and antiproliferative effects11. Recently, polydatin was
separated using column chromatography. Then the 4′-OH-
12
used to relieve the toxic and side effects of cisplatin
and
protected resveratrol was further chosen for the synthesis of
polydatin15(with a total yield of about 20%). Regioselectivity is
one of the typical problems that must be considered in
synthetic organic chemistry, especially in the synthesis of
glycosides16, 17. For stereo- and regioselective glycosylation,
the advantages of biocatalysis over chemical processes are
obvious16.
treat acute severe hemorrhagic shock 13. A phase II clinical
study of polydatin injection (HW6) is in progress
(clinicaltrials.gov: NCT01780129). A growing appreciation for
the important biological roles and the therapeutic potential of
polydatin has resulted in an increasing interest in its synthesis.
Selectivity is key for glycosylation of polyphenols. The 4
group of free resveratrol was verified to be important for
various biological activities 8, 9
′-OH
Selective biocatalytic glycosylation avoids using protective
group chemistry with exquisite stereoselectivity (α-anomer or
β-anomer according to the kind of enzyme). Therefore, it
presents a highly attractive route for polydatin synthesis.
Polydatin could be produced by plant culture, while the
stilbene glucoside derivatives were mixed18. In the culture of
Phytolacca americana cells, the ratio of 3-O-β-glucoside of
.
In the glycosylation of a compound with several possible
positions, the chemists should develop effective protective
group technology to achieve the synthesis of the target
glycoside14,
requiring
sequential
manipulation
of
protecting/deprotecting and resulting in relatively low “atom
resveratrol (polydatin) to 4′-O-β-glucoside of resveratrol was
about 8:519. The glycosylation of resveratrol was also achieved
by glycosyltransferase YjiC from Bacillus licheniformis DSM 13,
which resulted in a complicated mixture of four glucosides: 3-
a.College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech
University, 30 Puzhunan Road, Nanjing 211816, China.
b.School of pharmaceutical sciences, Nanjing Tech University, 30 Puzhunan Road,
Nanjing 211816, China.
c. Jiangsu National Synergetic Innovation Center for Advanced Materials, 30
Puzhunan road, Nanjing 211816, China.
† Correspondence and requests for materials should be addressed to Bingfang He
(email: bingfanghe@njtech.edu.cn) and Jianlin Chu (email: cjl2fl@126.com).
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
O-, 4′
sucrose
-O-, 3,5-O-, and 3,5,4
′
-O-β-glucoside20. However, the
TtSPP (R134A) from
phosphorylase
Thermoanaerobacterium thermosaccharolyticum was the most
effective for the 3-OH glycosylation of resveratrol21.
Nevertheless, the anomeric carbon was α-configuration, which
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the screening capacities. Nevertheless, at this point, a strategy
for protein engineering combined with structure-guided
alanine scanning and saturation mutations was performed to
shift the enzyme’s specificity toward polydatin formation (Fig.
1).
Results and discussion
UGTBL1 displayed a similar conformation to the OleI (Fig. S1),
with about 40% of amino acid sequence identity, and the
highly
conserved
backbone
structure.
In
the
glycosyltransferase UGTBL1, the substrate was accommodated
in the cleft formed between the N- and C-terminal domains28.
Cumulatively, some successful efforts to engineer
glycosyltransferases using structure-guided design are
evidence of the importance of amino acid residues close to the
substrate-binding pockets in regulating the function of the
enzymes. For example, the substrate promiscuity of
glycosyltransferase OleD from S. antibioticus was expanded by
the mutation of the amino acid residues around the binding
site 29, 30. Consequently, residues lining the glycosyl acceptor
substrate binding site were considered for potential
mutagenesis.
In the first round, alanine scanning mutation on the selected
19 residues nearing the glycosyl acceptor substrate binding
site was carried out to find the “hotspot” that could
significantly influence regioselectivity of UGTBL1. These 19
mutants (the primers, see Table S1) were constructed, and
their catalytic activity and regioselectivity to resveratrol were
analyzed (Fig. 2a). About one-third of these mutants retained
more than 80% activity compared with the wild type, while the
mutants H16A, Y134A, F236A, andE322A were almost inactive.
Most mutants showed no significant change in regioselectivity.
Fortunately, one of the mutants, I62A, significantly switched
Fig. 2 Activity and regioselectivity of mutants obtained from alanine scanning (a)
and Site-saturated mutagenesis of 62nd site (b) of UGTBL1. Regioselectivity: ratio of
resveratrol-3-O-glucoside (polydatin) to resveratrol-4′-O-glucoside.
4′
-O-β-glucoside to 3-O-β-glucoside of resveratrol. The ratio of
was different from the natural polydatin.
polydatin to resveratrol-4′-O-glucoside was approximately 3.
The enzymes derived from bacteria show diversity in
substrate specificity, but the development of enzyme
The key amino acid hot spot 62nd residue, which contributed
to the regioselectivity of UGTBL1 for polydatin synthesis was
identified in the first round. The site 62 was present in a loop
region (loop N3) following β-sheet 3 in the N-terminal domain
(Fig. S2)28. Coincidentally, the importance of residues within
the loop N3 with respect to urdamycin (an angucycline-type
antibiotic) specificity was shown in studies with the UrdGT1b
and UrdGT1c from S. fradiae.31 The amino acid residues
located in the loop N3 of UrdGT1b and UrdGT1c were
engineered and the engineering glycosyltransferases were
used to synthesize the new urdamycin derivatives. In the
mutational study of OleD (from S. antibioticus), the single
mutation (P67T) that contributed the most to substrate
specificity was also located within the loop N329. In this study,
the present glycosyltransferase mutagenesis highlighted the
importance of key residues within the loop N3, which might
respond to glycosyltransferase acceptor specificity. It was
greatly recommended as a potential focal point for future
glycosyltransferase engineering efforts and presented a new
scaffold for further (semi-)rational redesign.
engineering provides an efficient means for producing
designated products22,
23
.
In the engineering of
glycosyltransferases, some successes were achieved in
modulating the activity and substrate selectivity by structure-
guided evolution. For example, the semi-rationally designed
glycosyltransferase UGT51 from Saccharomyces cerevisiae
presented about1800-fold enhanced catalytic efficiency
(kcat/KM) for converting protopanaxadiol to ginsenoside Rh224.
The activity of mutant glycosyltransferase OleI (V150A) from
Streptomyces antibioticus was about 10% higher than that of
the wild type25.
In this study, a glycosyltransferase UGTBL1 (GenBank No.
AKA94108) from B. licheniformis ZSP0126,
27
was found to
catalyze the glycosylation of resveratrol, yielding resveratrol
-O-β-glucoside as the main product and polydatin in small
4′
amounts (Fig. S3). From this candidate enzyme, UGTBL1 was
engineered for 3-OH of resveratrol. However, unveiling the
mutants displaying improved properties among the
astronomically large sequence space to produce them
experimentally is wet-lab intensive and practically limited by
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Fig. 3 Time course of the glycosylation of resveratrol by UGTBL1 wild type (a) and I62G (b).
Then, the 62nd site of UGTBL1 was selected for further mutant I62G presented about sevenfold higher kcat/KM (377.2)
saturation mutation to further optimize the selected lead for 3-OH than for 4 -OH (kcat/KM: 52.0). According to kcat/KM,
catalyst. All of these mutants retained more than 80% activity the I62G mutant preferred polydatin formation over 4 -O-
′
′
compared with the wild type, except for the mutant I62S. From glucoside formation by roughly sevenfold, while the result of
the second screening, the mutants I62G, I62Q, I62D, I62E, and wild type UGTBL1 was just the opposite (Fig. S3). The
I62H also showed marked preference for the glycosylation of replacement of Ile to Gly at the 62nd site of UGTBL1 could
3-OH. The best mutant, I62G, displayed the largest significantly mediate the regioselectivity of glycosylation of
improvement in regioselectivity compared with I62A, and the resveratrol.
ratio of polydatin to resveratrol-4′-O-glucoside was more than
7. Fortunately, the mutant I62G markedly changed the
regioselectivity without abolishing but slightly improving the
activity (Fig. 2b). In the study of UGT71G1 from Medicago
truncatula, mutation resulted not only in a change of
regioselectivity to flavonol quercetin but also in a prominent
decrease in the total activity32.
Conclusion
In summary, two rounds of mutation (alanine scanning and
saturation mutation) were carried out for the residues near
the substrate binding site based on the semi-rational design
strategy. The “hot spot” was found and the exploratory
mutational experiments provided a mutant that enhanced the
regioselectivity and activity in one case. This study illustrated
the ability of structure-guided evolution to significantly alter
glycosyltransferase regioselectivity via engineering in the loop
N3 and showed that this region might have the potential for
the further engineering. It paved the way for a sustainable and
scalable biocatalytic process for producing polydatin.
The time courses of resveratrol glucosides catalyzed by
UGTBL1 wild-type and I62G were determined (Fig. 3). A
maximal production of glucosides were obtained after a 3-h
reaction catalyzed by mutant I62G with the same amount of
enzymes, while the reaction catalyzed by wild-type UGTBL1
needed more than 6 h to reach the reaction platform. In this
work, with an equimolar ratio of UDP-glucose and resveratrol,
the conversion of resveratrol reached an equilibrium at about
80%. The conversion rate of resveratrol could be enhanced
while increasing the ratio of UDP-glucose to resveratrol (data
Engineered
glycosyltransferase
exhibiting
enhanced
regioselectivity could be of interest for diverse glycoside
synthesis and drug discovery22, 23. The development described
in this study might stimulate the generation of novel
glycosylation catalysts using (semi-)rational design of the
active site pocket of the known glycosyltransferases.
Furthermore, the crystallization of UGTBL1 and its complex
with substrate is ongoing in our laboratory. The high-quality
protein crystal structure, in conjunction with kinetic analyses
of the wild type enzyme and point mutation variant, might
facilitate the exploration of the molecular mechanism
underlying glycosyltransferase regioselectivity glycosylation.
not shown). The ratio of polydatin/resveratrol-4′-O-β-
glucoside was strikingly different for mutant I62G compared
with that for the wild type. This can be explained by the
apparent kinetic parameters of these two enzymes (Table 1).
The wild-type UGTBL1 displayed a lower KM (0.29 mM) toward
4
′
-OH of resveratrol and a higher KM (0.37 mM) toward 3-OH
of resveratrol, indicating that the wild type UGTBL1 had a
greater affinity for 4 -OH. The mutant I62G displayed a lower
KM (0.29 mM) for 3-OH and a higher KM (0.85 mM) for 4 -OH;
the enormous increase in KM for 4 -OH reflected a significant
decrease in affinity for 4 -OH. The affinity of mutant I62G for 3-
OH was much higher than that for 4 -OH, suggesting that the
′
′
′
′
′
Experimental
selectivity of UGTBL1 was mainly determined by kinetics. The
evidence from enzyme kinetic characterization also supported
the idea that in terms of catalytic efficiency (kcat/KM). The
Materials
Resveratrol was obtained from Nanjing Zelang Medical
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Resveratrol
4’-OH
3-OH
Enzyme
Wild type
I62G
KM (mM) kcat (s-1) kcat/ KM KM (mM) kcat (s-1) kcat/ KM KM (mM) kcat (s-1) kcat/ KM
0.31
0.33
129.8
145.9
418.5
442.0
0.29
0.85
109.1
44.2
376.0
52.0
0.37
0.29
20.9
56.4
109.4
377.2
Table 1 Apparent kinetic parameters for wild type UGTBL1 and mutant I62G.
Technology Co.,Ltd, UDP-glucose was obtained from Sigma- sodium dodecyl sulphate–polyacrylamide gel electrophoresis
Aldrich. Other solvents and reagents used in this study were of (SDS-PAGE) (Fig. S4).
analytical grade from commercial sources.
Enzyme assays
Gene cloning, plasmid construction and mutagenesis
The reactions were performed in a typical volume of 100 μL
The wild-type UGTBL1 gene, ugtBL1 (Genbank No., Nucleotide: containing 2 mM resveratrol and 2 mM UDP-glucose, 50 μg/mL
KP123426; Protein: AKA94108), was introduced into the EcoR I enzyme, and 50 mM sodium phosphate buffer (pH 8.0) to
and Xho I sites of pET28a to generate pET28a-UGTBL1 the determine the activity and regioselectivity of UGTBL1 and its
expression plasmid.
mutant-catalyzed glycosylation. Enzymatic conversion was
The site-directed mutations were introduced using a carried out at 30 °C with an agitation rate of 500 rpm using a
modified whole plasmid polymerase chain reaction(PCR) thermo-shaker (DITABIS, Pforzheim, Germany). The reaction
method33. The plasmid pET28a-UGTBL1 was used as a template. was stopped by adding 900 μL methanol. The mixture was
The primers used in the site-directed mutagenesis are listed in centrifuged at 12,000 rpm for 10 min, and the supernatant was
the Table S1. The PCR cycles were as follows: 95 °C for 1 min, used for analysis by high-performance liquid chromatography
98 °C for 10 s, and 68 °C for 7.5 min, with steps 2–3 were run (HPLC).
for 30 cycles. The PCR system contained a 2 μL template (5
ng/mL), 1
The apparent kinetic parameters were analyzed by varying
×
2 μL forward and reverse primers (10 μM), 5 μL resveratrol at a fixed concentration of UDP-glucose.
primerSTAR buffer, 2.5 μL dNTP mixture (2.5 mM), and 0.25 μL
PrimeSTAR HS DNA polymerase (2.5 U/μL, TaKaRa, Dalian,
HPLC and HRMS analysis
China). The obtained PCR product was digested with Dpn I The supernatants of sample were filtered through a 0.22 μm
(TaKaRa, Dalian, China) and used for transformation to nylon filter, and analyzed by HPLC using a C18 column (4.6
Escherichia coli BL21 (DE3). All of the mutants were confirmed mm×250 mm, 5 μm, Kromasil, Bohus, Sweden). The column
by sequencing (Genewiz, Suzhou, China).
temperature and flow rate were 30 °C and 1.0 mL/min,
respectively. A binary gradient was used. The two solvents
were: H2O (mobile phase A) and methanol (mobile phase B).
Protein expression and purification
The recombinant E. coli harboring the appropriate plasmid The method was: 20% (4 min), 20–50% (11 min), 50% (3 min),
were cultured in the Lysogeny broth (LB) medium containing 50–100% (8 min), 100% (5 min), 100–20% (2 min), 20% (2 min).
50 μg/mL kanamycin at 37 °C and 200 rpm. When the OD600 UV detection was at 310 nm.
reached approximately 0.8, the protein expression was
The molecular weight of products in the HPLC peaks were
induced with 1.0 mM isopropyl-β-D-1-thiogalactopyranoside analyzed with high resolution mass spectrometry (HRMS) (Fig.
(IPTG) for 6-8 h at 30 °C. The cells were harvested via S5). The HRMS analysis was performed in positive ion mode on
centrifugation at 12,000 × g for 10 min. To purify the target Agilent 6520 Accurate-Mass Q-TOF LC/MS platform (Agilent
proteins. The cells were resuspended in buffer A (20 mM Tris- Technologies, Santa Clara, CA, USA).
HCl, 500 mM NaCl, 20 mM imidazole, and 10% glycerol; pH
Production, purification and identification of the product
7.9) and lysed by sonication to purify the target proteins. The
target
proteins
were
purified
by
nickel-affinity Upscaling of the transglycosylation reactions on resveratrol
chromatography on Ni-NTA resin (GE Healthcare, Chicago, IL, was performed up to 100 mL, contained 2 mM resveratrol,
USA) at AKTA prime plus system (GE Healthcare, Chicago, IL, 2mM UDP-glucose, 100 μg/mL enzyme, and 50 mM sodium
USA). The clear supernatant of cell lysate was loaded onto the phosphate buffer (pH 8.0). Threefold volumes of methanol
Ni-NTA column. The column was washed with five times were added and mixed. The mixture was centrifuged at 12000
volume of buffer B (20 mM Tris-HCl, 500 mM NaCl, 100 mM rpm for 10 min, and the supernatant was concentrated by
imidazole, and 10% glycerol; pH 7.9), and then eluted using rotary evaporation. The concentrate was further purified on a
buffer C (20 mM Tris-HCl, 500 mM NaCl, 150 mM imidazole, semi-preparative C18 reverse-phase column (20.0 mm×250
and 10% glycerol; pH 7.9). The fractions containing purified mm, 10 μm, SinoChrom, Dalian, China). The flow rate was 10
protein were concentrated, and the buffer was changed to 50 mL/min, and H2O was used as the mobile phase A and
mM sodium phosphate buffer (pH 7.0) using Amicon Ultra methanol was used as the mobile phase B. The percentage of
Centrifugal Filter Devices (10 kDa, Millipore, Billerica, MA, mobile phase B was manually increased from 10% by
USA). Protein concentrations were determined using the increments of 1% per minute until all products were eluted.
bicinchoninic acid (BCA) method with bovine serum albumin The collected factions with the same resveratrol glucoside
(BSA) as the standard. The purified proteins were confirmed by were pooled together and concentrated on
a rotary
evaporator. The residual water was removed by freeze-drying.
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11 C. E. Storniolo, P. Quifer-Rada, R. M. Lamuela-Raventos and J.
J. Moreno, Food Funct., 2014, , 2137-2144.
12 S. Ince, D. A. Acaroz, O. Neuwirth, H. H. Demirel, B. Denk, I.
The chemical structure of the purified products was
identified by the analysis of 1H NMR, 13C NMR and
5
Heteronuclear
Multiple-Bond
Correlation
(HMBC)
-O-β-
Kucukkurt and R. Turkmen, Food Chem. Toxicol., 2014, 72
147-153.
,
spectroscopy (polydatin: Fig. S6–S8; resveratrol-4
′
glucoside: Fig. S9–S11, Table S2). All NMR data were recorded
in dimethyl sulfoxide–d6 on a 400-MHz NMR spectrometer
(Bruker, Rheinstetten, Germany).
13 X. M. Wang, R. Song, Y. Y. Chen, M. Zhao and K. S. Zhao,
Expert Opin. Inv. Drug, 2013, 22, 169-179.
14 B. Yu, J. Sun and X. Yang, Acc. Chem. Res., 2012, 45, 1227-
1236.
15 Z. Zhang, B. Yu and R. R. Schmidt, Synthesis, 2006, 1301-1306.
Homology modeling
16 X. M. Zhu and R. R. Schmidt, Angew. Chem. Int. Ed., 2009, 48
,
Homology modeling was performed with the SWISS-MODEL
server34, 35 using the reported crystal structure (PDB No. 2IYA)
of glycosyltransferase OleI25 from Streptomyces antibioticus as
a template. The resulting model was superimposed onto the
crystal structure of the template OleI in complex with
substrate oleandomycin. The residues with 3.5 Å from
substrate amount to the resveratrol in homology model
UGTBL1, were selected for the mutagenesis in the first round of
screening. The amino acid residue that significantly affected
the regioselectivity of UGTBL1 was selected for saturation
mutagenesis in the second round of screening.
1900-1934.
17 J. Wang, G. Li and M. T. Reetz, Chem. Commun., 2017, 53
3916-3928.
18 D. Donnez, P. Jeandet, C. Clément and E. Courot, Trends
Biotechnol., 2009, 27, 706-713.
19 D. Sato, N. Shimizu, Y. Shimizu, M. Akagi, Y. Eshita, S.-i. Ozaki,
N. Nakajima, K. Ishihara, N. Masuoka and H. Hamada, Biosci.
Biotechnol. Biochem., 2014, 78, 1123-1128.
20 R. P. Pandey, P. Parajuli, J. Y. Shin, J. Lee, S. Lee, Y.-S. Hong, Y.
I. Park, J. S. Kim and J. K. Sohng, Appl. Environ. Microbiol.,
2014, 80, 7235-7243.
,
21 M. E. Dirks-Hofmeister, T. Verhaeghe, K. De Winter and T.
Desmet, Angew. Chem., 2015, 127, 9421-9424.
22 J. B. McArthur and X. Chen, Biochem. Soc. T., 2016, 44, 129-
142.
23 J. Schmid, D. Heider, N. J. Wendel, N. Sperl and V. Sieber,
Front. Microbiol., 2016, 7, 182.
Conflicts of interest
There are no conflicts to declare.
24 Y. Zhuang, G. Yang, X. Chen, Q. Liu, X. Zhang, Z. Deng and Y.
Feng, Metab. Eng., 2017, 42, 25-32.
25 D. N. Bolam, S. Roberts, M. R. Proctor, J. P. Turkenburg, E. J.
Dodson, C. Martinez-Fleites, M. Yang, B. G. Davis, G. J. Davies
and H. J. Gilbert, Proc. Natl. Acad. Sci. USA, 2007, 104, 5336-
5341.
26 B. Fan, T. Chen, S. Zhang, B. Wu and B. He, Sci. Rep., 2017, 7,
463.
27 S. Zhang, Z. Zhou, Z. Yao and B. He, Biochem. Eng. J., 2013,
71, 105-110.
28 S. A. Osmani, S. Bak and B. L. Moller, Phytochemistry, 2009,
70, 325-347.
29 G. J. Williams, C. Zhang and J. S. Thorson, Nat. Chem. Biol.,
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21506099 and 81673321). We also
acknowledge the fund sponsored by the Program for
Innovative Research Team in Universities of Jiangsu Province
(2015) and the Jiangsu Synergetic Innovation Center for
Advanced Bio-Manufacture.
2007, 3, 657-662.
30 R. W. Gantt, R. D. Goff, G. J. Williams and J. S. Thorson,
Angew. Chem. Int. Ed., 2008, 47, 8889-8892.
31 D. Hoffmeister, B. Wilkinson, G. Foster, P. J. Sidebottom, K.
Notes and references
1
2
3
4
J. A. Baur and D. A. Sinclair, Nat. Rev. Drug Discov., 2006, 5,
493-506.
L. Lu, S. Zhu, H. Zhang, F. Li and S. Zhang, RSC Adv., 2015, 5,
14114-14122.
Ichinose and A. Bechthold, Chem. Biol., 2002, 9, 287-295.
32 X.-Z. He, X. Wang and R. A. Dixon, J. Biol. Chem., 2006, 281
34441-34447.
,
33 J. Sanchis, L. Fernández, J. D. Carballeira, J. Drone, Y.
Gumulya, H. Höbenreich, D. Kahakeaw, S. Kille, R. Lohmer, J.
J.-P. Peyralans, J. Podtetenieff, S. Prasad, P. Soni, A. Taglieber,
S. Wu, F. E. Zilly and M. T. Reetz, Appl. Microbiol. Biotechnol.,
2008, 81, 387-397.
34 K. Arnold, L. Bordoli, J. Kopp and T. Schwede, Bioinformatics,
2006, 22, 195-201.
35 N. Guex, M. C. Peitsch and T. Schwede, Electrophoresis,
2009, 30, S162-S173.
Y. Qiao, J. Sun, S. Xia, X. Tang, Y. Shi and G. Le, Food Funct.,
2014, , 1241-1249.
5
A. Gracia, J. Miranda, A. Fernandez-Quintela, I. Eseberri, M.
Garcia-Lacarte, F. I. Milagro, J. A. Martinez, L. Aguirre and M.
P. Portillo, Food Funct., 2016,
J. Walker, K. Schueller, L.-M. Schaefer, M. Pignitter, L.
Esefelder and V. Somoza, Food Funct., 2014, , 74-84.
D. Vergara, P. Simeone, D. Toraldo, P. Del Boccio, V. Vergaro,
S. Leporatti, D. Pieragostino, A. Tinelli, S. De Domenico, S.
Alberti, A. Urbani, M. Salzet, A. Santino and M. Maffia, Mol.
7, 1680-1688.
5
6
5
Biosyst., 2012, 8, 1078-1087.
7
L. Biasutto, E. Marotta, A. Bradaschia, M. Fallica, A. Mattarei,
S. Garbisa, M. Zoratti and C. Paradisi, Bioorg. Med. Chem.
Lett., 2009, 19, 6721-6724.
8
9
A. Lepak, A. Gutmann, S. T. Kulmer and B. Nidetzky,
ChemBioChem, 2015, 16, 1870-1874.
I. Medina, D. Alcantara, M. J. Gonzalez, P. Torres, R. Lucas, J.
Roque, F. J. Plou and J. C. Morales, J. Agr. Food Chem., 2010,
58, 9778-9786.
10 H. Wen, X. H. Gao and J. H. Qin, Integr. Biol., 2014, 6, 35-43.
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