토양으로부터 Cryptococcus sp. CS-2의 분리 및 균주가 분비하는 Polygalacturonase의 특성에 관한 연구

강희경,문명님,임채영,양영기 한국미생물학회 1999 미생물학회지 Vol.35 No.2

제주도 밀감 과수원의 토양으로부터 polygalacturonase 고생산 효모를 분리하였다. 분리된 strain CS-2 의 생리화학적인 실험을 수행한 결과 분리균주는 Diazonium bule B color test 와 urease test에서 양성반응, acetic acid, citrate acid 생성 시험, gelatin 분해시험, fat 분해시험에서는 음성반응을 나타났고, 탄소원의 조사에서 galactose를 탄소원으로 이용할 수 없었고, 질소원은 모두 이용할 수 없었다. 또한 $30^{\circ}C$ 에서는 성장하는 반면, $35^{\circ}C$ 이상에서는 성장을 하지 못하였고 50% 이상의 glucose 농도, 0.01% cyclohexamide 그리고 1% acetic acid에서 성장을 하지 못하였다. 현미경을 이용한 형태학적 관찰 결과, 크기는 $1.3{\times}2.9$$\mu$m인 타원형으로 관찰되었다, ,multiple budding을 하며 ascospore는 존재하는 반면 pseudomycelium과 true mycelium 은 존재하지 않았다. 이와 같은 결과를 종합하여 Cryptococcus 속의 특징과 유사함을 알 수 있었다. 분리된 Cryptococcus sp. CS-2 의 polygalacturonase 는 유도적으로 합성되어 catabolic repression 에 의해 조절됨을 알 수 있었으며, 3일 배양시 가장 높은 활성도를 나타내었으며, 고유 활성도는 2.50~2.55 units/mg 으로 나타났다. 이러한 polygalacturonase를 SDS-PAGE, activity staining 그리고 단일 탄소원에 따른 단백질 양상의 변화를 비교하여 분자량 측정한 결과 약 46KDa으로 나타났다. A ploygalacturonase-produchg yeast was isolated from Cheju soil by selective eivichment media. One strain which has the highesl activity of polygalacturonase was selected. The characle~ishcs of the strain CS-2 were as follows: CS-2 utilized xylose. sucrose, maltose, u.ehalose, cellobiose. melibiose, lactose, raffinose, inosiiol, dulicilol, and dextrose, but did not utilized galactose, nitrate. nit~te, and lysine. Growth of CS-2 was inhibited by cyclohexamide, 1% acetic acid, and high concenaation (over 50%) of glucose. It grew at $30^{\circ}C$ but did 'IIOL $35^{\circ}C$. The cell size ofthe strain CS-2 was 2.9 p ~ n in length and 1.3 $\mu$ in diameter. Vegetable reproductmn was multiple budding and ascospre was present I to 4. Pseudomycelia or true myceliua formation were not observed In any of the cullureq. These results suggest that strain CS-2 is most likely a strain related Cryptococcus spp. (Cryptococcu spp. CS-2). When polygalacturonase or ihe yeast was induced by addition of polygalactoronic acid, polygalacturonase activity was detected in culture supernatent. There was a peak of specific activity a1 he mid-stationary phase(3 days culture) of growth. Polygalacturonase specific activity of Crylmcoccus sp. CS-2 was 2.96 unitsling. The molecular weighl ol'polygalacturonase was showed to be 46 KDa by both SDS-PAGE and activity stailling.

잿빛곰팜이병균 Botrytis cinera가 분비하는 Polygalacturonase의 부분정제와 특성

나유진,김재원,정영륜,허남응,조광연 한국식물병리학회 1994 Plant Pathology Journal Vol.10 No.3

Polygalacturonase (PG) produced by Botrytis cinerea in the culture broth containing citrus pectin as a carbon source was partially purified and characterized. PG was produced on a range of carbon sources such as starch, glycerol, cellobiose, and Na+-PAG with total activities of 34.8, 32.0, 29.2, 27.8 units, respectively. The specific activity was highest with 2316.7 units on Na+-PGA. Proteins of culture filtrate were concentrated with polyethylene glycol and acetone and applied to a hydroxyapatite column. Among three active fractions collected from the column, the reaction containing the highest PG activity was resolved by a Q-sepharose column. The active fraction from the Q-sepharose column was further purified by HPLC Mono Q column. The partially purified enzyme was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among a few protein bands revealed, the amount of the protein of which molecular weight estimated to be 43 kDa coincided with the PG activity. The partially purified PG had optimal temperatures between 35~55$^{\circ}C$ and pH between 4.5~5.5.

토양으로부터 Polygalacturonase를 생산하는 Cryptococcus spp. CS-2의 분리 및 동정

강희경,임채영,양영기 조선대학교 생명과학연구소 1998 생명과학 연구 Vol.6 No.-

제주도 북제주군 애월읍에 위치한 밀감 과수원의 토양으로부터 YM 배지를 이용하여 효모를 분리한 후, 1% polygalacturonic ac펀가 첨가된 배지에서 polygalacturonase 생성능력이 우수한 효모를 분리하였다. 분리된 strain CS-2의 생리화학적인 실험을 수행한 결과 분리균주는 Diazonium blue B(DBB) color test와 urease test에서 양성반응을, acetic acid, citrate acid 생성시험, gelatin과 fat 분해시험에서는 음성반응을 나타내어 Cryptococcus속과 비슷한 양상을 보였다. 탄소원의 조사에서 galactose를 탄소원으로 이용할 수 없었고, 질소원은 모두 이용할 수 없었다 또한 30℃에서는 성장하였으나, 35℃ 이상의 온도, 50% 이상의 glucose농도(w/w), 0.01% cyclohexamide 그리고 1% acetic acid에서는 성장을 하지 못하였다. 현미경을 이용한 형태학적인 관찰 결과, strain CS-2의 크기는 1.3×2.9μm인 타원형으로 관찰되었다. multiple budding을 하고, ascospore는 존재하는 반면, pseudornyceliurn과 true mycelium은 존재하지 않았다. 본 실험에서 사용된 strain CS-2의 형태학적, 배양학적, 생리학적인 여러 가지 결과를 종합하여 Cryptococcus속의 특징과 유사함을 확인할 수 있었기에 Cryptococcus spp. CS-2라 명명 하였다. A ploygalacturonase-producing yeast was isolated from Cheju soil by selective enrichment medium. One strain which has the highest activity of polygalacturonase was selected. The characteristics of the strain CS-2 were as follows: CS-2 utilized xylose, sucrose, maltose, trehalose, cellobiose, melibiose, lactose raffinose, inositol dulicitol, and dextrose, but did not utilize galactose, nitrate, nitrite, and lysine. Growth of CS-2 was inhibited by cyclohexamide, 1% acetic acid, and high concentration(over 50%) of glucose. It grew at 30℃ but did not at 35℃, The cell size of the strain CS-2 was 2.9㎛ in length and 1.3㎛ in diameter. Vegetable reproduction was multiple budding and ascospores was present 1 to 4. Pseudomycelia or true mycelia formation were not observed in any of the cultures. These results suggest that strain CS-2 is most likely a strain related Cryptococcus spp.(Cryptococcus spp. CS-2).

Polygalacturonase의 활성 증진 및 이를 이용한 식물 단세포 제조 방법

김혁화(Hyuk?Hwa Kim) 한국식품영양과학회 2007 한국식품영양과학회지 Vol.36 No.12

본 연구에서는 효소의 활성을 증진시킬 수 있는 새로운 방법의 개발을 위하여 미생물로부터 분리, 정제된 polygalacturonase(PGase)에 xanthan gum, guar gum, locust bean gum 등과 같은 교질물질을 첨가함으로써 효소의 안정성 외에 활성을 특이적으로 향상시킬 수 있는 새로운 방법 조사하였다. 정제된 PGase를 0.2%의 상술한 교질물질을 함유하는 동일한 완충용액에 잘 혼합시켜 30℃에서 배양하여 효소의 불활성화에 대한 일차반응 속도상수 k값을 구한 결과, 대조군의 k값이 0.0082 min<SUP>-1</SUP>인데 반해 xanthan gum 첨가 시는 0.0003 min<SUP>-1</SUP>, guar gum 첨가 시는 0.0001 min<SUP>-1</SUP>이하, locust bean gum 첨가 시는 0.0001 min<SUP>-1</SUP>로서 교질물질 첨가에 의해 효소의 안정성이 현저히 증가하였다. 또한 대조군에 비해 xanthan gum 첨가 시는 PGase의 상대활성이 89%가 증가되었으며, guar gum 첨가 시는 97%, locust bean gum 첨가 시는 90%가 증가되어 상술한 교질물질들이 효소의 활성 촉진제로서의 기능이 있음을 확인할 수 있었다. Guar gum 처리에 의해 약 2배의 활성이 증진된 상태로 당근 단세포 생성 반응을 수행한 결과 모든 반응시간에서 guar gum을 가했을 때가 PGase만을 가하여 단세포화 반응을 수행한 경우보다 높은 수율을 보였으며, 단세포화 반응 2시간 경과 후에 대조군 대비 13%의 가장 높은 수율 향상을 보였다. This study was carried out to enhance the stability and activity of polygalacturonase (PGase) purified from Kluyveromyces marxianus IFO 0288. Gums such as xanthan gum, guar gum, and locust bean gum were capable of increasing the catalytic stability and activity of the PGase. At 30℃, the rate constants for the inactivation of the PGase with xanthan gum, guar gum, and locust bean gum were estimated to be 0.0003 min<SUP>-1</SUP>, below 0.0001 min<SUP>-1</SUP>, and 0.0001 min<SUP>-1</SUP> respectively, whereas control was estimated to be 0.0082 min<SUP>-1</SUP>. The yield of the maceration reaction catalyzed by the PGase for the production of carrot single cells increased by 13% in the presence of guar gum, where the relative enzyme activity supplemented with guar gum was two-fold greater than that of the PGase alone.

Characterization of an Apple Polygalacturonase-Inhibiting Protein (PGIP) That Specifically Inhibits an Endopolygalacturonase (PG) Purified from Apple Fruits Infected with Botryosphaeria dothidea

( Dong Hoon Lee ),( Han Hong Bae ),( In Kyu Kang ),( Jae Kyun Byun ),( Sang Gu Kang ) 한국미생물생명공학회 2006 Journal of microbiology and biotechnology Vol.16 No.8

Polygalacturonase를 검출하기 위한 종이 기반의 효소결합 면역반응 센서 제작

황영국 ( Young-kug Hwang ),김지관 ( Ji-kwan Kim ),이영환 ( Young Hwan Lee ),최영수 ( Young-soo Choi ) 한국센서학회 2021 센서학회지 Vol.30 No.5

In this paper, we describe the fabrication of a paper-based enzyme-linked immunosorbent assay (ELISA) to detect polygalacturonase (PG), which is used as a biomarker to determine whether a plant is infected with a disease. The proposed paper-based ELISA can analyze the concentration of PG in a short time using a small sample compared to the traditional ELISA, which is generally performed using a well plate. To increase the resolution of the sensor, we optimized the dilution ratio of the HRP-conjugated goat anti-rabbit IgG antibody and the dilution ratio of the anti-PG and HRP-conjugated goat anti-rabbit IgG antibodies. Furthermore, for quantitative analysis of PG concentration, Delta RGB analysis was conducted to detect color changes in the sensing window displayed by the PG samples at various concentrations. Based on the experiment, the fabricated paper-based ELISA could measure at least 0.25 μg of PG and the measurement range was 0.25-2 μg. Therefore, the paper-based ELISA for detecting PG is expected to be able to determine the presence or absence of disease in crops at the infection stage in the future.

Escherichia coli에서 Neurospora crassa polygalacturonase를 암호화하는 합성 유전자의 발현

김지호, 김상래, 이병욱 고신대학교보건과학연구소 2020 보건과학연구소보 Vol.28 No.-

Escherichia coli에서 Neurospora crassa polygalacturonase를 암호화하는 합성 유전자의 발현

Characterization of an Apple Polygalacturonase-inhibiting Protein (PGIP) from Apple Fruits

이동훈,강상구,강인규,이윤경,최철,변재균,Lee, Dong-Hoon,Kang, Sang-Gu,Kang, In-Kyu,Lee, Yoon-Kyeong,Choi, Cheol,Byun, Jae-Kyun Korean Society of Life Science 2006 생명과학회지 Vol.16 No.4

사과 겹무늬썩음병균이(Botryosphaeria dothidea) 생성하는 세포벽 분해효소인 endopolygalaturonase를 억제하는 polygalacturonase-inhibiting protein (PGIP)를 사과 과실로부터 분리하였다. 분리되어진 사과 PGIP는 사과 겹무늬썩음병균이 생성하는 PG에 대하여 혼합형의 저해를 나타내었다. PGIP의 반응 최적온도는 $40^{\circ}C$이며 최적 pH는 5.0이었다. 이 효소는 $60^{\circ}C$까지는 비교적 안정하였으나 $70^{\circ}C$에서는 효소의 활성이 완전히 억제되었으며 pH 4.0에서 8.0까지는 안정하였다. PGIP는 $K^+$, $Cu^{2+}$, $Mg^{2+}$, $Ca^{2+}$ 과 $Zn^{2+}$ 등의 금속이온과 SDS 그리고 CDTA에 의해 효소의 활성이 저해되었다. An apple polygalacturonase-inhibiting protein (PGIP), that specifically inhibited endopolygalacturonase (PG, EC 3.2.1.15) from Botryosphaeria dothidea, was purified from B. dothidea infected apple (Malus domestica cv. Fuji) fruits. The apple PGIP was a mixed-type inhibitor of PG from B. dothidea. Optimal temperature for the maximum enzyme activity was $40^{\circ}C$, and optimum pH of the purified PGIP was pH 5.0. PGIP was stable up to temperature of $60^{\circ}C$ and was completely suppressed after heating at $70^{\circ}C$ for 10 min, PGIP was stable at pH between 4 and 8. Inhibition of PG by PGIP was reduced by $K^+$, $Cu^{2+}$, $Mg^{2+}$, $Ca^{2+}$ and $Zn^{2+}$ metal ion, sodium dodecyl sulfate (SDS) and 1,2-diaminocyclohexane tetra acetate (CDTA).

잿빛곰팡이병균 Botrytis cinerea가 분비하는 Polygalacturonase의 부분정제와 특성

나유진,김재원,정영륜,허남응,조광연 한국식물병리학회 1994 Plant Pathology Journal Vol.10 No.3

Characterization of the Gene Encoding Radish (Raphanus sativus L.) PG-inhibiting Protein

Hwang, Byung-Ho,Kim, Hun,Lim, Sooyeon,Han, NaRae,Kim, Jongkee Korean Society of Horticultural Science 2013 원예과학기술지 Vol.31 No.3

A radish (Raphanus sativus L.) polygalacturonase-inhibiting protein (PGIP) gene was cloned and compared to the PGIP gene (BrPGIP2) from Chinese cabbage (Brassica rapa ssp. pekinensis) in order to gain more information on controlling a disease and improving produce quality. To clone the radish PGIP gene, primers were designed based on conserved sequences of two PGIP genes (BnPGIP1 and BnPGIP2) from rape (B. napus L. ssp. oleifera), Chinese cabbage and Arabidopsis thaliana. PCR cloning was performed with cDNA from the stigma of radish 'Daejinyeoreum' as a template to confirm DNA fragments which were about 600 base pair in size. Sequence analysis revealed 84.1% homology with BrPGIP2 and 70.1% with BnPGIP1. DNA walking was conducted to confirm the open reading frame of 972 bp, and the gene was named RsPGIP1. RsPGIP1 consisting with 323 amino acids (aa) has a high leucine content (54/323) and contains 10 leucine-rich repeat domains, as do most BrPGIPs of Chinese cabbage. The gene expression of RsPGIP1 was induced by abiotic stresses and methyl jasmonate. It showed enrichment in the stigma and the primary root than a leaf. Cloning RsPGIP1 will aid to further apply practices on postharvest quality maintenance and disease control of the root.