• Journal of Microbiology
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• 제38권3호
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• pp.160-168
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• 2000

A-factor I a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. to identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by Streptomyces griseus IFO 13350 and its A-factor deficient mutant strain, Streptomyces griseus HH1, as well as Streptomyces griseus HH1 transformed with the afsA gene were sturdied. In general Streptomyces griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of Streptomyces griseus IFO 13350 was greatly enhanced more than twice compared with that of Streptomyces griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of Streptomyces griseus HH1 was greatly enhanced more than twice compared with that of Streptomyces griseus IFO 13350, and this observation was reversed in the presence of thiostreptione, However, Streptomyces griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between Streptomyces griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-amionpeptidase activity was 2 times higher in Streptomyces griseus HH1 than in strain IFO 13350 . Streptomyces griseus HH1 harboring afsA showed a similar level of enzyme activity , however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morpholofical differentiation, the formation of aerial meycelium and spores was delayed by two or three days.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1350-1355
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• 2004

The sprA and sprB genes, encoding the 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 S. griseus and were overexpressed in various strains of S. griseus. When the sprT gene was introduced into S. griseus, trypsin activity increased 2-fold in the A-factor deficient mutant strain, S. griseus HH1, and increased 4-fold in the wild strain, S. grise us IFO 13350. However, there was no detectable increase of chymotrypsin activity in the transformants of S. griseus with either sprA or sprB, in contrast to the results obtained from S. lividans as a heterologous host. To solve the negative gene dosage effects in S. griseus, either the sprA or the sprB genes with their own ribosome binding sites were linked to the downstream of the entire sprT gene, and the coexpression efficiency was examined in S. lividans and S. griseus. The transformants of S. lividans with either pWHM3-TA (sprT+sprA) or pWHM3­TB (sprT+sprB) showed 3-fold increase of trypsin activity over that of the control, however, only the transformant of pWHM3-TB demonstrated 7-fold increase in chymotrypsin activity, indicating that the pWHM3-TB has a successful construction for the overexpression of chymotrypsin in Streptomyces. When the coexpression vectors were introduced into S. griseus IFO 13350, the trypsin level sharply increased by more than 4-fold, however, the chymotrypsin level did not increase. These results strongly suggest that the overexpression of the sprA and sprB genes is tightly regulated in S. griseus.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.333-340
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• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• 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.

• 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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• 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.

• 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.

• 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.

• 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 $50^{\circ}C$.