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Biosynthesis of glycyrrhetinic acid-3-O-monoglucose using glycosyltransferase UGT73C11 from Barbarea vulgaris Xiaochen Liu, Liang Zhang, Xudong Feng, Bo Lv, and Chun Li Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b03391 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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Industrial & Engineering Chemistry Research

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Biosynthesis of glycyrrhetinic acid-3-O-

2

monoglucose using glycosyltransferase UGT73C11

3

from Barbarea vulgaris

4

Xiaochen Liu, Liang Zhang, Xudong Feng, Bo Lv*, Chun Li

5

Institute for Biotransformation and Synthetic Biosystem, Department of Biological Engineering,

6

Beijing Institute of Technology, Beijing 100081, PR China

7

KEYWORDS:

8

Glycosyltransferase, Glycosylation, Barbarea vulgaris UGT73C11, Glycyrrhetinic acid,

9

Glycyrrhetinic acid-3-O-monoglucose

10

ABSTRACT:

11

Glycyrrhetinic acid (GA) is the pentacyclic triterpenoid hydrophobic aglycone with many

12

pharmacological effects and biological activities. Glycosylation is often used to improve the

13

aglycone’s properties such as solubility, stability and pharmacological potency. UDP-

14

glycosyltransferases (UGTs) are main enzymes to catalyze this conversion via transferring

15

glycosyl moiety to corresponding acceptor substrates in nature. However, glycosyltransferase

16

which can transfer glucose to GA has not been reported yet. The glycosyltransferase UGT73C11

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from plant Barbarea vulgaris was reported with the glycosylation function to compounds which

2

are similar to GA in chemical structure. In this study, UGT73C11 was selected to express

3

functionally in Escherichia coli and purified as the biocatalyst for the glycosylation of GA. As a

4

result, the recombinant UGT73C11 catalyzed UDP-glucose and GA to produce a new compound

5

GA-3-O-monoglucose. The product GA-3-O-monoglucose was characterized by HPLC and LC-

6

ESI-MS spectrometry with an exact mass of 633, and the glucose was linked with O atom at GA

7

C-3 position with β-glycosidic bond by IR and NMR analysis. Under optimal reaction

8

conditions, the recombinant UGT73C11 showed the highest activity at 40> with pH of 7.0, and

9

the highest conversion was found at substrate molar ratio UDP-glucose/GA of 5:1. At last, 98%

10

of GA was converted into the corresponding GA-3-O-monoglucose under optimized conditions

11

at 6 h. GA-3-O-monoglucose improved significantly the solubility and bioactivity of the parent

12

GA according to data from the water solubility and antibacterial activity detestation. These

13

results indicate that the recombinant UGT73C11 was potentially exploited as biocatalyst for the

14

glycosylation of GA in industrial and pharmaceutical use.

15

INTRODUCTION

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GA, a well-known herbal medicine component widely used all over the word, is the major

17

bioactive pentacyclic triterpenoid hydrophobic aglycone obtained mainly from the roots and

18

rhizomes of licorice1, 2. GA is famous for a wide variety of pharmacological effects and

19

biological activities, such as anti-inflammatory3-5, antibacterial activities6, antitumor activities7, 8

20

and other chemotherapeutic agent4, 9. Because of the structural safety and widely therapeutic

21

activities, GA was modified as steroid backbone structure to improve or even create novel

22

pharmacological properties to cure special diseases10. For example, disodium succinoyl


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glycyrrhetinate synthesized from GA by adding a succinic acid moiety exhibited strong

2

antibacterial activity against several S. aureus strains6. Recently, a new GA derivative has been

3

synthesized as a highly selective inhibitor against human carboxylesterase 2 with the significant

4

inhibitory effect11. Glycyrrhetinic acid-cinnamoyl hybrids were demonstrated better anti-tumor

5

activity than GA via modification at the C-3 position of GA with cinnamoyl analogues12.

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Glycosylation has been demonstrated to be a powerful strategy in amplifying natural products 13, 14

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diversity and new drug discovery

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natural products aglycones such as increasing the water solubility, changing the chemical

9

structure diversification and stability, and improving the pharmacological potency and 15

, as glycosylation plays an important role in modifying

10

bioactivity properties

. In addition, the attached sugar moieties are critical for the

11

pharmaceutical and biological effects of GA-glycosides derivatives. For example, glycyrrhizin

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(GL) and glycyrrhetinic acid monoglucuronide (GAMG) are two glycosylated GA derivatives

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with glucuronidic acids. The two GA-glycosides derivatives are not only more soluble than GA

14

but also present special sweetener tastes and widely pharmacological activities16-18. With this

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strategy, a series of GA glycosides were synthesized utilizing methyl glycyrrhetinate and

16

different glycosyls moieties by chemical method to improve pharmaceutical and biological

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activities of hydrophobic aglycone GA19, 20.

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However, this chemical glycosylation of natural products often requires rigorous reaction

19

conditions, such as special temperature, catalyst and multiple steps of protection/deprotection to

20

control regioselectivity leading to the poor yield of final glycosylated product20-22. Alternatively,

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glycosyltransferase offer a potential solution to this problem via transferring glycosyl moiety

22

from sugar donor to corresponding position of acceptor substrates in the mild reaction

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conditions23, 24. Nowadays, more and more glycosyltransferases were found or engineered as


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biocatalysts to produce high value compound in fine chemistry24-27. It was reported that the novel

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isobavachalcone glucosides with anti-proliferative activities was synthesized by UDP-

3

glycosyltransferase (YjiC) from Bacillus licheniformis DSM-1328. Currently, several

4

glycosyltransferases have been reported for synthesizing triterpenoid saponins. The sapoggenins

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ginsenoside Rh1 was transformed into ginsenoside Rh1 glycosides by UDP-glycosyltransferases

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YojK1 and YjiC129. Glycosyltransferase UGT73C subfamily from Barbarea vulgaris catalyzed

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3-O-glucosylation of the sapogenins hederagenin and oleanolic acid with uridine diphosphate

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glucose(UDP-glucose) as glycosyl donor 30. Recently, a novel glucuronosyltransferase has been

9

reported to catalyze continuous two-step glucuronosylation of GA to yield glycyrrhizin31.

10

However, glycosyltransferase has been rarely reported to produce GA-glycoside derivatives with

11

glucose linkage.

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In this study, glycosyltransferase UGT73C11 from Barbarea vulgaris was recombinantly

13

expressed in E.coli after condon optimization and used for in vitro glycosylation of GA with

14

UDP-glucose as donor. We also successfully investigated the UGT73C11 biochemical

15

characterization and optimized the reaction conditions. In addition, the water solubility and

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antibacterial activity of GA glycoside product was tested to confirm the effect on biological

17

activity after adding glucose to GA.

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2. MATERIALS AND METHODS

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2.1. Chemicals

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Chemicals typically were of the highest purity available. GA (>98%) and UDP-glucose

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(>98%) and UDP-glucoronic acid (>98%) were purchased from Sigma-Aldrich (Shanghai,

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China).

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2.2 Plasmid construction and transformation


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The genes of UGT73C11 (GenBank: AFN26667.1) was chemically synthesized and inserted

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into pUC57 vector (Genewiz) with a codon usage optimization for expression in E. coli (Support

3

Information, Table S1). This sequence was cloned using the primers with the restriction enzymes

4

sites EcoR > and Sal > (F: CCGGAATTCATGGTTTCTGAAATCACCCACAAAT, R:

5

ACGCGTCGACGTTGTTAGACTGAGCCAGCTGCATGAT), then digested and ligated into

6

expression vectors pET-28a vector (stored in our lab) that had been previously digested by the

7

same restriction enzymes EcoR > and Sal >. The pET28a-UGT73C11 vector harbored a single

8

open-reading frame including the UGT73C11 protein and 6 His-tag at both N terminal and C

9

terminal of the UGT73C11 for further purification. The recombinant plasmid of pET-28a-

10

UGT73C11 was transferred into E. coli BL21 (DE3) competent cells (Biomed) using the

11

manufacturer’s protocol. Kanamycin (50 mg/L) was used for proper selection of clones, and the

12

transformants were picked out to screen the positive clones via colony PCR and sequencing

13

(Genewiz).

14

2.3 Enzyme heterologous expression and purification

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The positive transformants were inoculated into 5 mL LB liquid culture medium in the test

16

tube supplemented with Kanamycin (50 mg/L) and cultured at 37°C overnight with continuous

17

shaking at 170 rpm. Then 1% (v/v) of inoculum was added to 300 mL LB liquid culture medium

18

and incubated at 37°C until the OD600 reached 0.6, followed by adding isopropyl 1-β-D-

19

thiogalactopyranoside (IPTG) to the medium at a final concentration of 0.2 mM and further

20

incubation at 16°C for 18 h. The cells were harvested by centrifugation (8000 rpm for 10 min at

21

4°C) and resuspended to be sonicated in 20 mL binding buffer (50 mM PBS, pH 7.4 100 mM

22

NaCl, 20 mM imidazole). After cell debris was removed by centrifugation at 12000 rpm for 20

23

min at 4°C, the supernatant was yielded as crude enzyme containing the soluble UGT73C11


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protein. Protein UGT73C11 with 6

His-tag at N-terminal and C-terminal respectively was

2

purified by Ni-NTA affinity chromatography (GE Healthcare).The target bound protein was

3

eluted by elution buffer (50 mM PBS, pH 7.4 100 mM NaCl,100 mM imidazole). This

4

purification was performed by AKTA purifier system (GE Healthcare).Finally, purifier elution

5

buffer was exchanged to 50 mM PBS buffer pH 7.4 in 10 KDa Amicon Ultra centrifugal filters

6

(Merck) by centrifugation. Enzyme purity was assessed by SDS-PAGE32, and the protein

7

concentration was determined using NanoDrop 2000 (Thermo Scientific).

8

2.4 In vitro enzymatic glycosylation of GA

9

The glycosylation reaction was performed in 100 µL reaction buffer (50 mM PBS, pH 7.4)

10

containing 50 mg/L of UGT73C11 enzyme, 1 µM UDP-Glucose (Sigma-Aldrich), 1 µM GA

11

(Sigma-Aldrich). After 2 h incubation at 37°C, the reaction mixture was terminated by adding

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900 µL methanol and analyzed by HPLC and LC-MS.

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The optimum pH of UGT73C11 was investigated from pH 4.0 to11.0 in different buffer (100

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mM saline-sodium citrate (SSC) buffer, pH 4.0-5.0; 100 mM phosphate buffer, pH 6.0-7.0 Tris-

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HCl buffer, pH 8.0-9.0,NaHCO3-NaOH buffer, pH 10.0-11.0) at 40°C. The optimum

16

temperature of UGT73C11 was determined at pH 7.0 from 25°C to 55°C. We also detected the

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divalent metal ions at final concentration 5 mM and the substrates ratios effect on this

18

glycosylation reaction at 40°C pH 7.0. For kinetic studies of UGT73C11, the initial velocities of

19

the enzymatic reaction were examined by varying the concentration of UDP-glucose and GA.

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Values of the Michaelis constants (Km) and maximal velocity (Vmax) were obtained by the

21

Lineweaver-Burk plot.

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2.5 HPLC and LC-MS analysis


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The HPLC analysis was carried out with a Shimadzu LC 10AD instrument (Shimadzu Corp.,

2

Kyoto, Japan). The chromatographic separations were performed on a reverse-phase C18 column

3

(5 mL; 250 × 4.6 mm; Shimadzu) at 40 °C with UV detection at 254 nm. A gradient elution

4

method was employed with the mobile phase liquid consisting a mixture of methanol and 0.6%

5

acetic acid (84:16 v/v) at a flow rate of 1 mL/min. For LC-MS analysis, the product was

6

evaporated by vacuum concentrator system. The product was re-suspended with 20 µL methanol

7

and injected into an LC-MS (Agilent 6460 Triple Quad LC/MS).

8

2.6 Preparation and purification of GA-3-O-monoglucose

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For getting more GA-3-O-monoglucoses production, the reaction was enlarged as follows:

10

purified UGT73C11 was mixed with UDP-glucose (5 mM) in 25 mL PBS buffer (50 mM at pH

11

7.0) in Erlenmeyer flask. GA (1 mM) dissolved in 1 mL ethanol was batch adding to mixture

12

every two hours when the reaction mixture was stirred in water baths shaker for 18 h and

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temperature was controlled at 40°C. Subsequently, the products was extracted from reaction

14

mixture using twice the volume of butanol, then evaporated by rotary evaporator. The product

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was resuspended with 5 mL methanol and purified by semi-preparation LC10AR equipment

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(Shimadzu Corp., Kyoto, Japan) with a Shimadzu C18 column (20×250 mm). The elution was

17

performed with the mobile phase consisting of methanol and 0.6% acetic acid (80:20 v/v) at a

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flow rate of 5 ml/min. After collecting the target peak, the white power of GA-3-O-monoglucose

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was obtained by using rotary evaporator, freeze-drying and vacuum concentration. The purified

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compound was analyzed by FT-IR Tensor 27 (Bruker, Germany) and AVANCE III HD 700

21

MHz spectrometer (Bruker, Germany).

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2.7 Fourier transform infrared spectroscopy


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The FT-IR spectrum of GA and GA-3-O-monoglucose was recorded in the region of 400-4000

2

cm-1 on Bruker Tensor 27 spectrophotometer with a spectral resolution of 4 cm-1 using KBr

3

pellet technique. KBr pellet of solid sample was prepared from mixture of KBr and the sample in

4

400:1 ratio using a hydraulic press.

5

2.8 NMR spectroscopy

6

The 1H NMR spectrum were recorded on a Bruker AVANCE III spectrometer operating at 700

7

MHz. The measurements were done in methnol-d4 solution. The solution was prepared by

8

dissolving 8 mg of the samples in 0.5 mL of methnol-d4. Chemical shifts are reported in ppm

9

relative to TMS.

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2.9 Water solubility determination

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Excess compounds GA and GA-3-O-monoglucose was mixed with 0.5 mL of distilled water

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in Eppendorf tubes respectively at room temperature. After sufficient dissolution with the

13

sonication assistance and centrifugation at 15000 rpm for 15 min to remove insoluble material,

14

the supernatant solution was collected and pretreated with methanol as previously described to

15

subject to HPLC analysis at 254 nm. The HPLC peaks were integrated to calculate the sample

16

solution concentrations.

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2.10 Antibacterial activity assay

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Escherichia coli, Staphylococcus aureus and Bacillus subtilis were selected to be candidate

19

bacterial strains to be tested. Different concentrations of GA and GA-3-O-monoglucose were

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prepared as follow: 1 mM, 5 mM, 10 mM. The filter paper method was used to determine the

21

sensitivity of the bacterial species to these different concentrations per the inhibition zone. Each

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bacterial species of the above mentioned were cultured in LB medium respectively, and then

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inoculated in LB agar medium in petri dish. Filter papers contained the above-mentioned

24

compounds were individually placed in each inoculated plate and then incubated for an overnight


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at 37°C. The results were detected next day, and inhibition zone were measured by Vernier

2

caliper.

3

3. RESULTS AND DISCUSSION

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3.1 Recombinant expression and purification of UGT73C11

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UGT73C11 (GenBank: AFN26667.1) from Barbarea vulgaris encoding the enzyme with

6

glycosylation activity of saponions consists of 488 amino acids30. In this work, UGT73C11 gene

7

condon usage was optimized and chemically synthesized for its high-efficiency and soluble

8

expression in E.coli strain. The optimized UGT73C11 sequence was shown in Support

9

Information Table S1. After amplified by PCR using the forward and reverse primers (Figure

10

S1), the UGT73C11 sequence was successfully ligated into pET28a vector and expressed in E.

11

coli BL21 (DE3). The molecular mass of purified recombinant enzyme was about 62 kDa as

12

determined by SDS-PAGE (Figure 1).

13

E. coli was used here as an expression host strain for the overexpression of recombinant

14

UGT73C11 because of its well-known genetic background and simple operation. However,

15

inclusion bodies are sometimes formed when heterologous proteins are expressed in this

16

prokaryotic host due to the lack of post-translational modifications33. Multiple strategies was

17

established for efficiently enhancing solubility of recombinant proteins in E. coli such as

18

optimizing codon usage, fermentation optimization, expression with fusion protein tag and co-

19

expression with molecular chaperones34-36. In this study, the UGT73C11 codon usage was

20

optimized by replacing the codons rarely used in E. coli. The glycosyltransferase UGT73C11

21

with both N-terminal and C- terminal 6-histidine tags was expressed as soluble enzyme at first

22

try in E coli.

23

3.2 Glycosylation of GA and structural analysis of GA glycoside


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The purified recombinant UGT73C11 was used to catalyze the in vitro glycosylation of GA

2

(Scheme 1). As shown in Figure 2, after 2 h reaction, a new peak appeared at about 8.4 min

3

which was suspected to belong to the produced GA-glycoside. To verify this, the new product

4

was subjected to LC-ESI-MS spectrometry analysis. The new peak was further identified as GA-

5

monoglucose by the accurate molecular ion [M+H]+ at m/z 633.1(Figure 3) compared with the

6

theoretical molecular weight of 632. These evidences showed that the recombinant UGT73C11

7

transferred only one glucose to GA under the given reaction conditions which is consistent with

8

the previous report where hederagenin and oleanolic acid were used as acceptors

9

substrate specificity of UGT73C11 was evaluated by using UDP-glucuronic acid which was also

10

an important sugar donor, and no new peak was detected in HPLC, indicating that recombinant

11

UGT73C11 is strictly specific for the UDP-glucose.

12

30

. Then, the

Some GTs have been reported with emphasis on their acceptor promiscuity and donor

13

specificity27,

37, 38

14

saponins, as it was able to transfer glucose from UDP-glucose to C-3 hydroxyl of both oleanolic

15

acid hederagenin and betulinic acid30. Here, the recombinant UGT73C11 showed the

16

promiscuity to the pentacyclic triterpenoid aglycone GA which had the similar structure

17

compared with oleanolic acid hederagenin and betulinic acid.

. UGT73C11 had also been reported with the broad acceptor specificity to

18

FT-IR and NMR were employed to analyze the structure of GA-monoglucose and the

19

glycosylation position. The vibrational assignments for different functional groups from FT-IR

20

are shown in Figure 4. The carboxylic acid group at C-30 was both shown in substrate GA and

21

product GA-monoglucose at 2700-3000 cm-1. New peaks appeared at 1046-1143 cm-1 for product

22

which was related to the ether linkage functional group of GA-monoglucose in FT-IR. According


10

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1

to the results, we can draw the conclusion that the glucose moiety was transferred to the O atom

2

at C-3 position of GA by the recombinant UGT73C11.

3

1

H NMR data (Table 1) confirmed that the glucose from UDP-glucose was linked to substrate

4

GA. This was clearly indicated by the brand new chemical shift and multiplicity of the respective

5

sugar H in the GA-3-O-monoglucose NMR results in comparison with aglycone GA NMR data.

6

It has been reported that glycosyltransferases can catalyze the formation of glycosidic bond in

7

both α-type or β-type

8

glucose to form β-glycosidic bond by calculating the coupling content H1 and H2 of glucose.

9

The coupling content value is 7.8 HZ (Table 1), which means that the atomic distance between

10

H1 and H2 is relatively far and the dihedral angle between H1 and H2 is 180º. Therefore, the

11

GA-3-O-monoglucose bond formed between sugar C1 and O atom from GA C3 position must be

12

β-type.

23, 39

. In this study, we find that the recombinant UGT73C11 can transfer

13

3.3 Optimization of enzymatic reaction conditions

14

The glycosylation conditions catalyzed by recombinant UGT73C11 were optimized in this

15

section. As shown in Figure 5a, the optimum reaction temperature was determined to be 40>,

16

which is quite typical for glycosyltransferases26. Further, the highest enzyme activity in the

17

process of GA-3-O-monoglucose synthesis was found at pH 7.0, which is similar to other

18

reported GTs (Figure 5b). The UGT73C11 activity sharply declined as the decrease of pH at acid

19

environment, while more gently dismissed as the increase of pH value at base environment.

20

Metal irons especially various divalent metal ions were another important factors for GTs 38

. Many GTs utilize divalent metal ions such as magnesium (Mg2+) or manganese

21

functions

22

(Mn2+) as cofactor39. As shown in Figure 5c, the recombinant UGT73C11 activity was generally

23

independent of most metal divalent metal ions, and it was enhanced significantly by Mg2+ and


11


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strongly inhibited by Zn2+ and Cu2+. The UGT73C11 activity for glycosylation of GA was not

2

even detected in the presence of Cu2+, which showed the similar result in other

3

glycosyltransferase research in UGT73 family34, 38.

4

To evaluate the affinity and catalytic efficiencies of the recombinant UGT73C11 in GA

5

glycosylation, apparent kinetic parameters were determined at 40 > and pH 7.0. As shown in

6

Table 2, Km for substrate UDP-glucose and aglycone GA were 485.70 µM and 104.38 µM,

7

respectively, indicating that the recombinant UGT73C11 showed about 4 times higher affinity to

8

acceptor GA than donor UDP-glucose. Kcat/Km values for donor and acceptor substrates were

9

also shown in Table 2 for analyzing the catalytic efficiencies. It showed that the recombinant

10

UGT73C11 is more efficient to transform substrate GA to GA-3-O-glucose than substrate UDP-

11

glucose.

12

The optimal ratio of GA and UDP-Glucose was investigated by varying the UDP-Glucose

13

concentration ranging from 1 µM to 10 µM with a fixed GA concentration of 1 µM. As shown in

14

Figure 6, we achieved approximately 43% conversion in 2 h with the ratio of UDPG/GA at 5:1.

15

The conversion was nearly 45% when the ratio of UDP-Glucose/GA was 10:1. This result

16

provides an insight that two times the amount of UDP-Glucose only increased small amount of

17

the product. The result showed that 5 µM UDP-glucose was enough for the highest conversion of

18

1 µM GA to GA-3-O-monoglucose in 2 h, so the ratio of UDP-glucose/GA at 5:1 was selected to

19

be proper amount. The time dependent glycosylation of GA to GA-3-O-monoglucose by

20

recombinant UGT73C11 is shown in Figure 7. The conversion increased as the reaction

21

proceeded and became stable after 6 h with more than 98% yield. This result is higher than that

22

of the chemical glycosylation of GA in literature. For example, GA-glycoside derivative was

23

prepared in 65% yield using glycosyl bromide donors and silver zeolite as catalyst which was


12

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1

performed in dichloromethane with molecular sieves 4 Å at room temperature after 4 days20.

2

Monodesmosidic GA glycosides was synthesized with the maximum yield of 62% by applying

3

trifluoromethanesulfonate (TMSOTf) as promoter at -70> and 2 h stirring19. Therefore, the

4

glycosylation of GA catalyzed by glycosyltransferase UGT73C11 showed more preponderant by

5

comprehensive consideration of reaction conditions and yield.

6

Glycosylation of hydrophobic aglycone can potentially improve their pharmacokinetics,

7

enhance desired potency, facilitate membrane transport40. GA as the important active component

8

of traditional herbal licorice has been used in China for thousands of years with many therapeutic

9

effects1. However, the poor pharmacokinetic properties and low water solubility of cholesterol-

10

like aglycone have hampered further pharmaceutical developments and applications, as the

11

chemical structure of GA is a nonpolar pentacyclic triterpenoids aglycone skeleton41,

12

Enzymatic glycosylation is a good avenue to improve both their water solubility and

13

pharmacological activity43, 44. UGT73C11 from Barbarea vulgaris was found for the first time

14

for the glycosylation of GA to synthesize GA-3-O-monoglucose to increase water solubility and

15

improve pharmacokinetic properties. In addition, the glucose moiety introduced to GA increased

16

its sweetness by 218 times higher than sucrose45. This enzymatic method is green, specificity

17

and efficient with improved product yield comparing with organic synthesis method. It also

18

provided the potential driver of industrial production of green chemistry and sustainability in

19

production processes for GA glycoside. GA synthesis pathway in vivo has been reported as the

20

genes discovery of CYP88D6 and CYP72A15446, 47. We discovered UGT73C11 as biocatalyst

21

could transfer glucose to GA and produce GA glycoside derivative in vitro in this study. We

22

believed that this glycosyltransferase would pave a way to produce GA-3-O-monoglucose in vivo

23

with the help of synthetic biology and metabolic engineering.

42

.


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Page 14 of 32

1

3.4 Water solubility

2

To determine water solubility, GA-3-O-monoglucose and GA were mixed with distilled water

3

to dissolve a super-saturated aqueous solution. After centrifuged to remove insoluble material,

4

GA-3-O-monoglucose and GA water solution was analyzed by HPLC. The results showed that

5

the value of solubility of GA-3-O-monoglucose in water was 354.95 µmol·L−1 which was about

6

10.21 times higher than that of its parent compound GA of 34.76 µmol·L−1 (Table 3). As

7

expected, these data suggested that enzymatic biosynthesis of GA glucosides greatly enhanced

8

their water solubility comparing with GA aglycone. The water solubility played a major role in

9

therapeutic efficacy of natural products drugs, as lower solubility resulted in short retention time

10

in the intestine as well as its lower absorption of pentacyclic triterpenoid aglycone drugs48 .

11

Several reports have been shown that the water solubility improvement can be an effective way

12

to enhance the bioactivities of bioactive compounds49-51. Water solubility of GA-3-O-

13

monoglucose was obviously enhanced comparing with parent GA, and it may be a potential

14

effective solution to improve the retention time and absorption of GA in the intestine.

15

3.5 Antibacterial activity

16

The inhibition zone of three candidate bacterial strains after treatment with different

17

concentrations of GA-3-O-monoglucose and GA were shown in Table 4 and Figure S2. The

18

results showed that the two compounds had no effect on the Escherichia coli. However, GA-3-

19

O-monoglucose and GA showed inhibition activity to the Staphylococcus aureus and Bacillus

20

subtilis. Staphylococcus aureus is a major pathogen in humans and may cause various diseases

21

as an opportunistic infectious agent52. Therefore, GA-3-O-monoglucose and GA may have

22

potential for clinical use against Staphylococcus aureus infectious diseases. Antibacterial activity

23

of GA was reported in many research53, 54. From the data of diameters of inhibition zone, we can

24

see that antibacterial activity of GA-3-O-monoglucose was significantly enhanced to resist


14

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Staphylococcus aureus and Bacillus subtilis comparing with the parent aglycone GA in the same

2

concentration. It demonstrated that the introduction of glucose moiety to GA has been improved

3

antibacterial activities. Other bioactivities like anti-cancer and anti-inflammatory activities of

4

GA-3-O-monoglucose were worth further studying in next work.

5

4. CONCUSIONS

6

In this work, the recombinant UGT73C11 from Barbarea vulgaris was successfully used for

7

the glycosylation of GA to form β glycosidic bond at C-3 position by transferring a molecular

8

glucose from donor UDP-glucose. It showed high specificity to glycosyl donor and promiscuity

9

to acceptors like triterpenoid aglycone. In vitro reaction conditions were optimized to obtain a

10

maximum conversion up to 98% at 6 h. The glucose moiety introduction to GA improved

11

significantly the water solubility and antibacterial activity of the parent GA. The bioavailability

12

of the GA-3-O-monoglucose needs to be investigated to evaluate applicability as new functional

13

compound. The glycosyltransferase UGT73C11 is an attractive biocatalyst for cost effective

14

glycosylation of complex structure molecules like GA.

15

FIGURES

16

Figure 1. SDS-PAGE chromatograph. M: Protein marker; Lane 1: Purification of the

17

recombinant UGT73C11.

18

Figure 2. HPLC analysis of in vitro glycosylation reaction mixture. a: control including the

19

recommend UGT73C11 and acceptor GA without donor UDP-glucose after 2 h incubation at 37

20

>. Compound 1 is GA; b:test products catalyzed by the recommend UGT73C11 with GA and

21

UDP-glucose as substrate after 2 h reaction at 37 >.Compound 1 is GA, and compound 2 is GA-

22

glycoside, UDP-glucose cannot be detected in this HPLC condition.


15


Page 16 of 32

1

Figure 3.

LC-ESI-MS analysis of GA glycoside synthesized by UGT73C11-catalyzed

2

glycosylation reaction. The product eluted at 25.07 min was found to have a molecular ion

3

[M+H]+ at m/z 633.1, representing GA monoglucose, while substrate GA was found at 36.31 min

4

with a molecular ion [M+H]+ at m/z 471.3.

5

Figure 4. FT-IR spectrum. a: FT-IR spectrum of Substrate GA; b: FT-IR spectrum of product

6

GA monoglucose.

7

Figure 5. The effect of pH (a), temperature (b) and divalent metal ion (c) on recombinant

8

UGT73C11 activity. N.D. is short for not detect. Values are the average of three independent

9

replicates; error bars represent average ± one standard deviation. Values with different letter

10

subscripts are significantly different from each other (P

monoglucose Using Glycosyltransferase ... - ACS Publications

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