• Biotechnology and Bioprocess Engineering:BBE
• /
• v.6 no.3
• /
• pp.167-172
• /
• 2001

Staphylokinase (SAK) was produced in B. subtilis using two different promoter systems, i.e. the P43 and sacB promoters. To maximize SAK expression in B. subtilis, fermentation control strategies for each promoter were examined. SAK, under P43, a vegetative promoter transcribed mainly by $\sigma$(sup)B containing RNA polymerase, was overexpressed at low dissolved oxygen (D.O.) levels, suggesting that the sigB operon is somewhat affected by the energy charge of the cells. The expression of SAK at the 10% D.O. level was three times higher than that at the 50% D.O. level. In the case of sacB, a sucrose-inducible promoter, sucrose feeding was used to control the induction period and induction strength. Since sucrose is hydrolyzed by two sucrose hydrolyzing enzymes in the cell and culture broth, the control strategy was based on replenishing the loss of sucrose in the culture. With continuous feeding of sucrose, WB700 (pSAKBQ), which contains the SAK gene under sacB promoter, yielded ca. 35% more SAK than the batch culture. These results present efficient promoter-dependent control strategies in B. subtilis host system for foreign protein expression.

• KSBB Journal
• /
• v.16 no.4
• /
• pp.415-419
• /
• 2001

To produce staphylokinase (SAK) in B. subtilis WB700, media optimization was carried out and the operation of batch and fed-batch fermentation were compared. Tryptone is a good nitrogen source and its optimum concentration in modified super rich(MSR) media is 15 g/L. When glucose is used as a limiting carbon source in the MSR media, 5 g/L of an optimum glucose concentration was identified for the SAK production under the control of P43 promoter. As the expression of P43 promoter is controlled by the limitation of oxygen, the SAK production was controlled at the 30% DO level in the fed-batch fermentation. Unexpectedly, batch fermentation using MSR media showed 1.5 times higher yield of SAK than that of the fed-batch fermentation. The main cause of the results comes from not achieving higher cell concentration in the fed-batch fermentation and the optimum expression level of P43 promoter under oxygen or nutrient limitations. We could not achieve the increase in cell concentration by any means in batch culture as well as fed-batch culture. The highest yield in the batch culture was 2880 units of SAK activity and 455 mg/L of secreted SAK.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.7
• /
• pp.894-901
• /
• 2012

Based on their respective antitumor and thrombolytic activities, the superantigen staphylococcal enterotoxin C2 (SEC2) and staphylokinase (Sak) were chosen for the construction of the novel chimeric proteins Sak-linker-SEC2 and SEC2-linker-Sak using a linker composed of nine Ala residues. Both chimeric proteins possessed nearly the same PBMC proliferation stimulating activity and antitumor activity as SEC2 and thrombolytic activity as Sak. Neither the SEC2 or Sak component of each chimeric protein affected the activity of the other component. The results presented in this study provide a possible strategy to prevent and cure tumor thrombus.