64-fold increase compared to transformant containing the promoter-less xylanase/pAN-56-1. There was a significant change in the activity profile when wheat flour medium was used (Table 2). A8 showed the maximum change, with a 3.95-fold increase in the specific activity, whereas A5 showed the minimum change, with a 2.78-fold increase in the specific activity compared to the transformant harboring promoter-less xylanase/pAN-56-1. selleck kinase inhibitor The activity of the transformant K5 carrying the Pcat924/xylanase/pAN56-1
showed the maximum change, with a 10.3-fold increase in the specific activity compared to the transformant harboring the promoter-less xylanase/pAN-56-1, whereas transformant K2 showed the least, with a 2.91-fold increase in specific activity. The results clearly depicted that AlX was expressed 6.35-fold more under the Pcat924 promoter in comparison with Pcat300. GSK2118436 manufacturer The effect of inducers on AlX activity in K6 was examined. The inducers used in this study were H2O2, CaCO3 and a combination of both. The inducers were added to the seed media. Optimal concentration of the inducer was determined for the maximum activity of the reporter gene. 0.1, 0.15, 0.20 and 0.25% (v/v) of H2O2 were used to examine the enzyme production. The maximum increase of 9.62-fold in specific activity was observed at 0.20% (v/v) H2O2 (Fig. 4),
when compared to control 2 (transformant harboring promoter-less xylanase/pAN56-1) and a 2.61-fold increase in specific activity was observed when compared to control 1 (K6 transformant harboring Pcat(924)
xylanase/pAN56-1 but grown without inducer). Induction of the promoter by CaCO3 was also studied using various concentrations (1.5%, 2.5%, 3.5% and 4.5%) of CaCO3. There was an appreciable decrease in AlX activity when the concentration of CaCO3 was increased from 1.5% to 4.5% (Fig. 4). The maximum increase in specific activity of 8.11-fold compared to control 2 and 2.20-fold compared to control 1, was seen with 1.5% CaCO3. Combinations Vitamin B12 of H2O2 and CaCO3 (0.1% H2O2 + 1.5% CaCO3, 0.15% H2O2 + 2.5% CaCO3, 0.20% H2O2 + 3.5% CaCO3, 0.25% H2O2 + 4.5% CaCO3) were investigated. The maximum increase of 7.59-fold in specific activity compared to control 2 and 2.06-fold compared to the control 1 was observed at 0.20% H2O2 + 3.5% CaCO3 (Fig. 4). Therefore, it appears that each of the two inducers is involved in co-operative regulation of catR promoter. In this study, we sought to exploit catR promoter to produce recombinant protein. For this purpose, two promoters of different lengths. Pcat300 and Pcat924, were amplified and cloned in promoter-less xylanase/pAN56-1 vector. The ability drive the expression of alx gene was evaluated for both transformants harboring Pcat(300) xylanase/pAN56-1 and Pcat924bp xylanase/pAN56-1. Expression of AlX in all transformants suggested that Pcat(300) contained the sequences required to initiate the start of transcription.