인삼부산물 및 인삼밭 토양에서 분리한 9균주 중 β-Glucosidase 생성 균주 GYP-1과 GYP-3-3 균주를 선발하였다. 선발된 β-Glucosidase 생성 균주에 대하여 16S rRNA 유전자 염기서열과 ITS 염기서열을 기반으로 계통 분석을 실시한 결과, GYP-1 균주는 Rhodotorula 속에 속하며, GYP-3-3 은 Brachybacterium 속에 속하는 것으로 확인되었다. 특히, Rhodotorula sp. GYP-1 균주는 호기성 효모 종으로 biomass 생산량이 높아 최종 우수 균주로 선발하였다. Rhodotorula sp. GYP-1가 생성하는 β -Glucosidase의 온도 및 pH에 따른 효소 활성 및 안정성을 검정한 결과, 30 ℃에서 6.7 unit/ml로 가장 높은 활성을 나타내었고, 20 ℃ ∼ 40 ℃에서 효소 활성의 약 70 ％ 이상을 유지하는 것으로 확인하였다. pH에 따른 효소의 활성 및 안정의 경우, pH 5에서 6.8 unit/ml으로 가장 높은 활성을 나타내었고 pH 5∼ pH 8까지 93.3 % 이상의 효소 활성을 유지하는 것으로 확인하였다. Rhodotorula sp. GYP-1가 생성하는 β-Glucosidase는 ginsenoside Rb1 minor 진세노사이드로 분해하는 것으로 확인되었다. 또한, 인삼 뿌리 병원균(Botrytis cinerea)에 대해 항진균능을 갖는 것으로 확인되었다.

β-glucosidase is an enzyme that hydrolyzes glycosidic bonds to generate terminal non-reducing residues in β-D-glucosides and oligosaccharides. These enzymes are used to degrade the plant cell walls of plant biomass. The 170 lactic acid bacteria isolated from beachu kimchi were examined for their β-glucosidase activity(E.C. 3.2.1.21.). Among the tested lactic acid bacteria, 48 strains utilized cellobiose as a carbon source. The highest β-glucosidase activity was 0.0537±0.0021(U/mg protein) in MSE129. MSE129 was identified to be Weissella koreenisis using 16S rRNA gene sequence analysis.

The glucosidase inhibitors are currently interested owing to their promising therapeutic potential in the treatment of disorders such as diabetes, human immunodeficiency virus (HIV) infection, metastatic cancer, and lysosomal storage diseases. Among them, β-d-glucosidase inhibitors can be used to elucidate the mechanisms of various biochemical reactions and to treat the Gaucher disease as potential therapeutics. In this study, the natural β-glucosidase inhibitor was isolated from jujube leaf extract. The isolation and purification are vital for the structure analysis. The jujube leaf was extracted with hot water (95°C, 15 min). Then the natural β-glucosidase inhibitor was isolated using size exclusion chromatography and semi-preparative HPLC. In the first purification process, the 35 fractions were collected according to molecular size using PD-10 column packed with Sephadex G-25 medium. The strongest inhibition on the activity of β -glucosidase was observed at 6 and 7 fractions. The chromatogram of these samples showed that 3 peaks were observed comparing with baseline. For further purification, the strongest inhibitory activity peak was isolated using semi-preparative HPLC with VDS C18 column. Highly purified inhibitor, which can be used for structure analysis and characterization, was obtained. Further study is necessary to identify the natural β-glucosidase inhibitor in the jujube leaf extract.

팽화홍삼에 존재하는 major ginsenoside를 minorginsenoside로 생물전환하기 위하여 β-glucosidase 활성이높은 유산균주를 탐색하였다. 된장으로부터 분리한 YLB 8균주가 β-glucosidase 활성이 가장 높았으며 Leuconostocmesenteroides로 동정되었다. L. mesenteroides YLB 8을MRS 배지에서 배양하였을 경우 최대의 β-glucosidase 효소 활성을 나타내기 위한 최적 배양 조건은 glucose 첨가량 1%(w/v), 초기 pH 8.0, 배양온도 30oC, 배양시간 16시간이었다. L. mesenteroides YLB 8을 이용하여 팽화홍삼추출액을 생물전환하였을 경우 최적 온도는 30oC었으며,팽화홍삼 추출액의 농도는 12oBx이었다. 팽화홍삼 추출액내에 존재하는 major ginsenoside인 Rb1과 Rb2는 minorginsenoside인 Rg3로 전환되었으며, Re는 Rg1으로 전환되었으며, 이 중 일부는 Rh1과 Rh2로 전환되었다.

중고압 하에서 β-glucosidase 효소반응을 물리화학적 관점에서 연구하였다. 모델 기질 (p-nitrophenyl-β-D-glucopyranoside)에 대한 β-glucosidase 효소의 작용에 대한 압력 효과를 실험하였다. 즉, 압력 조건(25MPa, 50 MPa, 75 MPa, 100 MPa)과 시간 (10분, 60분, 1시간, 6시간, 24시간, 40시간)의 처리 조건에서 효소 활성도를 분광학적인 표준방법에 따라 측정하였다. 효소-기질 반응의 단계를 크게 kinetic 구간과 평형 구간으로 구분하여 물리화학적 모델을 적용하여, 정역반응속도 상수, 평형상수, 압력에 의한 부피 감소 등을 산출하였다. 대기압에서 100MPa까지 압력이 증가할수록 효소-기질 반응의 생성물이 더 많이 형성되었으며 전형적인 kinetic 구간과 평형 구간이 나타났다. 압력, 시간, 생성물농도 등의 데이터로부터 kinetic 구간과 평형에서의 생성물 예측 모델을 완성하였다. 결론적으로 중고압 처리에 의하여 효소-기질 반응이 촉진됨을 알 수 있었고, 임의의 압력 및 시간 조건에 따른 생성물의 농도를 예측할 수 있게 되었다.

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.

In this study, a biodegradation model of based on molecular cellulose was established. It is a mathematical, kinetic model, assuming that two major enzymes randomly break glycosidic bonds of cellulose molecules, and calculates the number of molecules by applying the corresponding probability and degradation reaction coefficients. Model calculations considered enzyme dose, cellulose chain length, and reaction rate constant ratio. Degradation increased almost by two folds with increase of temperature (5℃→25℃). The change of degradation was not significant over the higher temperatures. As temperature increased, the degradation rate of the molecules increased along with higher production of shorter chain molecules. As the reaction rates of the two enzymes were comparative the degree of degradation for any combinations of enzyme application was not affected much. Enzyme dose was also tested through experiment. While enzyme dose ranged from 1 mg/L to 10 mg/L, the gap between real data and model calculations was trivial. However, at higher dose of those enzymes (>15 mg/L), the experimental result showed the lower concentrations of reductive sugar than the corresponding model calculation did. We determined that the optimal enzyme dose for maximum generation of reductive sugar was 10 mg/L.

Background : Ginsenosides, the main ingredient of ginseng roots can be confirmed various physiological activity such as anticancer, antioxidant, a natural ginsenosides is there a structure to be absorbed into the body does not work well absorbed through this process biologically active thus a high conversion ginsenosides. β-glucosidase enzyme is observed in several of the microorganism with an enzyme that serves to convert a ginsenoside prosper that is absorbed into the body. Methods and Results : To view a primary β-glucosidase activity, the bacteria were innoculated in esculin agar medium and the color change of the media were measured by the time and degree of changing color. In the other method, 5 mM of p-nitrophenyl-β-D-glucopyranoside (pNPG) containing 25 mM phosphate buffer solution (pH 7.0) was added to 50 ul enzyme solution. Then the solution was added to 50 ul reaction for 5 min at 30°C. The amount of p-nitrophenol liberated measured at 405 nm absorbance. The experimental results showed higher β-glucosidase activity in Pediococcus pentosaceus, Leuconostoc mesenteroide, Leuconostoc mesenteroides subsp. cremoris, and Paenibacillus polymyxa by using esculin agar medium method. Similarly in second method, β-glucosidase activity was higher in P. pentosaceus 402.32±11.43 unit/l, L. mesenteroide 353.73±14.64 unit/l, Lactobacillus sakei 198.4±15.47 unit/l Lactobacillus plantarum subsp. plantarum 164.1±8.12 unit/l. Conclusion : The result that the β-glucosidase activity was higher in P. pentosaceus, L. mesenteroide, and L. plantarum subsp. plantarum as compared to tested microbes. Therefore selected bacteria can be used in the industry of functioned foods and beverage to improve human healths.