• 한국미생물·생명공학회지
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• 제26권2호
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• pp.122-129
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• 1998

최근 생물방제균으로 주목받고 있는 Enterobacter cloacae의 방제기작이 이 균에 의해 토양내에서 생산된 휘발성 ammonia이며 ammonia의 생산에는urease가 관계한다는 보고를 근거로 하여, 항생물질 생산성 균주로 선발된 우수한 길항균주에 암모니아 생성능, 즉 urease 유전자를 유전적으로 부가함으로써 항진균성 길항물질 생산과 암모니아 생산이 동시에 이루어 질 수 있는 새로운 다기능의 생물방제균을 유전적으로 육종하고자 하였다. 저병해 인삼경작지로부터 식물근부균 Fusarium solani의 생육을 강하게 억제하는 길항세균 한 균주 SH14균주를 분리, 선발하였으며, 분리된 균주를 동정한 결과 Bacillus subtilis이거나 그 근연종으로 추정되었다. 억제기작 실험을 통해 길항균주 B. subtilis SH14에 의해 생산되는 항진균성 길항물질은 외막가수분해효소와 같은 고분자 물질이 아니라 열에 안정한 저분자의 항생물질임을 알 수 있었다. 한편 ammonia 생산을 위한 urease의 유전자는 urease 생산력이 강력한 호알칼리성 Bacillus pasteurii의 urease 생산유전자를 E. coli-Bacillus shuttle vector인 pEB203에 subcloning하였고, 이어서 pGU 366으로 명명된 이 recombinant plasmid를 선발된 항진균성 길항균주 B. subtilis SH14에 PEG-induced protoplast transformation 방법으로 도입, 발현시켰으며, 최적조건을 조사하여 90분간의 lysozyme 처리과정 후 1.5 $\mu\textrm{g}$/$m\ell$의 DNA와 40% PEG4000의 첨가로 약 6.5$\times$$10^{-4}$의 형질전환율을 얻을 수 있었다. 아울러 암모니아 생성능이 부가된 생물방제균 B. subtilis SH14(pGU366)에 의해 식물근부균 F. solani에 대한 생육억제력이 증가되는지 여부를 억제거리 측정법과 균체중량법을 통해 확인한 결과 urease 유전자가 도입된 형질전환체 B. subtilis SH14(pGU366)의 근부균 생육억제능이 각각 36.7%, 44.0%정도로 숙주균주인 B. subtilis SH14에 비해 근부균 생육억제능을 보다 강하게 나타내었음을 알 수 있었다. 따라서 항진균성 항생물질 생산성 생물방제균 B. subtilis SH14에 외부의 urease유전자를 도입하여 ammonia 생성능을 부가함으로써 생물방제력의 상승효과를 거둘 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.532-534
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• 2005

6종 한약재의 70% 에탄올 추출물의 Helicobacter pylori의 위내 서식을 도와주는 것으로 알려진 urease의 활성억제능은 오매(Mume Fructus)의 에탄올(70%) 추출물에서 가장 높았으며, 소목 및 연교의 추출물도 높았다. 특히, 오매의 경우 한약재 추출물을 넣지 않은 대조군과 비교하여 90% 이상의 urease 활성억제 효과를 나타내었다.

• Advances in Traditional Medicine
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• 제7권1호
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• pp.79-84
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• 2007

Urease was immobilized with calcium alginate by entrapment method in the form of spherical beads and stored in Tris/acetate buffer (pH 7.3) at $4^{\circ}C$. Urease immobilized at different concentration of alginate beads (3%, 4% and 5%) showed higher apparent $K_{m}$ values than the soluble urease. Furthermore, $K_{m}$ has been shown to be corelated with effective diffusion coefficient (De) at different concentration of alginate gel. The present study showed that diffusion and reaction contribute to control the overall rate.

• 한국가금학회지
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• 제22권2호
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• pp.105-115
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• 1995

The balance of microbial populations in the gastrointestinal (GI) tract of all warm-blooded animals is critical to the maintenance of health and resistance to disease. The composition of the populations can be altered by diet and environment, making the host animal susceptible to disease, and reducing growth rate and feed efficiency. Some feed additives including antimicrobial agents, prohiotics or yucca extract have been used to promote growth and feed utilization. There is evidence that part of growth-promoting effect of those feed additives results from the suppression of microbial urease activity or ammonia production in the GI contents of animals. Over 200 microbial species have been known to produce urease and the product of urea hydrolysis, ammonia, is toxic to animals. Carefully tested probiotics or other urease-suppressing agents can be a possible alternative to antimicrobial agents including antibiotics as growth promotants used for animals feeds.

• Bulletin of the Korean Chemical Society
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• 제32권12호
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• pp.4377-4381
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• 2011

In this study, real-time and label-free methods for monitoring the enzymatic reaction of urease, which releases ammonia through the hydrolysis of urea in an aqueous solution, were developed using a liquid crystal (LC)-based pH sensor. Nematic liquid crystal 4-cyano-4'-pentylbiphenyl (5CB), doped with 4'-pentyl-biphenyl-4-carboxylic acid (PBA), exhibited a shift in optical appearance from bright to dark when it was in contact with ammonia generated from the enzymatic reaction between urease and urea. This optical change was attributed to the anchoring transitions of LCs caused by hydrophobic interactions between the tails of deprotonted PBA ($PBA^-$) molecules and the LCs at the aqueous/LC interface. This novel technique holds great promise for the sensitive detection of urease along with its substrates and inhibitors.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.700-704
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• 1998

Each of the recombinant structural genes of Helicobacter pylori urease, ureA and ureB, was cloned and overexpressed as inclusion bodies. Solubilization and renaturation of the inclusion bodies were carried out, to accelerate the pairing of sulfhydryl groups and the incorporation of nickel ions, which would lead to the native structure with high enzyme activity. Rates of urea hydrolysis were monitored as an indication of in vitro activation of renatured ureases. The activation of the apoprotein using 1 mM nickel ion, 100 mM sodium bicarbonate and a 10:1 ratio of reducing power resulted in a weak urease activity (about 11% of the native urease activity encoded by pTZ 19R/ure-l). When a sparse matrix screen method originally discovered for the crystallization of proteins was used, the activity increased higher than that obtained using glutathione. The effect of polyethylene glycol (PEG) on the activity was noticeable, giving two-fold increase in the specific activity (about 11 U/mg of protein corresponding to 22% of the native urease activity encoded by pTZ19R/ure-1).

• 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.

• Korean Chemical Engineering Research
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• 제51권5호
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• pp.591-596
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• 2013

전통식물한약재에 대한 데이터베이스, 한국식물자원에 대한 문헌 및 선행연구 결과에 근거해 선정한 6가지의 식물 한약재(연교, 소목, 형개, 황련, 소엽, 오매)의 에탄올(70%) 추출물 중에서 오매 추출물이 Helicobacter pylori의 urease 활성에 가장 강한 억제력을 나타내었다. 극성이 다른 용매를 사용한 회분식 용매추출과 칼럼 크로마토그래피를 이용한 3단계 분리공정을 통하여 오매 추출물에 함유된 유효성분을 분리 정제하였다. NMR 분석에 의해 정제된 오매 추출물에 함유된 활성성분이 ursolic acid로 확인되었다. 본 실험결과는 오매에 함유된 이 성분이 항생제를 대신하여 H. pylori 제거에 활용될 수 있음을 시사하였다.

• 공업화학
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• 제18권1호
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• pp.10-16
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• 2007

본 연구에서는 인체의 해로운 요소를 분해하고 분해 생성물인 암모니아($NH_3$)의 제거를 위한 우레아제 고정막 제조와 가수분해 특성에 관하여 검토되었다. 우레아제 고정막은 방사선 그라프트 중합법에 의해 다공성 중공사막에 음이온 교환기로서 DEA를 도입한 후 요소의 가수분해를 위해 우레아제를 고정시켜 제조하였다. 음이온 교환기가 도입된 막에 우레아제가 고정된 경우 그라프트율이 증가할수록 고정량이 증가하였다. 이것은 그라프트율이 증가할수록 그라프트 체인이 신장하여 단백질이 다층으로 고정되기 때문이다. 한편, 가교제의 첨가는 우레아제 고정막의 세척 과정에서 발생하는 탈리 현상을 억제할뿐 아니라 막의 반복사용도 가능하게 하였다. 5 h의 가교 반응을 통하여 탈리현상이 발생되지 않는 우레아제 고정막을 제조하였으며, 이때 제조된 우레아제 고정막은 1 mol과 4 mol의 요소 용액에서 각각 98%와 50% 이상의 가수분해 성능을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1456-1463
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• 2009

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.