• 생명과학회지
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• 제10권4호
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• pp.397-402
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• 2000

본 연구는 인산 축적능이 뛰어난 균주를 분자 육종하여 생물학적 폐수처리 및 토양의 인산 집적을 해결시키는 산업적 유용한 재료로 이용하기 위한 기초연구를 목표로 하고 있다. Polyphosphate kinase의 ATP의 phosphate를 단리하여 한분자씩 결합시키는 형태로 polyphosphate의 합성반응을 촉매한다. 인산 축적에 관한 대사과정의 분자적 이해를 위하여 Serratia marcescens균주로부터 Southern hybridization방법으로 ppk를 암호하는 유전자를 찾아내어 새조합시킨 pDH3를 구축하였다. pDH3으로부터 ppk를 암호하는 유전자 영역의 4.0 kb 단편을 가진 subclone을 작성하였다.Serratia marcescens의 polyphosphate kinase의 활성은 catabolite repression에 의한 조절을 받았다. 발현멕타에 삽입시킨 재조합 플라스미드를 대장균에 도입시킨 결과, polyphosphate kinase의 효소활성이 크게 증가됨을 확인 하였다. 또한 대량 발현시킨 결과를 SDS-PAGE를 통하여 75 KDa의 발현산물을 확인할 수 있었다.

• BMB Reports
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• 제44권6호
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• pp.387-392
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• 2011

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.584-590
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding ${\beta}-xylosidase$, a major xylanolytic enzyme, was previously cloned and sequenced by the present authors. Sequence analysis indicated that translation of the xylA gene was initiated from the noncanonical initiation codon UUG, confirmed by analyzing three different amber (UAG) mutants of the xylA gene. In the present study, the UUG initiation codon was mutated into AUG or GUG, and the effects of the mutations on the XylA synthesis were examined. The AUG initiation codon was found to direct the highest level of ${\beta}-xylosidase$ synthesis; three-fold and fourteen-fold more enzyme activity than the UUG codon in E. coli and B. subtilis cells, respectively. Surprisingly, contrary to other systems reported to date, the UUG start codon was found next to AUG in the relative order of translational efficiency in both organisms. In addition, a greater abundance of the xylA mRNA was detected with the AUG start codon in both of these host cells than with GUG or UUG. Northern blot and Toeprint assays revealed that this was due to enhanced stability of mRNA with the AUG initiation codon. As expected, the ${\beta}-xylosidase$ protein level in the bacterial cells containing mRNA with the AUC start codon was also much higher than the levels with the other two different mRNAs.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2016년도 제50회 동계 정기학술대회 초록집
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• pp.230.2-230.2
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• 2016

A new approach for antimicrobial is based on the overproduction of reactive nitrogen species (RNS), especially; nitric oxide (NO) and peroxinitrite ($ONOO^-$-) are important factors to deactivate the bacteria. Recently, non-thermal atmospheric pressure plasma jet (APPJ) has been frequently used in the field of microbial sterilization through the generation of different kinds of RNS/ROS species. However, in previous study we showed APPJ has combine effects ROS/RNS on bacterial sterilization. It is not still clear whether this bacterial killing effect has been done through ROS or RNS. We need to further investigate separate effect of ROS and RNS on bacterial sterilization. Hence, in this work, we have enhanced NO production, especially; by applying a 1% of HNO3 vapour to the N2 based APPJ. In comparison with nitrogen plasma with inclusion of water vapour plasma, it has been shown that nitrogen plasma with inclusion of 1% of HNO3 vapour has higher efficiency in killing the E. coli and different type of cancer cell through the high production of NO. We also investigate the enhancement of NO species both in atmosphere by emission spectrum and inside the solution by ultraviolet absorption spectroscopy. Moreover, qPCR analysis of oxidative stress mRNA shows higher gene expression. It is noted that 1% of HNO3 vapour plasma generates high amount of NO for killing bacteria and cancer cell killing.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2015년도 제49회 하계 정기학술대회 초록집
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• pp.159-159
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• 2015

A new approach for antimicrobial is based on the overproduction of reactive nitrogen species (RNS), especially; nitric oxide (NO) and peroxinitrite (ONOO-) are important factors to deactivate the bacteria. Recently, non-thermal atmospheric pressure plasma jet (APPJ) has been frequently used in the field of microbial sterilization through the generation of different kinds of RNS/ROS species. However, in previous study we showed APPJ has combine effects ROS/RNS on bacterial sterilization. It is not still clear whether this bacterial killing effect has been done through ROS or RNS. We need to further investigate separate effect of ROS and RNS on bacterial sterilization. Hence, in this work, we have enhanced NO production, especially; by applying a 1% of HNO3 vapour to the N2 based APPJ. In comparison with nitrogen plasma with inclusion of water vapour plasma, it has been shown that nitrogen plasma with inclusion of 1% of HNO3 vapour has higher efficiency in killing the E. coli through the high production of NO. We also investigate the enhancement of NO species both in atmosphere by emission spectrum and inside the solution by ultraviolet absorption spectroscopy. Moreover, qPCR analysis of oxidative stress mRNA shows higher gene expression. It is noted that 1% of HNO3 vapour plasma generates high amount of NO for killing bacteria.

• Journal of Periodontal and Implant Science
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• 제38권sup2호
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• pp.335-342
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• 2008

Purpose: Cytolethal distending toxin (CDT) considered as a key factor of localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis is composed of five open reading frames (ORFs). Among of them, the individual role of CdtA and CdtC is not clear; several reports presents that CDT is an AB2 toxin and they enters the host cell via clathrin-coated pits or through the interaction with GM3 ganglioside. So, CdtA, CdtC, or both seem to be required for the delivery of the CdtB protein into the host cell. Moreover, recombinant CDT was suggested as good vaccine material and antibody against CDT can be used for neutralization or for a detection kit. Materials and Methods: We constructed the pET28a-cdtC plasmid from Aggregatibacter actinomycetemcomitans Y4 by genomic DNA PCR and expressed in BL21 (DE3) Escherichia coli system. We obtained the antibody against the recombinant CdtC in mice system. Using the anti-CdtC antibody, we test the native CdtC detection by ELISA and Western Blotting and confirm the expression time of native CdtC protein during the growth phase of A. actinomycetemcomitans. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and generated the anti CdtC antibody against recombinant CdtC subunit expressed in E. coli system. Our anti CdtC antibody can be interacting with recombinant CdtC and native CDT in ELISA and Western system. Also, CDT holotoxin existed at 24h but not at 48h meaning that CDT holotoxin was assembled at specific time during the bacterial growth. Conclusion: In conclusion, we thought that our anti CdtC antibody could be used mucosal adjuvant or detection kit development, because it could interact with native CDT holotoxin.

• 한국축산식품학회지
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• 제37권1호
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• pp.134-138
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• 2017

Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (${\underline{s}}pheroplast$ ${\underline{p}}rotein$ ${\underline{y}}$). Spy is a chaperone protein expressed and localized in the periplasm of E. coli during spheroplast formation, or by exposure to protein denaturing conditions. spy expression in S. typhimurium was examined by constructing a spy-gfp transcriptional fusion. S. typhimurium spy transcription was strongly induced during spheroplast formation, and also when exposed to membrane-disrupting agents, including ethanol and the antimicrobial peptide polymyxin B. Moreover, spy induction required the activity of regulator proteins BaeR and CpxR, which are part of the major envelope stress response systems BaeS/BaeR and CpxA/CpxR, respectively. Results suggest that monitoring spy transcription may be useful to determine whether a molecule particularly cause envelope stress in Salmonella.

• 한국축산식품학회지
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• 제39권4호
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• pp.601-609
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• 2019

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

• Journal of Microbiology
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• 제44권3호
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.