• 한국미생물·생명공학회지
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• 제36권4호
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• pp.314-319
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• 2008

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.231-236
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• 2004

The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at $26^{\circ}C$. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa ($\alpha$) and 62 kDa ($\beta$) subunits were recovered by using IMAC with just C-terminus tagging of the $\beta$ subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

• 대한환경공학회지
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• 제29권7호
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• pp.833-838
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• 2007

본 연구에서는 양전하를 띤 여재 입자에서 박테리아의 부착 및 이동에 관한 연구를 위하여 회분 및 칼럼실험을 수행하였다. 본 실험에 사용된 박테리아는 E. coli ATCC 11105(길이: 2.2 ${\mu}m$ 직경: 0.6 ${\mu}m$)이었고, 여재는 석영모래(입경분포: 0.5-2.0 mm, 평균 입경: 1.0 mm)와 철코팅 모래이었다. 회분실험결과에 의하면 철코팅 함량이 증가함에 따라, 박테리아의 부착량이 증가하는 것으로 나타났다. 철코팅 모래의 함량 0%(석영모래 100%)에서는 약 46% 정도의 박테리아가 부착되었고, 철코팅 모래의 함량 100%(석영모래 0%)에서는 약 97% 정도의 박테리아가 부착되었다. 칼럼실험결과 또한 철코팅 함량이 증가함에 따라, 박테리아의 부착량이 증가하는 것을 보여주고 있다. 철코팅 모래의 함량이 0에서 100%로 증가함에 따라 박테리아의 부착량이 8에서 94%로 증가하였다. 본 연구의 실험결과를 통해 양전하를 띤 여재 입자가 박테리아의 이동에 영향을 미칠 수 있음을 알 수 있다.

• Environmental Engineering Research
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• 제13권3호
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• pp.141-146
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• 2008

Transport of Escherichia coli ATCC 11105 through porous media was investigated in this study using two sets of column experiments to quantify the attachment-related parameters (sticking efficiency, attachment rate coefficient and filter factor). The first set of experiments was performed in quartz sand under different ionic strength conditions (1, 20, 100, 200 mM) while the second experiments were carried out in quartz sand mixed with metal oxyhydroxide-coated sand (0, 5, 10, 25%). The breakthrough curves of bacteria were obtained by monitoring effluent, and then bacterial mass recovery and attachment-related parameters were quantified from these curves. The first experiments showed that the mass recoveries were in the range of 13.3 to 64.7%, decreasing with increasing ionic strength. In the second experiments, the mass recoveries were in the range of 15.0 to 43.4%, decreasing with increasing coated sand content. The analysis indicated that the sticking efficiency, attachment rate coefficient and filter factor increased with increasing ionic strength and coated sand content. The value of filter factor in the first experiments ranged from 1.45 e-2 to 6.72 e-2 1/cm while in the second experiments it ranged from 2.78 e-2 to 6.32 e-2 1/cm. Our filter factor values are one order of magnitude lower than those from other studies. This discrepancy can be attributed to the size of sand used in the experiment. The analysis demonstrated that the travel distance of bacteria estimated using the filter factor can be varied greatly depending on the solution chemistry and charge heterogeneity of porous media.