Pseudomonas sp. β-1,4-Glucosidase 유전자의 Escherichia coli와 Bacillus subtilis에의 Cloning 및 발현

김양우,전성식,김석재,정영철,성낙계 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.2

섬유성 biomass의 효소적 가수분해시에 endo-β-1,4-glucanase와 exo-β-1,4-glucanase의 작용으로 유리되는 cellobiose 또는 oligosaccharide를 glucose 단위로 절단하는 β-1,4-glucosidase 유전자를 분리하여 분자수준에서 정보를 얻고자 Pseudomonas sp. LBC505의 염색체 DNA를 EcoRI으로 절단하여 shot gun 방법으로 pUC19를 이용하여 E. coli에 형질전환시켜 5-bromo-4-chloro-3-indolyl-β-D-glucopylanoside(X-glu)가 함유된 배지에서 청색을 형성하는 4개의 colony 중에서 2개의 재조합체 pGL1과 pGL2를 분리하였다. 이들은 각각 1.2 kb와 4.2 kb EcoRI insert를 함유하고 있었는데 pGL1의 1.2 kb와 4.2 kb EcoRI insert를 함유하고 있었는데 pGL1의 1.2 kb 단편은 SacI 인식부위는 하나, AluI 인식부위는 두개 존재하였고 이 단편에 dioxigenin labeled deoxyuridin triphosphate을 반응시켜 얻은 probe와 Pseudomonas sp. LBC505 염색체 DNA를 hybridization하였을 때 강한 상동성이 인정되었고 또한 면역학적 실험에서도 침강선이 형성되어 cloning된 β-1,4-glucosidase 유전자는 Pseudomonas sp. LBC505 유래임이 확인되었다. E. coli에서 pGL1의효소활성은 모균주에 비하여 1.5배 증가되었고 pGL2는 비슷한 수준이었으나 두 재조합체 모두 세포내와 periplasmic 분획에서 거의 검출되었다. 그리고 pBD64에 subcloning된 pBGL40은 Bacillus subtilis에서 효소활성이 pGL1보다 약 1.6배 증가되었다. PGL1의 β-1,4-glucosidase의 효소적 특성은 모균주와 동일하였고 그리고 PNPG와 cellobiose 기질에서만 활성을 나타내었다. For the purpose of producing glucose from cellobiose or oligo saccharide and obtaining genetic information of β-1,4-glucosidase gene, a β-1,4-glucosidase gene of Pseudomonas sp. LBC505, potent cellulase complex and xylanase producing strain, was cloned in Escherichia coli and Bacillus subtilis into pUC19 and pBD64, respectively. Recombinant plasmid pGL1 contained 1.2 kb EcoRI fragment was isolated from transformants forming blue color around colony on LB agar plate containing 20 ㎍/ ㎖ of 5-bromo-4chloro-3indolyl-β-D-glucopyranoside(X-glu) and ampicillin. The recognition sites of the 1.2 kb fragment contains one SacI and two AluI sites. The pGL1 was hybridized to the pattern of EcoRI-digested chromosomal DNA from Pseudomonas sp. LBC505. Immunodiffusion assays revealed that pGL1-encouded β-1,4-glucosidase showed homology with that of host strains. Enzyme activity was not influenced by the reverse orientation of 1.2 kb insert in pGL1, indicating that it contained a promoter of β-1,4-glucosidase gene cloned. The biosynthesis of β-1,4-glucosidase of pGL1 was derepressed in the presence of glucose. β-1,4-Glucosidase activity was observed in intracellular (37%), periplasmic (51%) and extracellular (12%) of E. coli carrying pGL1, and its activity was about 1.5 times higher than that of original cell for the gene.

비병원성 야생효모, Apiotrichum sacrabaeorum WP49-1로부터 β-Glucosidase 생산

신유리, 장지은, 이종수 배재대학교 자연과학연구소 2016 自然科學論文集 Vol.32 No.1

콩에 함유되어 있는 식물성 여성 호르몬인 estrogen의 흡수를 증가시키기 위해 높은 활성의 β-glucosidase를 생산하는 비병원성 야생 효모의 선별과 β-glucosidase의 생산 조건을 조사하였다. 38종의 비병원성 야생효모 중 Apiotrichum scarabaeorum WP49-1의 무세포 추출물이 6.5 unit의 가장 높은 β-glucosidase 활성을 보였고 Zygosaccharomyces rouxii O-5-7도 5.9 Unit의 높은 활성을 보였다. 최종적으로 β-glucosidase 생성 효모 균주로 Apiotrichum scarabaeorum WP49-1 균주를 선별하였다. β-Glucosidase는 Apiotrichum scarabaeorum WP49-1을 yeast extract-peptone-dextrose 배지에 접종하여 30℃에서 72시간 배양했을 때 최대로 생성되었다. 선발 균주는 구형으로 출아에 의한 영양증식을 하였고 포자를 형성하지 않았다. 또한 비타민이 함유되어 있지 않은 배지에서도 생육하였고 5% 에탄올에 대하여 내성을 보였다

Bioconversion of major ginsenosides Rg₁ to minor ginsenoside F₁ using novel recombinant ginsenoside hydrolyzing glycosidase cloned from Sanguibacter keddieii and enzyme characterization

Kim, J.K.,Cui, C.H.,Yoon, M.H.,Kim, S.C.,Im, W.T. Elsevier Science Publishers 2012 Journal of biotechnology Vol.161 No.3

This study focuses on the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing glycosidase from Sanguibacter keddieii in order to biotransform ginsenosides efficiently. The gene, termed bglSk, consists of 1857bp and revealed significant homology to that of glycoside hydrolase family 3. The enzyme was over-expressed in Escherichia coli BL21 (DE3) using a GST-fused pGEX 4T-1 vector system. The over-expressed recombinant enzymes could convert six major ginsenosides Rb<SUB>1</SUB>, Rb<SUB>2</SUB>, Rc, Rd, Re and Rg<SUB>1</SUB> into more pharmacologically active rare ginsenosides such as C-Y, C-Mc, C-K, Rg<SUB>2</SUB>(S), and F<SUB>1</SUB>. Especially, BglSk could completely convert the Rg<SUB>1</SUB> into F<SUB>1</SUB>. The GST-fused BglSk was purified with GST.bind agarose resin and then characterized. The kinetic parameters for β-glucosidase had apparent K<SUB>m</SUB> values of 0.456+/-0.009 and 0.167+/-0.003mM and V<SUB>max</SUB> values of 30.2+/-0.7 and 4.1+/-0.1μmolmin<SUP>-1</SUP>mg of protein<SUP>-1</SUP> against p-nitrophenyl-β-d-glucopyranoside and Rb<SUB>1</SUB>, respectively.

Pseudomonas sp. Endo-1,4-β-Glucanase와 β-1,4-Glucosidase 유전자의 대장균 및 효모에서 동시 발현

김양우,전성식,정영철,성낙계 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.6

Two-cistron system을 이용하여 endoglucanase와 β-glucosidase를 E. coli 또는 S. cerevisiae에서 동시 생산을 시도하였다. 3종류의 two-cistron system, 5'-tac promoter---endoglucanase gene--β-glucosidase gene---3' 또는 5'-tac promoter---β-glucosidase gene-endoglucanase gene---3' 및 5'-tac promoter---endoglucanase gene--SD--β-glucosidase gene---3'를 구축하여 대장균과 효모에서 발현시켰을 때 두 종류의 효소가 동시에 생산되었으며, 효모에서는 대장균에 비하여 7∼8배 정도 활성이 낮았고 또한 생성된 효소의 44% 이상이 세포질에 존재하였다. 이들 재조합 DNA를 함유하고 잇는 효모에서 endoglucanase 또는 β-glucosidase의 생합성은 glucose 또는 cellobiose에 의해 저해되지 않았으며, 이 효모 배양액은 두 효소의 synergistic action으로 CMC를 glucose, cellobiose, oligosaccharide로 효율적으로 분해시켰다. 이런 결과는 형질전환체 효모를 이용하면 섬유질로부터 ethanol를 직접 생산할 수 있다는 것을 제시해준다. We attempted simultaneous expression of genes coding for endoglucanase and β-glucosidase from Pseudomonas sp. by using a synthetic two-cistron system in Escherichia coli and Saccharomyces cerevisiae. Two-cistron system, 5'--tac promoter-endoglucanase gene--β-glucosidase gene-3', 5'-tac promoter--β-glucosidase gene--endoglucanase gene--3' and 5'-tac promoter-endoglucanase gene--SD sequence--β-glucosidase gene--3, were constructed, and expressed in E. coli and S. cerevisiae. The E. coli and S. cerevisiae contained two-cistron system produced simultaneously endoglucanase and β-glucosidase. The recombinant genes contained the bacterial signal peptide sequence produced low level of endoglucanase and β-glucosidase in S. cerevisiae transformants: Approximately above 44% of two enzymes was localized in the intracellular fraction. The production of endoglucanase and β-glucosidase in yeast was not repressed in the presence of glucose of cellobiose. The yeast strain contained recombinant DNA with two genes hydrolyzed carboxymethyl cellulose, and these endoglucanase and β-glucosidase degraded CMC synergistically tow glucose, cellobiose and oligosaccharide. This result suggests the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.

Substrate specificity of β-glucosidase from Gordonia terrae for ginsenosides and its application in the production of ginsenosides Rg₃, Rg₂, and Rh₁ from ginseng root extract

Shin, K.C.,Lee, H.J.,Oh, D.K. Society for Bioscience and Bioengineering, Japan ; 2015 Journal of bioscience and bioengineering Vol.119 No.5

A β-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb<SUB>1</SUB> was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg<SUB>3</SUB> from Rb<SUB>1</SUB> by the enzyme were pH 6.5, 30<SUP>o</SUP>C, 20 mg/ml enzyme, and 4 mg/ml Rb<SUB>1</SUB>. Under these conditions, G. terrae β-glucosidase completely converted Rb<SUB>1</SUB> and Re to Rg<SUB>3</SUB> and Rg<SUB>2</SUB>, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg<SUB>1</SUB> to Rh<SUB>1</SUB> at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg<SUB>3</SUB>, 1.47 mg/ml Rg<SUB>2</SUB>, and 1.17 mg/ml Rh<SUB>1</SUB> from Rb<SUB>1</SUB>, Re, and Rg<SUB>1</SUB>, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30<SUP>o</SUP>C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg<SUB>2</SUB>, Rg<SUB>3</SUB>, and Rh<SUB>1</SUB> from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg<SUB>3</SUB>, Rg<SUB>2</SUB>, and Rh<SUB>1.</SUB>

Biotransformation of Major Ginsenoside Rb₁ to Rd by Dekkera anomala YAE-1 from Mongolian Fermented Milk (Airag)

( Gereltuya Renchinkhand ),( Soo-Hyun Cho ),( Young W. Park ),( Gyu-yong Song ),( Myoung Soo Nam ) 한국미생물 · 생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.10

Dekkera anomala YAE-1 strain separated from “airag” (Mongolian fermented mare’s milk) produces β-glucosidase, which can convert ginsenoside Rb₁ from Panax ginseng. Ginseng- derived bioactive components such as ginsenoside Rb₁ have various immunological and anticancer activities. Airag was collected from five different mare milk farms located near Ulaanbaatar, Mongolia. YAE-1 strains were isolated from airag to examine the hydrolytic activities of β-glucosidase on Korean Panax ginseng using an API ZYM kit. Supernatants of selected cultures having β-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb₁ at 40°C, pH 5.0. The YAE-1 strain was found to be nearly identical at 99.9% homology with Dekkera anomala DB-7B, and was thus named Dekkera anomala YAE-1. This strain exerted higher β-glucosidase activity than other enzymes. Reaction mixtures from Dekkera anomala YAE-1 showed great capacity for converting ginsenoside Rb₁ to ginsenoside Rd. The β-glucosidase produced by Dekkera anomala YAE-1 was able to hydrolyze ginsenoside Rb₁ and convert it to Rd during fermentation of the ginseng. The amount of ginsenoside Rd was highly increased from 0 to 1.404 mg/ml in fermented 20% ginseng root at 7 days.

젖산균 및 효모를 이용한 밀배아로부터 2-Methoxy-1,4-benzoquinone (2-MBQ) 및 2,6-Dimethoxy-1,4-benzoquinone(2,6-DMBQ)의 생산

유종길,김명동 한국산업식품공학회 2010 산업 식품공학 Vol.14 No.4

본 연구에서는 세포외 β-glucosidase를 생산하는 효모 37개 균주와 Lactobacillus 젖산균 5개 균주 중에서 세포외 β-glucosidase 효소활성이 우수한 Lactobacillus zeae와 Pichia pijperi을 선발하였고, 2-MBQ와 2,6-DMBQ 생산을 위한 밀배아 발효의 스타터로 사용하였다. P. pijperi 균주만을 이용하여 밀배아를 발효한 경우 2-MBQ는 거의 생성되지 않았으며, 2,6-DMBQ는 발효 개시 후 36시간과 48시간이 경과한 후 각각 0.02±0.01 mg/g 및 0.02±0.001 mg/g의 농도로 생성되었고 Lb. zeae 균주만을 이용하여 밀배아를 발효한 경우에는 발효개시 후 36시간과 48시간이 경과한 후 2-MBQ는 0.21±0.02 mg/g에서 0.28±0.06 mg/g으로 증가하였고 2,6-DMBQ 생성량의 유의적인 차이는 없었다. 효모와 젖산균을 동시에 사용하여 밀배아를 발효했을 때에는 발효 개시 후 36시간이 경과한 후 2,6-DMBQ의 생성량은 효모 및 젖산균을 단독으로 사용한 경우와 비교하여 유의적인 차이를 나타내지 않았지만 2-MBQ의 농도는 0.39±0.06 mg/g을 나타내었으며 발효 48시간이 경과하면 더욱 증가하여 0.46± 0.07 mg/g을 나타내었다. 이러한 결과는 젖산균인 Lb. zeae 균주가 효모인 P. pjperi보다 상대적으로 2-MBQ 및 2,6-DMBQ를 생산하는데 중요한 역할을 하며, 두 균주를 동시에 사용하면 각 균주를 단독으로 사용한 경우와 비교하여 2-MBQ 및 2,6-DMBQ를 생산하는데 상승효과가 있음을 알 수 있었다. Wheat germ contains the glycosylated forms of 2-methoxy-p-benzoquinone (2-MBQ) and 2,6-dimethoxy-p-benzoquinone (2,6-DMBQ), both of which have antimicrobial and immunostimulatory effects. Conversion of glycosylated 2-MBQ and 2,6-DMBQ to their more functional unglycosylated forms requires enzymatic action of β-glucosidase. We investigated the applications of lactic acid bacteria and yeast that produce β-glucosidase as starters for production of unglycosylated 2-MBQ and 2,6-DMBQ from wheat germ. Lactobacillus zeae and Pichia pijperi were selected through β-glucosidase enzyme assays for 37 yeast strains and five strains of lactic acid bacteria. Lb. zeae was more efficient than P. pijperi at producing 2-MBQ and 2,6-DMBQ from wheat germ. After 48 hr of fermentation with a mixed culture of Lb. zeae and P. pijperi, the concentration of 2-MBQ was 0.46±0.07 mg/g, indicating an approximately 1.6-fold higher concentration than that obtained by pure culture of Lb. zeae. However, the concentration of 2,6-DMBQ was not significantly enhanced by fermentation with a mixed culture of Lb. zeae and P. pijperi.

Molecular Orbital Theory on Cellulolytic Reactivity Between pNP-Cellooligosccharides and β-Glucosidase from Cellulomonas uda CS1-1

( Min Ho Yoon ),( Yun Kyu Nam ),( Woo Young Choi ),( Nack Do Sung ) 한국미생물생명공학회 2007 Journal of microbiology and biotechnology Vol.17 No.11

Purification and Characterization of a β-Glucosidase Capable of Hydrolyzing Soybean Isoflavone Glycosides from Pichia guilliermondii K123-1

소재현,김원찬,신재호,Choon-Bal Yu,이인구 한국식품과학회 2010 Food Science and Biotechnology Vol.19 No.5

A β-glucosidase, efficiently hydrolyzing isoflavone glycoside to isoflavone aglycone, was purified from Pichia guilliermondii K123-1, isolated from Korean soybean paste by ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, and fast protein liquid chromatogram (FPLC). The molecular mass of purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The optimum temperature for enzyme activity was 45℃ and it decreased dramatically above 50℃. The maximal activity was at pH 4.5 and more than 80% of the activity was retained for 24 hr in the pH range from 4.0 to 8.0 at 4℃. The N-terminal amino acid sequence of the enzyme was determined to be GLNWDYDNDK. Based on its substrate specificity and catalytic properties, the activity of the purified β-glucosidase was more effective when the sugar moiety of the glycoside was glucose and the size of the aglycone similar to that of the isoflavones. The purified β-glucosidase efficiently converts genistin and daidzin to genistein and daidzein 1.96 and 1.75 times more than almond meal β-glucosidase.

Purification and Characterization of Ginsenoside R_b1-Metabolizing β-Glucosidase from Fusobacterium K-60, a Human Intestinal Anaerobic Bacterium

PARK, Sun-Young,BAE, Eun-Ah,SUNG, Jong Hwan,LEE, Seung-Kwon,KIM, Dong-Hyun 경희대학교 동서의학연구소 2001 東西醫學硏究所 論文集 Vol.2001 No.-

Fusobacterium K-60, a ginsenoside R_b1-metabolizing bacterium, was isolated from human intestinal feces. From this Fusodobacterium K-60, a ginsenoside R_b1-metabolizing enzyme, β-glucosidase, has been purified. The enzyme was purified to apparent homogeneity by a combination of butyl-Toyopearl, hydroxyapatite ultragel, Q-Sepharose, and Sephacryl S-300 HR column chromatographies with a final specific activity of 1.52 μmol/min/mg. It had optimal activity at pH 7.0 and 40℃. The molecular mass of this purified enzyme was 320 kDa, with 4 identical subunits (80 kDa). The purified enzyme activity was inhibited by Ba^++, Fe^++, and some agents that modify cysteine residues. This enzyme strongly hydrolyzed sophorose, followed by pnitrophenyl β-D-glucopyranoside, esculin, and ginseno-side R_b1. However, this enzyme did not change 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901) to 20(S)-protopanaxadiol, while it weakly changed ginsenoside R_b1 to IH-901. These findings suggest that the Fusobacterial β-glucosidase is a novel enzyme transforming ginsenoside R_b1.