• 한국미생물·생명공학회지
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• 제30권4호
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• pp.305-311
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• 2002

Aspergillus ficuum 유래 acetyl xylan esterase(AXEase) 유전자(AXE)를 Pichia pastoris에서 과발현ㆍ분비 생산하기 위해 AOXI promoter와 mating factor $\alpha$-1 분비신호서열 하류에 AXE를 연결한 염색체 삽입 발현계(pPICZ$\alpha$C-AXE, 4.6 kb)를 구축하였다. 이것을 SacI으로 절단한 뒤 P. pastoris의 염색체 DNA 5'AOX1 부위에 삽입시켰다. 형질전환된 P. pastoris 균주를 메탄을 배지에서 플라스크 회분배양한 결과, 배양 36시간 때의 건조균체농도는 6 g-DCW/1, AXEase 총 발현량은 77 unit/ml이었다. 최적화된 methanol과 histidine 공급방법을 채용한 유가배양시 균체농도는 97 g-DCW/1, AXEase 총발현량은 930 unit/m1로 크게 증가하였다. 효소활성의 90% 이상은 배양 상등액에 존재하였으며, 상등액 단백질의 80%이상이 AXEase 단백질(33.5 kDa)였다. 이러한 결과는 9.8 g/l의 AXEase 단백질을 배양 상등액으로 대량 분비ㆍ생산할 수 있음을 의미한다.

• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.311-318
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• 2013

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1484-1489
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• 2014

A novel esterase gene, est01, was successfully unearthed from a biogas digester microbiota metagenomic library. The 1,194 bp est01 gene encodes a protein of 44,804 Da (designated Est01). The amino acid sequence of Est01 shows only moderate (33%) identity to a lipase/esterase. Phylogenetic analysis and biochemical characterization confirmed that Est01 is a new member of family VIII esterases. The purified Est01 from recombinant Escherichia coli BL21 (DE3) showed high hydrolytic activity against short-chain fatty acid esters, suggesting that it is a typical carboxylesterase rather than a lipase. Furthermore, the Est01 was even active at $10^{\circ}C$ (43% activity remained), with the optimal temperature at $20^{\circ}C$, and had a broad pH range from 5.0 to 10.0, with the optimal pH of 8.0. These properties suggest that Est01 is a cold-adaptive esterase and could have good potential for low-temperature hydrolysis application.

• Asian-Australasian Journal of Animal Sciences
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• 제33권6호
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• pp.949-956
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• 2020

Objective: This study was conducted to confirm the effects of new inoculants producing-antifungal or esterase substances on rye silage and its rumen fermentation indices by comparing wild with mutated types. Methods: Rye harvested at dough stage was ensiled into 3 L mini bucket silo (1 kg) for 90 d in triplicate following: distilled water at 20 μL/g (CON); Lactobacillus brevis 100D8 (AT) and its inactivation of antifungal genes (AT-m) at 1.2×10⁵ cfu/g, respectively; and Leuconostoc holzapfelii 5H4 (FD) and its inactivation of esterase genes (FD-est) at 1.0×10⁵ cfu/g, respectively. After silo opened, silage was sub-sampled for the analysis of ensiling quality and its rumen fermentation indices. Results: Among the wild type inoculants (CON vs AT vs FD), FD inoculant had higher (p<0.05) in vitro digestibilities of dry matter and neutral detergent fiber, the total degradable fraction, and total volatile fatty acid in rumen, while AT inoculant had higher (p<0.05) lactate, acetate, and lactic acid bacteria in silage. Silage pH and the potentially degradable fraction in rumen increased (p<0.05) by inactivation of antifungal activity (AT vs AT-m), but lactate, acetate, and lactic acid bacteria of silage decreased (p<0.05). In silage, acetate increased (p<0.05) by inactivation of esterase activity (FD vs FD-est) with decreases (p<0.05) of pH, ammonia-N, lactate, and yeast. Moreover, inactivation of esterase activity clearly decreased (p<0.05) in vitro digestibilities of dry matter and neutral detergent fiber, the total degradable fraction, and total volatile fatty acid in the rumen. Conclusion: This study concluded that FD inoculant confirmed esterase activity on rye silage harvested at dough stage, while AT inoculant could not be confirmed with antifungal activity due to the absence of mold in all silages.

• The Korean Journal of Ecology
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• 제20권2호
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• pp.103-109
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• 1997

Eleven phenolic compounds including caffeic acid were identified through analyzing the aqueous extracts of Pinus rigida by HPLC. Among them, protocatechuic acid was the maximum amount of 6.84 ppm. Seed germination of Cassia mimosoides var. nomame was significantly stimulated by the extract of P. rigida leaves in the proportion ot concentration. However, root growth was elevalted at a threshold concentration below 25%, but it was inhibited at high concentrations. In 50% extract of P. rigida, upward root tip of C. mimosoides var. nomame showed negageotropism which the root end showed necrosis. New isozyme bands were induced indicating concentration activity of peroxidase from the extract of C. mimosoides var. nomame, especially in the cathodic region. Although it reduced the mumber of isozyme bands of esterase, esterase activities were stimulated in the anodic region of C. mimosoides var. nomame. The activity of amylase was not remarkably different between control and treatment.

• 한국응용곤충학회지
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• 제43권4호
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• pp.317-322
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• 2004

배추좀나방의 발육단계별 indoxacarb의 살충활성, 침투이행성 및 잔효성을 조사하였고, esterase, acetylcholinesterase, glutathione S-transferase 등의 효소활성에 미치는 영향을 검토하였다. 배추좀나방 유충에 대해서 높은 살충효과를 나타내었으나, 알과 번데기에 대해서는 살충율이 $10\%$이하이었다. 엽면침투이행성과 근부침투이행성의 효과는 없었으며, 잔효성은 10일째까지 $80\%$의 살충효과를 유지하였다. Indoxacarb는 esterase와 glutathione S-transferase의 활성은 저해하지 않았지만 acetylcholinesterase의 활성을 저해하였다.

• 한국동물학회지
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• 제39권2호
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• pp.190-197
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• 1996

The optimal conditions and substrate specificity of whole body esterase (EST) activity, effects of inhibitors (Eserine, Paraoxon, p-HMB, DDVP, DFP) on the enzyme, and ontogenv of the isozymes were determined in Lucilio ilfustris Meisen. The optimal temperature was $45^{\circ}C$ regardless of kind of reacted substrate, $\alpha-naphthyl$ acetate $(\alpha-Nal,$ a.naphthvl butylate $(\alpha-N),$ and Pnaphthyl acetate $(\beta-Na),$ but the optimal pH showed some regioselectivitv to naphthvl group of the esters; PH 7.0 for Iform, pH 7.5 for a-form. The maximum reaction rate was recorded at about 2.5 $\times$ 10's M of PNa and etNa, but 1.0 $\times$ 10'S M of $\alpha-Nb.$ Among the five EST inhibitors tested, DDVP was the most powerful. However, distinction of the relative specificity of inhibitors between three body parts, head, thorax, and abdomen, was shouts, representing differences in the distribution and activity of isozvmes. Of 12 carboxyl-esterases (CE), 8 cholinesterases (ChE) and 2 arvlesterases (ArE) identified based on their inhibitor specificity throughout the development, two larval and prepupal stage specific ChEs, no pupal specific, and 2 CEs.2ChEs. and one ArE adult specific isozvmes were confirmed.

• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.37-42
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• 1997

A bacterial strain L1 producing a thermostable esterase was isolated from soil taken near a hot spring and identified as Bacillus stearothermophilus by its microbiological properties. The isolated thermostable esterase was purified by ammonium sulfate fractionation, ion .exchange and hydrophobic interaction chromatographies. The molecular weight of the purified enzyme was estimated to be 50,000 by SDS-PAGE. Its optimum temperature and pH for hydrolytic activity against PNP caprylate were $85^{\circ}C$ and 9.0, respectively. The purified enzyme was stable up to $70^{\circ}C$ and at a broad pH range of 4.0-11.5 in the presence of bovine serum albumin. The enzyme was inhibited by phenylmethylsulfonyl fluoride and diethyl p-nitrophenyl phosphate, indicating the enzyme is a serine esterase. The enzyme obeyed Michaelis-Menten kinetics in the hydrolysis of PNPEs and had maximum activity for PNP caproate ($C_6$) among PNPEs ($C_2-C_12$) tested.

• 한국응용곤충학회지
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• 제32권3호
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• pp.348-353
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• 1993

포장의 약제저항성 변동요인을 조사하기 위하여 복수아혹진딧물의 Carboxyl esterase(CE) 활서측정에 의한 저항성도의 변동상황을 조사하였다. 비닐하우스에서 고추육묘중 발생한 복수아혹진딧물의 CE활성 측정 결과 약제저항성도는 무처리구가 40이였으나 아세페이트를 처리한구는 78로 약제처리로 인한 저항성도가 38이 증가하였다. 또한 노천망실에서 약제처리를 하지 않고 재배한 케일에 발생한 복숭아혹진딧물의 CE활성에 의한 저항성도는 7월이 24이었으나, 8, 9월의 활성이 계속 증가하여 10월이 83으로서 최고에 달하였다가 11월에는 다시 81에서 79로 약천 떨어지고 있어 약제이외에 저항성도의 자연변동요인이 있음을 확인하였다. 전국적으로 18개장소에서 채집한 진딧물의 CE활성을 측정한 결과 저항성도의 평균이 여름은 50$\pm$14였으며 늦가을인 11월은 82$\pm$10으로서 여름보다 늦가을인 11월이 32가 높았으며 그중에도 약제를 살포사지 않은 곳 보다 약제살포 회수가 많은 곳일수록 높았다.

• 한국미생물·생명공학회지
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• 제45권2호
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• pp.149-154
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• 2017

본 연구에서는 Bacillus 유래 esterase를 생산할 수 있는 재조합 대장균을 사용하여 유가식 배양을 이용한 고농도 균체 배양을 통해 esterase 생산성을 극대화하고자 하였다. 유가식 배양 중 순수 산소의 공급을 통해 용존산소를 30% 이상 유지한 경우와 포도당농도를 1 g/l 이상 유지한 경우 각각 $OD_{600}$ 76 (35.8 g/l DCW)과 $OD_{600}$ 90 (42.4 g/l DCW)까지 균체량을 증가시킬 수 있었다. 포도당의 공급에도 불구하고 배양 후반에 세포의 성장이 정체되는 현상을 극복하기 위해 yeast extract가 강화된 추가 배지의 공급을 시도하였으며, 그 결과 $OD_{600}$ 185 (87.3 g/l DCW)까지 고농도 균체 배양이 가능함을 확인하였다. 단백질 생산 수율의 향상을 위해 성장 시기에 따라 induction에 의한 세포 성장과 esterase 생산성을 평가하였고, 그 결과 대수 성장기 후반에 induction을 유도한 경우 세포 성장 측면에서는 최대 $OD_{600}$ 190(89 g/l DCW)까지 고농도 균체 배양이 가능함을 확인하였다. Esterase 생산성 측면에서는 대수 성장기 초반에 induction 을 유도한 경우에 비해 최대 5.8배 생산성이 증가됨을 확인할 수 있었다. 따라서 본 연구를 통해 순수산소와 질소원의 공급을 통해 확립된 대장균 고밀도 배양방법을 기초로 IPTG 유도시간을 최적화 함으로써 Bacillus 유래 esterase의 최대 생산성을 확보할 수 있는 배양방법을 확립하였다.