• 한국미생물·생명공학회지
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• 제36권1호
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• pp.28-33
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• 2008

Streptomyces griseus trypsin (SGT)을 코드하는 sprT 유전자와 그 하류에 존재하는 두 개의 조절 유전자 rsgtR1 및 sgtR2를 동시에 갖고 있는 재조합 벡터 pWHM3-TR1R2를 S. lividans TK24 및 S. griseus IFO 13350에 도입하여, 트립신의 생산성을 더욱 증대시킬 수 있는 배지를 조사하였다. S. lividans TK24/pWHM3-TR1R2의 경우 배양 5일을 기준으로 R2YE에서 가장 높은 생산성(0.74 unit/mL)을 나타냈고, C5/L. (0.66 unit/mL), Livid (0.08 unit/mL), NDSK(0.06 unit/mL) 순으로 나타났다 S. griseus IFO 13350/pWHM3-TR1R2의 경우에는 전반적으로 배양 7일에 트립신 활성이 가장 높았으며, C5/L (1.518 unit/mL), R2YE(1.284 unit/mL), NDSK (0.932 unit/mL), Livid (0.295 unit/mL) 순으로 나타났다. S. griseus IFO 13350/pWHM3-TR1R2를 C5/L 배지에서 7일간 배양한 배양액으로부터 $25%{\sim}60%$ ammonium sulfate 침전, CM-sepharose 및 Sp-sepharose column chromatography를 통하여 트립신을 고순도로 정제할 수 있었다. 최종 purification fold는 6.5배, 순수 정제된 트립신의 specific activity는 69,252 unit/mg, 회수율은 1.4%이었다.

• BMB Reports
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• 제32권1호
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• pp.86-91
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• 1999

The sprT gene encoding Streptomyces griseus trypsin (SGT) was introduced into Streptomyces lividans TK24 and Streptomyces lividans 1326 to study which strain would be better to overexpress the extracellular proteinase. Various media with different compositions were also used to maximize the productivity of SGT in heterologous hosts. The SGT productivity was best when the transformants of S. lividans TK24 and 1326 were cultivated in R2YE medium, and their relative trypsin activity of the culture broth measured with an artificial chromogenic substrate, N-${\alpha}$-benzoyl-DL-arginine-${\rho}$-nitroanilide, were 382 units/ml and 221 units/ml, respectively. They produced high levels of SGT in GYE medium but relatively lower than those in R2YE medium, and negligible amount of SGT was produced in Ferm, RASF, LIVID, and NDSK media. Considering non-SGT associated activity in Pronase powder, it was estimated that the transformant of S. lividans TK24 can produce SGT in R2YE 3.5 times more than the amount by S. griseus 10137 from which the sprT gene had been originated. The growth of S. lividans reached the maximum level of cell mass at 5 d of culture, but SGT production started in the stationary phase of cell growth and kept increasing until the ninth day of culture in R2YE medium, but in GYE media the productivity reached at the maximum level at 7 d of cultivation.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1191-1196
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• 2009

The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and $50^{\circ}C$, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to $70^{\circ}C$. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

• 미생물학회지
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• 제41권4호
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• pp.255-261
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• 2005

Streptomyces griseus trypsin(SGT)을 코드하는 sprT 유전자를 포함하여 약 6.7 kb의 DNA 단편을 Streptomyces griseus ATCC 10137의 염색체 DNA로부터 클로닝 하여 염기서열을 결정하였다. 염기서열 분석 결과, 염색체 DNA를 EcoRI-HindIII 제한효소로 완전 분해하여 클로닝한 약6.7 kb의 단편에는 sprT유전자를 포함하여 총6개의 완전한 ORF (open reading frame)와 1개의 불완전한 ORF가 존재하는 것으로 밝혀졌으며, 순서대로 ORF1, SGT, ORF2, ORF3, ORF4, ORF5, ORF6로 명명하였다. 예상 단백질의 아미노산 서열 분석 결과, ORF1은 oxidoreductase, ORF3는 ArsR family의 transcription regulator, ORF4는 Listeria monocytogenes의 LPXTG motif를 갖는 peptidoglycan bound protein, ORF5는 transmembrane helix를 갖는 막단백질, ORF6는 Streptomyces avermitilis에서 보고된 lipoprotein과 높은 상동성을 보였으며, ORF2는 기능을 예측 할 수 없었다. 이와 같은 분석결과, sprT 유전자 주위에는 세포막이나 세포벽 구성성분을 코드하는 유전자가 존재하고 있으며, 따라서 SGT Pretense는 이러한 세포막 또는 세포벽 합성이나 분해과정에서 어떤 기능을 담당할 가능성 이 있는 것으로 추측되었다.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.

• 생약학회지
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• 제42권3호
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• pp.240-245
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• 2011

A high-performance liquid chromatography (HPLC) method was developed for quantitative analysis of liquiritin and glycyrrhizin in Sagunja-tang (SGT, Sijunzi-tang in Chinese), a traditional Korean medicine. HPLC analysis was performed using a Gemini C18 column operating at $40^{\circ}C$, and photodiode array (PDA) detection at 254 nm and 280 nm for quantification of the two components in SGT. The mobile phase using a gradient flow consisted of two solvent systems. Solvent A was 1.0% (v/v) aqueous acetic acid and solvent B was acetonitrile with 1.0% (v/v) acetic acid. Calibration curves were acquired with $r^2$ values > 0.9998, and the relative standard deviations (RSDs, %) for intra- and inter-day precision were not exceed 4.0%. The recovery of each component was in the range of 91.85 - 108.62%, with a RSD less than 4.0%. The contents of the two components in SGT were 7.94 - 13.83 mg/g.

• 한국작물학회지
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• 제59권3호
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• pp.341-349
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• 2014

경실로 인해 다른 두과작물에 비해 포장 입묘가 불량한 팥의 포장출현율의 효과적인 예측 방법을 개발하기 위해 인위로 노화 정도를 조절한 종자에 대해 실험실에서 몇 가지 종자세 검사를 실시하여 포장출현력과 비교하였다. 얻어진 종자세 지표들에 대해 포장출현력과의 상관분석을 통해 예측을 위한 효율적인 지표들을 도출하였다. 팥에서 종자세가 높을 경우에는 표준발아검사에서의 종자활력률이 포장출현력 예측에 효과적이며, 약간 노화된 종자의 경우에는 저온발아검사(CGT)에서의 비정상묘율과 배축의 길이는 포장출현력과 높은 상관관계를 보였으며, 전기전도도와 CSVT에서의 활력률과 테트라졸리움 검사에서 종자세와 예측발아율은 포장출현력과 5% 수준에서 상관관계가 인정되었다. 종자세가 낮은 종자에서는 CSVT에서 불발아종자율이 포장출현력과 5% 수준에서 상관을 보였다. 포장출현력 예측에 효과적인 몇 가지 종자세지수를 이용하여 stepwise multiple regression 분석을 실시한 결과 테트라졸리움(TZ) 검사에서의 예측발아율은 결정계수($R^2$)가 0.820으로 포장출현력 예측에 가장 효과적인 검사방법임을 알 수 있었다. 표준발아검사(SGT)에서의 종자활력률, 저온발아검사(CGT)에서의 정상묘율과 건물중을 회귀방정식에 추가함에 따라 86.9% 까지 포장출현력 예측효율을 증가시킬 수 있었다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 추계학술대회 및 정기총회
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• pp.26-26
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• 2015

Phytophthora infestans is the causal agent of potato and tomato late blight, one of the most devastating plant diseases. P. infestans secretes effector proteins that are both modulators and targets of host plant immunity. Among these are the so-called RXLR effectors that function inside plant cells and are characterized by a conserved motif following the N-terminal signal peptide. In contrast, the effector activity is encoded by the C terminal region that follows the RXLR domain. Recently, I performed in planta functional profiling of different RXLR effector alleles. These genes were amplified from a variety of P. infestans isolates and cloned into a Potato virus X (PVX) vector for transient in planta expression. I assayed for R-gene specific induction of hypersensitive cell death. The findings included the discovery of new effector with avirulence activity towards the Solanum bulbocastanum Rpi-blb2 resistance gene. The Rpi-blb2 encodes a protein with a putative CC-NBS-LRR (a coiled-coil-nucleotide binding site and leucine-rich repeat) motif that confers Phytophthora late blight disease resistance. We examined the components required for Rpi-blb2-mediated resistance to P. infestans in Nicotiana benthamiana. Virus-induced gene silencing was used to repress candidate genes in N. benthamiana and to assay against P. infestans infections. NbSGT1 was required for disease resistance to P. infestans and hypersensitive responses (HRs) triggered by co-expression of AVRblb2 and Rpi-blb2 in N. benthamiana. RAR1 and HSP90 did not affect disease resistance or HRs in Rpi-blb2-transgenic plants. To elucidate the role of salicylic acid (SA) in Rpi-blb2-mediated resistance, we analyzed the response of NahG-transgenic plants following P. infestans infection. The increased susceptibility of Rpi-blb2-transgenic plants in the NahG background correlated with reduced SA and SA glucoside levels. Furthermore, Rpi-blb2-mediated HR cell death was associated with $H_2O_2$, but not SA, accumulation. SA affects basal defense and Rpi-blb2-mediated resistance against P. infestans. These findings provide evidence about the roles of SGT1 and SA signaling in Rpi-blb2-mediated resistance against P. infestans.

• 한국작물학회지
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• 제45권5호
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• pp.305-309
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• 2000

Ultra-drying [＜5.0% seed moisture content (SMC)] storage technique is a cost-effective storage method for oily seeds. To decide proper ultra-drying condition for soybean seeds, drying rate was compared three silica gel to seed ratios, two seed sizes with varietal difference, two kinds of container, and three seed amounts per container under :t 23$\pm$1$^{\circ}C$. When the relative humidity (RH) was reduced at the rate of less than 0.1 % a day, silica gel was replaced with dry one by 47 days. Higher silica gel to seed ratios (3:1 and 2:1) dried faster than lower ratio (1:1) until 28 days, but not after 43 days of drying. Also, large seeded variety was dried faster than small seeded variety. Kinds of container and seed amounts per container didn't show differences in drying of soybean seeds. After completion of ultra-drying, percentage germination by standard germination test (SGT) was not different among silica gel to seed ratios, kinds of container, and seed amounts per container, except among seed sizes (varieties). Before SGT, soybean seeds were premoistened using saturated ${CaCl}_2$ for 48 hours and ${NH}_4$Cl for 24 hours in desiccators. To compare germinability between ordinary-dried seeds and ultra-dried seeds, the seeds of seven soybean varieties, which were varying in size from 8.1 to 34.9 g per 100 seeds, were dried using same amount of silica gel under 23$\pm$1$^{\circ}C$. After completion of 76 days of drying, SMCs were reduced to 3.13-3.45% from 7.86-8.82%. SMC after completion of drying was not correlated with 100-seed weight (r=0.556). Before germination tests, soybean seeds were premoistened using saturated salt solutions. Percentage germination was higher with ultra-dried seeds than ordinary-dried seeds in SGT and higher with ordinary-dried seeds than ultra-dried seeds in AAT at the beginning of storage and after 6 months storage, but general trend of percentage germination was not observed among varieties classified by 100-seed weight. From these results, we concluded that further studies are needed to improve ultra-drying storage method for soybean seeds.