Production of the Minor Ginsenoside F2 from the PPD-mix-type Major Ginsenosides Using a Novel Recombinant Glycoside Hydrolase from Novosphingobium aromaticivorans

주베르,박혜윤,김가련,Chang-Hao Cui,조영준,김선창,임완택 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.6

Background: The minor ginsenosides such as F2, C-K, Rh2, and Rg3 make up less than 1% of the total ginseng extract; however, many studies have shown that ginsenoside F2 has anti-cancer and antioxidant effects and improves dementia and atopic dermatitis. Therefore, the enhanced production of this minor ginsenoside is a promising approach for the pharmaceutical industry. In this study, we found and cloned a novel glycoside hydrolase gene for the gram-scale production of ginsenoside F2. Methods and Results: Novosphingobium aromaticivorans, which was isolated from deep-terrestrial-subsurface sediments, has shown ginsenoside-converting ability. From this bacterium, a novel glycoside hydrolase, named BglNar, was found that can efficiently biotransform the protopanaxatriol (PPD-mix-type) major ginsenosides (Rb1, Rb2, Rc, and Rd) into the minor ginsenoside F2. This enzyme was cloned, expressed in Escherichia coli BL21, and characterized. The BglNar comprises 439 amino acid and belongs to the glycoside hydrolase family 1. The Km value of p-nitrophenyl- β-D-glucopyranoside was 9.06 ± 0.28 and the Vmax value was 24.0 ± 0.34 μmol/min/mg of protein. For gram-scale production of minor ginsenoside F2, crude PPD-mix-type ginsenosides (2 g/400 mL) were treated with BglNar and 1.14 g of F2 with final purity of 82.5 ± 1.3%, was obtained after purification using a column packed with HP20 resin. Conclusion: Our preliminary data demonstrate that the gram production of ginsenoside F2 using a recombinant enzyme will enhance the health benefits of Panax ginseng. This is the first study describing the gram-scale production of F2 from PPD-mix using a single novel ginsenosidetransforming β-glucosidase of the GH family 1. Significance and Impact of Study: In most of the previous studies, researchers have been used the combination of enzymes for production of F2, while in this study we found the possibility of gram-scale production of F2 from PPD-mixtype major ginsenosides using a single recombinant enzyme, thus simplifying the production and reducing the cost.

Efficient Production of Various Minor Ginsenosides from PPD- and PPT-type Major Ginsenosides Using a Single Recombinant BglFc Isolated from Flavobacterium chilense

주베르,Sofiane Medjebouri,Qinzhen Liu,박혜윤,김가련,임완택 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.2

Background: Rare ginsenosides (F2, Rg3, Gyp- XVII, and C-K) are pharmaceutically active components of Panax ginseng, which are derived from the conversion of major ginsenosides through various transformation methods. To date, most studies have failed to identify a competent bacterial strain and recombinant enzyme for converting protopanaxadiol (PPD)- and protopanaxatriol (PPT)-type ginsenosides to target minor ginsenosides. Method: Our study identified and employed nine sets of clones from different glycoside hydrolase bacterial strains for major ginsenoside bioconversion. Among these nine clones, only BglFc was selected based on its strong biotransformation ability and capacity to generate complete minor ginsenosides. bglFc was cloned and expressed in Escherichia coli using the pGEX-4T-1 vector system, and the recombinant enzyme was used for efficiently producing minor ginsenosides. Results: Recombinant BglFc is 2,394 bp and 798 amino acid residues long, with a predicted molecular mass of 78.8 kDa. BglFc belongs to the glycoside hydrolase family 3, and demonstrates a promising ability to convert major ginsenosides into minor ones. The Km and Vmax values of pNPG were 0.81 ± 0.06 and 4.0 ± 0.2 mM·min-1·mg-1 of protein, respectively. Under optimal conditions (37°C, pH 7.0), the ginsenoside transformation pathways for BglFc were as follows: Rb1→Rd→Rg3(S)→Rh2(S); GypXVII →GypLXXV→C-K; GypLXXV→C-K; F2→C-K; Rb2 →C-O→C-Y; Rb3→C-Mx1→C-Mx; Rc→C-Mc1→CMc; Re→Rg2(S); and Rg1→Rh1(S), respectively. Conclusion: These results suggest that recombinant BglFc demonstrates a strong transformation activity for both PPD- and PPTtype major ginsenosides. Therefore, we conclude that BglFc can be used for gram unit production of various minor ginsenosides. Significance and Impact of Study: Previously, researchers have used a combination of enzymes for the production of minor ginsenosides. However, in this study, we found a favorable enzyme that can be used alone for the production of a different type of minor ginsenoside using the proposed conversion pathway.

Description of 39 unrecorded bacterial species in Korea, belonging to the class Alphaproteobacteria

주베르,김승범,조장천,윤정훈,조기성,성치남,배진우,장광엽,전체옥,임완택 국립생물자원관 2017 Journal of species research Vol.6 No.2

During an investigation of the biodiversity of bacterial species in Korea, we discovered many indigenous prokaryotic species. A total of 39 bacterial strains in the class Alphaproteobacteria were isolated from various environmental samples collected from marine organisms, sea water, fresh water, tap water, mud flats, activated sludge, mineral water, tidal flats, soil and decayed plants. From the high 16S rRNA gene sequence similarity (>98.7%) and formation of robust phylogenetic clades with the most closely related species, it was determined that each strain belonged to each independent and predefined bacterial species. There is no official report that any of these 39 Alphaproteobacteria species have been described in Korea. Specifically, 18 species in 11 genera in the order Sphingomonadales, 11 species in 10 genera in the order Rhizobiales, two species in two genera in the order Caulobacterales, six species in six genera in the order Rhodobacterales and two species in two genera in the order Rhodospirillales were found in Korea. Gram reaction, colony and cell morphology, basic biochemical characteristics, isolation source, and strain IDs are described in the species description section.

바쟁 무브

에르베 주베르- 로랑생(Hervé Joubert-Laurencin ) 동의대학교 영화·트랜스미디어연구소 2019 디스포지티프 Vol.6 No.-

Complete genome sequence of Niabella ginsenosidivorans BS26^T, a ginsenoside-converting bacterium, isolated from compost

이영우,시디키 무하마드 주베르,류청매,김대철,임완택,Lee, Young-Woo,Siddiqi, Muhammad Zubair,Liu, Qing-Mei,Kim, Dae-Cheol,Im, Wan-Taek The Microbiological Society of Korea 2018 미생물학회지 Vol.54 No.4

퇴비로부터 분리한 Niabella ginsenosidivorans $BS26^T$ 균주의 유전체서열을 분석하였다. 균주 $BS26^T$의 유전체는 G + C 비율이 44.48%이며, 4,800개의 유전자와 4,704개의 단백질 코딩 유전자, 85개의 위유전자 그리고49개의 RNA유전자를 포함한 단일 원형 염색체로 구성되었으면 그 크기는 5,627,734 bp였다. 균주 $BS26^T$는 인삼사포닌의 당 분해에 관여하는 여러 타입의 글라이코시다제 유전자를 가지고 있었다. 이러한 유전체 분석은 주요 진세노사이드 전환에 관여하는 유전자 특징을 이해하는데 큰 기여가 되었다. An orange-colored, rod-shaped strain, designated Niabella ginsenosidivorans $BS26^T$, was isolated from compost. Strain $BS26^T$ showed the ability to convert major ginsenosides to minor ginsenosides, and its whole genome was sequenced. The whole genome of N. ginsenosidivorans $BS26^T$ consists of a single circular chromosome of 5,627,734 bp with 44.48% G + C content. Based on the complete genome sequence of strain $BS26^T$, we found several glycosides hydrolase-encoding genes that might involve in the conversion of major ginsenosides into minor ginsenoside and deliberate its strong pharmacological effects.

Terrabacter ginsengisoli sp. nov., isolated from ginseng cultivating soil

Mei-Fang Jin,Xiao-Tian Quan,주베르,Qing-Zhen Liu,Hong Shan Yu,임완택 한국미생물학회 2018 The journal of microbiology Vol.56 No.5

A Gram-positive, strictly aerobic, nonmotile, yellowish, coccus- rod-shaped bacterium (designated Gsoil 653T) isolated from ginseng cultivating soil was characterized using a polyphasic approach to clarify its taxonomic position. The strain Gsoil 653T exhibited optimal growth at pH 7.0 on R2A agar medium at 30°C. Phylogenetic analysis based on 16S rRNA gene sequence similarities, indicated that Gsoil 653T belongs to the genus Terrabacter of the family Humibacillus, and was closely related to Terrabacter tumescens DSM 20308T (98.9%), Terrabacter carboxydivorans PY2T (98.9%), Terrabacter terrigena ON10T (98.8%), Terrabacter terrae PPLBT (98.6%), and Terrabacter lapilli LR-26T (98.6%). The DNA G + C content was 70.5 mol%. The major quinone was MK-8(H4). The primary polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidyl-ethanolamine. The predominant fatty acids were iso-C15:0, iso-C16:0, iso-C14:0, and anteiso-C15:0, as in the case of genus Terrabacter, thereby supporting the categorization of strain Gsoil 653T. However, the DNA-DNA relatedness between Gsoil 653T and closely related strains of Terrabacter species was low at less than 31%. Moreover, strain Gsoil 653T could be both genotypically and phenotypically distinguished from the recognized species of the genus Terrabacter. This isolate, therefore, represents a novel species, for which the name Terrabacter ginsengisoli sp. nov. is proposed with the type strain Gsoil 653T (= KACC 19444T = LMG 30325T).