Protein Engineering of Flavin-containing Monooxygenase from Corynebacterium glutamicum for Improved Production of Indigo and Indirubin

Hye Sook Jung(정혜숙),Hae Bin Jung(정혜빈),Hee Sook Kim(김희숙),Chang Gyeom Kim(김창겸),Jin Ho Lee(이진호) 한국생명과학회 2018 생명과학회지 Vol.28 No.6

향상된 인디고이드 생산능력을 갖는 코리네박테리움 유래의 변이 플라빈-함유 모노옥시게나제(cFMO)를 개발하기 위하여, cFMO 효소의 상동성모델을 이용하여 말토오스-결합단백질(MBP)과 융합된 4가지 변이체(F170Y, A210G, A210S, T326S)를 제작하고 그 생화학적 특징을 밝혔다. 정제된 MBP-T326S는 최적 활성을 위하여 야생형보다 고농도의 FAD (100 μM)를 요구하며, 100 μM의 FAD 첨가조건에서 kcat/Km이 3.8배 증가되었다. 인돌 옥시게나제 활성은 야생형의 63-77%를 나타냈다. MBP-T326S는 기질이 존재하지 않을 경우 쓸모없는 NADPH 산화효소활성이 매우 낮은 수준을 보여주었다(21-24%). T326S이외의 변이 단백질들은 야생형에 비하여 Km은 비슷하며 kcat/Km은 증가하였다. MBP-F170Y와 -A210S 변이단백질은 인돌 옥시게나제 활성이 각각 3.1배, 2.9배 증가하였다. 2.5 g/l의 트립토판을 함유한 LB배지에서 인디고이드 생산을 시험했을 때, 야생형 cFMO를 함유한 대장균은 684 mg/l의 인디고와 104 mg/l의 인디루빈을 생산한 반면, T326S를 함유한 세포는 1,040 mg/l의 인디고와 112 mg/l의 인디루빈을 생산하였다. 이전의 결과인 Methylophaga 유래의 FMO를 발현하는 대장균에서 가장 높은 수준인 920 mg/l의 인디고를 생산한 것과 비교하면, 본 연구결과는 인디고 생산이 13% 높은 수준이였다. 상동성 모델링에 기반한 cFMO의 단백질공학은 인디고이드 생산균을 개발하는데 보다 더 논리적인 전략을 제시하였다. Flavin-containing monooxygenases from Corynebacterium (cFMOs) were mutagenized based on homology modeling to develop variants with an enhanced indigoid production capability. The four mutants, F170Y, A210G, A210S, and T326S, which fused to a maltose-binding protein (MBP), were constructed, and their biochemical properties were characterized. Of these, purified MBP-T326S required a higher concentration of exogenous FAD (100 mM) than the wild-type MBP-cFMO for optimal activity and showed a 3.8-fold increase in the kcat/Km value at 100 μM FAD compared to that of MBP-cFMO at 2 μM FAD. The indole oxygenase activities of MBP-T326S decreased to 63-77% compared to that of the MBP-cFMO In addition, MBP-T326S displayed a very low level of futile NADPH oxidase activities (21-24%) in the absence of a substrate. Mutant proteins except for T326S displayed similar Km and increased kcat/Km values compared to the wild-type. MBP-F170Y and -A210S mutants showed elevated indole oxygenase activity higher than 3.1- and 2.9-fold, respectively, in comparison with MBP-cFMO. When indigoid production was carried out in LB broth with 2.5 g/l of tryptophan, Escherichia coli expressing cFMO produced 684 mg/l of indigo and 104 mg/l of indirubin, while cells harboring T326S produced 1,040 mg/l of indigo and 112 mg/l of indirubin. The results indicate that the production of indigo was 13% higher when compared to a previous report in which an E. coli expressing FMO from Methylophaga produced 920 mg/l of indigo. The protein engineering of cFMO based on homology modeling provided a more rational strategy for developing indigoid-producing strains.

환경스트레스와 관련된 indole-3-acetic acid 및 1-aminocylopropane-1-carboxylyic acid deaminase 활성을 갖는 박테리아의 분리와 특성 연구

김희숙 ( Hee Sook Kim ),김지윤 ( Ji-youn Kim ),이송민 ( Song Min Lee ),박혜정 ( Hye-jung Park ),이상현 ( Sang-hyeon Lee ),장정수 ( Jeong Su Jang ),이문현 ( Mun Hyon Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 한국미생물·생명공학회지 Vol.47 No.3

본 연구에서 부산, 창원, 제주도 일대에서 채취한 토양으로부터 분리한 미생물을 이용하여 식물 생장 촉진활성 및 식물병원성 곰팡이에 대한 길항능을 확인하고자 하였으며, 분리주 간에 비교우위를 통해 Pseudomonas plecoglossicida ANG14, Pseudarthrobacter equi ANG28, Beijerinckia fluminensis ANG34, Acinetobacter calcoaceticus ANG35를 최종 선정하였다. 이들은 ACC deaminase 생성능, IAA 생성능, 질소 고정능, 인산 가용화능 및 siderophore 생성능을 모두 가지며, 일부 식물병원성 곰팡이에 대해 길항능을 가지는 것을 확인하였다. 특히 ANG35의 경우에는 7가지 식물병원성 곰팡이 중 6가지에 대해 비교적 높은 억제율을 나타내어 식물 생장 촉진뿐만 아니라 방제활성을 가지므로 식물이 생장하는 데 도움을 줄 것으로 기대된다. 또한, 4균주간의 세포외 효소활성 synergy effect를 확인한 결과 단독배양을 했을 때보다 혼합배양 시 세포외 효소활성이 증가하는 것을 확인하였다. 따라서, 식물 생장촉진 활성 및 식물병원성 곰팡이에 대한 결과를 통해 새로운 생물학적 제제로서 이용 가능성을 제시한다. In this study, strains isolated from soil samples collected from Busan, Changwon, and Jeju Island were examined to verify their abilities of phosphate solubilization and nitrogen fixation, production of indole-3-acetic acid (IAA), siderophore, and 1-aminocylopropane-1-carboxylyic acid (ACC) deaminase in order to select strains that promote plant growth and play a role in biocontrol of pests or pathogens. According to the results of this study, most of the isolated strains were found to have ability of phosphate solubilization, nitrogen fixation, IAA production, siderophore production, and production of ACC deaminase. These isolated strains might help plant growth by directly improving absorption of nutrients essential for phosphate solubilization and nitrogen fixation. In addition, they can promote plant growth and control resistance to plant diseases through extracellular enzyme activity and antifungal activity. In addition, most of the selected strains were found to survive in various environmental conditions such as temperature, salinity, and pH. Therefore, Pseudomonas plecoglossicida ANG14, Pseudarthrobacter equi ANG28, Beijerinckia fluminensis ANG34, and Acinetobacter calcoaceticus ANG35 were finally selected through a comparative advantage analysis to suggest their potential as novel biological agents. Further studies are necessary in order to prove their efficacy as novel biological agents through formulation and optimization of effective microorganisms, their preservation period, and crop cultivation tests.

자궁경수술로 성공적으로 치료된 점막하 낭성자궁선근종 2예

김남숙 ( Nam Sook Kim ),김주명 ( Joo Myung Kim ),김미라 ( Mi La Kim ),이시원 ( Si Won Lee ),주관영 ( Kwan Young Joo ),김혜선 ( Hye Sun Kim ),김희숙 ( Hy Sook Kim ),홍성란 ( Sung Ran Hong ) 대한산부인과학회 2009 Obstetrics & Gynecology Science Vol.52 No.2

Two women presented with history of vaginal bleeding and abnormal transvaginal ultrasound findings. Saline infusion sonohysterography was done for preoperative evaluation and this imaging study revealed centrally located uterine mass with internal cystic portion. Our initial impression was submucosal myoma with cystic degeneration and hysteroscopic removal of the mass was performed. During the procedure, dark brownish cystic fluid was drained. The excised submucosal mass was pathologically diagnosed as adenomyosis and clinically as submucosal adenomyotic cyst. Submucosal adenomyotic cyst should be part of the differential diagnosis of submucosal uterine masses with cystic portion. We report two cases of submucosal adenomyotic cyst successfully treated with hysteroscopic resection and brief review on this topic.

실업자의 자살생각 영향요인

김혜영(Kim, Hye Young),김희숙(Kim, Hee Sook) 한국보건간호학회 2017 韓國保健看護學會誌 Vol.31 No.1

Sensitivity of AutoPap Primary Screening System with Location-Guided Screening in Uterine Cervical Cytology

최종순,장회숙,김희숙,전이경,김혜선,박지영,박인서,홍성란,Choi, Jong-Sun,Jang, Hoi-Sook,Kim, Hy-Sook,Chun, Yi-Kyeong,Kim, Hye-Sun,Park, Ji-Young,Park, In-Sou,Hong, Sung-Ran The Korean Society for Cytopathology 2003 대한세포병리학회지 Vol.14 No.2

Objective: The sensitivity of the AutoPap Primary Screening System with Location-Guided Screening (AutoPap LGS) for Identifying atypical cells in cervicovaginal smears was evaluated. Methods: Two hundred forty one slides with atypical cervical cytology randomly sampled were rescreened both manually and by the AutoPap LGS. The AutoPap LGS localized the atypical cells as 15 fields of view(FOVs), which were reexamined by manual review. The sensitivity was also evaluated in accordance with the cellularity of the smears. Results: The AutoPap LGS successfully processed 232 out of 241 slides. The sensitivity of the AutoPap LGS identifying the atypical cells in successfully processed slides was 97.4%(226/232). The false negative rate was 2.6%(6/232). There was no false negative case on high grade squamous intraepithelial lesion (HSIL) or squamous cell carcinoma(SCC) smears in the AutoPap LGS. The FOVs localized the diagnostic-atypical cells in 97.8%(221/226). The number of diagnostic-atypical FOVs was increased in higher-degree of atypical cytology. The AutoPap LGS localized the atypical cells in 100% of adequately cellular smears and in 92.5% even in low cellular smears. Conclusion: The AutoPap LGS showed relatively good sensitivity to detect atypical cells. It can be a valuable system to localize atypical cells, especially in HSIL or cancer slides, even in smears with low cellularity.

토양에서 분리한 Pseudomonas geniculata ANG3, Exiguobacterium acetylicum ANG40 및 Burkholderia stabilis ANG51의 식물 생장촉진 활성 및 식물병 방제활성

김지윤 ( Ji-youn Kim ),김희숙 ( Hee Sook Kim ),이송민 ( Song Min Lee ),박혜정 ( Hye-jung Park ),이상현 ( Sang-hyeon Lee ),장정수 ( Jeong Su Jang ),이문현 ( Mun Hyon Lee ) 한국미생물 · 생명공학회 2020 한국미생물·생명공학회지 Vol.48 No.1

본 연구는 토양으로부터 분리한 균주를 대상으로 식물병 방제활성 및 식물 생장촉진 활성을 확인하고자 하였다. 식물병원성 곰팡이에 대한 길항능을 통해 방제기능을 확인할 수 있었으며, 이는 siderophore 및 항생물질 생성 등에 기인되는 것으로 판단된다. 또한 ANG40의 경우에는 amylase, cellulase, xylanase와 같은 세포외 효소활성을 갖고 있음을 확인하였다. 이 외에도 질소 고정능, 인산 가용화능, siderophore 생성능 등 다양한 실험을 통해 식물 생장에 필수적인 질소, 인, 철 등을 식물이 흡수 가능한 형태로 변화시켜 식물 생장에 도움을 줄 수 있을 것으로 기대된다. 또한 6종의 분리균주는 모두 에틸렌 생성과 연관된 IAA를 생성하였으며, 그 중에서도 ANG51의 경우에는 ACC deaminase 활성도 갖고 있음을 확인하였다. 따라서, 최종 선별된 Pseudomonas geniculata ANG3, Exiguobacterium acetylicum ANG40, Burkholderia stabilis ANG51을 이용하여 식물 생장촉진 활성과 식물 병원성 곰팡이에 항진균 활성을 갖는 새로운 생물학적 제제로써 이용 가능성을 제시하였다. This study was conducted to investigate both plant growth-promoting and plant disease- controlling activities of bacterial strains isolated from soil. All the isolated strains were able to grow at various temperatures. All the strains, except ANG40, showed antagonistic effects against various phytopathogenic fungi. This antagonism can be ascribed to the production of siderophores and antibiotic substances. In addition, all the strains showed abilities such as nitrogen fixation, phosphate solubilization, and siderophore production. These results suggest that nitrogen, phosphorus, and iron can be converted into forms that can be easily absorbed by the plants for their growth. Analysis of the growth-promoting properties revealed that ANG51 produced 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and indole-3-acetic acid (IAA) both of which are related to ethylene production. In contrast, the other strains were found to have only IAA-producing ability. Therefore, this study suggests that Pseudomonas geniculata ANG3, Exiguobacterium acetylicum ANG40, and Burkholderia stabilis ANG51, which were selected through analysis of comparative advantages for both plant growth promotion and disease-controlling activity, may be used as biological agents.

자궁경부 세포진에서 ASCUS 진단의 세분화 - "ASCUS, R/O HSIL" : 세포학적 소견 및 조직학적 상관성

김혜선,김복만,김의정,김희숙,Kim, Hye-Sun,Kim, Bock-Man,Kim, Yee-Jung,Kim, Hy-Sook 대한세포병리학회 2002 대한세포병리학회지 Vol.13 No.1

Cytologic and histopathologic features and human papillomavirus (HPV) DNA detection associated with 101 cervicovaginal smears which are ciassified as 'atypical squamous cells of undetermined significance, rule out high grade squamous intraepithelial lesion(ASCUS, R/O HSIL)' were reviewed and compared to 89 smears of 'ASCUS, not otherwise specified(NOS)'. Cytologic fieatures of ASCUS, R/O HSIL included atypical single small cells(36.6%), hyperchromatic tissue flagments(35.6%), atypical metaplastic cells(18.8%), endometrial cell-like clusters(5.9%), and atypical parakeratotic cells(3.0%). A final diagnosis of HSIL on biopsy was assigned to 47(54.0%) of 87 women with ASCUS, R/O HSIL and to 13(14.6%) of 89 women with ASCUS, NOS (p=0.000). There was no difference in HPV DNA detection late between ASCUS, R/O HSIL and ASCUS, NOS smears. These data suggest that subclassification of ASCUS is helpful to manage patients because ASCUS, R/O HSIL is more often associated with an underlying HSIL on biopsy. Therefore, women with ASCUS, rule out HSIL should be actively managed with colposcopic examination.

유두상 미성숙 화생의 자궁경부 세포진 소견

김혜선,선미임,김의정,김희숙,Kim, Hye-Sun,Seon, Mee-Im,Kim, Yee-Jung,Kim, Hy-Sook 대한세포병리학회 2002 대한세포병리학회지 Vol.13 No.1

Papillary immature metaplasia (PIM) of the uterine cervix (Immature condyloma) is a subset of low grade squamous intraepithelial lesion (LSIL) which is frequently associated with human papilloma virus (HPV) types 6 and 11. The histologic features of PIM include filiform papillae lined by evenly spaced immature metaplastic-type cells with frequent nucleoli, mild anisokaryosis, and a low mitotic index. To characterize the cytologic changes associated with PIM, we analyzed 14 cases of PIM from our file. We reviewed biopsy slides and the cervicovaginal smears taken proximate to the time of blopsy. Histologically, nine cases had either flat condyloma (7 cases) or high grade squamous intraepithelial lesion (HSIL) (2 cases). Cytologic changes included cells in various stages of maturation with karyomegaly (14 cases), cells with irregularities in the nuclear membrane (13 cases), intermediate cells with karyomegaly(13 cases), cells with binucleatlon (13 cases), and aborted koliocytes (11 cases) Cervicovaginal smears from all cases were interpreted as atypical squamous cells of undetermined significance (ASCUS), NOS or ASCUS, rule out squamous intraepithelial lesion (SIL) or LSIL in two cases with flat condyloma or HSIL in a case with severe dysplasia. PIM is a distinct histologic entity that can present with a spectrum of cytologic findings, but cytologic findings may resemble variable reactive conditions and immature HSIL. Therefore, it is difficult to diagnose PIM by cytology alone. However, the meticulous efforts for making the cytologic diagnoses which can Induce active management of patients are recommended because PIM is a variant of LSIL and frequently has a flat condyloma or HSIL.

자궁내막암 환자의 수술 전 자궁경부 세포진 검사 - 임상병리학적 연관성

성석주(Seok Ju Seong),김태진(Tae Jin Kim),임경택(Kyung Taek Lim),정환욱(Hwan Wook Chung),이기헌(Ki Heon Lee),박인서(In Sou Park),심재욱(Jae Uk Shim),박종택(Chong Taik Park),김혜선(Hye Sun Kim),김희숙(Hy Sook Kim) 대한산부인과학회 2002 Obstetrics & Gynecology Science Vol.45 No.10

목적 : 자궁내막암 환자에서 수술 전에 악성 예후인자를 미리 알 수 있다면 치료 계획을 세우는데 많은 도움이 된다. 이 연구의 목적은 자궁내막암 환자에서 수술 전에 시행한 자궁경부 세포진 검사 결과와 악성 예후인자와의 상관관계를 알아보는 것이다. 연구 방법 : 1989년 1월부터 2000년 6월까지 삼성제일병원에서 수술을 시행받은 자궁내막암 환자 중에서 수술 전에 자궁경부 세포진 검사를 하였던 163예의 환자를 대상으로 하였다. 모든 환자에서 전자궁적출술 및 양측 부속기 절제술과 복강 내 세포진 검사를 시행하였고, 골반 림프절 절제술은 79예에서, 또 부대동맥 림프절 절제술은 81예에서 시행하였다. 이들 환자의 수술 전에 시행한 자궁경부 세포진 검사 결과를 정상, AGUS, adenocarcinoma로 나누어 자궁내막암의 예후인자와의 상관관계를 검토하였다. 통계는 카이 제곱 테스트를 이용하였다. 결과 : 163예 환자의 평균 나이는 49세 (24-75세)였고, 50세 미만이 65예 (39.9%)이고 50세 이상이 98예 (60.1%)였다. 수술 전 자궁경부 세포진 검사에서 72예 (44.2%)는 정상, 38예 (23.3%)는 AGUS, 53예 (32.5%)는 adenocarcinoma였다. 자궁경부 세포진 검사는 고령 (p=.014), 나쁜 조직학적 분화도 (p=.000), 자궁경부 침윤 (p=.015), 심부 자궁근층 침윤 (p=.000), 림프-혈관 침윤 (p=.000) 및 진행된 수술병기 (p=.049)와 통계적으로 유의한 상관관계를 보였고, 조직학적 분류 (p=.328), 복강내 세포진 검사 (p=.067), 자궁부속기 침범 (p=.602), 골반 림프절 침윤 (p=.266) 및 부대동맥 림프절 침윤 (p=.220)과는 통계적으로 유의하지 않았다. 결론 : 자궁내막암 환자에서 수술 전 자궁경부 세포진 검사상 발견된 AGUS나 adenocarcinoma는 예후인자 중 고령, 나쁜 조직학적 분화도, 자궁경부 침윤, 심부 자궁근층 침윤, 림프-혈관 침윤 및 진행된 수술병기와 통계학적으로 유의한 상관관계가 있었다. 따라서 자궁경부 세포진 검사는 자궁내막암 환자의 수술 전 평가방 법으로 중요시되어야 할 것이다. Objective : The aim of this study was to evaluate the correlation between preoperative Pap smears and known poor prognostic factors in patients with endometrial carcinoma. Methods : Between January 1989 and June 2000, preoperative evaluation of Pap smears were done in 163 patients with endometrial carcinoma who underwent total abdominal hysterectomy with bilateral salpingooophorectomy, peritoneal cytology, and pelvic and/or para-aortic lymphadenectomy. All Pap smears and histologic sections were reviewed. Pathologic parameters of hysterectomy specimens were evaluated and correlated with the findings of Pap smears. Chi-square test was used for statistical analysis. p-values<0.05 were considered significant. Results : The mean age of patients was 49 years with range between 24 and 75 years old. 72 patients (44.2%) had normal, 38 patients (23.3%) had atypical glandular cells of undetermined significance (AGUS), and 53 (32.5%) had adenocarcinoma on preoperative Pap smears. Statistically significant associations were found between Pap smears and age (p=.014), histologic grade (p=.000), cervical involvement (p=.015), depth of myometrial invasion (p=.000), lymph-vascular space invasion (p=.000), and surgical stage (p=.049). Patients with malignant cytology were more likely to have older age, poorly differentiated malignancies, deeper myometrial invasion, cervical metastases, lymphvascular invasion and higher surgical stage. However, histologic subtypes (p=.328), peritoneal cytology (p=.067), adnexal involvement (p=.602) and pelvic and/or para-aortic lymph node metastases (p=.266 and p=.220) were not statistically significant. Conclusion : This study revealed that preoperative abnormal Pap smears in patients with endometrial carcinoma were significantly associated with age, histologic grade, cervical involvement, depth of myometrial invasion, lymphvascular space invasion and surgical stage. Therefore, Pap smears could be an important part of the preoperative evaluation in patients with endometrial carcinoma.

자궁경부와 질에 발생한 연화판증의 세포소견 -1예 보고-

전이경,홍성란,김혜선,김지영,김복만,김희숙,Chun, Yi-Kyeong,Hong, Sung-Ran,Kim, Hye-Sun,Kim, Ji-Young,Kim, Bok-Man,Kim, Hy-Sook 대한세포병리학회 2008 대한세포병리학회지 Vol.19 No.2

Malakoplakia is an uncommon chronic granulomatous inflammation that usually involves the urinary and gastrointestinal tracts, but rarely affects the female genital tract. We experienced a case of malakoplakia in a cervicovaginal smear in a 54-year-old woman. Colposcopic examination showed a friable, easily bleeding tissue in the uterine cervix and the vaginal fornix. The cervicovaginal smear consisted of numerous isolated histiocytes, polymorphonuclear leukocytes, lymphocytes, and plasma cells. The histiocytes had an abundant, granular, and degenerated cytoplasm with inflammatory cell debris. Michaelis-Gutmann bodies were readily identified.