Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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An Efficient Method for the Release of Recombinant Penicillin G Amidase from the Escherichia coli Periplasm

대장균의 periplasm으로부터 재조합 PGA 단백질의 효율적이고 간단한 방출 방법

  • 이상만 (청주대학교 생명과학과)
  • Received : 2017.07.24
  • Accepted : 2017.10.23
  • Published : 2017.10.30
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Abstract

In this study, we report on a simple, efficient method for obtaining penicillin G amidase (PGA) from recombinant Escherichia coli using a formulation mixed with detergent and lysozyme. Research was conducted on the extraction efficiency of PGA from the periplasmic space in cells in terms of the type of detergent, detergent concentration, pH, reaction time, and temperature of permeabilization. The extraction yield of PGA in the formulated surfactant/lysozyme treatment was increased by approximately (55-65 U/ml) in comparison with that in the single surfactant treatment. The released PGA solution was concentrated and exchanged with buffer using an ultrafiltration (U/F) system. The yields of diatomite filtration, membrane filtration (M/F), and U/F were 69.7%, 93.8%, and 77.3%, respectively. A total of 212 KU of PGA was recovered. At the 25-L culture scale, the overall yield of extraction using the mixed surfactant/lysozyme method was 49.2%. The specific activity of extracted PGA was 11 U/mg in protein. The concentrated PGA solution was immobilized on microporous silica beads without further purification of PGA. The total immobilization yield of PGA on the resin was 48.7%, while the enzyme activity was 101 U/g. The immobilized PGA was successfully used to produce 6-APA from penicillin G. Our results indicated that a simple extraction method from periplasmic space in E. coli may be used for the commercial scale production of ${\beta}-lactam$ antibiotics using immobilized PGA.

세제에 의하여 대장균의 periplasm에서 penicillin G amidase (PGA)를 방출하는 방법을 연구하였다. 결과적으로 세제와 lysozyme의 혼합 작용이 효과적인 것으로 나타났다. 세포 투과성의 최적 조건을 알아보기 위하여 세제의 종류, 농도, pH, 반응 시간, 온도 등의 영향을 살펴보았다. 그리하여 대장균에서 재조합 PGA를 periplasm에서 방출하는 모델을 만들 수 있었고 방출된 PGA를 농축할 수 있었다. 실리카 구슬을 이용한 고정화 시스템으로 PGA 용액을 농축할 수 있었으며, 더 이상의 정제 과정 없이 순수하게 추출 할 수 있었다. 고정화된 PGA는 penicillin G 생성의 원료인 6-APA를 생산하는데 사용할 수 있었다. 이 방법은 대장균으로부터 재조합 단백질을 추출하는 간단한 방법이며 고정화 PGA를 이용하여 ${\beta}-lactam$ 항생물질의 산업적 생산 이용될 수 있을 것으로 사료된다.

Keywords

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