• Journal of Microbiology and Biotechnology
• /
• v.23 no.6
• /
• pp.826-832
• /
• 2013

We investigated the transgalactosylation reaction of chlorphenesin (CPN) using ${\beta}$-galactosidase (${\beta}$-gal)-containing Escherichia coli (E. coli) cells, in which galactose from lactose was transferred to CPN. The optimal CPN concentration for CPN galactoside (CPN-G) synthesis was observed at 40 mM under the conditions that lactose and ${\beta}$-gal (as E. coli cells) were 400 g/l and 4.8 U/ml, respectively, and the pH and temperature were 7.0 and $40^{\circ}C$, respectively. The time-course profile of CPN-G synthesis under these optimal conditions showed that CPN-G synthesis from 40 mM CPN reached a maximum of about 27 mM at 12 h. This value corresponded to an about 67% conversion of CPN to CPN-G, which was 4.47-5.36-fold higher than values in previous reports. In addition, we demonstrated by thin-layer chromatography to detect the sugar moiety that galactose was mainly transferred from lactose to CPN. Liquid chromatography-mass spectrometry revealed that CPN-G and CPN-GG (CPN galactoside, which accepted two galactose molecules) were definitively identified as the synthesized products using ${\beta}$-gal-containing E. coli cells. In particular, because we did not use purified ${\beta}$-gal, our ${\beta}$-gal-containing E. coli cells might be practical and cost-effective for enzymatically synthesizing CPN-G. It is expected that the use of ${\beta}$-gal-containing E. coli will be extended to galactose derivatization of other drugs to improve their functionality.

• Journal of Life Science
• /
• v.25 no.10
• /
• pp.1164-1168
• /
• 2015

Previous research showed that chlorphenesin galactoside (CPN-Gal), a preservative in cosmetics, was safer than CPN against human skin cells [9]. To establish a stable and long-term process for CPN-Gal production, we investigated the repeated-batch process. In this process, β-gal-producing recombinant Escherichia coli cells were immobilized in calcium alginate beads, and CPN was converted to CPN-Gal by the transgalactosylation reaction. The process was conducted in a 300 ml flask, which contained E. coli cell-immobilized alginate beads, 33.8 mM of CPN, and 400 g/l of lactose. The pH and temperature were 7.0 and 40℃, respectively. During the repeated-batch operation, four consecutive batch operations were conducted successfully until 192 hr. The conversion yield of CPN to CPN-Gal was 64% during 192 hr, which was higher than the values in previous reports [3, 13]. Thereafter, however, the conversion yield gradually decreased until the operation was finished at 336 hr. Western blotting of immobilized E. coli cells revealed that β-gal gradually decreased after 192 hr. In addition, alginate beads were cracked when the operation was finished. It is probable that, including this loss of E. coli cells by cracks, deactivation, and product inhibition of E. coli β-gal might lead to a gradual decrease in the production of CPN-Gal after 192 hr. However, as the purification of β-gal is not necessary with β-gal-producing recombinant E. coli cells, β-gal-producing E. coli cells might be a practical and cost-effective approach for enzymatically synthesizing CPN-Gal. It is expected that this process will be extended to long-term production process of CPN-Gal for commercialization.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.6
• /
• pp.818-825
• /
• 2013

We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.6
• /
• pp.2819-2822
• /
• 2012

Objective: To assess induction effects of Chlamydia pneumoniae (Cpn) on lung cancer in rats. Methods: A lung cancer animal model was developed through repeated intratracheal injection of Cpn (TW-183) into the lungs of rats, with or without exposure to benzo(a)pyrene (Bp). Cpn antibodies (Cpn-IgA, -IgG, and -IgM) in serum were measured by microimmunofluorescence. Cpn-DNA or Cpn-Ag of rat lung cancer was detected through polymerase chain reaction or enzyme-linked immunosorbent assay. Results: The prevalence of Cpn infection was 72.9% (35/48) in the Cpn group and 76.7% (33/43) in the Cpn plus benzo(a)pyrene (Bp) group, with incidences of lung carcinomas in the two groups of 14.6% (7/48) and 44.2% (19/43), respectively (P-values 0.001 and <0.000 compared with normal controls). Conclusions: A rat model of lung carcinoma induced by Cpn infection was successfully established in the laboratory for future studies on the treatment, prevention, and mechanisms of the disease.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.3
• /
• pp.542-549
• /
• 2010

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin $\beta$-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at $90^{\circ}C$ for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}$ and $50^{\circ}$, respectively. Specifically, the activity of malate dehydrogenase (MDH) at $85^{\circ}$ was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of pro-carboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

• The Transactions of the Korea Information Processing Society
• /
• v.6 no.11
• /
• pp.3070-3076
• /
• 1999

Petri net is one of the effective modeling techniques which analyzes and designs concurrent and asynchronous systems. CPN is an extended Petri net which has color tokens. In this paper, we propose a new test case generation method using CPN. It transforms Estelle Specification into CPN, which is applicable to Design/CPN. It also generates UIO and subtour from OG and descriptor, which are resulted from Design/CPN. Using the proposed method, we can get more improved test coverage than existing methods. Therefore, more effective protocol conformance testing could be conducted. The test case generating method will be the basis of the automatic testing environmented.

• Proceedings of the Korean Information Science Society Conference
• /
• 1998.10a
• /
• pp.258-260
• /
• 1998

CPN(Colored Petri Net)은 시스템을 분석하고 설계하는 모델링 기법인 페트리넷의확장형으로서, 토큰에 color을 부여하여 자료흐름까지 고려할 수 있다. 이 연구에서 Estelle 로 기술된 프로토콜 명세를 Design /CPN에 적용가능한 CPN으로의 변환 방법을 제안하였다. 그리고 변환된 CPN을 Design /CPN에 입력하여 얻은 OG(Occurrence Graph)와 각 노드에 대한 내부 정보인 descriptor를 이용하여, 자료흐름이 고려된 보다 간편하고 효율적인 테스트 케이스를 생성하였다.

• Journal of KIISE:Computing Practices and Letters
• /
• v.5 no.4
• /
• pp.457-466
• /
• 1999

원자력 발전소 계측 제어 시스템, 의료 관련 시스템, 항공 관련 시스템 등 실생활과 밀접한 시스템에 소프트웨어의 사용이 점차 증가하고 있다. 이러한 시스템에서 소프트웨어의 오류는 예기치 않는 사고를 유발하여 인명, 재산상의 심각한 타격을 줄 수 있다. 그러므로 고신뢰도 소프트웨어의 개발 시에는 반드시 시스템의 안전성을 보장해 주어야 한다. 역방향 안전성 분석 방법은 시스템의 안전성을 분석하는 한가지 방법으로서 시스템의 위험 상태를 정의하고 그 위험의 원인들을 추적, 분석함으로써 안전성에 대한 효율적인 분석을 수행할 수 있는 장점을 갖는다. 이 논문에서는 소프트웨어 개발 초기 단계에서 안전성을 분석할 수 있는 방법으로 Colored Petri Nets(CPN)에 기반을 둔 역방향 안전성 분석 방법을 제시한다. 또한 CPN 역방향 안전성 분석 도구인 SAC(Safety Analyzer for CPN)의 설계 및 구현에 대해 언급한다. SAC은 기존의 상용 CPN 모델링 도구인 Design/CPN과 연계하여 사용될 수 있으므로 CPN으로 모델링된 시스템의 안전성을 분석할 수 있다는 장점이 있다. 이 논문에서는 예제로 자동 교통 제어 시스템의 일부를 CPN으로 모델링하고 SAC을 이용한 분석 과정을 기술한다.Abstract In safety-critical systems such as nuclear power plants, medical machines, and avionic systems which are closely related with our livings, the usage of software in the controlling part is growing rapidly. Since software errors in safety-critical systems may cause serious accidents leading to financial or human damages, system safety should be ensured during and after development of a system. A backward safety analysis technique defines system hazards and tries to trace their causes by analyzing system states backward. In this paper, we provide a backward safety analysis technique based on Colored Petri Nets(CPN), which is applicable to the early software development phase. Also Safety Analyzer for CPN(SAC), the supporting tool, is designed and implemented. Since SAC is compatible with Design/CPN, a commercial tool for supporting CPN, it can be applicable to analyze safety in practical problems. As an example, we model a part of the traffic light control system using CPN and analyze safety properties of the model using the SAC tool.

• Journal of Life Science
• /
• v.25 no.2
• /
• pp.130-135
• /
• 2015

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At $25^{\circ}C$ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, $25^{\circ}C$ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

• The Journal of Korean Institute of Communications and Information Sciences
• /
• v.28 no.5B
• /
• pp.489-499
• /
• 2003

This paper is a study about Honey-pot Model using CPN(Colored Petri Nets) that is a method of intrusion detection. Suggested Honey-pot model consists of two parts : \circled1 security kernel module for active induction of hacker's intrusion, intrusion detection and behavior pattern analysis. \circled2 virtual module for activity of induced hackers. However, suggested model was compared and analysed with conventional Denning model and Shieh nodel. The Honey-pot model using CPN can classify the characteristic of intrusion pattern, modeling intrusion pattern and pattern matching procedure, detect DDoS attack through multi hosts, and provide basis of study model for analysing intrusion pattern, finally.