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Sustainable Production of Dihydroxybenzene Glucosides

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Sustainable Production of Dihydroxybenzene Glucosides J. Microbiol. Biotechnol. (2018), 28(9), 1447–1456 https://doi.org/10.4014/jmb.1805.05054 Research Article Review jmb Sustainable Production of Dihydroxybenzene Glucosides Using Immobilized Amylosucrase from Deinococcus geothermalis S Hun Sang Lee1†, Tae-Su Kim1†, Prakash Parajuli1, Ramesh Prasad Pandey1,2, and Jae Kyung Sohng1,2* 1Institute of Biomolecule Reconstruction (iBR), Department of Life Science and Biochemical Engineering, Sun Moon University, Asan 31460, Republic of Korea 2Department of Pharmaceutical Engineering and Biotechnology, Sun Moon University, Asan 31460, Republic of Korea Received: May 23, 2018 Revised: June 15, 2018 The amylosucrase encoding gene from Deinococcus geothermalis DSM 11300 (DgAS) was codon- Accepted: July 4, 2018 optimized and expressed in Escherichia coli. The enzyme was employed for biosynthesis of First published online three different dihydroxybenzene glucosides using sucrose as the source of glucose moiety. July 3, 2018 The reaction parameters, including temperature, pH, and donor (sucrose) and acceptor *Corresponding author substrate concentrations, were optimized to increase the production yield. This study Phone: +82-41-530-2246; demonstrates the highest ever reported molar yield of hydroquinone glucosides 325.6 mM Fax: +82-41-530-8229; E-mail: [email protected] (88.6 g/l), resorcinol glucosides 130.2 mM (35.4 g/l) and catechol glucosides 284.4 mM (77.4 g/l) when 400 mM hydroquinone, 200 mM resorcinol and 300 mM catechol, respectively, † These authors contributed were used as an acceptor substrate. Furthermore, the use of commercially available equally to this work. amyloglucosidase at the end of the transglycosylation reaction minimized the gluco- oligosaccharides, thereby enhancing the target productivity of mono-glucosides. Moreover, the immobilized DgAS on Amicogen LKZ118 beads led to a 278.4 mM (75.8 g/l), 108.8 mM (29.6 g/l) and 211.2 mM (57.5 g/l) final concentration of mono-glycosylated product of hydroquinone, catechol and resorcinol at 35 cycles, respectively, when the same substrate concentration was used as mentioned above. The percent yield of the total glycosides of hydroquinone and catechol varied from 85% to 90% during 35 cycles of reactions in an S upplementary data for this immobilized system, however, in case of resorcinol the yield was in between 65% to 70%. The paper are available on-line only at immobilized DgAS enhanced the efficiency of the glycosylation reaction and is therefore http://jmb.or.kr. considered effective for industrial application. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2018 by Keywords: Dihydroxybenzene glucosides, amylosucrase, amyloglucosidase, Deinococcus The Korean Society for Microbiology geothermalis, enzyme immobilization and Biotechnology Introduction transglycosyation of phenolic compounds. The biologically active phenolic compounds usually contain Amylosucrase (AS) is a glycosyltransferase (GT) structurally single or multiple functional groups such as hydroxyl and belonging to the glycoside hydrolase [1, 2]. Transglycosylation methyl moieties bonded to aromatic hydrocarbons. These reactions of AS are usually conjugations of single and/or hydroxyl groups are prone to subsequent modifications, multiple (gluco-oligosaccharides) sugar(s) with a great which alters their physiochemical functions compared with advantage compared with Leloir GTs. For production of the parent compounds. Glycosides of hydroquinone (para- bioactive phenolic glycosides [2, 3], AS has been used in the dihydroxybenzene), popularly known as skin-whitening synthesis of a variety of attractive biomaterials including agents and widely used in the cosmetic industry, inhibit amylose-like polymers, starch, dendritic nanoparticles, and melanin formation in the skin and lighten dark skin, microparticle encapsulation [4-6]. Although AS has potential freckles and age spots, as well as repair sun damage [7]. application in glycodiversification, it is rarely reported for Resorcinol and catechol are derivatives of meta- and ortho- September 2018 ⎪ Vol. 28⎪ No. 9 1448 Lee et al. dihydroxybenzenes, respectively. They also exhibit potent in 50 ml LB broth at 37oC for 24 h. The cells were harvested by biological activities and therefore have pharmaceutical as centrifugation at 840 ×g (for 10 min at 4oC and washed using a well as cosmeceutical applications. Resorcinol is used as a buffer containing 50 mM Tris-HCl and 10% glycerol (pH 7.5). skin-whitening agent and as an ointment to relieve pain These cells were re-suspended in the same buffer and disrupted and itching [8-10], while catechol is used as an anti-cancer with a sonicator in an ice bath. The supernatant and cell debris were separated by centrifugation at 13,475 ×g for 15min at 4oC. compound and as a cosmeceutical agent [4, 11]. Although Expressed protein was analyzed by sodium dodecyl sulfate- these phenolic compounds are highly useful, their polyacrylamide gel electrophoresis (SDS-PAGE) with 12% (w/v) applications have been limited due to issues related to acrylamide gel. The supernatant was purified using nickel stability, poor solubility, and short life in a photosensitive nitrilotriacetic acid (Ni-NTA) affinity column chromatography environment. Bulky sugar attachment to these compounds (Qiagen Inc., USA) and eluted at various concentrations (10, 100, has been shown to resolve these issues of water solubility, 200, and 500 mM) of imidazole, respectively, as previously reported stability, and biological properties [12]. [15, 16]. In this study, three dihydroxybenzenes (hydroquinone, catechol and resorcinol) were glycosylated using the Enzymatic Reaction Conditions for DgAS and Amyloglucosidase amylosucrase enzyme prepared using a codon optimized The transglycosylation reaction was performed using gene of Deinococcus geothermalis DSM 11300 (DgAS). The dihydroxybenzenes (hydroquinone, resorcinol and catechol) oligosaccharide-conjugated phenolic glycosides were also (Sigma-Aldrich, USA) as acceptor molecules in the presence of sucrose as glucose donor catalyzed by amylosucrase (DgAS). The generated during transglycosylation. The productions of these 200 μl of the total reaction mixture contained 20 mM dihyroxybenzene, phenolic mono-glucosides were enhanced by optimization 2 mM ascorbic acid, 200 mM sucrose, 0.4 mg/ml amylosucrase of various reaction parameters, enzyme immobilization enzyme and 200 mM Tris-HCl buffer at pH 7. The reactions were and treatment with commercial amyloglucosidase from incubated at 37oC for 30 min and stopped by keeping the reaction Aspergillus niger. Previous studies reported the synthesis of mixture in a boiling water bath for 10 min. The reaction mixtures α-arbutin and its potential use in the cosmeceutical industry were treated with 13 U/ml of commercial amyloglucosidase [13, 14]. (Sigma-Aldrich, USA) at 40oC for 3 h. The samples were treated with chilled methanol to stop further reaction and centrifuged at Materials and Methods 13,475 ×g for 15 min to separate them from protein precipitates. The reaction mixtures were analyzed by high performance liquid Chemicals and Reagents chromatography-photo diode array (HPLC-PDA) and confirmed Hydroquinone, resorcinol, catechol, ascorbic acid, sucrose, and by electrospray ionization mass spectrometry (ESI/MS) and amyloglucosidase (from Aspergillus niger) were purchased from nuclear magnetic resonance (NMR) analyses. The production Sigma-Aldrich (USA). High performance liquid chromatography yield was enhanced by the addition of 10% of various antioxidants (HPLC) grade acetonitrile and water were purchased from (ascorbic acid) to the reaction mixtures. Mallinckrodt Baker (USA). Restriction enzymes were purchased from Takara Bio (Japan) and Promega (USA). Amicogen LKZ118 Analytical Methods beads were purchased from Amicogen (Korea) Reverse-phase HPLC-PDA analyses was performed at 280 nm UV absorbance using a C18 column packed with YMC-Pack ODS- Bacterial Strains, Cloning and Culture Conditions AQ; 4.6 mm internal diameter, 150 mm long and 5 μm particle size Escherichia coli XL1 blue was used as a host for all DNA long, connected to photo diode array (PDA) on binary condition manipulation while E. coli BL21 (DE3) (Novagen, Darmstadt, consisting of solvent A (0.05% trifluoroacetic acid buffer-TFA), Germany) was used for the expression and production of DgAS water, and 100% acetonitrile maintained at a flow rate of 1 ml/min enzyme using pHCE IIB (NdeI) vector. Recombinant strains were for a 25-min program. The acetonitrile was set throughout as 0 - grown in Luria-Bertani (LB) agar plate and broth medium 40% (0-15 min), 40-75% (15-20 min), 75-0% (20-25 min). The supplemented with ampicillin (100 μg/ml) at 37oC. The nucleotide purification of compounds was carried out by preparative HPLC sequence of amylosucrase (GenBank Accession No. ABF44874.1) with a C18 column (YMC-PACK ODS-AQ; 150-250 mm long, sourced from Deinococcus geothermalis DSM 11300 was codon- 20 mm internal diameter, 10 μm particle size) connected to a UV optimized and synthesized by General Biosystems (USA). The detector using a 36-min binary program. The flow was 10 ml/min 1,984-bp gene fragment was flanked by NdeI and HindIII throughout the program as acetonitrile flowed for 0-20 min restriction sites. (40%), 20-30 min (75%), 30-36 min (0%). The high resolution quadruple-time of flight electrospray Enzyme Expression and Purification ionization-mass spectrometry (HRQTOF-ESI/MS) was performed Recombinant E. coli BL21 (DE3) harboring DgAS was cultured in positive- ion mode using Acquity
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