Studied On Aspartic Acid Protease Inhibitor From Coriolus Versicolor By Submerged Fermentation

The research mainly focused on the following aspects about aspartic acid protease inhibitor from Coriolus versicolor by submerged fermentation, including fermentation conditions, extracting, purification and the basic properties.Compared the mycelium growth rate by plate culture, Coriolus versicolor and Gctnoderma lucidum G-8 grew the most rapidly, about 13.7mm/d, and the mycelium of Coriolus versicolor was the most dense, Gantheram Lucidum grew the most slowly, about 5.0mm/d.Compared the biomass of different fungi by submerged fermentation, the mycelium dry weight of Coriolus versicolor is the most heavy 15.06g/L, is about 2.83 times of Gantheram Lucidum; Dried the fermentation broth by rotary vacuum drying and vacuum freeze drying into powder, and then by hot water extraction, ethanol precipitation, eventually, compared the inhibition rate of extract effect on pepsin, Coriolus versicolor is still the best one, the inhibition rate of extract from powder by vacuum freeze drying effect on pepsin was about 29.3%, and about 20% by rotary vacuum drying.Compared the inhibition rate of Coriolus versicolor broth by pre-and post cell disruption effect on pepsin, after cell disruption the inhibition rate improved from 0.53% to 37.2%, it was suggested that the intracellular of Coriolus versicolor contains the pepsin inhibitor. Studied on ultrasonic time, ultrasonic power, ultrasonic treatment capacity and ultrasonic temperature, the best ultrasonic conditions was determined as 10 minutes of ultrasonic time, 300W of ultrasonic power, 40mL of ultrasonic treatment capacity and 20℃of ultrasonic temperature.One kind of aspartic acid protease inhibitor CVPI was purified, The purification was carried out by shaking flask fermentation-cell disruption-decoloration by macroporous resin D101-DEAE52 ion exchange chromatogram-UltroGel ACA54 gel-filtration chromatogram, respectively.The molecular mass of CVPI was 23442 as estimated via gel filtration; The inhibitor showed a remarkable heat stability, it retained its initial activity even at up to 100℃for 30 min; The pH stability range of CVPI is about 3.5～7.5; The inhibitor rate improved quickly in 2 hours time of inhibitory reaction, and inproved hardly after 2 hours latter; By investigating the interaction between this inhibitor and variety of proteinases, it was indicated that the inhibitor was more specific against aspartic acid proteinase (such as pepsin and yeast proteinase A) than other proteinases; The dissociation constants (Ki) and concentration required for 50% inhibition (IC50) for pepsin were 0.82μmol/L and 15μg/mL, and the dissociation constants (Ki) and concentration required for 50% inhibition (IC50) for proteinase A were 1.32μmol/L and 29μg/mL; Kinetic studies (Lineweaver-Burk method) showed CVPI is a kind of noncompetitive and anti-compeititve inhibitor to pepsin while competitive and anti-competitive inhibitor to yeast proteinase A.The main impact factors were determined as inoculum size, initial pH and medium volume using single factor experiment method. The response surface methodology and single-factor tested for the best culture conditions indicated that fermentation temperature is 26℃, fermentation time is 120 h, rotate speed is 170r/min, medium volume is 71.31 mL/250mL, initial pH is 5.97, inoculum size is 13.96%; The inhibitor units were improved from 3.72IU/mL to 10.15IU/mL.