인간 wellness를 위한 제약용 효소의 개발

이상만(Sang-Mahn Lee) 한국엔터테인먼트산업학회 2015 한국엔터테인먼트산업학회논문지 Vol.9 No.3

항생물질은 지난 60년 동안 인간의 건강을 증진시키고 질병 없는 wellness를 구현하는데 지대한 공헌을 하였다. 그 가운데 천연 penicillin의 화학적 변형에 의하여 생산된 반합성 penicillin은 의약 산업에 획기적인 발전을 가져오게 되었다. Penicillin G acylase (PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid (PAA)와 6-aminopenicillanic acid (6-APA)로 분해하는 효소로서 항생물질 산업 분야의 발전에 크게 기여한 매우 중요한 효소이다[12]. PGA는 페니실린-지를 페닐아세트산과 6-아미노페니실란산으로 분해하고,6--아미노페니실란산은 페니실린계 항생물질 생산을 위한 전구물질로 사용된다[17]. 본 연구에서는 대장균에서 주변부로 발현시키고 이곳으로부터 PGA를 간단하고 신속하게 추출하는 방법을 최적화하고 최적화한 조건에서 배양액 25 L 규모로 PGA를 추출하고 추출한 PGA를 특별한 정제 과정 없이 수지에 고정화하였다. 추출한 PGA를 이용한 고정화 효율은 45.5%였다. 또한 반복사용의 가능성을 확인하기 위하여 고정화한 PGA를 이용하여 10회 연속으로 반응시켜 그 활성을 조사했을 때 효소활성은 거의 소실되지 않았다. 이러한 결과는 생물전환반응에 사용하는 효소를 대장균에서 간편하게 추출하고 추출액을 한외여과를 통하여 농축한 뒤 바로 고정화하여 사용할 수 있다는 것으로 생물전환반응을 통하여 유용 물질을 생산하는데 유용하게 사용할 수 있을 것으로 생각한다. Penicillin G Acylase (PGA, benzylpenicilliaminohydrolase, EC 3.5.1.11) is important enzyme which converts penicillin G to 6-aminopenicillanic acid(6-APA) and penylacetic acid(PAA). 6-APA is used as a precursor for synthesis of a series of penicillinanic antibiotics for the human health. In this study, the active PGA was successfully secreated into periplasmic space in E.coli BL21(DE3) harboring pET-pga plasmid. A efficient extraction process for PGA from periplasm in recombinant E.coli was found out. At the optimized condition, the extracted PGA solution was immobilized on Amberlite ZAD-7 without further purification procedure. The immobilized yield for PGA was 45.5%. Also, in order to confirm possibility of reuse, multi-batch operation of immobilized PGA was performed. The immobilized PGA was used to produce from penicillin G to 6-APA without loss of activity during 10-times operation. These results mean the enzyme extration procedure from periplasm in E.coli may be valuable for the industrial scale production of useful materials using immobilized enzyme.

Penicillin G Amidase생산을 위한 재조합 대장균의 유가배양에 관한 연구

이상만 ( Sang Mahn Lee ) 한국미생물생명공학회 2008 한국미생물·생명공학회지 Vol.36 No.4

An Efficient Method for the Release of Recombinant Penicillin G Amidase from the Escherichia coli Periplasm

Sang-Mahn Lee(이상만) 한국생명과학회 2017 생명과학회지 Vol.27 No.10

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 β-lactam 항생물질의 산업적 생산 이용될 수 있을 것으로 사료된다. 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 β–lactam antibiotics using immobilized PGA.

Salmonella typhimurium 의 Nucleotide reductase 를 코드하는 nrd 유전자의 클로닝

김순옥(Soon Ok Kim),이상만(Sang Mahn Lee),장종수(Jong Soo Chang),송방호(Bang Ho Song) 경북대학교 생명공학연구소 1989 遺傳工學硏究所報 Vol.4 No.1

N/A

Salmonella typhimurium 의 cytidine deaminase의 효소학적 특징

이상만 청주대학교 산업과학연구소 2002 産業科學硏究 Vol.19 No.2

The cytidine deaminase was partialy purified with sephadex G-200 and the characteristics of the enzyme were clarified. The molecular mass of the plasmid-encoded protein was identified as about 27kDa in a minicell system. The native enzyme was estimated to have the molecular mass of 60kDa by gel filtration. This indicates that the native enzyme may exist as a dimer composed of two identical subunits. The enzyme was reasonably stable in the pH range of 6 to 9, and was labile under high temperature above 50℃. Mercaptoethanol, pCNB, mercury and copper were found to inhibit the enzyme activity. The cytidine analogues of bromo- and iodo-(deoxy)-cytidine were also found to inhibit the activity, while fluorodeoxycytidine and azacytidine were found to activate it. The Km values in deoxycytidine, cytidine, methyldeoxycyodine and ara-C were 0.01, 0.08, 0.13 and 0.15nM respectively. The cytidine mononucleotide of CMP and dCMP activated the enzyme activity while the tnnucleotide of CTP and dCTP inhibited it. Considenng that gene expression is parallel to the exponential phase, Salmonella cytidine deaminase has a role as a pyrimdine salvage enzyme in scavenging nucleotides.

Salmonella typhimurium cytidine deaminase를 암호하는 cdd 유전자의 클로닝

이상만 청주대학교 산업과학연구소 2002 産業科學硏究 Vol.19 No.2

The Salmonella typhimurium cdd gene encoding cytidine deaminase(cytidine/2'-deoxycy-tldine aminohydrlase, EC 3.5.4.5.) was isolated through shotgun cloning by complementation of the E.coli cdd mutation. By subsequent deletion and subcloning from the original 3.7Kb of EcoR Ⅰ insert(pSAM1), the precise region of the cdd structural gene is located around the BgⅢ site in the middle part of 1.7Kb of NruⅠ_(2)/PvuⅠ_(2) segment The cdd gene expression in E.coli JF611/pSAM1 was amplified about 50 fold compared to that of the wild type, and the gene expression was increased about 160 fold by the aid of the lac-Z promoter by insertion of the gene into the multicloning site of the pUC vector. The cdd gene expression was maintained in the stationary phase after reaching the peak in the late logarithmic phase.

16S rRNA 분석에 의한 토양에 분포하는 셀룰로스 분해세균의 동정

이상만 청주대학교 2014 産業科學硏究 Vol.31 No.2

The cellulase producing bacteria was isolated from soil. The bacteria was identified as one of genus Flavobacterium by 16S rRNA gene sequence and phylogenetic tree. Flavobacerium genus is the major group among the Bacteroide pilum. The bacteria was isolated with varius environmental conitions such as water and soil..

Salmonella typhimurium의 cytidine deaminase 생산 최적화 조건에 관한 연구

이상만 청주대학교 산업과학연구소 2004 産業科學硏究 Vol.21 No.2

This paper studies the production of enzyme by adding a variety of factors of nutrient to E.Coli strain which contains cdd gene of Salmonella typhimurium in order to optimize its condition for producing enzyme. In the case of a minimal medium where a variety of carbon sources are added, enzyme is produced best when glycerol is added. And enzyme is produced in optimum when the concentration of glycerol is 25mg/㎖ Regarding the impact of nitrogen sources, enzyme is produced best when (NH_4)_2 SO_4 is added And enzyme IS produced in optimum when the concentration of (NH_4)_2 SO_4 is 10mg/㎖. Concerning the impact of various precursors of nucleic acid, cytidine and deoxycytidine induce the production of enzyme. The optimum concentration of them is 200㎍/㎖. By adding a variety of cytosine analogue, iodocytidine, ara-C and bromo(deoxy)cytidine increase the enzyme product.

Pseudomonas sp. DJ-12의 5-carboxymethyl-2-hydroxy-muconic semialdehyde dehydrogenase를 coding하는 hpaE gene의 유전자 분석

이상만 청주대학교 산업과학연구소 2006 産業科學硏究 Vol.23 No.2

Pseudomanas sp. DJ-12 is able to metabolize 4-hydroxybenzoate and protocatechuate as the sole carbon and energy source. The nucleotide sequence of the hpaE gene encoding 5-carboxymethyl-2-hydroxy-muconic semialdehyde dehydrogenase which converted to the 5'carboxymethyl-2-hydroxy-muconate was determined. The fragment included an open reading frame of 567 bp which has ATG initiation codon and TAA termination codon and GGAA ribosomal binding site. The predicted amino acid sequence of the enzyme consists of 189 amino acid. The deduced amino acid sequence of the hpaE enzyme exhibited 66% identity with that of the corresponding enzyme in Burkholderia fungorum.

Alkaline Protease 생성 미생물의 분리 및 효소 생산에 관한 연구

이상만 청주대학교 산업과학연구소 1998 産業科學硏究 Vol.15 No.3

A bacterium JS-39 producing a alkaline protease under alkaline condition was isolated from soil the bacterium showed maximum production of alkaline protease at 40℃, pH 9, and cultivation time of 50 hours. In alkaline protease productivity, mannose, soluble starch and glucose among tested carbon sources were very effective. Casamino acid was found to be the most effective nitrogen sourse in productivity.