Synthesis of α-cichoriin Using Deinococcus geothermalis Amylosucrase and Its Antiproliferative Effect

Moon, Keumok,Park, Hyun Su,Lee, Areum,Min, Jugyeong,Park, Yunjung,Cha, Jaeho The Korean Society for Microbiology and Biotechnol 2022 한국미생물·생명공학회지 Vol.50 No.2

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.

Enzymatic Synthesis of Resveratrol α-Glucoside by Amylosucrase of Deinococcus geothermalis

( Keumok Moon ),( Seola Lee ),( Hyunsu Park ),( Jaeho Cha ) 한국미생물 · 생명공학회 2021 Journal of microbiology and biotechnology Vol.31 No.12

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.

[지상10]The effect of stress on the cell proliferation and differentiation of the dentate gyrus in ovariectomized mice hippocampus.

Keumok Cho,Min Ho Moon,Sua Nam,Chan Park,Junghye Kim,Heekyung Ahn 대한체질인류학회 2004 대한체질인류학회 학술대회 연제 초록 Vol.47 No.-

Synthesis of Aesculetin and Aesculin Glycosides Using Engineered Escherichia coli Expressing Neisseria polysaccharea Amylosucrase

( Soyoon Park ),( Keumok Moon ),( Cheon-seok Park ),( Dong-hyun Jung ),( Jaeho Cha ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.4

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.

Saci_1816: A Trehalase that Catalyzes Trehalose Degradation in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

( Junho Lee ),( Areum Lee ),( Keumok Moon ),( Kyoung-hwa Choi ),( Jaeho Cha ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.6

Previously, a cytosolic trehalase (TreH) from the hyperthermophilic archaeon Sulfolobus acidocaldarius was reported; however, the gene responsible for the trehalase activity was not identified. Two genes, saci_1816 and saci_1250, that encode the glycoside hydrolase family 15 type glucoamylase-like proteins in S. acidocaldarius were targeted and expressed in Escherichia coli, and their abilities to hydrolyze trehalose were examined. Recombinant Saci_1816 hydrolyzed trehalose exclusively without any help from a cofactor. The mass spectrometric analysis of partially purified native TreH also confirmed that Saci_1816 was involved in proteins exhibiting trehalase activity. Optimal trehalose hydrolysis activity of the recombinant Saci_1816 was observed at pH 4.0 and 60℃. The pH dependence of the recombinant enzyme was similar to that of the native enzyme, but its optimal temperature was 20-25℃ lower, and its thermostability was also slightly reduced. From the biochemical and structural results, Saci_1816 was identified as a trehalase responsible for trehalose degradation in S. acidocaldarius. Identification of the treH gene confirms that the degradation of trehalose in Sulfolobus species occurs via the TreH pathway.

Identification and Characterization of a Novel Thermostable GDSL-Type Lipase from Geobacillus thermocatenulatus

( Eunhye Jo ),( Jihye Kim ),( Areum Lee ),( Keumok Moon ),( Jaeho Cha ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 Journal of microbiology and biotechnology Vol.31 No.3

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50oC and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and in silico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

Effects of Aspergillus oryzae and Bacillus subtilis Fermentation on the Metabolic Composition of Soybean Residues

Hyejin Hyeon,Cheol Woo Min,Keumok Moon,Jaeho Cha,Sun Tae Kim,Jae Kwang Kim 한국식품영양과학회 2019 한국식품영양과학회 학술대회발표집 Vol.2019 No.10

E. coli 발현 시스템에 의해 생산된 recombinant human bone morphogenetic protein-2의 정제와 생물학적 활성

최경희,문금옥,김수홍,윤정호,장경립,조규성,Choi, Kyung-Hee,Moon, Keumok,Kim, Soo-Hong,Yun, Jeong-Ho,Jang, Kyung-Lib,Cho, Kyoo-Sung 대한치주과학회 2008 Journal of Periodontal & Implant Science Vol.38 No.1

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

아욱 잎에서 분리한 Bacillus velezensis MV2의 유전체 염기서열 분석과 항균활성능 연구

이현주 ( Hyeonju Lee ),조은혜 ( Eunhye Jo ),김지혜 ( Jihye Kim ),문금옥 ( Keumok Moon ),김민지 ( Min Ji Kim ),신재호 ( Jae-ho Shin ),차재호 ( Jaeho Cha ) 한국미생물생명공학회(구 한국산업미생물학회) 2021 한국미생물·생명공학회지 Vol.49 No.1

본 연구에서는 국내 자생 식물인 아욱으로부터 새로운 균주를 분리 및 동정하였고 해당 미생물이 생산하는 항미생물질의 활성과 관련 생합성 유전자들을 확인하고자 하였다. 16S rRNA 유전자 서열 정보를 토대로 비교한 결과, 아욱에서 분리된 균주는 Bacillus velezensis이었으며 strain은 MV2라고 명명되었다. 유전체 염기서열 분석을 통해 전체 유전정보를 확인할 수 있었으며, 45.57% GC 함량을 가지는 4,191,702 bp 크기의 1개 컨티그(contig)가 존재하는 것으로 확인되었다. B. velezensis MV2가 정지기에 생산하는 물질 중 항균 활성이 확인된 소수성 물질 분획을 이용하여 항균 활성 스펙트럼 테스트를 진행한 결과, 그람음성균보다 그람 양성균에서 더 높은 억제능이 확인되었다. 6종의 곰팡이를 이용한 항진균 활성 테스트에서는 모든 진균에 대해 강한 저해 활성을 보였으며, 특히 F. fujikuroi와 F. graminearum에 대한 항진균 활성이 매우 강하게 나타났다. 세균에 대한 항균물질의 작용 기작 분석을 통해 해당 항균물질은 균을 용해시키는 살균(bactericidal) 특성을 가진 것으로 추측할 수 있었다. B. velezensis MV2의 유전체 염기서열 정보를 통해 이차대사산물 생합성 유전자 cluster를 탐색한 결과 총 47가지 이차대사산물 생산이 예측되었으며, 기존에 밝혀져 있는 물질들과 유사도 80% 이상인 물질은 14개로 확인되었다. 앞서 확인된 내용들을 바탕으로 B. velezensis MV2에서 생성되는 항균물질은 비리보솜 펩타이드성 물질로 예상되며, 향후 항균물질의 동정과 활용 가능성에 대한 추가적인 연구가 필요할 것으로 생각되었다. 기존에 상업적으로 이용되었던 곰팡이에 대한 활성이 낮은 항균물질 생물제제들과 함께 복합기능성 미생물제로 활용하여 식품산업 및 농업에서의 이용 가능성이 있음을 보여주었다. A bacterial strain isolated from a Malva verticillata leaf was identified as Bacillus velezensis MV2 based on the 16S rRNA sequencing results. Complete genome sequencing revealed that B. velezensis MV2 possessed a single 4,191,702-bp contig with 45.57% GC content. Generally, Bacillus spp. are known to produce diverse antimicrobial compounds including bacteriocins, polyketides, and non-ribosomal peptides. Antimicrobial compounds in the B. velezensis MV2 were extracted from culture supernatants using hydrophobic interaction chromatography. The crude extracts showed antimicrobial activity against both gram-positive bacteria and gram-negative bacteria; however, they were more effective against gram-positive bacteria. The extracts also showed antifungal activity against phytopathogenic fungi such as Fusarium fujikuroi and F. graminearum. In time-kill assays, these antimicrobial compounds showed bactericidal activity against Bacillus cereus, used as indicator strain. To predict the type of antimicrobial compounds produced by this strain, we used the antiSMASH algorithm. Forty-seven secondary metabolites were predicted to be synthesized in MV2, and among them, fourteen were identified with a similarity of 80% or more with those previously identified. Based on the antimicrobial properties, the antimicrobial compounds may be nonribosomal peptides or polyketides. These compounds possess the potential to be used as biopesticides in the food and agricultural industry as an alternative to antibiotics.