• 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.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.563-570
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• 2016

S. griseus wild type에서 dasA 유전자의 과발현에 의해 유도된 기저균사의 ectopic sporulation 관련 유전자를 알아보기 위해서, empty vector가 삽입된 균주와 dasA가 과발현된 균주의 전사체를 DNA microarray법으로 비교하였다. DNA microarray 결과를 토대로 dasA 유전자 과발현 균주에서 2배이상 발현량이 증가되었으며 p-value가 0.05 미만(p-value < 0.05)인 유전자들 중에서 false positive 를 제외시키는 작업을 통하여 최종적으로 4개의 유전자(SGR794, SGR2469, SGR3656, SGR3657)와 3개의 cluster (SGR795-797, SGR2377-2378, SGR6997-6998)를 선발하였다. 이들의 전사량은 low resolution Sl nuclease mapping 법을 통하여 dasA 유전자 과발현 균주에서 증가된 것을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1125-1129
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• 2005

Cloning of a 6.6-kb BamHI digested chromosomal DNA from S. griseus IFO13350 revealed the presence of an additional gene encoding a novel trypsin-like enzyme, named SprU. The SprU protein shows a high homology ($79\%$ identity, $88\%$ similarity) with the SGT protease, which has been reported as a bacterial trypsin in the same strain. The amino acid sequence deduced from the nucleotide sequence of the sprU gene suggests that SprU is produced as a precursor consisting of an amino-terminal presequence (29 amino acid residues), prosequence (4 residues), and mature trypsin consisting of 222 amino acids with a molecular weight of 22.94 kDa and a calculated pI of 4.13. The serine, histidine, and aspartic acid residues composing the catalytic triad of typical serine proteases are also well conserved. When the trypsin activity of the SprU was spectrophotometrically measured by the enzymatic hydrolysis of the artificial chromogenic substrate, N-${alpha}$-benzoyl-DL-arginine-p-nitroanilide, the S. lividans transformant with pWHM3-U gave 3 times higher activity than that of control. When the same recombinant plasmid was introduced into S. griseus, however, the gene dosage effect was not so significant, as in the cases of other genes encoding serine proteases, such as sprA, sprB, and sprD. Although two trypsins, SprU and SGT, have a high degree of homology, the pI values, the gene dosage effect in S. griseus, and the gene arrangement adjacent to the two genes are very different, suggesting that the biochemical and biological function of the SprU might be quite different from that of the SGT.

• 한국미생물·생명공학회지
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• 제35권2호
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• pp.112-117
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• 2007

Protein kinase는 진핵생물과 원핵생물을 포함하는 모든 생명체에서 세포생존에 절대적으로 중요한 조절 기능을 담당한다. 일반적으로 원핵생물은 histidine 과 aspartic acid kinase로 구성된 bacterial two-component regulatory system에 의하여 환경변화에 따른 유전자의 발현이 조절되지만, 방선균을 비롯한 고등 원핵생물에서는 진핵생물성의 serine/threonine kinase들이 세포분화와 같은 분화과정을 조절하고 있다. Streptomycin 생산균인 Streptomyces griseus 균주에서도 다양한 serine/threonine kinase들이 존재하는 것으로 추정되며, 이들의 기능을 밝히는 것은 생명현상을 이해하는 중요한 열쇠를 제공해 줄 것으로 기대된다. 따라서, S. griseus로부터 protein kinase 를 동정하는 연구를 실시하였으며, 기존의 복잡한 chromatography법의 단점을 보완하기 위해 anti-phosphothreonine, anti-phosphoserine, anti-phosphotyrosine antibody를 이용한 immunoaffinity column chromatography 방법을 도입하였다. 실험 결과 약 14, 29, 31, 35, 40, 52, 56, 60 kDa의 단백질을 효과적으로 동정 할 수 있었으며, nonradioactive protein kination assay 방법으로 이들의 인산화능을 확인하였다.

• Journal of Microbiology
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• 제41권4호
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• pp.289-294
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• 2003

Gelatinase is a proteolytic enzyme that hydrolyzes gelatin. Gelatinolytic activity was detected from culture broths of Streptomyces griseus IFO13350 and HH1 by paper disc assays on 0.5% agar plates containing 1% gelatin. The concentrated extracellular protein from the S. griseus was analyzed by SDS polyacrylamide gel, and two proteins, with molecular weights of 30 and 28 kDa, respectively, were identified to have gelatinase activity by gelatin zymography. The protein with a molecular weight of 28 kDa was confirmed to be S. griseus trypsin (SGT). The effects of metal ions and metal chelators on the protease activity of the SGT were studied. Of the metal ions tested, only manganese was found to enhance the protease activity, 2.6 times, however, $Co^{2+},\;Cu^{2+},\;and\;Zn^{2+}$, and metal chelators, such as EDTA and EGTA, inhibited the SGT activity. When the protease activity of the SGT was measured at various pHs, in the presence of 5 mM $MnCl_2$, its highest activity was at pH 11.0, whereas only 60% of the maximum activity was observed between pHs 4.0 and pH 6.0, and almost 80% activity between pHs 7.0 to pH 10.0. The protease activity was measured at various temperatures in the presence of 5 mM $MnCl_2$. The SGT was found to be stable up to $60^{\circ}C$ for 30 min, while only 16% of the enzyme activity remained at $60^{\circ}C$, and at $80^{\circ}C$ almost all the activity was lost. The optimal temperature for the protease activity was $50^{\circ}C$.

• Journal of Microbiology
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• 제37권2호
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• pp.102-110
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• 1999

Genetic determinant for the secondary metabolism was studied in heterologous expression in Streptomyces lividans TK-24 using Streptomyces griseus ATCC 10137 as a donor strain. Chromosomal DNA of S. griseus was ligated into the high-copy number Streptomyces shuttle plasmid, pWHM3, and introduced into S. lividans TK-24. A plasmid clone with 4.3-kb BamHI DNA of S. griseus (pMJJ201) was isolated by detecting for stimulatory effect on actinorhodin production by visual inspection. The 4.3-kb BamHI DNA was cloned into pWHM3 under the control of the strong constitutive ermEp promoter in both directions (pMJJ202); ermEp promoter-mediated transcription for coding sequence reading right to left: pMJJ203; ermEp promoter-mediated transcription for coding sequence reading left to right) and reintroduced into S. lividans TK-24. The production of actinorhodin was markedly stimulated due to introduction of pMJJ202 on regeneration agar. The introduction of pMJJ202 also stimulated production of actinorhodin and undecylproidigiosin in submerged culture employing the actinorhodin production medium. Introduction of pMJJ203 resulted in a marked decrease of production of the two pigments. Nucleotide sequence analysis of the 4.3-kb region revealed three coding sequences: two coding sequences reading left to right, ORF1 and ORF2, one coding sequence reading right to left, ORF3. Therefore, it was suggested that the ORF3 product was responsible for the stimulation of antibiotic production. The C-terminal region of ORF3 product showed a local alignment with Myb-related transcriptional factors, which implicated that the ORF3 product might be a novel DNA-binding protein related to the regulation of secondary metabolism in Streptomyces.

• 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.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.169-177
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• 1992

토양으로부터 alkaline protease 생성능이 강한 Streptomyces griseus HC-1141을 분리하였으며, 효소생산의 최적 배양조건은 0.5 casein, 0.05 ammonium chloride, 0.1 ferrous sulfate, 2.0의 lactose, pH 8.0에서 84시간 배양했을 때이다. 효소의 정제는 ammonium sulfate 침전, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, crystallization으로 하여 53.23배 정제할 수 있었으며 polyacrylamide gel 전기영동상 단일밴드를 나타내었다.

• 생명과학회지
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• 제19권5호
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• pp.676-679
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• 2009

미생물들 사이에 존재하는 수평적 유전자 전달을 이용하여 Jurkat T cell에 대하여 새로운 항생물질을 생산하기 위해 토양박테리아 AY2000과 여러 종류의 항생물질 생산 균주인 Streptomyces griseus의 공배양을 수행하였다. MTT assay를 수행하여 세포 독성을 실험을 하였을 때 공배양 상등액은 각각 배양 하였을 때보다 높은 세포독성을 보였고 또한 48시간 배양 하였을 때 가장 높은 활성을 나타내는 것으로 나타났다. 더욱이 DAPI 염색을 하였을 때 Jurkat T cell 세포의 세포핵의 변화도 관찰 되었다. 이런 결과는 공배양 상등액에 새로운 항생물질이 생성되었음을 보여주었고 이런 방법으로 새로운 항생물질 생산에 이용되어 질수 있음을 의미한다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권1호
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• pp.18-24
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• 2001

The advantage of using Streptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystalline chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75-99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 dyas of cultivation with 99% deacetylated chitosan. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)$_3$, (GlcN)$_4$and (GlcN)(sub)5 were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)$_3$was homogeneous and those of (GlcN)$_4$and (GlcN)(sub)5 were heterogeneous.