• 한국식품과학회지
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• 제47권5호
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• pp.593-598
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• 2015

This study determined the thermal resistance of Bacillus cereus, Escherichia coli O157:H7, and Listeria monocytogenes in multi-grain soymilk and proposes processing conditions that meet the national standard for retort food products in Korea. D and z values were calculated from thermal inactivation kinetic curves after heating at 55, 60, and $65^{\circ}C$. The D value for B. cereus at $55^{\circ}C$ was the highest (22.8 min), followed by that for E. coli O157:H7 (18.8 min) and L. monocytogenes (17.6 min). At $60-65^{\circ}C$, the order was L. monocytogenes ($D_{60-65^{\circ}C}=3.4-0.9min$), E. coli O157:H7 (3.0-0.3 min), and B. cereus (1.2-0.3 min). The z values for these species were 5.2, 5.5, and $7.7^{\circ}C$, respectively. The Korean national standard for retort food products was achieved by thermal processing at $124{\pm}2^{\circ}C$ for 0.3-2.2 min. This study provides useful data for ensuring both the microbiological safety and product quality of multi-grain soymilk products.

• Current Research on Agriculture and Life Sciences
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• 제17권
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• pp.59-63
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• 1999

벼의 엽록체 small HSP의 고온 스트레스 하에서의 기능을 밝히기 위하여 Oshsp21 cDNA를 pET 발현 vector에 도입하였다. 형질전환된 대장균 배양액에 IPTG를 첨가하여 단백질 발현을 유도시킨 다음 고온 stress 하에서의 생존율을 대조구와 비교하였다. 그 결과 대조균주의 경우 $50^{\circ}C$에서의 생존율이 크게 감소하였으나 Oshsp21이 발현된 대장균의 경우 70% 이상의 생존율을 나타내었다. 또한 대장균 단백질을 $55^{\circ}C$에서 30분간 열처리한 후, 대장균 단백질을 가용성과 비가용성 단백질로 분획한 다음, 각각의 비율을 조사한 결과, 대조균주의 경우 총 단백질의 약 60%가 비가용성 단백질로 변성되었으나, Oshsp21을 발현시킨 대장균의 경우 총 단백질의 약 35%만이 비가용성 단백질로 나타났다. 이러한 결과는 벼의 엽록체 small HSP는 세포내에서 분자 chaperone으로 기능하여 고온내성을 부여할 수 있음을 나타낸다.

• Toxicological Research
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• 제12권2호
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• Journal of Microbiology and Biotechnology
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• 제19권1호
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• pp.72-77
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• 2009

Here, we demonstrate that the overexpression of the GroELS chaperone system, which assists the folding of intracellular proteins and prevents aggregation of its biological targets, can enhance the thermotolerance of Escherichia coli strains and facilitate the production of recombinant protein under thermal stress. The overexpression of GroELS led to an about 2-fold higher growth rate of E. coli XL-1 blue than control at $45^{\circ}C$ and induced the growth of the strain even at $50^{\circ}C$, although the growth was not sustained in the second-round culture. The effect of GroELS overexpression was also effective on other E. coli strains such as JM109, $DH5{\alpha}$, and BL21. Finally, we have shown that coexpression of GroELS allows us to produce recombinant protein even at $50^{\circ}C$, a temperature at which the protein production based on E. coli is not efficient. This study indicates that the employment of the GroELS overexpression system can expand the range of environmental conditions for E. coli.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.305-309
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• 1998

Human cytochrome P450 1A2 is one of the major cytochrome P450s in human liver. It is known to be capable of activating a number of carcinogens such as arylamines and heterocyclic amines. In order to develop the new bacterial mutagenicity test system with human P450, a full length of human P450 1A2 cDNA inserted into pCW bacterial expression vector was introduced to Escherichia coli WP2 uvrA strain which is a well-known E. coli strain for bacterial reverse mutagenicity assay. Expressed human P450 1A2 showed typical P450 hemoprotein spectra. Maximum expression was achieved at 48 hrs after incubating at $30^{\circ}C$ in terrific broth containing ampicillin, IPTG and other supplements. High level expression of P450 1A2 in E. coli WP2 uvrA membranes was determined in SDS-PAGE. The well-known mutagens 2-aminoanthracene and MElQ increased the revertant colonies of E. coli WP2 uvrA expressing human P450 1A2 without an exogenous rat hepatic post-mitochondrial supernatant (S9 fraction) in a dose-dependent manner. The results show that the functional expression of human P450 in bacterial mutagenicity tester strain will provide a useful tool for studying the mechanism of the mutagenesis and carcinogenesis of new drugs and environmental chemicals.

• Journal of Ginseng Research
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• 제5권1호
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• pp.65-72
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• 1981

In this experiment, observations were made on the effects of ginseng saponin, one of the major components of Korean ginseng (Panax ginseng, C. A. Meyer) root, on the membranes of microorganism (E. coli K-12), the concentration of intracelluar and extracellular cycle AMP therein, and uptake of U-14C-glucose. When the E. coli were grown on media containing 0.1% ginseng saponin, the growth was faster than for that of the control by about 30 minutes. The lysis of E. coli grown on the ginseng saponin medium increased to about double that of the control in the stationary phase. And the amount of protein and lipopolysaccharides in the outer cell meberances increased 25% and 80% respectively in comparison with the control. By electron microscope observation, it was shown that the periplasmic region of the E. coli grown on the ginseng saponin medium was widened it was observed that the cellular cyclic AMP content of the E. coli increased significantly to the hightest levels between the late exponential phase and early stationary phase. The total cyclic AMP content of E. coli grown on the ginseng saponin medium decreased about 50% when compared to that of the control.

• Journal of Microbiology and Biotechnology
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• 제1권3호
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• pp.157-162
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• 1991

Conditions for glutathione production in E. coli cells which possess pGH501 (2 gshI+gshII) were studied. In terms of ATP supply for the glutathione synthesis, two different systems have been constructed and compared. When the acetate kinase reaction of E. coli was used for ATP generation, 20 mM of L-cysteine was completely converted to glutathione by toluene-treated E. coli cells (100 mg/ml) harboring pGH501 within 2 h at $37^{\circ}C$. However, considering the economical aspects, the glycolytic pathway of yeast was chosen as a better system for ATP generation. The optimal concentrations of reactants for glutathione production were determined to be as follows; 80 mM L-glutamate, 20 mM L-cysteine, 20 mM glycine, 20 mM $MgCl_2$, 50 mM potassium phosphate buffer (pH 7.5), 400 mM glucose, polyoxyethylene stearylamine ($5\;\mul/ml$), toluene-treated E. coli HB101/pGH501 (100 mg/ml), and dried yeast cells (400 mg/ml). The conversion ratio of L-cysteine to glutathione was 80% (about 5 mg/ml) under optimal condition within 6 h at $37^{\circ}C$.

• 한국식품영양과학회지
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• 제35권7호
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• pp.935-939
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• 2006

V. parahaemolyticus와 E. coli를 접종한 생굴을 대상으로 초고압처리 조건을 달리하여 살균효과를 측정하였다. 처리조건에 따른 V. parahaemolyticus의 균수 변화를 보면, 무처리한 굴의 초기 배양균수는 $3.8{\times}10^5\;CFU/mL$이었는데, $22^{\circ}C$/10분에서 $100{\sim}300\;MPa$로 처리한 결과 처리합력의 증가에 따라 감소하였으며 200 MPa 이상의 처리로 완전히 사멸되었다. $22^{\circ}C/150\;MPa$에서 $5{\sim}30$분으로 처리한 결과 처리시간의 증가에 따라 지속적으로 감소하였다가 25분 이상 처리하였을 때 모두 사멸되었다. 100 MPa,에서 0, $10^{\circ}C$에서 각각 20분과 25분 처리하였을 때 완전히 사멸되었다. 처리조건에 따른 E. coli의 균수변화를 보면, 무처리한 굴의 초기 배양균수는 $4.0{\times}10^7\;CFU/mL$이었는데, $22^{\circ}C/15$분에서 $150{\sim}400\;MPa$로 처리한 결과 처리압력 250 MPa까지는 거의 변화가 없었으나 그 이상의 압력에서는 급혁히 감소하였으며 375 MPa 이상에서는 완전히 사멸되었다. 300 MPa/15분에서 0, 10, $22^{\circ}C$로 처리한 결과 동일한 처리압력과 처리시간에서 처리온도가 낮을수록 살균효과가 더 높았다. $10^{\circ}C$/350 MPa에서 $5{\sim}25$분으로 처리한 결과 처리시간의 증가에 따라 균수가 급격히 감소하였으며, 350 MPa에서 5분처리로 약 4 log cycle 감소하였고 15분 이상의 처리로 검출한계 이하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.550-553
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• 2003

$Hisx_6-GFPuv-Cyt$ c fusion protein을 E. coli 균주 JM109과 BL21 각각에서 발현시킨 결과 발현온도는 $30^{\circ}C$보다 $37^{\circ}C$에서, BL21보다 JM109에서의 발현량이 더 많았다. 그러나 JM109, $37^{\circ}C$에서 발현시킨 fusion protein의 $Ni^{2+}-IDA-agarose$ purification결과 약 45kDa 부근의 fusion protein의 density가 감소되었음을 SDS-PAGE analysis을 통해 알 수 있었다. 또한 western blotting analysis를 통해 이 impurity가 degraded fusion임을 확인 할 수 있었다. degraded fusion은 BL21 균주에서 발현시킨 fusion protein에서도 생성됨을 확인하였다. 모든 결과를 종합해 볼때 $Hisx_6-GFPuv-Cyt$ c fusion protein의 발현은 IM109, $37^{\circ}C$에서 더 많았지만, BL21, $37^{\circ}C$에서 expression시킨 fusion protein이 보다 안정하다고 판단 되어진다. 향후 fusion protein이 bioelectronic device에 적용되려면 degraded fusion protein의 생성을 줄여 activity를 유지하도록 안정한 형태로 발현되어 순수하게 분리정제 되어야 하겠다.

• 대한임상검사과학회지
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• 제50권4호
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• pp.422-430
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• 2018

퀴놀론 항균제가 사람과 동물에게 부적절하고 광범위하게 사용될 경우 항균제내성인자의 출현 및 확산이 가속화 될 수 있다. 본 연구에서는 돼지의 직장면봉 검체(N=40) 및 임상 검체로(N=25)부터 분리된 총 65균주의 nalidixic acid 내성 대장균을 대상으로 quinolone 내성 기전을 조사하였다. 항균제 감수성은 디스크 확산법에 의해 결정되었다. Quinolone 내성과 관련된 유전자와 돌연변이를 조사하기 위해 PCR 및 DNA sequencing이 수행되었다. 총 65균주의 nalidixic acid 내성 대장균 중 62균주가 gyrA, parC, parE 유전자에 돌연변이를 포함하고 있었는데, gyrA 유전자에 돌연변이를 포함하고 있는 균주는 62균주(95.4%)였고, 35균주(53.8%)가 parC 유전자에 돌연변이를 갖고 있었으며, 7균주(10.8%)가 parE 유전자에 돌연변이를 포함하고 있었다. 35균주는 gyrA 와 parC 유전자에 모두 돌연변이를 가지고 있는 것으로 나타났다. 총 65균주의 대장균을 대상으로 plasmid-mediated quinolone resistance (PMQR) determinants를 조사하였다. 65균주의 nalidixic acid 내성 대장균 중 13균주에서 qnrS 유전자가 검출되었으나 이 중 10균주는 gyrA, parC, parE 유전자에 돌연변이를 포함하고 있는 것을 나타났다. 본 연구에서는 사람과 돼지로부터 분리된 대장균이 quinolone 계열 항균제에 내성을 나타내는데 중요한 역할을 하는 기전이 gyrA, parC, parE 유전자에 염색체 돌연변이가 발생하는 경우임을 확인하였는데 이 돌연변이들은 치료목적 또는 동물의 성장촉진을 위한 항균제의 과다사용으로 유발될 수 있다.