• Journal of Microbiology
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• v.38 no.3
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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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• v.14 no.6
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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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• v.8 no.4
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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.

• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.28-33
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• 2008

The expression vector (pWHM3-TR1R2) for sprT gene encoding Streptomyces griseus trypsin (SGT) followed by two regulatory genes, sgtR1 and sgtR2, was introduced into Streptomyces lividans TK24 and Streptomyces griseus IFO 13350. Various media with different compositions were used to maximize the productivity of SGT in the recombinant trains. he SGT productivity was best when the transformant of S. lividans TK24 was cultivated in R2YE medium (0.74 unit/mL) at 5 days of cultivation. C5/L (0.66 unit/mL) medium also gave a good productivity, but Livid (0.08 unit/mL) and NDSK (0.06 unit/mL) yielded poor productivities. S. griseus IFO 13350/pWHM3-TR1R2 produced SGT by 1.518 unit/mL (C5/L), 1.284unit/mL (R2YE),0.932 unit/mL (NDSK), and 0.295 unit/mL (Livid) at 7 days of cultivation, which was much higher than those from S. lividans TK24/TR1R2. The SGT protein was purified from the culture broth of S. griseus IFO 13350/pWHM3-TR1R2 in C5/L to homogeneity via ammonium sulfate fractionation, and CM-sepharose and SP-sepharose column chromatographies. The specific activity of purified SGT was 69,252 unit/mg, and the final purification fold and recovery yield were 6.5 and 1.4%, respectively.

• BMB Reports
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• v.32 no.1
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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.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.255-261
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• 2005

A 6.7kb DNA fragment containing the sprT gene encoding Streptomyces griseus trypsin (SGT) was cloned from Streptomyces griseus ATCC 10137, and the complete nucleotide sequence was determined. Nucleotide sequence and deduced amino acid or the EcoRI-HindIII fragment revealed the presence or the six complete ORFs containing the sprT gene and one incomplete ORF, which were named ORF1, SGT, ORF2, ORF3, ORF4, ORF5, and ORF6, respectively. ORF1 has homology with the oxidoreductases from several organisms. ORF2 and ORF3 show similarity with unknown proteins and transcription regulator that belongs to the ArsR family, respectively. ORF4 and ORF5 show homology with the peptidoglycan bound protein with LPXTG motif from Listeria monocytogenes and the membrane protein with transmembrane helix from several organisms, respectively. The last ORF, ORF6, shows homology with the lipoprotein from Streptomyces avermitilis.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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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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• v.11 no.6
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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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• v.15 no.5
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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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• v.41 no.4
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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 $50^{\circ}C$.