• 생명과학회지
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• 제21권11호
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• pp.1631-1635
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• 2011

재조합 아밀로수크라아제의 폴리페놀 배당체를 합성하는 능력을 검사하였다. 이 효소의 효소작용 특성에 근거하여 설탕을 기질로 사용하였으며 21 종류의 각기 다른 폴리페놀 화합물들이 수용체로 사용되었다. 당 전이 반응은 사용한 폴리페놀에 따라 하나 또는 두 개의 주요 폴리페놀 배당체를 합성하였다. 합성된 폴리페놀 배당체들은 박막 크로마토그래피법을 이용하여 확인되었고, 새로이 합성된 배당체의 구조는 당 전이 작용 특성에 근거하여 예측되었다. 수용체로 가능한 폴리페놀의 구조적 특징들이 평가되었으며, 이러한 결과는 Deinococcus geothermalis 유래 아밀로수크라아제가 식품, 화장품, 및 제약산업에서 높은 잠재성을 갖는 폴리페놀 배당체의 효소적 합성에 매우 효율적인 촉매로 활용될 수 있다는 것을 보여준다.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.566-570
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• 2018

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High-performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor, and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared with 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1692-1700
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• 2021

Glycosylation of resveratrol was carried out by using the amylosucrase of Deinococcus geothermalis, and the glycosylated products were tested for their solubility, chemical stability, and biological activities. We synthesized and identified these two major glycosylated products as resveratrol-4'-O-α-glucoside and resveratrol-3-O-α-glucoside by nuclear magnetic resonance analysis with a ratio of 5:1. The water solubilities of the two resveratrol-α-glucoside isomers (α-piceid isomers) were approximately 3.6 and 13.5 times higher than that of β-piceid and resveratrol, respectively, and they were also highly stable in buffered solutions. The antioxidant activity of the α-piceid isomers, examined by radical scavenging capability, showed it to be initially lower than that of resveratrol, but as time passed, the α-piceid isomers' activity reached a level similar to that of resveratrol. The α-piceid isomers also showed better inhibitory activity against tyrosinase and melanin synthesis in B16F10 melanoma cells than β-piceid. The cellular uptake of the α-piceid isomers, which was assessed by ultra-performance liquid chromatography (UPLC) analysis of the cell-free extracts of B16F10 melanoma cells, demonstrated that the glycosylated form of resveratrol was gradually converted to resveratrol inside the cells. These results indicate that the enzymatic glycosylation of resveratrol could be a useful method for enhancing the bioavailability of resveratrol.

• 한국미생물·생명공학회지
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• 제50권2호
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• pp.218-227
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• 2022

Glycosylation of aesculetin was performed using amylosucrase from the hyperthermophilic bacterium Deinococcus geothermalis DSM 11300 to improve the solubility and biological activity of aesculetin. A newly synthesized aesculetin glycoside was identified as α-cichoriin (aesculetin 7-α-D-glucoside) by nuclear magnetic resonance analysis. The solubility of α-cichoriin was 11 times higher than that of aesculetin because of the attached glucose moiety. Aesculetin and α-cichoriin had no significant effect on the proliferation of normal cells, such as RAW 264.7, but they showed a cell proliferation inhibitory effect on B16F10 melanoma cells. Unlike treatment with aesculetin and α-cichoriin, aesculin (aesculetin 6-β-D-glucoside) showed no antiproliferative activity in B16F10 cells. Based on the molecular structures of aesculin and α-cichoriin, the position where glucose binds to aesculetin and the anomeric configuration between glucose and aesculetin are thought to be important for exerting an antiproliferative effect on the B16F10 cell line. Based on these results, we propose that α-cichoriin, the α-glycosylated form of aesculetin, may serve as a model for developing phytochemical analogs with therapeutic potential for the treatment of diseases associated with tumor cell proliferation without cytotoxicity to normal cells.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1447-1456
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• 2018

The amylosucrase encoding gene from Deinococcus geothermalis DSM 11300 (DgAS) was codon-optimized and expressed in Escherichia coli. The enzyme was employed for biosynthesis of three different dihydroxybenzene glucosides using sucrose as the source of glucose moiety. The reaction parameters, including temperature, pH, and donor (sucrose) and acceptor substrate concentrations, were optimized to increase the production yield. This study demonstrates the highest ever reported molar yield of hydroquinone glucosides 325.6 mM (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, were used as an acceptor substrate. Furthermore, the use of commercially available 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 immobilized system, however, in case of resorcinol the yield was in between 65% to 70%. The immobilized DgAS enhanced the efficiency of the glycosylation reaction and is therefore considered effective for industrial application.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1253-1257
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• 2012

A gene (acas) designated as ${\alpha}$-amylase was cloned from Arthrobacter chlorophenolicus A6. The multiple amino acid sequence analysis and functional expression of acas revealed that this gene really encoded an amylosucrase (ASase) instead of ${\alpha}$-amylase. In fact, the recombinant enzyme exhibited typical ASase activity by showing both sucrose hydrolysis and glucosyltransferase activities. The purified enzyme has a molecular mass of 72 kDa and exhibits optimal hydrolysis activity at $45^{\circ}C$ and a pH of 8.0. The analysis of the oligomeric state of ACAS with gel permeation chromatography revealed that the ACAS existed as a monomer.

• Journal of Microbiology and Biotechnology
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• 제26권11호
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• pp.1845-1854
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• 2016

The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1698-1704
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• 2012

Resveratrol, or its glycoside form piceid, is a dietary antioxidant polyphenolic compound, found in grapes and red wine that has been shown to have protective effects against cardiovascular disease. However, very low water solubility of the compound may limit its application in the food and pharmaceutical industries. The amylosucrase (AMAS) of Alteromonas macleodii Deep ecotype was expressed in Escherichia coli and showed high glycosyltransferase activity to produce the glucosyl piceid when piceid was used as an acceptor. The conversion yield of piceid glucoside was 35.2%. Biotransformation using culture of the E. coli harboring the amas gene increased the yield up to 70.8%. The transfer product was purified by reverse phase chromatography and recycling preparative HPLC, and the molecular structure of the piceid glucoside was determined using NMR spectroscopy. The piceid glucoside was identified as glucosyl-${\alpha}$-($1{\rightarrow}4$)-piceid. The solubility of glucosyl piceid was 5.26 and 1.14 times higher than those of resveratrol and piceid, respectively. It is anticipated that dietary intake of this compound is more effective by enhancing the bioavailability of resveratrol in the human body because of its hydrophilic properties in the intestinal fluid.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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• pp.1436-1442
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• 2020

Amylosucrase (ASase, E.C. 2.4.1.4) is capable of efficient glucose transfer from sucrose, acting as the sole donor molecule, to various functional acceptor compounds, such as polyphenols and flavonoids. An ASase variant from Deinococcus geothermalis, in which the 226^th alanine is replaced with asparagine (DgAS-A226N), shows increased polymerization activity due to changes in the flexibility of the loop near the active site. In this study, we further investigated how the mutation modulates the enzymatic activity of DgAS using molecular dynamics and docking simulations to evaluate interactions between the enzyme and phenolic compounds. The computational analysis revealed that the A226N mutation could induce and stabilize structural changes near the substrate-binding site to increase glucose transfer efficiency to phenolic compounds. Kinetic parameters of DgAS-A226N and WT DgAS were determined with sucrose and 4-methylumbelliferone (MU) as donor and acceptor molecules, respectively. The k_cat/K_m value of DgAS-A226N with MU (6.352 mM^-1min^-1) was significantly higher than that of DgAS (5.296 mM^-1min^-1). The enzymatic activity was tested with a small phenolic compound, hydroquinone, and there was a 1.4-fold increase in α-arbutin production. From the results of the study, it was concluded that DgAS-A226N has improved acceptor specificity toward small phenolic compounds by way of stabilizing the active conformation of these compounds.

• Journal of Microbiology and Biotechnology
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• 제29권4호
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• pp.562-570
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• 2019

${\beta}$-Glucosylglycerol (${\beta}-GG$) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, ${\beta}-GG$ and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae ${\beta}$-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of ${\beta}-GGs$ [${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol and ${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}2$)-$\text\tiny{D}$-glycerol]. In the second transglycosylation reaction, ${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of ${\alpha}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}4$)-${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol and 28% of ${\alpha}$-$\text\tiny{D}$-maltopyranosyl-($1{\rightarrow}4$)-${\beta}$-$\text\tiny{D}$-glucopyranosyl-($1{\rightarrow}1/3$)-$\text\tiny{D}$-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of ${\beta}-GG$ was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.