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Valorizing dairy waste: thermophilic biosynthesis of a novel ascorbic acid derivative Jingwen Yang, Bianca Perez, Sampson Anankanbil, Jingbo Li, Ye Zhou, Renjun Gao, and Zheng Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03173 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Valorizing dairy waste: thermophilic biosynthesis of a novel ascorbic acid derivative

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Jingwen Yanga,b,c, Bianca Pérezb, Sampson Anankanbilb, Jingbo Lib, Ye Zhoua,b, Renjun Gao*a, Zheng Guo*b

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a

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Science, Jilin University, Changchun 130012, China;

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b

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c

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*Corresponding Authors: Zheng Guo; email: [email protected] ; Renjun Gao; email: [email protected]

Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life

Department of Engineering, Aarhus University, Gustav Wieds Vej 10, Aarhus 8000, Denmark;

School of Biological and Medical Engineering, Hefei University of Technology, Anhui 230009, China.

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ABSTRACT

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L-ascorbic acid (L-AA) is an essential nutrient that is extremely instable and cannot be synthesized

15

by the human body. Therefore, attempts have been done to develop biological active L-AA

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derivatives with improved stability. This work presents a facile, scalable and efficient enzymatic

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transgalactosylation of lactose to L-AA using β-glucosidase (TN0602) from Thermotoga

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naphthophila RKU-10. β-Glucosidase TN0602 displayshigh transgalactosylation activity at pH 5.0,

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75°C and L-AA/lactose ratio 2/1, to form a novel L-AA derivative (2-O-β-D-Galactopyranosyl L-

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Ascorbic Acid, L-AA-Gal) with a maximal productivity of 138.88mmol L-1 in 12h, which is higher

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than most reports of enzymatic synthesis of L-AA-α-glucoside. Synthetic L-AA-Gal retains most of

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L-AA antioxidant capability and presents dramatically higher stability than L-AA in oxidative

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environment(Cu2+). In conclusion, this work report a new way to valorize dairy waste lactose into a

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novel molecule L-AA-Gal, which could be a promising L-AA derivative to be used in a wide range

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of applications.

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KEYWORDS: L-ascorbic acid; transgalactosylation; β-glucosidase; Thermotoga naphthophila; 2-

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O-β-D-Galactopyranosyl L-Ascorbic Acid (L-AA-Gal); oxidative stability.

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INTRODUCTION

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L-ascorbic acid (L-AA) promotes cell growth through an activation of collagen synthesis1-2. Since

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L-AA cannot be synthesized by the human body, it is widely used as a nutrient in the

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pharmaceutical and cosmetic industries, as well as a preservative, by virtue of its excellent

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antioxidant activity3. However, L-AA is easily degraded and its instability greatly limits its

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application3. Therefore, the generation of new biological active L-AA derivatives with increased

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stability has been a research challenge for the scientific community4.

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Modification at 2-OH of L-AA to yield 2-O-α-D-glucopyranosyl L-ascorbic acid (L-AA-Glu) has

38

proved to stabilized L-AA5. L-AA-Glu presents a high degree of non-reducibility, excellent

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antioxidant ability, and ready release of L-AA and glucose. Thus L-AA-Glu has shown great

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potential for industrial application5. L-AA-Glu is commonly produced by transglycosylation of

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cyclodextrin glycosyltransferasein the presence of L-AA and cyclodextrin6. So far, five enzymes

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have been used for L-AA-Glu synthesis (See Table S1), namely α-glucosidase7, cyclodextrin

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glycosyltransferase3, 6, amylase8, sucrose phosphorylase9, and α-isomaltosyl glucosaccharide-

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forming enzyme10. Among them, cyclodextrin glycosytransferase can achieve the highest yields

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(38.46-423.08 mmol L-1 vs 14.66 mmol L-1, Table S1). However, many intermediate products, such

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as L-AA-Glun (n means the number of glycosyls attached to L-AA), are produced along with L-AA-

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Glu, and thus, further treatment with glucoamylase is necessary3. Moreover, the high cost and low

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solubility of cyclodextrin make this synthetic pathway not suitable for large-scale production of L-

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AA-Glu4, 11. Therefore, the selection of an easy and inexpensive method for L-AA derivatives is

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also important.

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Lactose, a naturally occurring sugar found mainly in milk, is produced in large amounts as a by-

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product from the dairy industry12; about 150–200 million tons of lactose are generated each year


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from liquid whey13. Recycling lactose to produce some sugar conjugate can make the lactose

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reclaimed, and hence reduce food waste. In view of the above, the enzymatic transgalactosylation

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with lactose as a donor and L-AA as an acceptor might be of great interest for industrial application.

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Therefore, herein we report the enzymatic transgalactosylation from lactose and L-AA using β-

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glucosidase (TN0602) from Thermotoga naphthophila RKU-10 to yield a very stable L-AA

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derivative. Moreover, DPPH free radical scavenging activity of L-AA-Gal was measured to

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evaluate the antioxidant efficiency of the compound in question. Furthermore, the stability in

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oxidative environment of L-AA and L-AA-Gal were also investigated with Cu2+ ion.

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MATERIALS AND METHODS.

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Materials. All the reagents were purchased from Sigma-Aldrich (St.Louis, MO, USA).T4PNK

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phosphorylase, and protein molecular weight standards were purchased from Takara Biotechnology

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Co., Ltd. (Dalian, China). DNA polymerase and protein molecular weight standards (Blue Plus

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Protein Marker and Blue Plus II Protein Marker) were supplied by Beijing TransGen Biotech Co.,

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Ltd. (Beijing, China). AxyPrep DNA gel extraction kit was purchased from Oxygen Biotechnology

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Ltd. (Hangzhou, China). Silica gel plates were purchased from Merck Ltd. (Darmstadt, Germany).

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Preparation of β-glucosidase. The protocol applied to produce β-glucosidase from Thermotoga

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naphthophila RUK-10 was previously described14. Briefly, the gene TN0602 encoding β-

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glucosidase (1341 bp) form Thermotoga naphthophila RUK-10 was amplified by PCR with

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Easytaq DNA polymerase. Based on the DNA sequence of the β-glucosidase form Thermotoga

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naphthophila reported in GeneBank (accession number CP001839.1), two oligonucleotides, 5’-

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forward

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CTGACACATATGAACGTGAAAAAGTTCCCT-3’) and 3’ reverse primer with an EcorRI site

primer

containing

a

restriction

site


for

NdeI

(5’-


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(5’-CTGCACGAATTCTTAATCTTCCAGACTG-3’), were designed, respectively. The amplified

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DNA fragment was purified by virtue of AxyPrep DNA gel extraction kit and digested with both

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NdeI and EcorRI endonucleases. The digested DNA fragment was purified and inserted into the

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pET-28b (+) plasmid digested with the same restriction enzymes by means of T4 DNA ligase kit.

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Escherichia coli strain BL21 (DE3) was transformed with the ligation mixture and plated on Luria-

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bertani (LB) agar containing 10 g L-1 kanamycin. The E. coli BL21 (DE3) cells were pre-incubated

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in LB medium (with 10 g L-1 kanamycin) with 180 rpm agitation at 37°C. After the OD600 of the

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culture liquid reached 1.0, IPTG was added to induce enzyme expression, and the cells were grown

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at 25°C for 12 h with 150 rpm agitation. The induced cells were harvested by centrifugation and

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washed with 50 mmol L-1 sodium phosphate buffer (pH 7.0) and disrupted by sonication. The

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cellular debris was removed by centrifugation (15,000 rpm for 15 min at 4°C) to obtain a crude

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extract. The crude extract was incubated in a water bath for 10 min at 80°C to denature the E. coli

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proteins. The extract was then centrifuged to separate the crude enzyme from the heat-denatured

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cellular components and proteins. The supernatant was loaded onto a Ni2+-NTA agarose resin

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column. The enzyme was eluted with a linear gradient of 0-200 mmol L-1 imidazole in sodium

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phosphate buffer. The purity was determined by 12% sodium dodecyl sulcate-polyacrylamide gel

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electrophoresis. Finally, the pure protein was lyophilised for subsequent experiments.

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Enzyme assay. The hydrolytic activity of β-galactosidase (TN0602) was determined by using 5%

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(w/w) lactose in 50 mmol L-1 sodium phosphate buffer (pH 7.0) as substrate. The hydrolytic

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reaction was started by adding the enzyme (1 g L-1) and letting the reaction run at 75°C with

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agitation (250 rpm) for 30 min.

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Analysis of the samples was carried out by high performance liquid chromatography (HPLC, find

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the conditions in the section of “Synthesis and analysis of 2-O-β-D-Galactopyranosyl L-Ascorbic


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Acid”.) and hydrolytic activity was calculated on the basis of galactose concentration. One unit of

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enzymatic activity was defined as the amount of enzyme required to liberate 1 µmol of galactose

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per minute under the experimental conditions14.

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Synthesis and analysis of 2-O-β-D-Galactopyranosyl L-Ascorbic Acid. The original reaction

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mixture contained L-AA (galactosyl acceptor), lactose (galactosyl donor), and TN0602. The pH (5-

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10) was adjusted using sodium carbonate or sodium citrate. The reaction mixture was incubated at

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50-75°C for 24 h in the dark.

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The reaction mixture was preliminarily analyzed using thin-layer chromatography (TLC) with n-

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butanol-acetic acid-water (3:1:1, v/v) as a developing solvent and ran for two times. The developed

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TLC plates were stained with α-naphthol (2.56 g L-1) in an ethanol-sulfuric acid mixture (90:10,

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v/v). All the carbohydrates were visualized by heating the plate at 100°C for 5 minutes.

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L-AA-Gal was measured by HPLC using a Luna 5u NH2 100A column (Phenomenex, Torrance,

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California, USA). Samples were filtered through a 0.45 µm membrane before injection. The assay

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conditions were a detection wavelength of 245 nm, mobile of 20 mmol L-1 NaH2PO4/ H3PO4 (pH

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4.0), and a flow rate of 1.0 mL min-1.

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Enzyme assay and quantitative analyses of trisaccharide, lactose, and galactose were determined by

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HPLC using a SUPELCOGELTM Ca column (Sigma-Aldrich, St. Louis, MO, USA) conjugated

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with an evaporative light scattering detector (ELSD). The mobile phase was deionized water and

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the flow rate was kept constant at 0.5 mL min-1. The column temperature was kept at 80°C.

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Structural identification of enzymatically synthetic 2-O-β-D-Galactopyranosyl L-Ascorbic

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Acid. L-AA-Gal from enzymatic reaction mixtures were separated and purified through NH2 TLC

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plates (Merck, Darmstadt, Germany) using chloroform-methanol-acetic acid-water (12:6:1:1, v/v),

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and the resulting products were subject to structural identification. The products were characterized



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through NMR and mass spectroscopy analysis. 1H NMR and 13C NMR were recorded on a Bruker

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AVANCE III 500 MHz and 100 MHz spectrometer using D2O as solvent, respectively. MS spectra

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were obtained on a Bruker Maxis Impact electrospray ionization quadrupole time-of-flight mass

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spectrometer (ESI-QTOF-MS).

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2-O-β-D-Galactopyranosyl L-Ascorbic Acid. Rf (n-butanol-acetic acid-water (3:1:1, v/v)): 0.19.1H

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NMR (400 MHz, D2O) δ/ppm 3.49–3.54(d, 1H), 3.67–3.70 (dd, 1H), 3.75–3.80 (m, 5H), 3.85–3.90

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(m, 1H), 3.99–4.02 (m, 1H), 4.09–4.13 (m, 1H), 5.57–5.58 (m, 1H);

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δ/ppm 172.4, 163.2, 117.5, 98.8, 76.5, 73.0, 72.5, 71.0, 68.9, 68.8, 62.0, 60.0; HR-MS: exact mass

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calculated for C12H18O11 [M-H]-: 337.0815 m/z, found 337.0678 m/z.

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Stability against oxidation and determination of radical scavenging activity. The purified L-

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AA-Gal was dissolved (10 mmol L-1) in the solution containing 10 µmol L-1 copper chlorideat room

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temperature15, L-AA was used as control. The decrease in absorbance was monitored with a UV

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Cary-50 spectrophotometer at 265nm.

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The reducing activities of the transfer products were determined using a stable radical, 1,1-

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diphenyl-2-picrylhydrazyl (DPPH)16. Different volumes (1-50 µL) of the L-AA and L-AA-Gal were

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added to 100 mmol L-1 DPPH solution in ethanol, and the absorbance at 517 nm was measured after

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30 min. As a control reaction, 100 mmol L-1 DPPH solution in ethanol without any sample was used

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instead. The difference in absorbance between the control and the sample was considered the

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radical scavenging activity of the sample.

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C NMR (100 MHz, D2O)

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RESULTS AND DISCUSSION

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Effect of pH regulators on the synthesis of L-AA-Gal. Transglycosylation with maltotriose or

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acarbose as a donor and L-AA as an acceptor in 25 mmol L-1 sodium citrate buffer at pH 6.0 has


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been previously reported8. The resulting product was a mixture of 6-α-glucosyl ascorbic acid and 6-

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α-maltosyl ascorbic acid, or 6-α-acarviosine-glucosyl ascorbic acid and 2-α-acarviosine-glucosyl

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ascorbic acid8. Sodium citrate was used as a conjugate base of a weak acid17, and as a pH regulator

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to adjust pH to 5.0 in the reaction system for L-AA-Glu synthesis6, 8. In L-AA, the ascorbate anion

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is stabilized by electron delocalization, in terms of resonance between two canonical forms. For this

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reason, L-AA is much more acidic than would be expected, consequently, the high concentration of

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sodium citrate that would be needed will decrease the enzyme activity and the solubility of

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substrate, leading to a low production of L-AA derivatives. Hence since sodium carbonateis a

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relatively strong base to adjust pH, the effect of both sodium carbonate and sodium citrate on the

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reaction kinetics of TN0602 for L-AA-Gal synthesis was evaluated from 50°C to 75°C. The

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reaction was carried out at a fixed ascorbic acid concentration, and sodium carbonate and sodium

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citrate were added directly to the reaction systems in solid forms, so that the volume of the reaction

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system did not change much. The amount of lactose was varied from 0.58 mol L-1 to 1.17 mol L-1

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using 1.14 mol L-1 L-AA and 20 g L-1 of TN0602 for 6 hours. The detailed kinetic parameters of the

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two pH regulator reaction systems are listed in Table 1. It could be seen that in all of the three

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temperatures (50, 70 and 75°C) the maximal reaction rate (Vmax) of sodium carbonate system is

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higher than that of sodium citrate system (0.37 mmol L-1 min-1 vs 0.055 mmol L-1 min-1; 0.46 mmol

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L-1 min-1 vs 0.11 mmol L-1 min-1; 0.61 mmol L-1 min-1 vs 0.49 mmol L-1 min-1; respectively).

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Meanwhile, the kcat values also perform the same trend as Vmax (0.039 s-1 vs 0.0058 s-1; 0.049 s-1 vs

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0.012 s-1; 0.065 s-1 vs 0.051 s-1, correspondingly). Moreover, the activation energy of sodium

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carbonate system (16.4 kJ mol-1) is much lower than that of sodium citrate system (39.01 kJ mol-1),

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suggesting that a much greater energy barrier needs to be overcome in the sodium citrate system. In

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addition, the Km of TN0602 decreased with the increase of temperature (50-75°C) in both two



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reaction systems (sodium carbonate: 0.36-0.27 mmol L-1; sodium citrate: 0.89-0.49 mmol L-1),

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indicating that the affinity of TN0602 to lactose is getting higher with the temperature (50-75°C).

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Furthermore, the kinetic results suggested that the affinity and catalytic efficiency of sodium

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carbonate system was higher compared to the sodium citrate system. This might be due to the

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stronger basicity of sodium carbonate compared to sodium citrate, thus the pH of the reaction

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system (0.2 g mL-1 L-AA) could be adjusted with a smaller amount of sodium carbonate (~0.06 g

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mL-1) than sodium citrate (~0.25 g mL-1). Therefore, sodium carbonate was selected as the pH

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regulator for further experiment.

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Effect of reaction time on the synthesis of L-AA-Gal. Since TN0602 can also catalyze other

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transgalactosylation reactions, (Scheme 1; e.g. transgalactosylation with lactose as both a galactosyl

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acceptor and a galactosyl donor) the effect of reaction time on the production of L-AA-Gal was also

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evaluated with 0.2 g mL-1 lactose (0.58 mol L-1) and 0.2 g mL-1 L-AA (1.14 mol L-1) at 75 °C and

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pH 5.0, 20 g L-1 of TN0602. As shown in Fig. 1a, in the first 5 hours of the reaction a rapid decrease

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in the lactose concentration is observed with a simultaneous increase in the production of L-AA-

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Gal, trisaccharide, glucose and galactose. A remarkable difference to produce L-AA-Gal and

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trisaccharide is that the L-AA-Gal concentration increased in a more gradual manner, leveling out

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until a maximum 84.89 mmol L-1 at a time when the enzymatic reactions has reached an

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equilibrium (6 h). This concentration is dramatically higher when compared to previous work where

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mutants of cyclodextrin glycosyltransferase from Paenibacillus macerans and glucoamylase and L-

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AA and maltodextrin were used for the synthesis L-AA-Gul, using and the highest titer of L-AA-

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Gul obtained was 1.92 g L-1 (~5.7 mmol L-1)18.

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Effect of reaction pH on the synthesis of L-AA-Gal. It is also known that enzymes are affected by

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changes in pH; i.e. pH can affect the ionization state of the enzyme residues19, the shape of the


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enzyme, and the substrate affinity. Hence to further optimize reaction conditions for the synthesis of

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L-AA-Gal, the effect of different pH (4.0-8.0) was investigated with 0.2 g mL-1 lactose (0.58 mol L-

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1

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demonstrated that TN0602 showed the highest transgalactosylation activity at pH 7.0 with lactose

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as the galactosyl acceptor14. Nevertheless, the highest L-AA-Gal production was observed in the pH

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range from 4 to 6 (pH 5 L-AA-Gal production=57.95 mmol L-1; Fig. 1b). However, comparing

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these results with the production of trisaccharide in the absence of L-ascorbic acid, it can be

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inferred that pH 4.0 could be considered as the optimum pH of TN0602 for the synthesis of L-AA-

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Gal since at this pH less trisaccharide is expected to form as by-product (pH 3.8, trisaccharide

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concentration=0, Fig. S1), and almost as much of L-AA-Gal as the main product (pH 4.0 L-AA-Gal

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concentration=53.51 mmol L-1, Fig. 1b). Moreover, using the pH below 6.0 can also decrease the

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side reaction (synthesis of trisaccharide), since the optimal pH range for the synthesis of

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trisaccharide in the absence of ascorbic acid was from 6.0 to 7.0 (Fig. S1). On the contrary at pH

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7.0, the concentration of L-AA-Gal significantly dropped (Fig. 1b), suggesting that the enzyme and

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L-AA were unstable at these pH values.

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Effect of enzyme concentration on the synthesis of L-AA-Gal. The rate of reaction also depends

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on enzyme concentration20. Thus the effect of increasing the enzyme concentration on the reaction

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rate was also evaluated. Different concentrations of TN0602 (10-25 g L-1) were respectively added

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to the mixture of L-AA (0.2 g mL-1, 1.14 mol L-1) and lactose (0.2 g mL-1, 1.14 mol L-1) for

210

incubation at 75°C and pH 5.0 for 24h. Results showed that L-AA-Gal production increased with

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the increase of TN0602 concentration up to 8h of reaction time, at which point the maximum L-

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AA-Gal production is reached. Accordingly, 20 g L-1 of enzyme was needed to yield the highest

) and 0.2 g mL-1 L-AA (1.14 mol L-1) at 75°C for 3 h, and 20 g L-1 of TN0602. Previous work



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concentration of L-AA-Gal (84.89 mmol L-1; (Fig. 1c).Thus, 20 g L-1 was selected for further

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understanding/optimization of the reaction system.

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Effect of reaction temperature on the synthesis of L-AA-Gal. Other parameter that can affect the

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reaction rate is the temperature; the lower the temperature of the reaction system, the lower kinetic

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energy. Accordingly, the effect of temperature (50-80°C) on the production of L-AA-Gal was

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evaluated using 0.2 g mL-1 lactose (0.58 mol L-1), 0.2 g mL-1 L-AA (1.14 mol L-1), and 20 g L-1 of

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TN0602 at pH 5.0. As seen in Fig 1d, the optimal temperature of the TN0602 for the synthesis of L-

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AA-Gal was 75°C (Concentration of L-AA-Gal=84.89 mmol L-1). At temperatures of 50°C up to

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75°C, L-AA-Gal formation was enhanced by approximately 40.5%. The latter agrees with the

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kinetic values (Vmax and kcat) obtained for the sodium carbonate reaction system when performing

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the reaction from 50°C to 70°C (Table S1). Overalls, TN0602 demonstrated to yield a high catalytic

224

activity at 70-80°C and short reaction time (6 h). Furthermore, 75°C is the highest temperature

225

reported so far for the enzymatic glucosylation of L-AA. The high reaction temperature can reduce

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the microbial contamination and the viscosity of the reaction system, and consequently increase the

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solubility and the diffusion coefficient of substrate21-22.

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Effect of substrate ratio on the synthesis of L-AA-Gal. In enzymatic transgalactosylations, the

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acceptor is commonly used in a suitable molar excess over the donor to suppress secondary

230

reactions (e.g. hydrolysis)23. To corroborate these findings and further optimize reaction parameters,

231

the effects of the substrate ratio (L-AA/lactose) on the enzymatic L-AA-Gal production were

232

examined with different concentrations of substrates (Table 2) using 20 g L-1 of TN0602 at 75°C

233

and pH 5.0. Table 2 shows the effect of varying substrate concentration on the formation of L-AA-

234

Gal. As expected, results showed that L-AA-Gal production improved when increasing the substrate

235

ratio (L-AA/Lactose). The highest productivity (Concentration of L-AA-Gal=133.38 mmol L-1 in


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12 h) was attained using a 2:1 ratio. A positive correlation between increasing L-AA loading and

237

enhanced production of L-AA-Gal is observed, for example, increasing the L-AA concentration

238

leads to an approximately 41-44% increase in the L-AA-Gal production (e.g. Entry 1 and 3-4). In

239

addition, the production of the secondary product trisaccharide decreased by 16-25% when

240

increasing molar ratio from 2:1 to 4:1 L-AA/Lactose. Therefore, increasing the concentration of L-

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AA will increase the relative probability of enzyme access to L-AA and thus reduce the production

242

of GOS.

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Comparison of antioxidant and oxidative stability of L-AA and L-AA-Gal. Previous work7

244

reported that the transglycosylation activity of glucoside hydrolase using maltose (355 µmol L-1)

245

and L-AA (355 µmol L-1) as substrates at 50°C and pH 5.3 for 5 h yielded a very stable and non-

246

reducing form of glucosylated L-AA. Thus, the antioxidant activity of L-AA-Gal, the new L-AA

247

derivative here presented, was examined monitoring the free radical scavenging activity24. As

248

shown in Figure 2a, an obvious linear relationship between the reducing absorbance and sample

249

volume with the same concentration is observed, that is, the absorption of 517 nm significantly

250

decreased by increasing of L-AA and L-AA-Gal concentration. However, the concentration of L-

251

AA (1-5 µL, black x-axis) was 10 times less than L-AA-Gal (10-50 µL, red x-axis). Since DPPH

252

radical is scavenged by the electron donated from the antioxidant, resulting in decolonization and

253

decrease in absorbance,25 it can be inferred that L-AA lost some of antioxidant activity after the

254

transgalactosylation. In addition, the stability of L-AA-Gal against an oxidative environment was

255

measured in the presence of Cu2+ ion15. As shown in Figure 2b, L-AA-Gal exhibited its stability

256

against Cu2+ ion as 100% was maintained even after 24 h in 10 µmol of Cu2+ ion, whereas L-AA

257

was degraded by oxidation after 10 h (~30% left). This result clearly indicated that L-AA-Gal was

258

more stable than L-AA.



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Modification at 2-OH of L-AA (AA-2G) has proved to stabilize L-AA. Thus, this derivative is of

260

great interest for industrial application5. For instance, 2-O-α-D-glucopyranosyl L-ascorbic acid is

261

used as an active ingredient in skin products. Still, even though this derivative possessed less

262

antioxidant capability than L-AA, glycosyl-(1, 2)-L-AA displayed higher stability than L-AA8, 15.

263

The L-AA derivatives here synthesized presented similar characteristic to that of glycosyl-(1, 2)-L-

264

AA. Comparing the

265

Glucopyranosyl-Lascorbic acid previously reported6, to the

266

derivative here presented, it can be confirmed that the hydroxyl group in position 2 of ascorbic acid

267

was modified. Figure S5 describes a potential mechanism of TN0602 to yield 2-O-β-D-

268

Galactopyranosyl L-Ascorbic Acid.

269

In summary, this work developed a new transgalactosylation reaction that lead to biosynthesis of a

270

novel derivative of L-Ascorbic Acid (L-AA-Gal), using β-Glucosidase TN0602 as biocatalyst and

271

lactose as substrate feedstock. Sodium carbonate was used to adjust the pH of the reaction mixture

272

to get higher substrate solubility and reaction rate; which results in L-AA-Gal=133.38 mmol L-1 in

273

12 h under optimal conditions. This biosynthetic pathway demonstrated to have potential value for

274

the industrial production of L-AA derivatives as it is a straightforward and relatively low cost

275

reaction system with high regiospecificity. Besides, the product L-AA-Gal is demonstrated to

276

possess antioxidant capability and to be more stable than L-AA in an oxidative environment.

277

Moreover, compared to other previous reported derivatives of L-AA6, 8, L-AA-Gal is easier to

278

produce as only one reaction-step is needed.

13

C NMR of ascorbic acid (Fig. S3) and the 13

C NMR of 2-O-α-D-

C NMR spectra of the L-AA

279 280

13

Supporting Information.


Page 15 of 24


281

Synthesis of L-AA-Gal using different glycosidases as reported by various authors; Effect of pH on

282

trisaccharide production; MS result; NMR result.

283 284

ACKNOWLEDGENTS.

285

The authors are grateful to the National High Technology Research and Development Program

286

(“863” Program) of China (No. 2013AA102104) for the financial support. Financial support from

287

Novo Nordisk Foundation (NNF16OC0021740) in Denmark is also acknowledged. B.P. thanks the

288

Danish Council for Independent Research for her postdoctoral grant 5054-00062B.

289



290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334

REFERENCES 1. Tajima, S.; Pinnell, S. R., Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA. Biochem Biophys Res Commun 1982, 106 (2), 632-7. 2. Hata, R.; Sunada, H.; Arai, K.; Sato, T.; Ninomiya, Y.; Nagai, Y.; Senoo, H., Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts. Eur J Biochem 1988, 173 (2), 261-7. 3. Zhang, Z. C.; Li, J. H.; Liu, L.; Sun, J.; Hua, Z. Z.; Du, G. C.; Chen, J. A., Enzymatic transformation of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) by immobilized alphacyclodextrin glucanotransferase from recombinant Escherichia coli. J Mol Catal B-Enzym 2011, 68 (3-4), 223-229. 4. Han, R.; Liu, L.; Li, J.; Du, G.; Chen, J., Functions, applications and production of 2-O-Dglucopyranosyl-L-ascorbic acid. Applied microbiology and biotechnology 2012, 95 (2), 313-20. 5. Kumano, Y.; Sakamoto, T.; Egawa, M.; Tanaka, M.; Yamamoto, I., Enhancing effect of 2-Oalpha-D-glucopyranosyl-L-ascorbic acid, a stable ascorbic acid derivative, on collagen synthesis. Biol Pharm Bull 1998, 21 (7), 662-6. 6. Gudiminchi, R. K.; Towns, A.; Varalwar, S.; Nidetzky, B., Enhanced Synthesis of 2-O-alphaD-Glucopyranosyl-L-ascorbic Acid from alpha-Cyclodextrin by a Highly Disproportionating CGTase. Acs Catalysis 2016, 6 (3), 1606-1615. 7. Yamamoto, I.; Muto, N.; Nagata, E.; Nakamura, T.; Suzuki, Y., Formation of a stable Lascorbic acid alpha-glucoside by mammalian alpha-glucosidase-catalyzed transglucosylation. Biochim Biophys Acta 1990, 1035 (1), 44-50. 8. Bae, H. K.; Lee, S. B.; Park, C. S.; Shim, J. H.; Lee, H. Y.; Kim, M. J.; Baek, J. S.; Roh, H. J.; Choi, J. H.; Choe, E. O.; Ahn, D. U.; Park, K. H., Modification of ascorbic acid using transglycosylation activity of Bacillus stearothermophilus maltogenic amylase to enhance its oxidative stability. Journal of Agricultural and Food Chemistry 2002, 50 (11), 3309-3316. 9. Kwon, T.; Kim, C. T.; Lee, J. H., Transglucosylation of ascorbic acid to ascorbic acid 2glucoside by a recombinant sucrose phosphorylase from Bifidobacterium longum. Biotechnology Letters 2007, 29 (4), 611-615. 10. Mukai, K.; Tsusaki, K.; Kubota, M.; Fukuda, S.; Miyake, T., Process for producing 2-0alpha-d-glucopyranosyl-l-ascorbic acid. 2014. 11. Han, R. Z.; Li, J. H.; Shin, H. D.; Chen, R. R.; Du, G. C.; Liu, L.; Chen, J., CarbohydrateBinding Module-Cyclodextrin Glycosyltransferase Fusion Enables Efficient Synthesis of 2-O-DGlucopyranosyl-L-Ascorbic Acid with Soluble Starch as the Glycosyl Donor. Applied and environmental microbiology 2013, 79 (10), 3234-3240. 12. Hassan, N.; Geiger, B.; Gandini, R.; Patel, B. K. C.; Kittl, R.; Haltrich, D.; Nguyen, T. H.; Divne, C.; Tan, T. C., Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis. Appl Microbiol Biot 2016, 100 (8), 3533-3543. 13. Smithers, G. W., Whey and whey proteins - From 'gutter-to-gold'. Int Dairy J 2008, 18 (7), 695-704. 14. Kong, F.; Yang, J.; Zhen, Z.; Liang, T.; Zhu, D.; Gao, R.; Xie, G., Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10: an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides. Chemical Research in Chinese Universities 2015, 31 (5), 774-780. 15. Yamamoto, I.; Muto, N.; Murakami, K.; Suga, S.; Yamaguchi, H., L-ascorbic acid alphaglucoside formed by regioselective transglucosylation with rat intestinal and rice seed alpha-


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glucosidases: its improved stability and structure determination. Chem Pharm Bull (Tokyo) 1990, 38 (11), 3020-3. 16. Li, J. B.; Lin, J. H.; Xiao, W. J.; Gong, Y. X.; Wang, M. M.; Zhou, P. F.; Liu, Z. H., Solvent extraction of antioxidants from steam exploded sugarcane bagasse and enzymatic convertibility of the solid fraction. Bioresource Technology 2013, 130, 8-15. 17. Daniels, R. S., CITRATED FOLIC ACID COMPOSITIONS AND METHODS FOR DELIVERING FOLIC ACID TO USP DISSOLUTION SPECIFICATIONS. 2015. 18. Han, R.; Liu, L.; Shin, H. D.; Chen, R. R.; Li, J.; Du, G.; Chen, J., Systems engineering of tyrosine 195, tyrosine 260, and glutamine 265 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance maltodextrin specificity for 2-O-(D)-glucopyranosyl-(L)ascorbic acid synthesis. Applied and environmental microbiology 2013, 79 (2), 672-7. 19. Shao, S. F.; Zhang, G. J.; Zhou, H. J.; Sun, P. C.; Yuan, Z. Y.; Li, B. H.; Ding, D. T.; Chen, T. H., Morphological evolution of PbS crystals under the control Of L-lysine at different pH values: The ionization effect of the amino acid. Solid State Sciences 2007, 9 (8), 725-731. 20. Mevkh, A. T.; Basevich, V. V.; Iarving, I.; Varfolomeev, S. D., [Inactivation of prostaglandin endoperoxide synthetase from the microsomal fraction of human platelets during the reaction]. Biokhimiia 1982, 47 (11), 1852-8. 21. Becker, P.; Abu-Reesh, I.; Markossian, S.; Antranikian, G.; Markl, H., Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil. Applied microbiology and biotechnology 1997, 48 (2), 184-90. 22. Kumar, S.; Nussinov, R., How do thermophilic proteins deal with heat? Cell Mol Life Sci 2001, 58 (9), 1216-1233. 23. Goedl, C.; Sawangwan, T.; Mueller, M.; Schwarz, A.; Nidetzky, B., A High-Yielding Biocatalytic Process for the Production of 2-O-(alpha-D-glucopyranosyl)-sn-glycerol, a Natural Osmolyte and Useful Moisturizing Ingredient. Angewandte Chemie-International Edition 2008, 47 (52), 10086-10089. 24. Bhat, R.; Liong, M. T.; Abdorreza, M. N.; Karim, A. A., Evaluation of Free Radical Scavenging Activity and Antioxidant Potential of a Few Popular Green Leafy Vegetables of Malaysia. International Journal of Food Properties 2013, 16 (6), 1371-1379. 25. Ibrahim, M. F.; Hussain, F. H. S.; Zanoni, G.; Vidari, G., Antioxidant and Free RadicalScavenging Activity of Tulipa Systola Roots, Leaves and Flowers Collected in the Kurdistan Region of Iraq. 2015, 34, 13-19.

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369 370 371 372 373 374 375 376 377 378 379 380 381

Figure captions Scheme 1. β-glucosidase (TN0602) catalyzed synthesis of 2-O-β-D-Galactopyranosyl L-Ascorbic Acid and side reaction pathways. Figure 1. Optimization of reaction parameters for the synthesis of L-AA-Gal. Effect of a) time course, b) pH, c) enzyme concentration, and d) temperature on L-ascorbic acid β-galactoside yield in β-glucosidasecatalyzed transgalactosylation reaction. (General reaction conditions: L-AA: 0.2 g mL-1, lactose: 0.2 g mL-1, pH 5.0, enzyme: 20 g L-1, temperature: 75˚C). Figure 2. Characterization of L-AA and L-AA-Gal. a) Effect of L-AA and L-AA-Gal on DPPH scavenging activity. (Reaction conditions: L-AA or L-AA-Gal: 10 mmol L-1 in water, DPPH: 100 mmol L-1 in ethanol, reaction time: 30 min, temperature: 25˚C, absorbance: 517 nm); b) Stability of L-AA and L-AA-Gal against oxidation in the presence of 10 µmol L-1 Cu2+ ion. (Reaction conditions: L-AA and L-AA-Gal: 10 mmol L-1, temperature: 25˚C).


Page 18 of 24

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382 383


Figures Scheme 1

384 OH HO

HO

HO

OH

HO O

+ O

O

OH

HO

O HO

OH

L-ascorbic acid

HO O OH OH

OH

Transgalactosylation HO Hydrolysis

O

Side reaction OH O HO

O

HO

HO O

OH HO

385 386

OH

OH Trisaccharide

+

OH

O OH O 2-O-β-D-Galactopyranosyl L-Ascorbic Acid

Lactose

HO

HO

OH HO O

O HO

O OH OH

+

OH HO

O OH OH

Glucose


OH HO

O OH OH

Glucose


387

Figure 1

388 389 390 391 392


Page 20 of 24

Page 21 of 24

393


Figure 2

394 395



396

Tables.

397

Table 1. Apparent kinetic parameters for the transgalactosylation of TN0602 Reaction Temp (˚C) Vmax(mmol L-1 Km (mmol L-1) kcat (s-1) system min-1) Using sodium 50 carbonate to 70 adjust pH.

0.37 (±0.033)

0.36 (±0.038)

0.039 (±0.0035)

0.46 (±0.049)

0.28 (±0.014)

0.049 (±0.0052)

75

0.61 (±0.058)

0.27 (±0.019)

0.065 (±0.0062)

0.055 (±0.0054)

0.89 (±0.068)

0.0058 (±0.00057)

0.11 (±0.01)

0.67 (±0.058)

0.012 (±0.0011)

0.49 (±0.044)

0.5 (±0.048)

0.051 (0.0046)

Using sodium 50 citrate to adjust pH. 70 75 398 399

a

Ea of the reaction system was measured at 50-75˚C


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Ea (kJ mol-1)a 16.4 (±1.65)

39.01 (±2.94)

Page 23 of 24

400

401 402


Table 2 Influence of the substrate molar ratio on L-AA-Gal synthesis.a Entry

Molar ratio Reaction (L-AA to system lactose)

1

2:1

2

1:1

3

4:1

4

2:1

a

Max L-AA- Time Gal (h) production (mM) 1.14 M L- 84.89 (±4.7) 6 AA, 0.58 M lactose 1.14 M L- 92.86 9 AA, 1.17 (±0.87) M lactose 2.27M L- 119.85 9 AA, 0.58 (±6.54) M lactose 2.27 M L- 133.88 12 AA, 1.17 (±16.68) M lactose

L-AA-Gal productivity (mg mL-1 h1 ) 4.78 (±0.26)

GOS3 production (mM)

3.49 (±0.033)

257.34 (±29.68)

4.5 (±0.25)

72.96 (±2.58)

3.77 (±0.48)

192.1 (±10.86)

Reaction conditions: temperature: 75˚C, pH 5.0, enzyme: 20 g L-1.


86.69 (±4.66)


403

404 405 406 407

TOC Graphic

Graphic Synopsis: A facile, scalable and efficient enzymatic transgalactosylation of lactose from dairy waste to L-ascorbic acid (L-AA) using thermophilic β-glucosidase as a biocatalyst, yielding a novel L-AA derivative with improved stability.

408


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Thermophilic Biosynthesis of a Novel Ascorbic ... - ACS Publications

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