흰쥐 담즙울체간의 α-D- 및 β-D-Gluccosidase와 β-D-Glucuronidase의 활성도

박은미,곽춘식 啓明大學校 醫科大學 1992 계명의대학술지 Vol.11 No.1

A study was made on the changes of α-D-glucosidase, β-D-glucosidase,activities in cholestatic rat liver. The lysosomal α-D-and β-D-glucosidase,cytosolic broad-specificity β-D-glucosidase, and total β-D-glucosidase and microsomal β-D-glucosidase activities were determined in the cholestatic rat liver induced by common bile duct ligation over a period of forty two days. The activities of α-D-glucosidase,broad-specificity β-D-glucosidase,and β-D-glucosidase in the serum were measured together with the above determination of hepatic glycosidases. The activity of lysosomal α-D-glucosidase in the cholestatic rat liver increased strikingly between the seventh and the forth-second days after ligation, and that of the serum strikingly increased between the fourteenth and forth-second days after ligation. The lysosomal acid β-D-glucosidase activity in the cholestatic rat liver markedly increased between the twenty-eighth and the forth-second days after ligation. But the cytosolic broad-specificity β-D-glucosidase in the cholestatic liver showed a slight increased between the seventh and the forty-second day after ligation, and that of the serum strikingly increased between the first and the forty-second days after ligation. The total β-D-glucosidase and the microsomal β-D-glucosidase activities in the cholestatic liver in creased considerably between the second and the forty-second days after ligation and β-D-glucosidase activity of serum showed a marked increase between the fourteenth and the forth-second days after ligation. According to the above results,α-D-glucosidase, β-D-glucosidase, β-D-glucosidase in the liver with cholestasis seem to be increasing activities of enzymes with a seemingly cause of the development of synthesis. Especially, when the cholestasis occurred, the activities of these enzymes of serum are higher than those of the enzymes to leak into the blood in great quantity.

흰쥐 재생간의 α-D- 및 β-D-Mannosidase와 α-D-및 β-D-Glucosidase의 활성도

김익,곽춘식 啓明大學校 醫科大學 1992 계명의대학술지 Vol.11 No.3

A study was made on the changes of -D-mannosidase, -D-mannosidase, -D-glucosidase and -D-glucosidase activities in regenerating rat liver. The cytosolic, lysosomal, Golgi and mecrosomal -D-mannosidase, lysosomal -D-mannosidase, lysosomal -D-glucosidase, cytosolic broad specificity -D-glucosidase and lysosomal acid -D-glucosidase activities were determined in the regenerating liver after 70% (median and left lateral lobers) partial hepatectomy in rats over a period of ten days. The activities of cytolic, lysosomal and Golgi -D-mannosidase, lysosomal -D-mannosidase sidase and lysosomal -D-glucosidase in the serum were measured together with the above determination of hepatic glycosidases. The activities of cytotoslic -D-mannosidase of the serum and regenerating liver decreased slightly between the second and the third days after partial hepatectomy. The activity of Golig -D-mannosidase in the regenerating liver decreased significantly on the third day after partial hepatectomy. However, no significant change in the serum Golgi -D-mannosidase activity was noted throughout the experiments. The microsomal -D-mannosidase activity in the regenerating liver had a slight diminution between the second and the sixth days after partial hepatectomy. The lysosomal -D-mannosidase activity in the regenerating liver significantly decreased on the second day after partial hepatectomy. That of serum lysosomal -D-mannosidase showed a slight decrease from the second day after operation. The lysosoml -D-glucosidase activity in the regenerating liver showed a slight decrease between the first and the third days after partial hepatectomy, while the serum lysosomal -D-glucosidase activity did not change. The cytosolic broad-specificity -D-glucosidase activity in the regenerating liver slightly decerased between the second and the third days after partial hepatectomy. The lysosomal acid -D-glucosidase activity in the regenerating liver considerably decreased between the first and the third days after operation. Summarizing the results, -D-mannosidase, -D-mannosidase, -D-glucosidase and -D-glucosidase in the regenerating liver thought to be the enzymes decreasing their activities in the regenerating stage.

Production of α-Glucosidase Inhibitor by β-Glucosidase Inhibitor-Producing Bacillus lentimorbus B-6

( Kyoung Ja Kim ),( Yong Joon Yang ),( Jong Kee Kim ) 한국미생물생명공학회 2002 Journal of microbiology and biotechnology Vol.12 No.6

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.

담즙울체 쥐에서 간 라이소솜의 α-D-Glucosidase와 β-D-Glucuronidase 활성도에 미치는 Taurocholate의 정맥 내 투여의 영향

박소경,김여희,곽춘식 啓明大學校 醫科大學 2002 계명의대학술지 Vol.21 No.1

이 연구는 담즙울체간에서 라이소솜의 α-D-glucosidase와 β-D-glucuronidase의 활성도 증가 기전의 일부를 알아내기 위하여 쥐에게 총담관 결찰로 담관을 폐쇄시킨 직후 TCA 또는 TUDCA를 정맥 내에 주입시킨 다음 경시적으로 간 라이소솜의 α-D-glucosidase 및 β-D-glucuronidase와 혈청의 라이소솜 α-D-glucosidase, 혈청의 총 β-D-glucuronidase의 활성도를 측정하여 이 효소들의 활성도 변동에 대한 이들 담즙산의 효과를 알아 보았다. 쥐에게 총담관 결찰을 시킨 직후 TCA를 주입시켜 1일 및 2일 경과시켰을 때 간 라이소솜의 α-D-glucosidase 및 β-D-glucuronidase 활성도와 혈청의 라이소솜 α-D-glucosidase 및 혈청의 총 β-D-glucuronidase 활성도는 대조군인 총담관 결찰만 시킨 군보다 통계학적으로 유의한 증가를 나타내었다. 이상의 결과로 보아 담즙울체간에서 활성도가 증가되는 라이소솜의 α-D-glucosidase 및 β-D-glucuronidase 활성도 증가는 담즙산 중 TCA에 의해 이들 효소의 합성이 증가되어 나타난 결과로 생각되며 아울러 담즙울체시 라이소솜 α-D-glucosidase 및 총 β-D-glucuronidase의 혈중 활성도 증가는 TCA에 의한 간의 괴사로 간 세포막의 투과성이 항진되어 나타난 결과로 생각된다. The effects of intravenously administered of high concentration of taurocholic acid (TCA) on α-D-glucosidase and β-D-glucuronidase activities in rat liver lysosomes were studied. These liver lysosomal enzymes, and serum lysosomal α-D-glucosidase and total β-D-glucuronidase activities were also determined in experimental rats with common bile duct ligation (CBDL). The activities of liver lysosomal α-D-glucosidase and β-D-glucuronidase as well as the activities of serum α-D-glucosidase and total β-D-glucuronidase were found to be significantly increased in the CBDL plus TCA injected group than in the control group such as CBDL alone group. On the other hand, these serum and hepatic enzymes activities did not change in the CBDL plus tauroursodeoxycholic acid injected group. The above results suggest that TCA induces biosynthesis of the lysosomal α-D-glucosidase and β-D-glucuronidase in the liver and that the elevated lysosomal α-D -glucosidase and total β-D-glucuronidase activities in the serum are most likely due to increased hepatocyte membrane permeability caused by TCA mediated liver cell necrosis.

주정 중독 흰쥐에서 총담관 결찰이 혈청 및 간의 α-D-및 β-D-Glucosidase 활성에 미치는 영향

곽춘식,김여희,임종술 啓明大學校 醫科大學 1993 계명의대학술지 Vol.12 No.2

이 연구는 간담도 질환시 음주의 해로움에 대한 생화학적 배경의 일단을 밝히고자 시행한 실험으로서 급성 및 만성 주정 중독을 시킨 흰쥐에게 총담관결찰로 담즙울체를 야기시켜 혈청과 간의 α-D-glucosidase와 β-D-glucosidase의 활성도를 측정한 것이다. 쥐간의 lysosomal α-D-glucosidase 활성도는 총담관만 결찰한 군보다 만성 주정 중독 후 총담관을 결찰한 군이 총담관 결찰 후 7일부터 14일까지 덜 현저한 증가를 나타내었다. 총담관을 결찰한 군과 만성 주정 중독 후 총담관을 결찰한 군에서 간의 lysosomal acid β-D-glucosidase 및 cytosolic broad specificity β-D-glucosidase 활성도를 비교했을 때는 양군간에 별 차이가 없었다. 총담관을 결찰하고 14일 경과한 군과 총담관 결찰 14일 후 급성 주정 중독을 시킨 군에서 간의 lysosomal α-D-glucosidase, lysosomal acid β-D-glucosidase 및 cytosolic broad specificity β-D-glucosidase의 활성도를 비교했을 때는 양군간에 별 차이가 없었다. 쥐 혈청의 lysosomal α-D-glucosidase와 broad specificity β-D-glucosidase 활성도를 총담관을 결찰한 군과 만성 주정 중독 후 총담관을 결찰한 군을 비교했을 때는 만성 주정 중독 후 총담관을 결찰한 군이 총담관만 결찰한 군보다 더 현저한 증가를 나타내었다. 그러나 총담관을 결찰하고 14일 경과한 군과 총담관 결찰 후 급성 주정 중독을 시킨 군에서 혈청의 이들 효소의 활성도를 비교했을 때는 broad specificity β-D-glucosidase만 총담관 결찰 후 14일 경과한 군보다 총담관결찰 14일 후 급성 주정 중독을 시킨 군이 그 활성도가 더 높았다. 이들 성적 중에서 혈청의 lysosomal α-D-glucosidase와 broad specificity β -D-glucosidase의 활성도가 총담관만 결찰했을 때 보다 만성 주정 중독 후 총담관을 결찰했을 때 더 증가되고 아울러 혈청의 broad specificity β-D-glucosidase 활성도가 총담관 결찰 14일 후 급성 주정 중독을 시켰을 때는 총담관만 결찰한 후 14일 경과했을 때보다 그 활성도가 높은 것은 만성 주정 중독 및 급성 주정 중독시 담즙울체가 야기되면 담즙울체만 있을 때보다 간손상이 증폭된다는 것을 나타낸 결과라 생각되며 이성적은 간손상의 증폭으로 간에서 이들 효소의 누출일 증가된 것이라 할 수 있다. 따라서 이 성적은 담즙울체로 간손상이 있을 때는 음주를 해서는 안된다는 것을 뒷받침하는 자료라 하겠다. The activities of the liver and serum α-D and β-D-glucosidase were studied for cholestasis induced by common bile duct ligation and chronic ethanol intoxication developed, or cholestasis after acute ethanol intoxication for manifestation of the biochemical background of alcohol intoxication in hepatobiliary disease. The groups that received common bile duct(CBD) ligation after being chronically intoxicated with ethanol showed a marked increase at the 7th and 14th day after the ligation in the liver lysosomal α-D-glucosidase activities. However, the activities showd a lower degree than groups of the CBD lgation. The groups that received CBD ligation after being chronically intoxicated with ethanol showed a slight increase after the ligation in the liver lysosomal acid β-D-glucosidase and cytosolic broad specificity β-D-glucosidase activities, and the same was seen in the groups of the CBD ligation. At the 1.5th and 24th hours after the acute intoxication with ethanol which was done after 14 days of the CBD ligation, the rats showed a considerable increase in the liver lysosomal α-D-glucosidase, lysosomal acid β-D-glucosidase and cytosolic broad speciticity β-D-glucosidase activities, and the same was seen in the group sacrificed on the 14th day after the CBD ligation. The groups that received CBD ligation after being chronically intoxicated with ethanol showed a marked increase after the ligation in the serum lysosomal α-D-glucosidase and broad specificity β-D-glucosidase activities. However, the activities showed a far higher degree than groups of the CBD ligation. For the groups of acute intoxication with ethanol done after 14 days of the CBD ligation, the serum broad specificity β-D-glucosidase activities increased markadly, but the activities showed a higher degree than the group with the 14th day after the CBD ligation. In summary, especially, when the acute and chronic ethanol intoxication with cholestasis occurred, the serum lysosomal α-D-glucosidase and broad specificity β-D-glucosidase are higher than in cholestasis because of increased liver cell damage, which causes the enzyme to leak into the blood in great quantity. Accordingly, these results will be the data supporting the alcoholic drink is enzymologically harmful in hepatobiliary disease.

Growth and β-Glucosidase Activity of Bifidobacterium

( Yun Jung Choi ),( Chul Jai Kim ),( So Young Park ),( Young Tae Ko ),( Hoo Kil Jeong ),( Geun Eog Ji ) 한국미생물생명공학회 1996 Journal of microbiology and biotechnology Vol.6 No.4

김치에서 분리된 젖산균의 β-glucosidase 활성 탐색

장미희,김명동 한국산업식품공학회 2010 산업 식품공학 Vol.14 No.3

β-Glucosidase 효소활성이 높은 균주를 선발하기 위하여 다양한 김치에서 분리된 젖산균의 β-glucosidase 활성을 탐색하였다. 김치에서 분리된 156개의 젖산균 중 134개의 균주만이 cellobiose를 탄소원으로 대사하였으며, 세포내 β-glucosidase 활성이 세포외 활성보다 현저히 높았다. 배추김치에서 분리된 W. cibaria KFRI88010 균주가 3.7±0.5 unit/mg protein으로서 가장 높은 세포내 β-glucosidase 효소활성을 나타내었으며, 효소활성은 pH 5, 37oC 반응조건에서 가장 높게 나타났다. Mn2+를 비롯한 금속이온은 효소활성을 크게 저해하였다. W. cibaria KFRI88010 균주를 배양할 때 사용한 탄소원 중, fructose는 cellobiose나 glucose와 비교하여 약 2.5배 이상의 높은 세포내 β-glucosidase 효소활성을 나타내었다. The β-glucosidase (E.C. 3.2.1.21) production capabilities of lactic acid bacteria isolated from a variety of kimchi (fermented vegetables) were examined. When grown in a medium containing cellobiose as carbon source, most lactic acid bacteria showed significantly higher intracellular levels of β-glucosidase than the extracellular levels. A maximum intracellular β-glucosidase activity of 3.7±0.5 (unit/mg protein) was obtained in the case of Weissella cibaria KFRI88010 isolated from kimchi. The optimum reaction conditions for W. cibaria KFRI88010 β-glucosidase activity were pH 5.0 and 37oC, and addition of divalent cations to the reaction mixture resulted in a notable decrease in enzyme activity. The β-glucosidase activity was enhanced twofold when W. cibaria KFRI88010 was grown in a medium containing fructose as compared with to a medium containing glucose or cellobiose.

김치에서 분리된 젖산균의 β-glucosidase 활성 탐색

장미희 ( Mi Hee Jang ),김명동 ( Myoung Dong Kim ) 한국산업식품공학회 2010 산업 식품공학 Vol.14 No.3

β-Glucosidase 효소활성이 높은 균주를 선발하기 위하여 다양한 김치에서 분리된 젖산균의 β-glucosidase 활성을 탐색하였다. 김치에서 분리된 156개의 젖산균 중 134개의 균주만이 cellobiose를 탄소원으로 대사하였으며, 세포내 β-glucosidase 활성이 세포외 활성보다 현저히 높았다. 배추김치에서 분리된 W. cibaria KFRI88010 균주가 3.7±0.5 unit/mg protein으로서 가장 높은 세포내 β-glucosidase 효소활성을 나타내었으며, 효소활성은 pH 5, 37℃ 반응조건에서 가장 높게 나타났다. Mn2+를 비롯한 금속이온은 효소활성을 크게 저해하였다. W. cibaria KFRI88010 균주를 배양할 때 사용한 탄소원 중, fructose는 cellobiose나 glucose와 비교하여 약 2.5배 이상의 높은 세포내 β-glucosidase 효소활성을 나타내었다. The β-glucosidase (E.C. 3.2.1.21) production capabilities of lactic acid bacteria isolated from a variety of kimchi (fermented vegetables) were examined. When grown in a medium containing cellobiose as carbon source, most lactic acid bacteria showed significantly higher intracellular levels of β-glucosidase than the extracellular levels. A maximum intracellular β-glucosidase activity of 3.7±0.5 (unit/mg protein) was obtained in the case of Weissella cibaria KFRI88010 isolated from kimchi. The optimum reaction conditions for W. cibaria KFRI88010 β-glucosidase activity were pH 5.0 and 37℃, and addition of divalent cations to the reaction mixture resulted in a notable decrease in enzyme activity. The β-glucosidase activity was enhanced twofold when W. cibaria KFRI88010 was grown in a medium containing fructose as compared with to a medium containing glucose or cellobiose.

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.