• 산업기술연구
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• 제39권1호
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• pp.15-19
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• 2019

This work focused on characterization of the starch degradation activity from extremophile strain Deinococcus geothermalis. Glucoamylase gene from D. geothermalis was cloned and overexpressed by pET-21a vector using E. coli BL21 (DE3). In order to characterize starch degrading activity of recombinant glucoamylase, enzyme was purified using HisPur Ni-NTA column. The recombinant glucoamylase from D. geothermalis exhibited the optimum temperature as $45^{\circ}C$ for starch degradation activity. And highly acido-stable starch degrading activity was shown at pH 2. For further optimization of starch degrading activity with metal ion, various metal ions ($AgCl_2$, $HgCl_2$, $MnSO_4{\cdot}4H_2O$, $CoCl_2{\cdot}6H_2O$, $MgSO_4$, $ZnSO_4{\cdot}7H_2O$, $K_2SO_4$, $FeCl_2{\cdot}4H_2O$, NaCl, or $CuSO_4$) were added for enzyme reaction. As results, it was found that $FeCl_2{\cdot}4H_2O$ or $MnSO_4{\cdot}4H_2O$ addition resulted in 17% and 9% improved starch degrading activity, respectively. The recombinant glucoamylase from D. geothermalis might be used for simultaneous saccharification and fermentation (SSF) process at high acidic conditions.

• 미생물학회지
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• 제46권3호
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• pp.308-312
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• 2010

대표적인 중호열성 방사선저항성 미생물인 Deinococcus geothermalis에는 다른 Deinococcus 속과 비교해 당대사 작용에 관여하는 유전자가 풍부하게 존재하는 특징이 있다. 본 연구에서는 D. geothermalis를 이용하여 xylan의 최적 분해조건을 확인하였고, beechwood xylan, birchwood xylan 및 oat spelt xylan의 최종 분해산물을 HPLC를 이용하여 분석하였다. 기질의 종류에 따른 당화율을 비교한 결과 beechwood xylan, birchwood xylan 그리고 oat spelt xylan 순서로 당화율이 높게 나타났다. D. geothermalis를 이용한 xylan의 최적 분해조건인 $40^{\circ}C$, pH 8.0 그리고 마그네슘 이온을 첨가하였을 때 당화율이 7.5배 증가하였다. Beechwood xylan과 birchwood xylan의 최종 분해산물은 xylose, xylobios, xylotriose, xylotetraose, xylopentaose, 그리고 xylohexalose였으며, xylose의 함량이 가장 높았다. 또한 oat seplt xylan의 최종 분해산물은 xylose, xylopentaose 그리고 xylohexalose가 생성되었다. Xylan의 효율적인 당화를 위하여 전처리 방법으로서 방사선조사를 하였고, 방사선조사 후 D. geothermalis에 의한 beechwood xylan, birchwood xylan 그리고 oat spelt xylan의 당화율이 증가하였다. 본 연구를 통하여 D. geothermalis 및 방사선을 이용한 전처리 방법이 xylooligosaccharides를 생산하는데 유용함을 확인하였다.

• 한국식품과학회지
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• 제43권3호
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• pp.369-374
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• 2011

D. geothermalis의 dgeo_0475 유전자로부터 만들어지는 효소를 정제하여 그 특성을 확인하였다. DGMA는 분자량이 약 68 kDa 크기의 효소로서 ${\beta}$-CD, soluble starch 및 pullulan을 가수분해하는 CD 분해 효소임을 확인하였다. 효소의 최적 온도는 $40^{\circ}C$ 최적 pH 는 6.0이며 대부분의 기질들을 glucose와 maltose로 가수분해 하였고 pullulan 및 soluble starch로부터 미량의 panose를 생성하였다. ${\beta}$-CD를 가장 잘 가수분해하나 기질간 상대적 활성차이는 다른 CD 분해효소에 비하여 크지 않았다.

• 방사선산업학회지
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• 제4권3호
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• pp.289-295
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• 2010

The bacterium Deinococcus radiodurans is one of the most resistant organisms to the effects of ionizing radiation and other DNA-damaging agents. In this study, distributions of 10 ddr (DNA damage response) genes were investigated in 8 species of Deinococcus by polymerase chain reaction (PCR). We have compared the sequences of ddr genes of D. radiodurans, D. geothermalis and D. deserti, and selected primers which are suitable for the detection of ddr in different species of Deinococcus. A sequence homology search and PCR assay showed that ddrO, which encodes a global regulator of the radiation-desiccation response, was most well conserved in the Deinococcus lineage.

• 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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• 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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• 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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• 제20권12호
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• pp.1637-1646
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• 2010

The bacterium Deinococcus radiodurans is one of the most resistant organisms to ionizing radiation and other DNA-damaging agents. Although, at present, 30 Deinococcus species have been identified, the whole-genome sequences of most species remain unknown, with the exception of D. radiodurans (DRD), D. geothermalis, and D. deserti. In this study, comparative genomic hybridization (CGH) microarray analysis of three Deinococcus species, D. radiopugnans (DRP), D. proteolyticus (DPL), and D. radiophilus (DRPH), was performed using oligonucleotide arrays based on DRD. Approximately 28%, 14%, and 15% of 3,128 open reading frames (ORFs) of DRD were absent in the genomes of DRP, DPL, and DRPH, respectively. In addition, 162 DRD ORFs were absent in all three species. The absence of 17 randomly selected ORFs was confirmed by a Southern blot. Functional classification showed that the absent genes spanned a variety of functional categories: some genes involved in amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, and DNA metabolism were not present in any of the three deinococcal species tested. Finally, comparative genomic data showed that 120 genes were Deinococcus-specific, not the 230 reported previously. Specifically, ddrD, ddrO, and ddrH genes, previously identified as Deinococcus-specific, were not present in DRP, DPL, or DRPH, suggesting that only a portion of ddr genes are shared by all members of the genus Deinococcus.

• 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.