Isolation and characterization of an aspartic protease from Salpichroa origanifolia fruits.

This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0-4.5 with hemoglobin and 5.5-6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin ß-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R.

BACKGROUND: The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related alpha-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans. RESULTS: CGTase activity produced from Bacillus circulans DF 9R was optimised in shake flasks using a combination of conventional sequential techniques and statistical experimental design. Effects of nutrients, including several carbon, nitrogen and mineral sources, were assayed. The selected minimal medium consisted of 1.5 % cassava starch, 0.4 % ammonium sulphate, 0.1 M phosphate buffer, 0.002 % MgSO4 and 0.002 % FeSO4. The optimal concentrations of carbon and nitrogen sources were determined using a central composite design. Maximum CGTase activity obtained in supernatants was 5.8 U/mL in 48 h of incubation. Optimal conditions for enzyme production also included an initial pH of 8.3 and 37 degrees C as the incubation temperature.Cell growth and CGTase production profile were not linked to each other, suggesting that enzyme production/secretion is not growth-associated but mainly a late-log phase event. CONCLUSION: We have screened conditions for optimal CGTase production. The selected minimal medium contained starch, ammonium, Mg2+ and Fe2+ as essential nutrients. As an additional advantage, this medium does not require complex nitrogen sources with varying and unknown composition.