• Journal of Microbiology and Biotechnology
• /
• 제14권3호
• /
• pp.576-583
• /
• 2004

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules and shows antimicrobial activities against Gram-positive bacteria. Deoxysugar biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-l55 genome was cloned. Four orfs were identified and a putative orf presumed to be the dTDP g]ucose-4,6-dehydratase gene was designated as gerE. GerE was expressed in E. coli by using a recombinant expression vector pHJ1. The expressed protein was purified from E. coli cell lysate by using ammonium sulfate fractionation, and DEAE-sepharose CL-6B and hydroxylapatite column chromatography. The molecular mass of the expressed protein correlated with the predicted mass that was deduced from the cloned gene sequence data. The recombinant protein was a homodimer with a subunit relative molecular weight of 39,000 Dalton. It was found to have dTDP-glucose 4,6-dehydratase activity and also found to be highly specific for dTDP-glucose as a substrate. The values of $K_{m} and V_{max}$ for dTDP-g]ucose were $32\mu$M and 335 nmol $min^{-1}$ (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of the protein. $NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• 한국식품과학회지
• /
• 제36권1호
• /
• pp.105-110
• /
• 2004

전국 각지에서 채집한 토양과 두엄에서 분리한 25종의 내열성 균주 중 내열성 단백질 가수분해효소 활성을 갖는 균주 DF 218을 선별하였다. 본 균주는 Gram 양성 간균의 특징을 나타냈으며 Bergey's manual of systematic bacteriology와 Biochemical tests for identification of medical bacteria에 준하여 생화학적 특성을 검토한 결과 catalase 양성, 포자형성, motility 양성, glucose 발효, hemolysis ${\beta}$균임을 나타내어 Bacillus sp.으로 추정되었다. 165 rDNA sequence 분석 결과 DF 218 균주는 Bacillus flexus과 sequence가 95% 일치하는 유사성을 보였으나 gene bank data base 상에서 165 rDNA sequence가 일치하는 균주는 검색되지 않았다. 이 같은 실험 결과에 따라 strain DF 218은 기존에 발표되지 않은 새로운 균주로 판단되어 Bacillus sp. DF 218로 명명하였다. Bacillus sp. DF 218은 1% trypton, 1% NaCl, 1% glucose의 배지조성과 배양은도 $60^{\circ}C$에서 32시간동안 배양하였을 때 최대의 단백질 분해효소를 생산하였다. Bacillus sp. DF 218로부터 단백질 분해효소를 acetone으로 침전시키고 DEAE-sepharose column chromatography를 통하여 효소를 정제하고 정제된 단백질을 SDS-PAGE를 통해 분석한 결과 61kDa 크기의 단일 band를 확인할 수 있었다. 이 효소의 최적 반응온도는 $60^{\circ}C$이었으며 최적 pH는 7.5로 측정되었다.

• Journal of Microbiology and Biotechnology
• /
• 제25권11호
• /
• pp.1944-1953
• /
• 2015

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80℃ in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50℃ and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni²⁺, Mn²⁺, and Cu²⁺ to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The K_m and V_max values were estimated to be 35.7 mg/ml, and 5 × 10⁴ U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

• 한국잠사곤충학회지
• /
• 제44권2호
• /
• pp.64-68
• /
• 2002

본 연구는 곤충 유전자를 이용한 항세균성 펩타이드 생산 및 농업용 소재로서의 응용에 관한 연구로서 항세균성 단백질 누에신 유전자를 베큘로 바이러스 발현계(BEVS)를 이용하여 곤충세포주에서 발현한 후 누에신 단백질의 농업용 소재로서의 가능성을 모색하기 위해 농작물을 가해하는 감자 고추의 무름병을 일으키는 Pectobacterium carotovorum subsp. carotovorum, 가지 및 고추의 풋마름병을 일으키는 Ralstonia solanacearum, 양송이 버섯의 세균성 갈색 무늬 병을 일으키는 Pseudomonas tolaasii 및 무와 배추의 검은썩음병을 일으키는 Xanthomonas campestris pv. campestris 에 대해서 항세균 활성을 관찰하였다. 그 결과 Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum 및 Pseudomonas tolaasii에 대해 높은 활성을 나타내었으며 Xanthomonas campestris pv. campestris에는 활성을 나타내지 않았다. Ion exchange 및 gel filtration chromatography 를 수행하여 약 20 kDa의 성숙 누에신 단백질을 순수 분리하여 pH 및 온도에 대한 안정성을 조사한 결과, pH 2~12 완충액에서 30분간 처리하였을 때에도 항세균 활성이 그대로 유지되었고 10$0^{\circ}C$에서 2시간 처리시에는 활성이 안정되었으며 4시간 처리시에도 80% 정도로 유지됨을 확인함으로써 누에신 단백질은 pH 및 온도에 대한 안정성이 있음을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제17권4호
• /
• pp.604-610
• /
• 2007

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at $30^{\circ}C$, and was unstable at temperatures higher than $30^{\circ}C$, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24h incubation at $4^{\circ}C$. The addition of $Ca^{2+}\;and\;Mg^{2+}$ enhanced the enzyme activity of LipA1, whereas the $Cd^{2+},\;Zn^{2+},\;CO^{2+},\;Fe^{3+},\;Hg^{2+},\;Fe^{2+},\;Rb^{2+}$, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate $(C_{14}\;acyl\; groups)$.

• Journal of Microbiology and Biotechnology
• /
• 제12권2호
• /
• pp.189-195
• /
• 2002

Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and calcium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at $28^{\circ}C$ for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate precipitation and DEAE-Sepharose CL-6B column chromatography, which produced a major band of 57 kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reaction temperature was elevated to $60^{\circ}C$. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as emulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.

• 미생물학회지
• /
• 제33권2호
• /
• pp.142-148
• /
• 1997

제한통성 메탄올 자호세균인 Methylovorus sp. strain SS1은 최적 성장 조건하에서는 소량의 세포외 다당류(EPS)를 생산하였지만, 질소원이 결핍된 성장 조건하에서는 성장속도는 느렸지만 다량의 EPS를 생산하였다. EPS는 배지내의 탄소대 질소 비율이 5.2일 때 가장 많이 생산되었다. EPS 생산을 위한 최적 온도는 30^{\circ}C.$이고 최적 pH는 6.5였다. EPS는 탄수화물과 단백질 및 약간의 피루브산으로 구성되어 있었고, 환원당으로는 다량의 포도당과 소량의 mannose가 존재하였다. 에탄올을 처리한 EPS(EPSae)에는 에탄올을 처리하지 않은 EPS(EPSbe)에 존재하던 피루브산이 존재하지 않았고, EPS보다 단백질의 양도 적고 점성도 낮았다. EPSbe의 점성은 NaCl에 의해 큰 영향을 받았는데, 0.5%(w/v) 농도의 NaCl 용액에서도 점성이 크게 떨어졌으며, 높은 온도에서는 점성이 비가역적으로 크게 증가하였다. Gel filtration 방법으로 조사한 EPSae의 분자량은 $2.5{\times}$10^6$ - $3.5{\times}$10^6$이었다. 냉동건조한 다당류를 전자현미경으로 관찰하였을 때, EPSbe는 섬유모양을 하였고, EPSae는 벌집모양의 망상구조를 하고 있었다.

• 한국수산과학회지
• /
• 제35권5호
• /
• pp.519-523
• /
• 2002

국내에서 양식되는 가시파래 (E. prolifera)의 효율적 이용과 새로운 기능성 식품소재로서 사용하기 위한 기초자료를 얻기 위해 가시파래의 수용성 다당을 추출 후 CPC와 음이온 교환수지로 분획하여 화학적 특성을 조사하였다. 가사파래를 열수로 3회 반복하여 추출하여 얻은 수용성 다당의 수율은 $23.7\%$였고 총당, 단백질, 황산기의 함량이 각각 $68.8\%$, $6.7\%$, $23.7\%$였다. 수용성 다당을 CPC처리하여 얻은 산성다당인 CPC-PS는 주 구성성분이 황산기, 우론산, rhamnose, xylose였으며 주요 구성당인 xylose : rhamnose : glucose의 몰비는 1 : 3.1 : 0.2 이었다. CPC-PS를 음이온 교환수지로 분획하였을 때 3개의 fraction (Fr-1, Fr-2, Fr-3)이 얻어졌으며 각 획분간의 화학적 조성상 큰 차이점 없었다. CPC-PS의 분자량은 620,000이었고, 이를 이온교환수지로 분획한 획분인 Fr-1은 710,000 ($84\%$), 90,000 ($14\%$), 70,000 ($2\%$), Fr-2는 510,000, Fr-3은 830,000 daltons으로 Fr-1을 제외한 나머지 획분은 단일피크로 대칭성을 나타내어 분자량이 균일함을 알 수 있었다. 그리고 이 획분들을 전기영동 하였을 때 Fr-1과 Fr-3은 단일 band를 나타내었다.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1997년도 춘계학술대회
• /
• pp.95-95
• /
• 1997

Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.

• Parasites, Hosts and Diseases
• /
• 제35권1호
• /
• pp.55-62
• /
• 1997

톡소포자충(ToxopLasmn gondii)은 다양한 항원을 가지고 있으며, 이들 항원의 분석은 세 포매개성 면역반응 및 톡소포자충증의 면역학적 진단방법의 연구에 매우 중요하다 본 연구는 톡소포자충의 여러 단백질중 대부분의 충주(strain)에 존재하는 분자량 30 kDa의 단백질을 단클론 항체를 이용하여 분리한 후. 30 kDa 항원의 면역학적 특성을 초음파 추출 조항원과 비교 평가하였다. 톡소포자충의 세포막 항원으로 면역한 마우스 비장세포와 마우스 Sp2/0-Agl4 골수종세포를 융합하여 8개의 단클론 항체를 Western blot으로 확인하였다 이들 단클론 항체는 높은 특이성을 보였으며, $IgG_{2b}가{\;}5개,{\;}IgG^{1}이{\;}2개,{\;}IgG_{2a}$가 1개였다. 간접형광항체법으로 충체내 위치를 관찰한 결과. 30 kDa 항원은 tachyzoite의 표면 세포막에 주로 분포하였다. 단클론 항체와 CNBr-activated Sepharose 4B를 coupling하여 만든 immunoafrnity chromatography를 이용 하여 30 kDa 항원을 분리하였다. 분리한 30 kDa 항원으로 자극시킨 마우스 복강대식세포의 $NO_2^{-}$ 생산량은 초음파 추출 조항원 사용군에 비해 유의하게 증가하였으나 대식세포의 탐식능은 유의한 차이가 없었다. 또한 ELISA로 톡소포자충증을 진단시, 톡소포자충 30 kDa 항원 사용군은 조항원 사용군에 비해 민감도의 변화는 없었으나 특이성은 증가하였다 이상으로 보아 톡소포자충 30 kDa 항원은 감염 방어 면역 효과가 있었으며 진단에 이용시 특이성을 더 높일 수 있었다.