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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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Journal of Agricultural and Food Chemistry

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%

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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

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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

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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

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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

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HDBs

hydrogen-bond donors

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PD-MNPs

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THF

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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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Figure Captions

375

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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

378

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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100

12

90 80

10

70

8

60 50

6

Initial reaction rate Yield

4 2

40 30 20

0

5

10

15

20

VA/DMY (mol/mol) Figure 1


25

Conversion(%)

Initial reaction rate (mM/h)

14


100 90

12

80

10

70 60

8

50

6

Initial reaction rate

4 2

Yield

40 30 20

20

30

40

50

60

Enzyme dosage (U) Figure 2


70

Conversion(%)

Initial reaction rate (mM/h)

14

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Relative activity (%)

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100 90 80 70 60 50 40 30 20 10 0

1

2

3

4

5 6 Batch

7

8

Figure3


9

10

DPPH radical scavenging rate (%)


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100 80 60 40 Vitamin C DMY DMY-16-acetate

20 0 0

2

4

6

8

Antioxidant concentration (mg/mL)

Figure4


10


Hydroxyl radical scavenging rate (%)

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100 80 60 40 Vitamin C DMY DMY-16-acetate

20 0 0.0

0.5

1.0

1.5

2.0

2.5


Figure 5


3.0


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Iron-chelating rate (%)

100 80 60 40 EDTA DMY DMY-16-acetate

20 0 0.0

0.1

0.2

0.3

0.4

0.5


Figure 6


0.6

Page 25 of 25


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