Study On Preparation And The Characteristics Of Two Aspartic Protease Inhibitors

In this thesis, two aspartic protease inhibitors (CVPI, PPI) were purified from versicolor fermentation broth and potatoes, and characterization of two inhibitors and the role of the basic mechanism were been studied, the submerged fermentation CVPI of versicolor conditions were optimized, and the application potential of the two inhibitors were preliminarily investigated. Study carbon and nitrogen sources in versicolor fermentation medium composition and though the orthogonal design to optimize the concentration of carbon and nitrogen source medium of coriolus versicolor submerged fermentation, The best concentration of carbon and nitrogen source as follow: 1% glucose, 1.5% potatoes juice, 1% soybean residue powder and 0.4% peptone respectively. On the basis of single factor experiments in the best culture conditions: initial pH6.0, liquid volume 70mL/250mL, rotation speed 160r/min, 10% inoculum, the fermentation temperature 28℃, optimized inhibitors yield 13.1IU/mL, compared with 2.01 times before optimization.CVPI was pretreated by decolorization, ultrofilition, and then isolated by ion-exchange chromatography DEAE-52 and MonoQ, the purification fold was 21.1 and the inhibition of activity was 295.6 IU /mg, identified by SDS-PAGE as a single subunit with molecular weight of about 22.3 kDa. The PPI was purified by cut potato homogenate, ultrafiltration, ethanol precipitation, DEAE-52 and Mono Q , the purification fold was 36.6. Identified by SDS-PAGE was a single band with molecular weight of 16.1 kDa.PPI analysis showed that acidic amino acids of PPI were higher in the amino acids, accounting for 32.6%; Inhibition of the best response time is 1 h; The high thermostability of PPI has also been observed at 20℃~ 80℃; pH stability range was 3.0 to 7.0; attribute analysis showed that PPI itself is not a protease and can not be hydrolyzed by pepsin; the PPI is more specific against pepsin than other proteinases; Concentration required for 50% pepsin inhibition (IC50) were 26.26μg/mL; it were non-competitive and competitive mixed type inhibitor.Used MALDI-TOF MS peptide mapping of CVPI and PPI analysis, database search, we found that two inhibitors of the amino acid level with other known aspartic protease inhibitors without a certain similarity, is an inhibitor type of the database not reported.CD spectra analysis pepsin combined with CVPI is accompanied by type, that is the inhibitor molecules do not occupy the binding sites of target enzyme, but tied with the enzyme molecule, and enzyme activity in the group with the formation of hydrogen bonds, while blockade of enzyme and substrate binding site, making it inactive. Type of PPI with the combination of pepsin may belong to "coverage", it may be similar to the linear molecule in the form of coverage to the target area around the active site, thereby preventing the enzyme's active site and substrate contacts.Fluorescent probe CVPI and PPI interaction with DNA, based on the results of this preliminary judgments on the basis of two may have some anti-cancer effect. Anti-oxidation experiments show that PPI has a strong scavenging effect on hydroxyl radical, can be used as antioxidants to slow the potential of hydroxyl radical damage to the human body.