• Journal of Microbiology and Biotechnology
• /
• v.14 no.6
• /
• pp.1350-1355
• /
• 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
• /
• v.15 no.5
• /
• pp.1125-1129
• /
• 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 and Biotechnology
• /
• v.11 no.6
• /
• pp.1077-1086
• /
• 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 Ginseng Research
• /
• v.45 no.2
• /
• pp.218-227
• /
• 2021

Background: Panax ginseng is one of the most important medicinal plants and is usually harvested after 5 to 6 years of cultivation in Korea. Heavy metal (HM) exposure is a type of abiotic stress that can induce oxidative stress and decrease the quality of the ginseng crop. Siderophore-producing rhizobacteria (SPR) may be capable of bioremediating HM contamination. Methods: Several isolates from ginseng rhizosphere were evaluated by in vitro screening of their plant growth-promoting traits and HM resistance. Subsequently, in planta (pot tests) and in vitro (medium tests) were designed to investigate the SPR ability to reduce oxidative stress and enhance HM resistance in P. ginseng inoculated with the SPR candidate. Results: In vitro tests revealed that the siderophore-producing Mesorhizobium panacihumi DCY119^T had higher HM resistance than the other tested isolates and was selected as the SPR candidate. In the planta experiments, 2-year-old ginseng seedlings exposed to 25 mL (500 mM) Fe solution had lower biomass and higher reactive oxygen species level than control seedlings. In contrast, seedlings treated with 10⁸ CFU/mL DCY119^T for 10 minutes had higher biomass and higher levels of antioxidant genes and nonenzymatic antioxidant chemicals than untreated seedlings. When Fe concentration in the medium was increased, DCY119^T can produce siderophores and scavenge reactive oxygen species to reduce Fe toxicity in addition to providing indole-3-acetic acid to promote seedling growth, thereby conferring inoculated ginseng with HM resistance. Conclusions: It was confirmed that SPR DCY119^T can potentially be used for bioremediation of HM contamination.

• Proceedings of the Korean Society of Precision Engineering Conference
• /
• 2010.11a
• /
• pp.853-854
• /
• 2010

• Proceedings of the Korean Society of Precision Engineering Conference
• /
• 2010.11a
• /
• pp.789-790
• /
• 2010

• The Korean Journal of Food And Nutrition
• /
• v.15 no.4
• /
• pp.364-369
• /
• 2002

Streptomyces shows a eukaryotic characteristic that vegetative cell can grow into mycelial form and has morphological and physiological differentiation at a certain period during its life cycle. Streptomyces has been used for the production of many biologically active compounds, such as antibiotics and pronase. Production of second metabolites and differentiation of the vegetative cell share the certain period of its lift cycle. Therefore, second metabolites may affect the differentiation of the vegetative cell. One of the microbial hormone, called A-factor, regulates the production of second metabolites, sporulation and differentiation of the cells. Streptomyces griseus produces streptomycin as well as many different kinds of proteinase. As mentioned, period of proteinases production overlaps with the period of differentiation of the vegetative cells. Protease may play a important role for the differentiation of the cells. In this paper, function of the SGPD gene cloned from S. griseus IFO 13350 tested whether it affects for the differentiation of A-factor mutated S. griseus HH1 and S. griseus IFO13350. pWHM3 and pWHM3-sprD plasmid was transformed into S. griseus HH1 and S. griseus IFO13350. Chymotrypsin activity of the cultured medium of the transformants with pWHM3-sprD plasmid didn't show any change with that of the transformants with plasmid only. The transformants with pWHM3-sprD plasmid didn't show the increase of the production of actinorhodin as well as morphological change in S. griseus IFO 13350 and HH1, as well. The promoter sequences of the SGPA and SGPB gene which encode chymotrypsin-like protease, were compared with that of SGPD gene. Regulatory mechanism of gene expression of proteinase genes will be studied for the development of high production system for protease as well as the function of the proteases.

• Korean Journal of Microbiology
• /
• v.41 no.4
• /
• pp.255-261
• /
• 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
• /
• v.8 no.4
• /
• pp.333-340
• /
• 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.

• Interaction and multiscale mechanics
• /
• v.6 no.2
• /
• pp.257-270
• /
• 2013

The use of lightweight materials has been steadily increasing in the automotive industry, and presents new challenges to material joining. Among many joining processes, self-piercing riveting (SPR) is particularly promising for joining lightweight materials (such as aluminum alloys) and dissimilar materials (such as steel to Al, and metal to polymer). However, to establish a process window for optimal joint performance, it often requires a long trial-and-error testing of the SPR process. This is because current state of the art in numerical analysis still cannot effectively resolve the problems of severe material distortion and separation in the SPR simulation. This paper presents a coupled meshfree/finite element with a moving boundary algorithm to overcome these numerical difficulties. The simulation results are compared with physical measurements to demonstrate the effectiveness of the present method.