• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.534-542
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• 1992

Trypsin inhibtor produced by Streptomyces sp. S-217 was purified by solvent extraction and various column chromatographies. and physico-chemical properties of the inhibitor were investigated. Inhibitor complex was formed for incubation of 10 min. Streptomyces 5-217 showed the highest production of trypsin inhibitor when it was cultivated at $37^{\circ}C$ for 66 hr in the medium containing 2% mannitol & 0.9% peptone, pH 7.0. Trypsin inhibitor was purified by column chromatography and high performance liquid chromatography. Trypsin inhibitor indicated the maxium wavelength at 215 nm and solubilities in water, methanol and dimethyl sulfoxide were 95, 70 and 75%, respectively. The concentration of 50% inhibition ($IC^{50}$) was 15 $\mu$g/ml. The inhibitor was stable on heating at $100^{\circ}C$ for 60 min in pH 5~9 and was more stable in alkaline region than acidic region.

• Molecules and Cells
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• v.37 no.10
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• pp.727-733
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• 2014

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-Omethylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong $ermE^*$ promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.