• BMB Reports
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• 제31권3호
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• pp.240-244
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• 1998

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and in a potent immunogen. In order to express the recombinant urease at a higher level, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a high copy-number vector. The recombinant H. pylori urease expressed in an E. coli strain that was grown in a rich medium supplemented with added nickel was purified to near homogeneity by using DEAE-Sepharose, Superdex HR200, and Mono-Q (FPLC) columns and the purified enzyme possessed the specific activity of 1255 U/mg. Polyclonal antibodies raised against the purified recombinant H. pylori urease were shown to be very specific when subjected to Western blot analysis, in which crude extracts from the H. pylori ATCC strain and the recombinant E. coli strains expressing various bacterial ureases were exnmined for cross-reactivity.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권2호
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• pp.140-145
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• 2006

The behaviour of alginate immobilized and soluble watermelon (Citrullus vulgaris) urease in water miscible organic solvents like, acetonitrile, dimethylformamide (DMF), ethanol, methanol, and propanol is described. The organic solvents exhibited a concentration dependent inhibitory effect on both the immobilized and the soluble urease in the presence of urea. Pretreatment of soluble enzyme preparations with organic solvents in the absence of substrate for 10 min at $30^{\circ}C$ led to rapid loss in the activity, while similar pretreatment of immobilized urease with 50% (v/v) of ethanol, propanol, and acetonitrile was ineffective. Time-dependent inactivation of immobilized urease, both in the presence and in the absence of urea, revealed stability for longer duration of time even at very high concentration of organic solvents. The soluble enzyme, on the other hand, was rapidly inactivated even at fairly lower concentrations. The results suggest that the immobilization of watermelon urease in calcium alginate make it suitable for its application in organic media. The observations are discussed.

• Applied Biological Chemistry
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• 제32권2호
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• pp.109-115
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• 1989

퇴비(堆肥) 및 비료(肥料) 시비방법별(施肥方法別)로 무비구외(無肥區外) 7처리(處理)로 21년간(年間) 시험한 미사식양질계(微砂埴壤質系) 보통답(普通畓)에서 urease, L-glutaminase, phosphatase 활성(活性)을 조사하였다. 총(總) urease 활성(活性)(TUA)과 미생물(微生物) 분필(分泌) urease 활성(活性)(MUA)은 처리간(處理間)에 차이가 심하였으나, 토양(土壤)에 축적(蓄積)된 urease 활성(活性)(AUA)은 처리간(處理間)에 차이가 경미(輕微)하였다. 특히 MUA는 3요소(要素)와 퇴비(堆肥), 3요소(要素)와 규산(硅酸) 시용구(施用區)에서 가장 높았으며 토양중(土壤中) 인산(燐酸), 가리(加里), 규산(硅酸) 함량(含量)과도 고도(高度)의 유의(有意)한 상관관계(相關關係)가 있었다. 총(總) L-glutaminase 활성(活性)(TGA)과 토양(土壤)에 축적(蓄積)된 L-glutaminase 활성(活性)(AGA)은 처리 간에 차이가 심하였으나, 미생물(微生物) 분필(分泌) L-glutaminase 활성(活性)(MGA)은 처리간에 차이가 경미(輕微)하였으며 활성정도(活性程度)는 AGA와 비슷하였다. 또한 AGA는 토양 중 규산(硅酸), 인산(燐酸) 함량(含量)과 pH와는 고도(高度)의 유의(有意)한 상관(相關)이 있었다. 토양 중 phosphatase 활성(活性)은 toluene 처리와 무처리간에 차이가 경미하였으나, 퇴비(堆肥)와 비료(肥料) 시비방법(施肥方法)에 따른 차이는 심하여서 3요소구(要素區), 3요소(要素)+퇴비구(堆肥區), 3요소(要素)+규산구(硅酸區)에서 활성(活性)이 높게 나타났다. 그리고 phosphatase 활성(活性)과 토양 PH, 규산(硅酸), 인산(燐酸), 칼슘 함량(含量)과는 유의성(有意性)이 있었다.

• 산업식품공학
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• 제13권4호
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• pp.251-261
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• 2009

In vitro physiological functions such as jack bean (Canavalia ensiformis) urease inhibitory activity and retarding effect of glucose/bile acid of Aloe vera gel concentrated by the optimized DIS (Dewatering Impregnation & Soaking) process conditions were examined. Urease inhibitory activity of DIS aloes ranged from 84.6 to 94.4%, which was similar to or higher than 86.3% of fresh aloe. Also, urease inhibitory activity of DIS aloes was maintained at initial levels after heat treatment (90$^{\circ}C$, 10 min.) and drying treatment (freeze or hot air drying). Urease inhibition pattern from Lineweaver-Burk plot indicated general non-competitive inhibition, and inhibition constants ($K_{IE}$ and $K_{IES}$) of DIS aloes were 41-149 and 87-163 $\mu$L/mL, respectively. DIS(glucose) and DIS(polyethylene glycol) exhibited the highest retarding effect of glucose and bile acid. Their retarding effects were about 1.6 and 1.8 folds higher than that of fresh aloe after 0.5 and 1 hr of the dialysis, respectively. Conclusively, the above in vitro physiological functions of Aloe vera gel concentrated by DIS process suggested that aloe products treated with DIS would have the potential benefits for protection against Helicobacter pylori and reduction of blood glucose and cholesterol levels.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.108-112
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• 1994

The genetic organization of the urease gene cluster from an alkalophilic Bacillus pasteurii was determined by subcloning and Tn5 transposon mutagenesis of a 10.7 kilobasepair cloned fragment. A region of DNA between 5.0 and 6.0 kb in length is necessary for urease activity. In vitro transcription-translation analysis of transposon insertion mutants of the cloned urease genes demonstrated that the major ($M_r$ 67,000) and minor ($M_r$ 20,000) structural peptides of urease are encoded at one end of the urease gene cluster and at least 3 additional polypeptides are encoded by adjacent DNA sequences.

• Journal of Life Science
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• 제9권1호
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• pp.5-8
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• 1999

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and a potent immunogen. Deletion mutageneses were performed in the H. pylori urease accessory genes by using combinations of restriction enzymes and other DNA modifying enzymes in order to assess the function of these accessory gene products in the expression of the active urease. Selective disruptions in the accessory gene regions resulted in complete abolishment of the urease activity, which is consistent with other bacterial ureases. Interestingly, deletions in ureE-containing regions caused reduced expression of the structural enzyme subunits.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.

• Fisheries and Aquatic Sciences
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• 제7권2호
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• pp.58-63
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• 2004

Five urease-positive Vibrio parahaemolyticus strains were isolated from Kamak Bay in Yeosu in 2002 and 2003. V. parahaemolyticus YKB4 and YKB14 were isolated from seawater, YFB20 from black rockfish (Sebastes schlegeli), and YFO2l and YFO22 from olive flounder (Paralichthys olivaceus). The five urease-positive strains (YKB4, YKB14, YFB20, YFO21, and YFO22) did not show hemolysin and protease activity, while they did alter in color (to red) as the bacteria grew in the urea broth medium. All samples showed identical biochemical characteristics as a reference strain, V. parahaemolyticus KCTC2471, except in urease production. The five urease-positive strains showed urease activities at a mid stationary phase, and their activity was maximal in the late stationary phase of their culture supernatant. The addition of urea to the Luria-Bertani (LB) broth medium significantly affected the initial production of urease of V. parahaemolyticus isolates. Mortality by urease-positive V. parahaemolyticus YKB4, YKB14, YFO2l, and YFO22 was significantly high, being$60-80\%$, while YFB20 only reflected a rate of $20\%$. Protease-positive V. parahaemolyticus FM39 and FM50 showed a $40\%$ and $60\%$ mortality rate, respectively. However, hemolysin-positive V. parahaemolyticus had no mortality, like the non-pathogenic V. parahaemolyticus KCTC2471, while V. vulnificus resulted in a $40\%$ mortality rate. Injection with urease-positive V. parahaemolyticus strains showed mortality within 12 hrs in mice, and the strains could be isolated from the dead mice.

• 생명과학회지
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• 제13권1호
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• pp.67-72
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• 2003

위염, 위궤양 및 위암의 원인 균인 Helicobacter pylori 가균체 표면에 다량 함유하며 주된 생존 인자이며 병원성 인자인 urease를 대장균에서 발현시키고 이 효소에 대한 항체 또는 기질과의 특이 상호 작용을 이용하여 두 단계의 간편한 방법에 의하여 정제하였다. 우선 anti-H. pylori Urease IgG-Sepharose column과 urea-Sepharose column을 각각 제조하고 DEAE-Sepharose 음이온 교환수지를 통하여 1차 정제한 시료를 각각 적용하고 제반 조건에서 용출시켰다. Anti-H. pylori urease IgG-Sepharose column의 경우에는 urease 시료가 너무 강력하게 결합함으로써 극단적인 pH조건에서만 용출이 가능함이 관찰되었으므로, 100 mM 탄산 완충액(pH 10.5)으로 최종 용출하였을 때 비교적 순수한 효소를 얻었으나, 비활성이 다소 감소된 것으로 나타났다. 한편, urea-Sepharose에 적용시킨 시료는 100 mM urea-HEB 완충액(pH 7.5)으로 비교적 용이하게 용출되어 비교적 높은 순도와 비활성의 urease 효소를 얻을 수 있었으나 이 경우에는 urease의 smaller subunit인 UreA peptide band의 강도가 다소 감소한 것이 관찰되었다.

• Applied Biological Chemistry
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• 제22권4호
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• pp.217-220
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• 1979

요소(尿素)를 첨가(添加)한 토양(土壞)과 첨가(添加)하지 않은 토양(土壞)에 함질소(含窒素) 제초제(除草劑) asulam, dimetametryne, linuron을 처리(處理)하여 $25^{\circ}{\pm}1^{\circ}C$에서 항온배양(恒溫培養)하면서 효소(酵素) urease activity의 변화(變化)를 경시적(經時的)으로 검사(檢討)하였던 바 다음과 같은 결과(結果)를 얻었다. 요소(尿素)를 첨가(添加)하지 않은 토양(土壤)에서 dimetametryne 및 linuron 약제(藥劑) 처리(處理)에서는 urease의 활성(活性)이 상당히 높았으며, 배양기간(培養期間)이 경과(經過)하면서 약제처리(藥劑處理) 농도(濃度)가 높음에 따라 효소활성(酵素活性)이 다소(多少) 높게 지속(持續)되어 갔다. 반면(反面) 요소(尿素)를 첨가(添加)한 토양(土壤)에서는 asulam과 dimetametryne 약제(藥劑)에 의(依)해서는 효소활성(酵素活性)이 상당히 억제되었으나 linuron 약제(藥劑)는 이와 다르게 요소단독처리구(尿素單獨處理區)보다 다소(多少) 높은 효소활성(酵素活性)을 보였다.