Streptomyces. orientalis에 의해 생산된 vancomycin의 정제

정형무,현형환 한국외국어대학교 기초과학연구소 2001 기초과학연구 Vol.11 No.-

vancomycin은 glycopeptide계 항생제로서 세포벽에 작용하기 때문에 외부로 분비되지만 일부는 자신의 세포벽에 결합되어 있다 따라서 발효액으로부터 균체를 제거할 때 vancomycin 회수율을 향상시키기 위해 발효액의 pH 및 회석배율에 따른 vancomycin 회수율을 조사한 결과, 발효액을 10배 이상 희석하고 pH 2.0으로 보정하여 균체를 제거 하였을 때 가장 높은 vancomycin 회수율을 나타내었다. 그러나 전체적인 정제 공정을 고려하여 2-3배 희석하고 pH 2.0으로 보정할 때 발효원액 자체를 여과할 때보다 75% 높은 회수율을 나타냈다. Vancomycin을 정제하기 위해 강산성 양이온 교환수지인 Deuolite C20과 약염기성 음이온 교환 수지인 Deuolite A56l 및 강염기성 음이온 교환수지 Diaion PA406에 대하여 탈색, 순도 및 수율을 조사하였다. 탈색, 수율 및 순도에서 C20(Na+)과 PA406(Cl-)이 효과적이었다. 양이온 교환 수지 및 음이온 교환 수지를 통과한 액을 비이온성 흡착수지 HP-20에 통액하여 vancomycin을 흡착시킨 후, 30% 메탄올로 용출하였을 때, 순도 94%의 vancomycin을 수율 77.5%로 분리할 수 있었다. HP20으로부터 분리한 vancomycin 분획에 남아 있는 color물질 및 pyrogen을 제거하기 위해 vancomycin 활성분획을 alumina에 통액한 결과, 발효 여액대비 수율은 90%이었고 순도는 91%로 증가하였으며 endotoxin은 0.312EU/mg-vancomycin 이었다. Alumina의 vancomycin 분획을 50mg/ml로 농축하여 CM-Toyopearl(NH4+)에 통액하고 0.5N HCl로 vancomycin을 용출시킨 결과, 발효 여액에 대한 수율은 87%였고 순도 99%까지 향상시킬 수 있었으며 endotoxin은 0.184EU/mg-vancomycin로 감소하였다. Vancomycin 결정을 얻기 위해 농축 농도 및 온도에 대하여 조사한 결과, 80mg/ml 이상 농축하여 30-40℃에서 결정화하였을 때 수율 및 순도가 가장 높았다. 따라서 CM-Toyopearl의 vancomycin 활성분획을 80mg/ml로 농축하고 10% NaOH로 pH8.2가 되도록 조정한 후, 30℃에서 1시간 교반하여 vancomycin을 석출시켰다. 석출된 vancomycin은 여과, 세정하여 건조한 결과 최종 정제 수율은 64%였으며 순도는 99.4%이었으며 endotoxin은 0.180EU/mg 이었다. Purification of vancomycin Ion exchange resins, DC-20, DA-561 and PA-406 were applied for discoloration and improvement of purity and yield in vancomycin purification. Cation exchange resin DC-20(Na+) and anion exchange resin DA-561 (Cl-) appeared to be most efficient for pre-treatment of culture supernatant for purification of vancomycin. The filtrate passed through the ion exchange columns, DC-20(Na+) and DA-56(Cl-) was then loaded on a nonfunctional type adsorption column HP20 and eluted with 30% methanol. of which the yield and purity was 94% and 77.5% respectively. In order to remove the color impurities of HP20 active fractions, active carbon and alumina were applied, and as the result, alumina was more efficient in terms of decoloration and purification yield than active carbon. When the active fractions passed through the HP20 column were treated with alumina. The purity was improved from 77.5% to 91% with 90% of yield, CM-toyopearl 650M (NH4+), a cation exchange resin was used as a next adsorption column to increase the purity of vancomycin. When the active fractions passed through alumina column were loaded on a CM-toyopearl column at the flow rate of 1.5bv/h, after concentrating to 50mg/ml of vancomycin using a reverse osmosis membrane system, the yield and purity was 87% and 99%, respectively. Optimum concentration and temperature for crystallization of vancomycin were also investigated. Then, the active fractions of CM-toyopearl column were collected and concentrated to 80mg/ml of vancomycin for crystallization. The highest yield and purity were obtained when crystallization was conducted at pH 8.2 and 30-40℃. By a series of purification processes, the crystallized vancomycin with 99.4% purity was obtained at 64% yield.

Vancomycin 생산 균주의 역가 향상 및 공정연구

정형무,현형환 한국외국어대학교 기초과학연구소 2000 기초과학연구 Vol.10 No.-

Vancomycin을 생산하는 균주인 Amycolatopsis orientalis로부터 자연 돌연 변이를 통해 vancomycin 생산역가가 7.2∼7.5g/ℓ인 균주 A. orientalis 17을 분리하였다. Vancomycin 고생산 균주를 개발하기 위해서는 순화고정된 균주 A. orientalis 17 대하여 UV를 변이원으로하여 인공 돌연변이 처리를 수행한 결과, 모균주보다 vancomycin 생산역가가 각각 13%, 21%향상된 변이주 177-1과 177-2를 분리하였다. Vancomycin 생산을 위한 최적 발효 조건을 조사한 결과, 탄소원으로는 dextrin이 균체 생장 및 Vancomycin 생산에 가장 효과적이었으며, 배양 중 온도 및 교반 속도는 각각 35℃와 700rpm일 때 가장 높은 Vancomycin 생산 역가를 나타냈다. 탄소원, 온도 및 교반속도를 최적화하여 7ℓ 발효조에서 배양하였을 때, 모균주 및 변이주 177-1, 177-2는 각각 7.6g/ℓ, 8.5g/ℓ, 8.9g/ℓ의 최종 vancomycin 생산성을 나타냈다.

대한적십자사 원주신청사

한일호(Han, Ilho),정형무(Herman Jung) 현대건축사 2011 월간 CONCEPT Vol.- No.149

재조합 대장균을 이용한 재조합 Glucagon-like Peptide-1 생산 조건 최적화

전유찬,권덕호,이유진,이상훈,권성훈,정형무,하석진 한국생물공학회 2023 KSBB Journal Vol.38 No.3

Obesity is well known as a metabolic abnormality and potentially life-threatening condition associated with several chronic diseases. Various peptide drugs have been developed to treat obesity. In this study, optimization of expression conditions were performed to produce glucagon-like peptide- 1 (GLP-1), which is used as an obesity treatment in recombinant E. coli. After the construction of expression vector system and optimization of expression conditions, the productivity of GLP-1 was increased to 11.18 mg/L·h in flask cultivation. Through fermentation conditions were optimized for bioreactor, the GLP-1 productivity was increased to 16.80 mg/L·h in bioreactor, which was 50.27% improvement as compared to the flask cultivation. When 1.00 mM lactose was used as an inducer instead of IPTG, 17.39% higher expression was shown than that with 0.01 mM IPTG.

다발성경화증 치료를 위한 인터페론 베타의 산업적 생산

손다진(Da-Jin Son),김종석(Jong-Seok Kim),박재범(Jae-Bum Park),권덕호(Deok-Ho Kwon),정형무(Hyung-Moo Jung),한상인(Sang-In Han),홍억기(Eock-Kee Hong),하석진(Suk-Jin Ha) 한국생물공학회 2018 KSBB Journal Vol.33 No.2

Multiple sclerosis is a chronic disorder commonly occurred in the central nervous system and is an autoimmune disease. Corticosteroids are used as a short-term treatment for multiple sclerosis, but they cause side effects. Therefore, interferon beta having antiviral and anti-inflammatory effects, is used for long-term treatment. Interferon beta-1a, produced in Chinese hamster ovary cells, is glycosylated upon biosynthesis and forms glycoform, which is similar to naturally occurring protein. However, its process has disadvantages such as slow cell growth, low yield and high contamination possibility. Also, recombinant Escherichia coli is widely used for the production of interferon beta-1b due to its rapid growth and easy process scale up, but it lacks post-translational modification, leading to low activity. In addition, the produced Interferon beta-1b might cause protein aggregation due to misfolding. Alternatively, yeast can be used as a production host possessing N-glycosylation activity. Therefore, the choice of expression system in the production of interferon beta should be carefully selected in relation to the quality and yield.

KTX 제동패드 성능확인을 위한 비상제동거리 측정

소진섭(Jin-Sub So),김대식(Dae-Sik Kim),김진우(Jin-Woo Kim),김주원(Ju-Won Kim),정형일(Hyeong Il Jeong),정연일(Yeon Il Jeong),김영훈(Young Hoon Kim),양정무(Jeong-Moo Yang) 한국철도학회 2013 한국철도학회 학술발표대회논문집 Vol.2013 No.11

철도차량안전기준의 비상제동거리 세부기준에 따르면, 부하조건으로 300 km/h 에서 제동거리가 3,300 m 이내가 되도록 제시되어 있다. 서울역을 출발하여 천안아산역까지 운행중 KTX 제동패드 신품 교환(약 4 만 km 운행)후 성능확인을 위해 2 개 편성에 대해 측정하였다. 1 차 스톱워치로 확인, 2 차 KP 지점을 체크, 3 차 ATESS(열차속도, 발견된 신호, 열차상태 등 운행상황 기록)분석하였다. The emergency braking distance in load conditions is under 3300m at 300km/h, according to the Railway vehicle safety standards. The performance verification of braking pads on the KTX from Seoul to Cheonan-Asan was measured. First, we analyzed the primary stopwatch data and checked KP points. We finally performed ATESS analysis..

코돈 최적화된 유전자를 이용한 재조합 대장균으로부터 인간 인터페론 베타 발현

김종석(Jong-Seok Kim),장승원(Seung-Won Jang),박재범(Jae-Bum Park),권덕호(Deok-Ho Kwon),장영준(Young-Jun Chang),정형무(Hyung-Moo Jung),한상인(Sang-In Han),홍억기(Eock-Kee Hong),하석진(Suk-Jin Ha) 한국생물공학회 2017 KSBB Journal Vol.32 No.1

The multiple sclerosis caused by multiple inflammatory disease or immune system disorder, is usually treated by interferon β through adjusting the abnormal immune reactions. For high production of human interferon β using recombinant E. coli, codon optimized and wild type genes were synthesized. When pET-15b or pET-21a vector was used as an expression vector with each gene, there was no target protein expression. When pQE30 vector was used as an expression vector, human interferon β was expressed by recombinant E. coli XL1-blue and E. coli JM109. Using the codon optimized gene, the expression of human interferon β was slightly increased as compared to that from wild type gene. However, most of expressed human interferon β was insoluble form.