The arrows indicate the expressed forms of MCAP protein when the initial pH value of the medium was 5.0 and the lines indicate the expressed forms of MCAP at initial pH of 7.0. None Selleckchem AZ 628 of the other recombinants analyzed in this study
was able to produce MCAP. It is possible that P. pastoris containing plasmid pGAPZα+MCAP (data not shown) was unable to cleave the MCAP gene intron sequence. Such a situation has been shown in S. cerevisiae that did not secrete R. niveus aspartic proteinase as it contained an intron sequence . In the case of strain containing pGAPZα+MCAP-2 and pGAPZα+MCAP-3 (Figure 3, lanes 4, 5, respectively), the start codon of α-MF secretion signal and start codon of MCAP are each very close to the promoter, which might have caused some inhibition of transcription. The unsuccessful result of X-33/pGAPZα+MCAP-SP
(Figure 3, lanes 6) could have been due to Selleck SBI-0206965 the deleted part of MCAP proenzyme sequence, which is very important for its conversion to the mature form. Effect of glucose concentration, temperature and initial pH on MCAP production Glucose concentration The activity of the MCAP produced by the recombinant X-33/pGAPZα+MCAP-5 grown in two concentrations of glucose as the sole carbon source in the YPD medium at pH 5.0 and 24°C was compared. When glucose was used at 20 g L-1 the relative activity of MCAP decreased to 40% compared to a glucose concentration of 40 g L-1 . The time course of MCAP production by X-33/pGAPZα+MCAP-5 (Figures 5 and 6A) showed that after 24, 48, 72 and 96 h of growth the activity of the crude enzyme was 13 (7 mg L-1), 172 (54 mg L-1), 257 (110 mg L-1) and 181 MCU mL-1 Calpain (100 mg L-1), respectively. Therefore, it was concluded that the maximum enzyme activity of 257 MCU mL-1 of fermentation broth was after approximately 72 h of cultivation when culture cells were in their late exponential growth phase and decreased after 96 h when the cells reached the stationary phase. The increase in activity was due to the quality of enzyme produced (Figures 5 and 6A). Furthermore, when the original MCAP gene was adapted to the optimal codon usage of P. pastoris, the expression of aspartic proteinase
in P. pastoris (X-33/pGAPZα+SyMCAP-6) increased by nearly 40%. The amount of MCAP produced after 72 h of cultivation was 186 mg L-1 and the maximum enzyme activity was 580 MCU. The amount of MCAP in the culture supernatant was estimated as the difference between the calculated proteins produced from the recombinant P. pastoris and wild-type P. pastoris, as well as by selleck chemical considering the band intensities on SDS-PAGE. Figure 6 Extracellular production of MCAP from recombinant P. pastoris X- 33/pGAPZα+MCAP-5. A) Time course in YPD medium containing 4% glucose at 24°C. B) Production of aspartic proteinase after 72 hours in YPD medium containing 4% glucose. The values shown are the mean activity with standard deviation obtained from three sets of experiments.