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Highly Efficient Enzymatic Acylation of Dihydromyricetin by the Immobilized Lipase with Deep Eutectic Solvents as Co-solvent Shi-Lin Cao, Xiao Deng, Pei Xu, Zi-Xuan Huang, Jian Zhou, Xuehui Li, Minhua Zong, and Wenyong Lou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00011 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Highly Efficient Enzymatic Acylation of Dihydromyricetin by the Immobilized Lipase with Deep Eutectic Solvents as Co-solvent Shi-Lin Cao1,4#, Xiao Deng1#, Pei Xu1, Zi-Xuan Huang2, Jian Zhou2, Xue-Hui Li2, Min-Hua Zong1,2,Wen-Yong Lou1,3,*, 1

Lab of Applied Biocatalysis, School of Food Science and Engineering, South China

University of Technology, No. 381 Wushan Road, Guangzhou 510640, China. 2

School of Chemistry and Chemical Engineering, South China University of

Technology, No. 381 Wushan Road, Guangzhou 510640, China. 3

State Key Laboratory of Pulp and Paper Engineering, South China University of

Technology, No. 381 Wushan Road, Guangzhou 510640, China. 4

Department of Food Science, Foshan University, No. 18 Jiangwan Yi Road, Foshan

528000, China. *Corresponding author. Tel.: +86-20-22236669; fax: +86-20-22236669.

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Abstract: Novel deep eutectic solvent (DES)-DMSO co-solvent system has been, for

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the first time, successfully used as the reaction medium for the enzymatic acylation of

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dihydromyricetin (DMY) catalyzed by the immobilized lipase from Aspergillus niger

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(ANL). The co-solvent mixture, ChCl:Glycerol-DMSO (1:3, v/v) proved to be the

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optimal medium. With the newly developed co-solvent, the initial reaction rate of

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enzymatic acylation of DMY achieved 11.1 mM/h and the conversion of DMY was

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91.6%. ANL@PD-MNPs is stable and recyclable in this co-solvent, offering 90%

8

conversion rate after repeated use of 5 times. The lipid-solubility of DMY-16-acetate

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was 10 times higher than that of its raw materials DMY. The results showed that the

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DMY-16-acetate product exhibits good antioxidative activity. The present research

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illustrated that the use of DES-DMSO co-solvent may become a feasible alternative

12

for the synthesis of DMY ester.

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Keywords: dihydromyricetin; deep eutectic solvent; Aspergillus niger lipase;

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acylation; antioxidant ability

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INTRODUCTION

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Dihydromyricetin (DMY), also known as ampelopsin, is a natural flavanonol and

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shows numerous bioactivities including antioxidant, anti-inflammatory, analgesic,

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antitussive, antibacterial, anti-thrombotic and anti-tumor activities1-3. However, the

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highly hydrophilic nature of DMY significantly limits its potential application.

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Compared with DMY, DMY-acetate was reported to have much higher liposolubility

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as well as comparable and even improved antioxidative ability4. According to the

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previous literature, the useful DMY fatty acid esters were industrially synthesized by

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chemical routes using acid or alkaline catalysts5. But there are a lot of drawbacks with

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these chemical methods such as unsatisfactory yields, low regioselectivities, harsh

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reaction conditions as well as time-consuming and arduous purification processes4. In

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this aspect, the competitive enzyme-catalyzed organic synthesis, for example, using

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lipase to replace chemical catalysts, has attracted growing interest in the hydrolysis6,

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epoxidation7, aldol addition8, acylation4, alcoholysis9, because of the excellent

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regioselectivities, wide substrate specificity, environmentally friendly nature and mild

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reaction conditions 10. Moreover, the stability, reusability and catalytic performances

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of enzyme can be generally enhanced through immobilization of enzyme 11.

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In the enzymatic acylation of DMY, a major problem in choosing solvents is the

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incompatibility between keeping a high enzyme activity and dissolving substrates

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well4. This polar substrate is scarcely soluble in enzyme-friendly weak-polar and

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non-polar solvents such as hexane, while the enzymes are prone to deactivation in

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high-polar solvents in which the substrates can be scarcely soluble12. Hence, the

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development of a suitable reaction medium is critical for the enzymatic acylation of 2 ACS Paragon Plus Environment

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DMY13.

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Lipase-catalyzed synthesis of DMY fatty acid esters has been carried out in

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traditional organic solvents including acetonitrile, acetone, tetrahydrofuran (THF),

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t-butanol, dimethyl sulfoxide (DMSO)14. DMSO is a polar solvent that has the ability

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to dissolve both polar and nonpolar compounds. Therefore, it is usually used as the

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solvent or co-solvent for enzymatic synthesis of biodiesel and ester derivatives of

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polar compounds15. Deep eutectic solvents (DESs) are considered as green solvents9

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and catalysts16-17. DESs can be prepared in high purity from low-cost starting

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materials, typically by mixing choline chloride (ChCl) with an hydrogen donor, e.g.

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an amine, amide, alcohol, or carboxylic acid18. To date, some investigations on

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enzyme-catalyzed biotransformations in DES-containing systems have been reported,

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including lipase-catalyzed transesterification19 and alcoholysis9, protease-catalyzed

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peptide synthesis20-21, epoxide hydrolase-catalyzed asymmetric hydrolysis of

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1,2-epoxyoctane22. Due to the non-toxicity, biodegradability and low-cost of DES, it

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is of great interest to investigate the lipase-catalyzed acylation of DMY by using

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DESs as alternative solvents or co-solvents 19, 23.

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In this work, we for the first time reported a new and green co-solvent system

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consisting of DMSO and DES (choline chloride: glycerol) as the reaction medium for

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the efficient enzymatic acylation of DMY with Aspergillus niger lipase (ANL). In this

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novel co-solvent system, not only DMY had moderate solubility, but also the

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immobilized ANL showed much higher catalytic activity than that in either DMSO or

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DES solvent alone. Besides, the DMY ester product has better lipid solubility and

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excellent antioxidant activity. Therefore, the newly developed biocatalytic system

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with the immobilized ANL and the DES-based co-solvent medium is very promising

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for efficient enzymatic acylation of DMY.

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EXPERIMENTAL SECTION

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Materials & Methods

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Dopamine hydrochloride was purchased from Aladdin (Shanghai, China). Ferric

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chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O)

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were obtained from Guangzhou Chemical Reagent Co. Ltd. Lipase from Aspergillus

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niger (2.11 U/g) was from Shenzhen Leveking Bioengineering Co. Ltd., (Shenzhen,

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China).Vinyl esters used as acyl donors (vinyl acetate, vinyl propionate, vinyl butyrate,

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vinyl caprylate, vinyl benzoate, vinyl 10-undecenoate, vinyl laurate and vinyl stearate)

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were purchased from Sigma-Aldrich and TCI Co. Ltd. (Shanghai, China).

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Dihydromyricetin (>99%) was purchased from Aladdin (Shanghai, China). Other

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chemicals, purchased from Guangzhou Chemical Reagent Co. Ltd, were of analytical

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grade unless stated otherwise.

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Immobilization of Aspergillus niger lipase

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The ANL (Aspergillus niger lipase) was immobilized according to our previous

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study24. First, the magnetic nanoparticles (MNPs) were prepared according to the

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conventional co-precipitation method with some modifications24. 15 mL of Dopamine

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hydrochloride (2.5 mg/mL) was added to the MNP suspension. The pH of the mixture

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was adjusted to 8.5 by the addition of NaOH solution (0.1mol/L). After vigorous

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stirring for 1 h, the polydopamine-coated magnetic nanoparticles (PD-MNPs) were

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separated by an external magnet and washed three times with deionized water. In

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order to immobilize ANL, an aqueous solution of ANL (1.5 mg/mL) was prepared

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first by dissolving the ANL powder in sodium phosphate solution (50 mmol/L,

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pH=8.0). Then the freshly prepared PD-MNPs solution was added to the ANL

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solution at 4°C in an ice bath. After stirring at 100 rpm for 12 h, the immobilized ANL

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(ANL@PD-MNPs) was washed with deionized water and then collected.

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Synthesis of DESs

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In this work, three kinds of DESs based on choline chloride were used as the

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co-solvents in enzymatic acylation of DMY. And three different hydrogen-bond

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donors (HBDs), such as glycerol, urea and xylitol, were selected for the synthesis of

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these DESs. A 150-mL jacketed glass vessel and a magnetic stirrer were employed to

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mix choline chloride with different HBDs in different molar ratios at 80 °C and 300

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rpm until a homogeneous and colorless liquid formed23. The above-mentioned

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procedure was performed under nitrogen atmosphere.

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Enzyme activity assay

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Lipase activity was assayed as described as followed6: Briefly, the enzyme was

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added into a solution containing 0.6 mL phosphate buffer (50 mM, pH 8.0) and 0.1

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mL p-nitrophenyl acetate (80 mM) in 2-propanol. The reaction was carried out at

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40 °C and 200 rpm for 5 min, and then stopped by adding 5.3 mL ethanol. The

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mixture was centrifuged at 12,000 g for 5 min (4 °C) and the absorbance of the

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supernatant at 405 nm was measured. The control experiments were carried out to

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determine the spontaneous hydrolysis of p-nitrophenyl acetate under the

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above-mentioned conditions without enzyme 6.

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Enzymatic acylation of DMY

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In a typical experiment, 60 U of the ANL@PD-MNPs was mixed with 2 mL pure

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DES or DMSO-DES co-solvent system. Then, DMY (20 mM) and vinyl acetate (200

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mM) were added to the above reaction system. The enzymatic acylation of DMY was

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performed at 40 oC in a shaking water bath (200 rpm). Samples (20 µL) were

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withdrawn at specified time intervals from the reaction mixture and diluted 20-fold

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with the methanol solution mobile phase of HPLC prior to HPLC analysis. In order to

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investigate the effect of the molar ratio of vinyl acetate to DMY, the enzymatic

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reaction was conducted as follows: 60 U of the ANL@PD-MNPs was mixed with 2

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mL Ch-Gly/DMSO (1/3, v/v) co-solvent system. Subsequently, DMY (20 mM) and

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vinyl acetate (50-500 mM) were added, and the reaction was performed at 40 oC in a

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shaking water bath (200 rpm). For the effect of the enzyme amount, the enzymatic

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reaction was conducted as follows: 20-70 U of the ANL@PD-MNPs was mixed with

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2 mL Ch-Gly/DMSO (1/3, v/v) co-solvent system. Then, DMY (20 mM) and vinyl

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acetate (200 mM) were added, and the reaction was carried out at 40 oC in a shaking

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water bath (200 rpm). Samples (20 µL) were withdrawn and determined by HPLC

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according to the above description.

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RESULTS AND DISSCUSSION

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The enzymatic acylation of DMY in the ChCl:Glycerol (1:2)(Ch-Gly) and DMSO

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co-solvent system

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In the present study, the recovery of lipase activity after immobilization was

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around 83.6% and the enzyme protein loading of the ANL@PD-MNPs was about 138

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mg protein /g PD-MNPs carrier. The ANL@PD-MNPs exhibit spherical morphology

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with the diameter < 30 nm (Figure S1).

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Reaction media plays an important role in enzymatic reaction. For the enzymatic

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acylation of DMY, the key is to find a suitable reaction medium that provides

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appropriate solubility for both the polar DMY and the non-polar acyl donors at the

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same time. The solubility of DMY in Ch-Gly, Ch-Xylitol and Ch-Urea were 418, 180

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and 102 mM (Table S1), respectively. The solubility of DMY in Ch-Gly was

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1.73-fold higher than that in DMSO media (242 mM)4. However, the DMY could not

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be acylated with vinyl acetate in 100% DESs media. This might be attributed the fact

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that the non-polar acyl donors vinyl acetate cannot dissolve in the DES media.

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Moreover, DMSO was a suitable solvent for vinyl acetate. According to the previous

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research, co-solvent reaction medium consisting of two solvents with different

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polarity can improve the initial and conversion rate of the enzymatic acylation25-26.

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Thus, the ANL@PD-MNPs catalytic acylation of DMY in Ch-Gly / DMSO

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co-solvent with different Ch-Gly volume fraction were performed (Table S2). When

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the Ch-Gly volume ratio decreased from 3/1 to 1/3, the initial reaction rate (V0)

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increased from 1.9 to 11.2 mM/h and conversion rate of DMY-16-acetate (CDMY-A)

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increased from 18.8% to 91.3%. The optimal Ch-Gly / DMSO co-solvent was consist

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of Ch-Gly/DMSO (1/3, v/v) co-solvent system. This result was superior to the

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enzymatic reaction by using DMSO as reaction medium (7.5 mM/h and 86.2%),

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according to our previous literature4.

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Moreover, the ANL@PD-MNPs catalytic DMY acetylation reaction with higher

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concentration of DMY (300 mM) in Ch-Gly/DMSO (1/3, v/v) co-solvent system. As

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shown in Table S3, in the Ch-Gly/DMSO (1/3, v/v) co-solvent system, the V0 and

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CDMY-A was 55.2 mM/h and 85.3%, respectively, which were significantly higher than

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that in DMSO (34.9 mM/h and 78.8%). These results might be attributing to the high

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solubility of DMY in the mixed solvent.

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In order to investigate the effect of substrate molar ratio between vinyl acetate

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and DMY on ANL@PD-MNPs catalytic DMY acetylation reaction in Ch-Gly/DMSO

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(1/3, v/v) co-solvent system, the concentration of DMY was fixed of 20 mM and the

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concentration of vinyl acetate changed from 50 to 500 mM. As shown in Figure 1, the

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substrate molar ratio had a significant impact on the initial reaction rate and

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conversion. The initial reaction rate and conversion rate increased with the increasing

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substrate molar ratio. When it reached 10:1 (vinyl acetate: DMY), the conversion rate

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and initial reaction rate were optimal, 91.3% and 11.2 mM/h respectively.

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The effect of the amount of ANL@PD-MNPs on the ANL@PD-MNPs catalytic

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DMY acetylation reaction was also investigated. The results were shown in Figure 2,

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when the enzyme amount was less than 50 U, the initial reaction rate and conversion

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rate increased rapidly with the increasing amount of enzyme. For example, when the

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amount of enzyme was 20 U, the initial reaction rate was 4.8 mM/h and the 8 ACS Paragon Plus Environment

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conversion rate was 70.6%. As the amount enzyme increased to 50 U, the initial

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reaction rate was 11.1 mM/h and the conversion rate was 91.6% respectively. When

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the amount of enzyme was more than 50 U, the conversion rate remained the same,

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the initial reaction rate increased slightly. According to our previous study24, the

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conversion of the lipase-catalyzed regioselective acylation of DMY was about 79.3%

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in DMSO. In contrast, a remarkable enhancement in the conversion was observed in

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the DES/DMSO co-solvent system (91.6%). This demonstrated that the addition of

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DES into DMSO could significantly improve the enzymatic regioselective acylation

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of DMY, which was attributable to the following reasons: (1) both the substrate DMY

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and the examined acylating reagents could be well dissolved in the DES-based

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co-solvent system; (2) the DES showed good biocompatibility with the enzyme.

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The operational stability (recycle-ability) of ANL@PD-MNPs was studied in the

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Ch-Gly/DMSO (1/3, v/v) co-solvent system. As shown in Figure 3, the immobilized

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lipase ANL@PD-MNPs retained more than 90% of relative activity after successive

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reuse of 5 cycles, exhibiting excellent recycle-ability. Moreover, ANL@PD-MNPs

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still kept above 56.7% of relative activity even after being repeatedly used for 10

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cycles. Certainly, the partial leakage of lipase from the support materials was

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observed with increasing batch of ANL@PD-MNPs re-use from 5 to 10 cycles,

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resulting in the gradual decrease of the relative activity. Also, the thermal stability of

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ANL@PD-MNPs was investigated by incubating ANL@PD-MNPs in the

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Ch-Gly/DMSO (1/3, v/v) co-solvent system at the operational temperature (40 oC) for

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24 hour, and it was found that ANL@PD-MNPs maintained more than 98% of its

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initial activity, indicating the relative good thermal stability under the operational 9 ACS Paragon Plus Environment

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conditions. Accordingly, the ANL@PD-MNPs-catalyzed acetylation of DMY has a

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good prospect of industrial application.

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The lipid-solubility determination of DMY and DMY-16-acetate

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The solubility of DMY and acetylated products in the oil phase was shown in

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Table S4. The lipid-solubility of DMY-16-acetate was 0.635g/100g oil, 10 times

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higher than the solubility of DMY (0.067 g/100g oil).

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The antioxidant ability of DMY and DMY-16-acetate

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As shown in Figure 4, the effect of DMY and DMY-16-acetate DPPH free

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radicals clearance was presented. It showed that DPPH radical scavenging rate of both

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the DMY and DMY-16-acetate increased constantly during the concentration range of

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0.25-9 mg/mL. When the concentration of standard control Vc was 4.00 mg/mL,

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DPPH free radical clearance reached 100%. In terms of IC50 (the concentration of

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antioxidants when DPPH clearance rate was 50%), DMY and DMY-16-acetate were

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3.71 mg/mL and 4.16 mg/mL, respectively. Both of them were higher than the IC50 of

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standard control Vc, 0.51 mg/mL. It is interesting to note that after DMY acetylation,

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its ability to remove DPPH free radical was improved.

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Hydroxyl radical has high electronegativity and strong oxidizing ability27. With

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phenolic hydroxyl group, a strong electron donating group, DMY and their acetylated

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products can reduce hydroxyl radicals. As shown in Figure 5, with the increase of

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DMY and DMY-6-acetate concentration, the clearance rate of hydroxyl radical

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increased accordingly. When the DMY and DMY-16-acetate concentration reached 10 ACS Paragon Plus Environment

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2.7 mg/mL, hydroxyl radical clearance rate was 94.6% and 98.1%, respectively.

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Comparing with DMY and DMY-16-acetate, the effect of the Vc on the hydroxyl

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radicals clearance has a different pattern. When the concentration of Vc was less than

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1.5 mg/mL, the hydroxyl radical clearance rate increased rapidly. When the

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concentration of Vc reached 1.5 mg/mL, all of the hydroxyl radicals were basically

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eliminated. The IC50 of Vc, DMY and DMY-16-acetate were 0.68 mg/mL, 1.73

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mg/mL and 1.79 mg/mL respectively. This result showed that the hydroxyl radical

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clearance ability of the product DMY-16-acetate remained stable after the acetylation

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of DMY. What’s more, DMY and DMY-16-acetate offered better efficiency in

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removing hydroxyl radicals than DPPH free radical.

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Fentons reagent, consisting of Fe2+ and H2O2, can oxidize organic compounds to

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inorganic compounds28. Therefore, it is helpful to reduce the concentration of Fe2+ in

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Fenton reaction in order to limit the oxidative damage in the human body. Figure 6

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illustrated the Fe2+-chelating ability of the substrate DMY and the product

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DMY-16-acetate, with EDTA as the control. As the concentration of EDTA, DMY

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and DMY-16-acetate increased, their Fe2+-chelating ability increased accordingly.

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The IC50 of EDTA, DMY and DMY-16-acetate Fe2+ chelating were 0.18 mg/mL, 0.10

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mg/mL and 0.13 mg/mL respectively. It showed that DMY and DMY-16-acetate

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exhibited better Fe2+-chelating ability than EDTA. The lower chelating ability of

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DMY-16-acetate may be attributed to the reduction of its hydroxyl groups after

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acetylation 29.

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In this paper, we have demonstrated that the co-solvent mixture of Ch-Gly DES

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and DMSO is much more suitable for the enzymatic acylation of DMY than the

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traditional organic solvents. Moreover, this novel co-solvent system can also be used

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for improving enzymatic synthesis of ester derivatives of polar polyhydroxylated

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compounds except DMY esters. In the co-solvent, DMSO played an important role in

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the enhancement of enzymatic acylation by increasing the affinity between enzyme

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and substrates. Moreover, the Ch-Gly DES, mainly promoted the dissolution of the

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polar substrate and prevented the inactivation of the enzyme in high concentration of

245

strong-polar organic solution. This co-solvent mixture is apparently superior to the

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recently reported pure organic reaction media for the enzymatic synthesis of DMY

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esters, since the use of DMSO usually leads to significant inactivation of the enzyme

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as well as tedious work-up procedures in the downstream purification. The

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DMY-16-acetate product exhibits good oxidation resistance, the IC50 of DPPH

250

scavenging, hydroxyl radical scavenging and chelating Fe2+ was 4.16 mg/mL, 1.79

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mg/mL and 0.13 mg/mL, respectively. Given the above advantages, the novel Ch-Gly

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DES and DMSO co-solvent developed in this work may find promising application in

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enzymatic catalysis.

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ASSOCIATED CONTENT

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ABBREVIATIONS USED

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ANL

Aspergillus niger lipase

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ANL@PD-MNPs

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ChCl

immobilized Aspergillus niger lipase

choline chloride 12 ACS Paragon Plus Environment

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DESs

deep eutectic solvents

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DMSO

dimethyl sulfoxide

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DMY

dihydromyricetin

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Gly

glycerol

263

HDBs

hydrogen-bond donors

264

PD-MNPs

265

THF

266

polydopamine-coated magnetic nanoparticles

tetrahydrofuran

ACKNOWLEDGEMENT

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We wish to thank the Program of State Key Laboratory of Pulp and Paper

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Engineering (2017ZD05), the National Natural Science Foundation of China

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(21676104; 21336002; 21376096), the Key Program of Guangdong Natural Science

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Foundation (S2013020013049), and the Open Funding Project of the State Key

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Laboratory of Bioreactor Engineering for partially funding this work. We also thank

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SCUT Doctoral Student Short-Term Overseas Visiting Study Funding Project.

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SUPPORTING INFORMATION DESCRIPTION

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The method: HPLC analysis of DMY and DMY-16-acetate; Larger-scale

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synthesis of DMY esters for the purification and structure determination of DMY

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esters; The lipid-solubilities and antioxidant abilities of DMY and DMY-16-acetate.

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Supplementary Tables: Effect of different DESs on solubility of DMY and the

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conversion of the ANL@PD-MNPs-catalytic acylation of DMY (Table S1). Effect of

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different Ch-Gly/DMSO volume ratio in Ch-Gly/DMSO cosolvent on acetylation of

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DMY by ANL@PD-MNPs(Table S2). Enzymatic acetylation of DMY by

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ANL@PD-MNPs at DMY concentration of 300 mM (Table S3). Solubility of DMY

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and DMY-16-acetate in oil (Table S4).

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Supplementary Figure: Scanning electron micrograph of the immobilized lipase (Figure S1).

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REFERENCE

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Shen Y., Lindemeyer A.K., Gonzalez C., Shao X.M., Spigelman I., Olsen R.W., Liang J. Dihydromyricetin as a Novel Anti-Alcohol Intoxication Medication. J. Neurosci., 2012, 32: 390-401 Liu J., Shu Y., Zhang Q.Y., Liu B., Xia J., Qiu M.N., Miao H.L., Li M.Y., Zhu R.Z. Dihydromyricetin Induces Apoptosis and Inhibits Proliferation in Hepatocellular Carcinoma Cells. Oncol. Lett., 2014, 8: 1645-1651 Hou X.L., Tong Q., Wang W.Q., Shi C.Y., Xiong W., Chen J., Liu X., Fang J.G. Suppression of Inflammatory Responses by Dihydromyricetin, a Flavonoid from Ampelopsis Grossedentata, Via Inhibiting the Activation of Nf-Kappa B and Mapk Signaling Pathways. J. Nat. Prod., 2015, 78: 1689-1696 Li W., Wu H., Liu B., Hou X., Wan D., Lou W., Zhao J. Highly Efficient and Regioselective Synthesis of Dihydromyricetin Esters by Immobilized Lipase. J. Biotechnol., 2015, 199: 31-37 Matsumoto T., Tahara S. Ampelopsin, a Major Antifungal Constituent from Salix Sachalinensis, and Its Methyl Ethers. J. Agr. Chem. Soc. Japan, 2001, 75: 659-667 Cao S.L., Huang Y.M., Li X.H., Xu P., Wu H., Li N., Lou W.-Y., Zong M.-H. Preparation and Characterization of Immobilized Lipase from Pseudomonas Cepacia onto Magnetic Cellulose Nanocrystals. Sci. Rep., 2016, 6: 20420 Svedendahl M., Carlqvist P., Branneby C., Allnér O., Frise A., Hult K., Berglund P., Brinck T. Direct Epoxidation in Candida Antarctica Lipase B Studied by Experiment and Theory. ChemBioChem, 2008, 9: 2443-2451 Branneby C., Carlqvist P., Hult K., Brinck T., Berglund P. Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations. J. Molecul. Catal. B: Enzym., 2004, 31: 123-128 Durand E., Lecomte J., Baréa B., Piombo G., Dubreucq E., Villeneuve P. Evaluation of Deep Eutectic Solvents as New Media for Candida Antarctica B Lipase Catalyzed Reactions. Process Biochem., 2012, 47: 2081-2089 Cao S.L., Li X.H., Lou W.Y., Zong M.H. Preparation of a Novel Magnetic Cellulose Nanocrystal and Its Efficient Use for Enzyme Immobilization. J. Mater. Chem. B., 2014, 2: 5522-5530 Cao S.L., Xu P., Ma Y.Z., Yao X.X., Yao Y., Zong M.H., Li X.H., Lou W.Y. Recent Advances in Immobilized Enzymes on Nanocarriers. Chinese J. Catal., 2016, 37: 1814-1823 Yu C.Y., Li X.F., Lou W.Y., Zong M.H. Cross-Linked Enzyme Aggregates of Mung Bean Epoxide Hydrolases: A Highly Active, Stable and Recyclable Biocatalyst for Asymmetric Hydrolysis of Epoxides. J. Biotechnol., 2013, 166: 12-19 Puri M., Barrow C.J., Verma M.L. Enzyme Immobilization on Nanomaterials for Biofuel Production. Trends Biotechnol., 2013, 31: 215-216 Sheldon R.A. Enzyme Immobilization: The Quest for Optimum Performance. Adv. Syn. Catal., 2007, 349: 1289-1307 Ge J., Lu D.A., Wang J., Liu Z. Lipase Nanogel Catalyzed Transesterification in Anhydrous Dimethyl Sulfoxide. Biomacromolecules, 2009, 10: 1612-1618 Tran P.H., Nguyen H.T., Hansen P.E., Le T.N. An Efficient and Green Method for Regioand Chemo-Selective Friedel-Crafts Acylations Using a Deep Eutectic Solvent ([Cholinecl][Zncl2]3). RSC Adv., 2016, 6: 37031-37038 Nguyen H.T., Tran P.H. An Extremely Efficient and Green Method for the Acylation of Secondary Alcohols, Phenols and Naphthols with a Deep Eutectic Solvent as the Catalyst. RSC Adv., 2016, 6: 98365-98368 Smith E.L., Abbott A.P., Ryder K.S. Deep Eutectic Solvents (Dess) and Their Applications. Chem. Rev., 2014, 114: 11060-11082

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342 343

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363 364 365 366

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Gorke J.T., Srienc F., Kazlauskas R.J. Hydrolase-Catalyzed Biotransformations in Deep Eutectic Solvents. Chem. Comm., 2008, 1235-1237 Cao S.L., Xu H., Li X.H., Lou W.Y., Zong M.H. Papain@Magnetic Nanocrystalline Cellulose Nanobiocatalyst: A Highly Efficient Biocatalyst for Dipeptide Biosynthesis in Deep Eutectic Solvents. ACS Sus. Chem. Eng., 2015, 3: 1589-1599 Maugeri Z., Leitner W., de Maria P.D. Chymotrypsin-Catalyzed Peptide Synthesis in Deep Eutectic Solvents. Eur. J. Org. Chem., 2013, 2013: 4223-4228 Cao S.L., Yue D.M., Li X., Smith T.J., Li N., Zong M.H., Wu H., Ma Y., Lou W.Y. Novel Nano/Micro-Biocatalyst: Soybean Epoxide Hydrolase Immobilized on Uio-66-Nh2 Mof for Efficient Biosynthesis of Enantipure (R)-1, 2-Octanediol in Deep Eutectic Solvents. ACS Sus. Chem. Eng., 2016, 4: 3586-3595 Zhao B.Y., Xu P., Yang F.X., Wu H., Zong M.H., Lou W.Y. Biocompatible Deep Eutectic Solvents Based on Choline Chloride: Characterization and Application to the Extraction of Rutin from Sophora Japonica. ACS Sus. Chem. Eng. 2015, 3: 2746-2755 Deng X., Cao S.-L., Li N., Wu H., Smith T.J., Zong M.-H., Lou W.-Y. A Magnetic Biocatalyst Based on Mussel-Inspired Polydopamine and Its Acylation of Dihydromyricetin. Chinese J. Catal., 2016, 37: 1-2 Ferrer M., Cruces M.A., Bernabe M., Ballesteros A., Plou F.J. Lipase-Catalyzed Regioselective Acylation of Sucrose in Two-Solvent Mixtures. Biotechnol. Bioeng., 1999, 65: 10-16 Adnani A., Basri M., Malek E.A., Salleh A., Rahman M.B.A., Chaibakhsh N., Rahman R. Optimization of Lipase-Catalyzed Synthesis of Xylitol Ester by Taguchi Robust Design Method. Ind. Crop. Prod., 2010, 31: 350-356 Zeng Q.H., Zhang X.W., Xu X.L., Jiang M.H., Xu K.P., Piao J.H., Zhu L., Chen J., Jiang J.G. Antioxidant and Anticomplement Functions of Flavonoids Extracted from Penthorum Chinense Pursh. Food Func., 2013, 4: 1811-1818 Jia S.P., Liang M.M., Guo L.H. Photoelectrochemical Detection of Oxidative DNA Damage Induced by Fenton Reaction with Low Concentration and DNA-Associated Fe2+. J. Phys. Chem. B, 2008, 112: 4461-4464 Klibanov A.M. Why Are Enzymes Less Active in Organic Solvents Than in Water? Trends Biotechnol., 1997, 15: 97-101

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Figure 1 Effect of substrate molar ratio on acetylation of DMY by ANL@PD-MNPs.

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Figure 2 Effect of enzyme dosage on ANL@PD-MNPs-catalyzed acetylation of

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DMY.

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Figure 3 Recycling ability of ANL@PD-MNPs in the Ch-Gly/DMSO (1/3, v/v)

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co-solvent system.

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Figure 4 Effect of antioxidant concentration on DPPH radical scavenging rate.

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Figure 5 Effect of antioxidant concentration on hydroxyl radical scavenging rate.

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Figure 6 Effects of antioxidant concentration on iron-chelating rate.

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Figure3

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Iron-chelating rate (%)

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