Earticle

4종 한약재에 대한 70% 에탄올 추출물의 Helicobacter pylori 세포 내 urease 활성억제능은 한약재 추출물을 넣지 않은 대조군과 비교하여 소목 (Caesalpinia sappan L.) 추출물의 경우 84%, 연교 (Forsythiae Fructus)의 추출물의 경우 72%, 애엽 (Artemisia asiatica Nakai) 추출물의 경우 31%이었다. 그러나 애엽의 80% 메탄올 추출물을 2차로 물, 에틸아세테이트 및 부탄올로 분획하였을 때 에틸아세테이트 분획에서 urease 활성억제 능력이 90%로 향상되었다.

Ethanol (70%) extract of Caesalpinia sappan L. and Forsythiae suspensa Vahl. showed 84% and 72% of anti-urease activity against Helicobacter pylori, respectively. Among the fraction of Artemisia asiatica Nakai extract using methanol (80%), water, ethyl acetate and butanol ethyl acetate fraction showed 90% of the anti-urease activity.

14종 한약재의 70% 에탄올 추출물의 Helicobacter pylori에 대한 항균활성은 황련 (Coptis japonica Makino)이 가장 뛰어났으며, 소엽 (Perilla frutescens var. acuta KUDO), 소목(Caesalpinia sappan L.) 및 형개 (Schizonepeta tenuifolia Briq.)의 경우에도 높은 항균활성을 나타내었다. 그러나Helicobacter pylori의 위내 서식을 도와주는 것으로 알려진 urease 활성억제능은 연교 (Forsythiae Fructus)의 메탄올(80%) 추출물에서 가장 높았으며, 소목 및 형개의 추출물도 높았다. 특히 연교의 경우 한약재 추출물을 넣지 않은 대조군과 비교하여 80% 이상의 활성이 억제되었다. 연교의 메탄올 추출물을 물, 에틸아세테이트 (ethyl acetate), 부탄올 순으로 분획하여 각 분획물에 대한 urease 활성억제능을 검색한 결과 에틸아세테이트 분획에서 92%의 urease 활성이 억제되었다.

Among 14 medicinal plants selected for the study ethanol (70%) extract of Coptis japonica Makino showed the highest anti-microbial activity against Helicobacter pylori followed by Perilla frutescens var. acuta KUDO, Caesalpinia sappan L. and Schizonepeta tenuifolia Briq. However, anti-urease activity of methanol (80%) extracts was best for Forsythiae Fructus followed by Caesalpinia sappan L. and Schizonepeta tenuifolia Briq. In the second fractionation using water, ethyl acetate and butanol more than 90% of the anti-urease activity was detected in the ethyl acetate fraction.

Contamination by radioactive strontium (90Sr) is a significant environmental problem. In this study, biomineralization based microbially induced calcite precipitation (MICP) seems to be a promising technique to sequestration of strontium from contaminated environments. Fifty-two ureolytic bacterial strains were newly isolated from various environments. From these, three strains were selected based on significant qualitative urease production by observing enzyme activity on urea agar plates and identified by 16S rDNA sequencing. The 3 strains (WJ-10, TB-22, TB-15) and positive control (Sporosarcina pasteurii KCTC3558) against groundwater environmental stresses such as salt, temperature, pH, starvation and strontium stresses. S. pasteurii showed a lower resistance capacity. On the other hand, the 3 strains was strongly resistance capacity and high strontium concentration (20 mM). Microbially induced calcite precipitation (MICP) process in sequestration was further confirmed by XRD analyses. XRD spectra showed presence of various sequestration products such as strontianite in samples.

메탄올을 사용한 1차 추출물의 경우 추출물을 넣지 않은 대조군과 비교하여 O.D. 값이 62% 감소하였다. 물과 부탄올, 에틸아세테이트를 이용한 2차 추출에서는 대조군과 비교하여 O.D.값이 각각 64%, 52% 및 90% 감소하였다. 따라서 에틸아세이트 분획물 중에 urease 활성억제 물질이 가장 많이 포함되어 있음을 알 수 있다.

Urease activity was lowered by 62% when the methanol extract of Artermisia asiatica Nakai was applied. Secondary extract of water, butanol and ethyl acetate decreased the activity by 64%, 52%, and 90%, respectively indicating that the fraction from ethyl acetate extraction contains the highest content of the inhibitory compounds.

연교를 메탄올로 1차 추출한 물질의 물, 부탄올, 에틸아세테이트 분획에서 물과 부탄올 분획은 한약재 추출물을 넣지 않은 대조군과 O.D. 값의 차이가 크지 않았으나 에틸아세테이트 분획물의 경우에는 대조군과 비교하여 O.D. 값이 70% 감소하였다. 이것으로 보아 연교의 에틸아세테이트 분획물 중에 urease 활성억제 물질이 포함되어 있음을 확인 할 수 있다. 연교로부터 분리된 betulinic acid와 oleanolic acid 모두 기존 항생제에 비하여 강한 urease 활성억제 효과가 있으며, 특히 butulinic acid가 oleanolic acid보다 강한 활성억제 효과가 있었다.

Betulinic and oleanolic acids isolated from Forsythia suspensa Vahl. Showed stronger urease inhibition activity compared with popular antibiotics such as amoxicilin, clarithromycin, metronidazole, and tetracycline. Betulinic and oleanolic acids lowered urease activity by 20% and 25%, respectively compared with the control experiments without the addition of the two acids.

In this work is reported the urease immobilized on magnetically separable polyaniline nanofibers to improve the enzyme activity. Immobilized urease shows good activity as 5times more than free urease. The immobilized urease showed improved stability and recyclability. PAMP-urease showed only a 3~5% decrease in activity after 1 months of vigorous shaking conditions (200 rpm) and at room temperature (25oC). After 10 times recycle, the residual activities of PAMP-urease was 92%.

Vibrio parahaemolyticus possessing urease-positive property is relatively rare, but such strains consistently exhibit the TDH-related hemolysin (TRH) gene. In this study, we examined the effects of incubation temperature on urease activity expression, using the TH3996 and AQ4673 strains where the enzyme activity is known to be temperature-dependent and -independent, respectively. In the TH3996 strain, $\beta$-galactosidase activity was 4.4-fold lower after $30^{\circ}C$ cultivation than after $37^{\circ}C$ in a ureR-lacZ fusion strain, but temperature dependency was not found in ureD- or nikA-lacZ fusion strains. However, ureR-, ureD-, and nikA-lacZ fusions of the AQ4673 strain was not influenced by incubation temperature. We compared the promoter sequences of ureR between the above two strains. Intriguingly, we detected mismatches of two nucleotides between the two strains located at positions -66 and -108 upstream of the methionine initiation codon for UreR. Additionally, urease activity was not affected by culture temperature at either $30^{\circ}C$ or $37^{\circ}C$ by allelic introduction of the AQ4673 ureR gene into the TH3996 ureR deletion mutant. Taken together, our study demonstrates that the transcriptional factor UreR is involved in the temperature dependency of urease activity, and two nucleotides within the ureR promoter region are of particular importance for the urease activity dependency of V. parahaemolyticus.

Purpose: The role of the Ferric Uptake Regulator (FUR) in the acid resistance of Helicobacter pylori (H. pylori)has been thought to be independent of urease. However, we demonstrated in this study that Fur influences urease activity. Materials and Methods: A fur knockout mutant of H. pylori was constructed by replacing the Fur gene with a kanamycin resistant marker gene. The wild-type H. pylori and fur mutant were compared for survival. The integrity of the inner membrane of the bacteria was evaluated by confocal microscopy using membrane-permeant and -impermeant fluorescent DNA probes. Urease activity of intact H. pylori was measured between pH 3 and 8. Real time PCR of both strains was performed for urease genes including ureI, ureE, ureF, ureG, and ureH. Results: The fur deletion affected the survival of H. pylori at pH 4. The urease activity curve of the intact fur mutant showed the same shape as the wild-type but was 3-fold lower than the wild-type at a pH of less than 5. Real time PCR revealed that the expression of all genes was consistently down-regulated in the fur mutant. Conclusion:The results of this study showed that fur appears to be involved in acid resistant H. pylori urease activity.

In this study, gamma-irradiated mackerel (Scomber japonicus) meat was stored in a refrigerator for 20 days to examine the physicochemical changes related to fishy smell. The effect of gamma irradiation on the inhibition of the activity of crude urease extracted from Vibrio parahaemolyticus was also evaluated. Increased levels of trimethylamine (TMA) and volatile basic nitrogen (VBN) content, which are the main components causing fishy smell, were significantly reduced by day 20 of storage after gamma irradiation, indicating that freshness was maintained during storage. The ammonia nitrogen contents of 3, 7, 10, and 20 kGy gamma-irradiated groups were significantly decreased by 6.5, 15.2, 17.4, and 23.9%, respectively, compared to non-irradiated groups on day 20 of storage. In addition, urease activity decreased in a gamma irradiation intensity-dependent manner. Volatile organic compounds (VOCs) were measured during the storage of gamma-irradiated mackerel meat. The contents of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavors due to spoilage of fish, were significantly reduced by day 20 of storage. Therefore, gamma irradiation can be considered useful for inhibiting urease activity and reducing fishy smell during fish storage.

In this study, the physicochemical changes related to fishy smell were determined by storing high hydrostatic pressure (HHP)-treated mackerel (Scomber japonicus) meat in a refrigerator for 20 days. The inhibition of crude urease activity from Vibrio parahaemolyticus using HHP treatment was also investigated. The mackerel meat storage experiment demonstrated that production of trimethylamine (TMA) and volatile basic nitrogen (VBN), the main components of fishy smell, was significantly reduced on the 20th day of storage after the HHP treatment compared to the untreated mackerels. The results demonstrated that the increased ammonia nitrogen rates in the 2000, 3000, and 4000 bar, HHP-treated groups decreased by 23.8%, 23.8%, and 31.0%, respectively, compared to the untreated groups. The enzyme activity of crude urease was significantly reduced in the HHP-treated group compared to that in the untreated group. Measurement of the volatile organic compounds (VOCs) in mackerel meat during storage indicated that the content of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to spoilage, were significantly reduced by HHP treatment. Collectively, our results suggested that HHP treatment would be useful for inhibiting the activity of urease, thereby reducing the fishy smells from fish and shellfish.

본 실험에서는 요소(尿素) 엽면시비(葉面施肥)에 의한 잎내 urease 효소의 활성도(活性度)에 미치는 영향을 콩, 라이맥, 토마토, 무 및 배추를 공시재료(供試材料)로 사용하여 조사하였다. urease 활성도(活性度)의 정확한 측정을 위하여 효소조액(酵素粗液)에 포함되어 있는 암모니아 이용 효소 중에서 열(熱)에 약한 효소의 불활성화(不活性化)를 꾀하고져 효소 반응액을 $60^{\circ}C$에서 3시간 동안 처리시켰다. 뇨소 처리를 받은 잎에서 urease 비활성도(比活性度)가 뇨소 무처리구(無處理區)에서 보다 엽면살포 후 일반적으로 2 내지 4일 동안에는 약간 높은 경향(傾向)을 보였다. 뇨소 처리구와 무처리구 간의 효소비활성도 차이는 어느 식물에서나 뇨소 처리 후 48시간 경과하였을 때 가장 두드러지게 나타났다. 콩과 라이맥의 경우에는 각가 6%와 10% 정도 높았고 토마토와 무에서는 약 20%정도, 배추에서는 60%정도의 차이가 있었다. 단백질의 함량은 콩과 토마토의 경우에서는 뇨소 처리구에서 엽면살포 후 약5일 동안 대조구(對照區)에 비해 높은 수준(水準)을 유지했으나 라이맥, 우무 및 배추에서는 본 실험기간 동안에는 큰 차이를 보이지 않았다. 잎내의 암모니아 농도는 urease 활성이 증가된 뇨소 처리구에서 오히려 약간 낮은 값을 보였다. 이상(以上)의 실험결과들은 뇨소의 엽면살포시 식물의 잎속으로 흡수(吸收)되어 들어간 뇨소가 urease의 생합성을 유도(誘導)하여 빨리 질소원(窒素源)인 뇨소를 식물 성장에 필요한 다른 대사물로 전환시켜 결과적으로 단기간내에 식물의 생육상태(生育狀態) 개선(改善)을 촉진시킨다고 사료된다.

Changes of urease activity in plant leaves following foliar application of urea were investigated with soybean, rye, tomato, radish and cabbage which were actively growing in a field. In this experiment, the procedure of the enzyme assay included incubation of the reaction mixture at $60^{\circ}C$ for 3 hr in order to inactivate heat unstable enzmes which may utilize ammonia produced by urease. The leaves with urea application showed somewhat higher urease specific activities for 2-4 days immediately after the foliar spray as compared with controls. The most difference of the specific activies between urea treatment and control was usually observed 2 days after urea application regardless of the plants. The difference of the specific activities disappeared completely 4 or 5 days following urea treatment. Protein contents in the leaves of soybean and tomato were increased for about 5 days after urea treatment, while no significant difference was found with rye, radish and cabbage. Urea application showed slightly lower ammonia concentration in the leaves which had higher urease activities. These results suggest that foliar spray of urea is very effective when nitrogen supply is required for rapid growth.

영남지역(嶺南地域) 답토양(畓土壤)의 이화학적(理化學的) 성질(性質)과 토양중(土壤中)의 urease 활성(活性)과의 관계(關係)를 검토(檢討)해 본 결과(結果) ; 1. 토양(土壤) 화학적(化學約) 성분(成分)과 urease 활성(活性)과는 유효인산(有效燐酸)($r=0.844^{**}$), 가리활성도비(加里活性度比)($r=0.762^{**}$), 유효규산(有效珪酸)($r=0.580^{**}$), $SiO_2$/O.M비(比)($r=0.591^{**}$) 등(等)과는 유의(有意)한 정상관(正相關) 관계(關係)였다. 2. 토양화학성분중(土壤化學成分中) 유기인산(有機燐酸)이 urease 활성(活性)에 미치는 기여도(寄與度)가 가장 컸다. 3. 토성별(土性別) urease의 활성(活性)은 사질토(砂質土)에서 가장 낮았으며, 토성(土性)이 식질화(埴質化)됨에 따라 증가(增加)하였다. 4. 토양(土壤) 배수등급별(排水等級別) urease 활성(活性)은 배수(排水)가 양호(良好)할수록 높았으며 배수(排水)가 불량(不良)한 답(畓)에서 낮은 경향(傾向)이었다. 5. Urease 활성(活性)이 높은 토양(土壤)이 상위등급지(上位等級地)의 답(畓)에 속(屬)하였다.

Paddy soil samples were collected from the plow layers of 19 soil series distributed in Yeongnam district to determine the relationship between soil urease activity and other soil characteristics. The results obtained were summarized as follows: 1. Simple correlation analyses showed that the urease activity was positively related with available $P_2O_5(r=.844^{**})$, potassium activity ratio($r=0.762^{**}$), available $SiO_2(r=.580^{**})$ and $SiO_2$/O.M ratio($r=0.591^{**}$). 2. Among soil chemical properties which had positive linear correlations with soil urease activity, the content of available $P_2O_5$ in soil had the highest contribution to the multiple regression equation of soil urease activity. 3. The activity of soil urease was especially lower in sandy texture than in clayey paddy soils, and a tendency was observed that the heavier soil textures the higher activity of soil urease. 4. Relatively well drained soils had the higher activity of soil urease while the soils in "poorly drained" had remarkably lower activity of soil urease. 5. The soils in higher classes of paddy soil equitability group had higher activities of soil urease.

Urease 활성이 중금속 이온에 의하여 저해되는 현상을 알아보기 위하여 발색시약을 사용하는 광학적인 방법으로 효소반응에 의한 암모니아 발생량을 측정하였다. 암모니아 농도에 따른 cuvette assay 시의 흡광도 중가는 암모니아 농도 3.0 mg/l 까지 직선성을 보였으며 이 때의 상관계수는 0.998(r)이었다. 중금속 이온에 의한 urease의 저해활성을 알아보기 위하여 기질에 단일 중금속 이온의 농도별 용액을 가하여 흡광도를 측정한 결과 효소의 저해도는 Hg(II)>Pb(II)>Cu(II)>Cd(II)>Zn(II) 이온의 순으로 나타났다. 한 가지의 중금속 이온 농도를 고정하고 다른 중금속 이온을 각기 다른 농도로 가하면서 urease활성을 측정하였을 때 효소의 저해도는 대체로 개별 중금속 이온에 의한 저해의 총합으로 나타났다. 상기의 결과는 본 연구의 방법이 Hg(II) 이온에 대한 선택적 검출법으로서 뿐만 아니라 여러 형태의 시료 중에 존재가능한 중금속 이온들을 정성적으로 판별하는 방법으로 활용될 수 있음을 보여주었다.

To determine the urease inhibitory activity of various heavy metal ions, a photometric cuvette assay for measuring ammonia production was developed. In this assay, the absorbance values at 630 m were linearly increased according to the ammonia concentrations up to 3.0 mg/l (r : 0.998). The urease inhibitions upon addition of a single species of heavy metal ions were in the decreasing order of Hg(II) > Pb(II) > Cu(II) > Cd(II) > Zn(II) ions. As expected, the urease inhibitions at a fixed concentration of a single species and at varying concentrations of other species occurred in the additive way. The above results show the applicability of the current method to the selective detection on Hg(II) ions as well as the screening of heavy metal ions possibly present at various samples.