• Journal of Microbiology and Biotechnology
• /
• 제24권7호
• /
• pp.925-935
• /
• 2014

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the ${\alpha}/{\beta}$-class of protein consisted of a central six-stranded ${\beta}$-sheet flanked by eight ${\alpha}$-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were $15^{\circ}C$ and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at $50^{\circ}C$ or $60^{\circ}C$ for 6 h, and 89% of its enzyme activity when preincubated at $70^{\circ}C$ for 1 h. The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by $Na^+$ and $Mg^{2+}$ ions but was strongly inhibited by $Cu^+$ and $Hg^{2+}$ ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a $K_m$ of 0.278 mM and a $k_{cat}$ of $1.9s^{-1}$. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

• 한국응용곤충학회지
• /
• 제31권2호
• /
• pp.144-152
• /
• 1992

재래종 밤나무와 저항성 품종 밤나무(축파, 단택)에 기생하는 밤나무 혹벌의 유충에서 유약 호르몬과 유약호르몬 특이에스테라제의 활성도를 Gas liquid chromatohraphy (R ller et al.1965), Galleria cuticle wax 검사 (de Loof & van Loon 1981) 및 Liquid scintilation counter (Hammock & Sparks 1977)로 비교 측정한 결과는 다음과 같다. 유충에서 GLC로 분리한 유약호르몬은 양군 모두에서 유약호르몬 I(JH-I)로 동정되었다. 저항성 품종(축파, 단택)에 기생하는 밤나무 혹벌의 유약호르몬 함량은 35,800 GU/g와 30,900 GU/g이고, 재래종에 기생하는 밤나무 혹벌의 유약호르몬의 함량은 28,600 GU/g으로 나타났다. 유약호르몬 특이 에스테라제 활성도는 저항성 품종(축파, 단택)에 기생하는 밤나무 혹벌은 1.48 n mole/min/ml와 1.63 n mole/min/ml, 그리고 재래종에 기생하는 밤나무 혹벌은 1.89 n mole/min/ml로 측정되었다. 유약호르몬 함량은 재래종 밤나무에 기생하는 밤나무 혹벌보다 저항성 밤나무(축파, 단택)에 기생하는 밤나무 혹벌에서 더 높았으나, 유약호르몬 특이에스테라제 활성도는 저항성 밤나무(축파, 단택)보다 재래종 밤나무에 기생하는 혹벌에서 더 높게 나타나 서로 역비례 경향을 나타냈다.

• 한국응용곤충학회지
• /
• 제33권2호
• /
• pp.74-80
• /
• 1994

바퀴에 대한 dichlorvos(DDVP) 약제로 11세대 누대 도태한 계통에 대한 저항성 유발정도, 교차저항성 및 효소활성을 조사한 결과는 반수치사농도 비교시 감수성에 비하여 8.58배 증가하였고 교차정항성은 chlorpyrifos, propowur, fenvalerate 와 permethrin 약제에서 각각 3.35, 4.09, 2.83 및 2.00배의 저항성을 보였으며 효소활성은 filter paper testdptjrka수성 대비 33%가 증가하였고 전기영동시험에서는 4개의 band인 esterase-1,2,4 및 7 band 가 확인되었다.

• Journal of Microbiology and Biotechnology
• /
• 제21권7호
• /
• pp.667-674
• /
• 2011

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to the class IV family. The purified enzyme worked optimally at $50^{\circ}C$ and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate ($C_4$), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.

• 농약과학회지
• /
• 제11권3호
• /
• pp.179-185
• /
• 2007

북방수염하늘소(M. saltuarius) 성충 장(gut)내에 존재하는 소화액에 대한 에스테라제(esterase)의 활성 변화를 in vitro에서 조사하였다. ${\alpha}$-naphthyl acetate와 ${\alpha}$-naphthyl butyrate 기질을 이용한 실험에서는 4개의 밴드가 관찰되었다. Carbofuran과 methomyl을 비교 처리한 결과 methomyl에서 강한 효소활성 저해가 관찰되었다. 특히 Est1은 carbofuran과 methomyl에 의해 모두 저해되는 것으로 나타났다. Chlorpyrifos, methidation, phenthoate를 비교 처리한 결과 chlorpyrifos에 의해 모든 밴드들이 저해되는 것을 볼 수 있었다. 특히 Est1은 chlorpyrifos에 의해 효소활성이 완전히 저해되었고, methidation 처리시 약 70% 정도의 효소활성이 저해 되었다. Eserine을 처리한 결과 Est1은 약 70% 정도의 활성 저해를, Est2, Est3, Est4에서는 미미한 효소활성 저해가 나타났다. ${\alpha}$-pinene에서는 뚜렷한 효소활성 저해는 관찰할 수 없었으나 bornyl acetate와 camphor에서는 미미한 효소활성 저해가 관찰되었다.

• 한국초지조사료학회지
• /
• 제11권3호
• /
• pp.158-161
• /
• 1991

Horizontal starch gel electrophoresis, follow by enzyrne-specific staining, separate and visualize several legume esterase isozyme. Using extracts prepared from cotyledon, radicle and plumule of legume seedlings germinated 5 days. The results were as follows. 1. The number and staining intensity of esterase isozyme bands varies depending on the plant species. tissues and developmental stage. 2. Bands in the cotyledon of field bean seedling expressed 4 and 1 in radicle. 3. In soybean cultivars, cotyledon of IIwangkum-kong had 3 bands and 1 band in the examined tissues of Paldal-kong and Jangkyung-kong seedling. 4. The cotyledon and radicle of french bean seedling had 3 bands, respectively. 5. The highest esterase isozyme activity appears to be expressed in the cotyledon and radicle of french bean, as indicated by intensity of stain, with the Paldal-kong particulary active.

• Journal of Microbiology and Biotechnology
• /
• 제6권3호
• /
• pp.173-178
• /
• 1996

Synergic effects among endo-xylanase, $\beta$-xylosidase, and acetyl xylan esterase of Bacillus stearothermophilus in the hydrolysis of xylan were studied by using birchwood, oat spelt, and acetylated xylan as substrates. Synergism between endo-xylanase and $\beta$-xylosidase was observed on all three substrates tested, indicating that $\beta$-xylosidase enhanced the production of xylose by relieving the end-product inhibition upon endo-xylanase conferred by xylooligomers. Endo-xylanase and $\beta$-xylosidase also showed synergism with acetyl xylan esterase in the hydrolysis of birchwood and acetylated xylan, while no synergic effect was detected in oat spelt xylan hydrolysis. Thus, the hydrolysis of xylan containing acetic acid side chains required the action of acetyl xylan esterase, which eliminated the steric hindrance of the side chains, leading to the better hydrolysis by endo-xylanase and $\beta$-xylosidase , and the acetyl xylan esterase activity was also enhanced by endo-xylanase and $\beta$-xylosidase for the latter enzymes provided acetyl xylan esterase with shorter xylan oligomers, the better substrate for the enzyme.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.625-628
• /
• 2001

토양으로부터 특이적인 광학 이성질체에 활성을 갖는 두 효소원을 선별한 후, 동정하여 Pseudomonas sp. S34와 B. stearothermophilus JY144로 명명하였고, genecloning과 sequencing을 통해 유전자의 특성 및 관련효소들과의 유연관계를 규명하였다. 규명된 정보의 분석과 비교를 통해 ketoprofen ethyl ester에 활성을 갖는 효소들의 구조적 특성을 추론할 수 있었고, 이를 바탕으로 발현시스템을 구축하였다. 대량생산된 효소를 활용한 반응의 결과 높은 수율과 순도를 갖는 각각의 광학이성질체를 경제적으로 생산할 수 있었다.

• Journal of Plant Biology
• /
• 제36권1호
• /
• pp.51-57
• /
• 1993

The microtuber was efficiently formed on SH medium containing 9% sucrose from the in vitro propagated shoot of potato (Solanum tuberosum cv. Sumi). In order to investigate gene expression depending on the development stage of microtuber, we examined the changes of peroxidase and esterase activities, and their isozyme patterns as well. Peroxidase and esterase activities were the highest at the 7 day-culture of the microtuber and subsequently decreased on the stage of microtuberization, whereas esterase activity increased at the stage of 60 day-culture. However, their activities in the ordinary tuber were higher than those of 60 day-cultured microtuber. In addition, in the peroxidase isozyme pattern two new bands of pI 7.05 and pI 4.65 were appeared at the 15- day and 60 day-cultures, respectively, as shown by isoelectric focusing. Various bands in the sterase isozyme pattern were shown at the 7 day-culture, and the band patterns were a large difference, comparing those of shoot and tuber. New bands in the esterase isozyme pattern also appeared at the 15 day- (pI4.52) and 60 day-cultures (pI 4.48). These results suggest that the changes of peroxidase and esterase activities and isozyme patterns are an important factor in the differentiation and development of potato.

• 한국임상수의학회지
• /
• 제7권1호
• /
• pp.429-438
• /
• 1990

건강한 개의 혈액세포와 골수세포의 세포화학적 특성을 조사하였다. 이 실험에서 혈액과 골수 시료에 항응고제를 일체사용하지 않았으며 시료채취후 즉시 도말표본을 작성하여 30분 이내에 반응을 실시하였다. 이 실험의 결과는 아래와 같다. 1. alkaline phosphatase의 활성은 호산구계통과 간혹 전골수구에서 양성반응을 나타내었다. 2. acid phosphatase의 활성은 대부분의 계통의 세포에서 양성반응을 나타내지만 tartrate로 억제하면 호산구계만 양성반응을 나타내었다. 3. peroxidase 활성은 골수구계통의 모든 세포에서 양성반응을 나타내며 단구에서는 미약한 양성의 미세과립을 나타내었다. 4. naphthyl-AS-D-chloroacetate esterase활성은 호중구계 세포에서만 양성을 나타낸다. 5. $\alpha$-naphthyl acetate esterase활성은 단구와 일부의 임파구에서 양성을 나타낸다 6. Sudan black B 염색은 골수구계 세포와 단구계 세포에서 양성을 나타내었다. 7. $\beta$-glucuronidase 활성은 적혈구계를 제외한 모든 세포에서 양성반응을 나타내었다.