Screening Of A Strain Producing Carboxypeptidase, Purification And Properties Of The Enzyme

This thesis consists of three parts: 1) preparation of soy peptides substrate for the carboxypeptidase.2) screening, identification of the strain producing carboxypeptidase, properties of enzyme producing, and mono-factors affecting the enzyme production of the strain producing carboxypeptidase. 3) purification of the carboxypeptidase. The achievements made are as follows:1. Substrate (soy peptides) was prepared from the SPI hydrolyzed by pepsin and proper soy peptides was obtained from the hydrolysates of SPI at 10h under the condition of the test. Bitter peptides were extracted from the hydrolysate of SPI by n-butanol and the molecular weight of the bitter peptides were between 300-1500D. The ratio of the bitter peptides covers 24.1% of the whole soy peptides.2. A strain was obtained from a large amount of bacteria, yeast and aspergillus screened from a lot of soil samples. When culture filtrate of the strain incubated with the substrate for an hour, the products were analyzed by amino acid auto-analyzer, according to the results, the strain was identified to be a strain producing carboxypeptidase named CP-1. The carboxypeptidase can release a lot of hydrophobic amino acid and reduce bitterness of bitter peptides. The strain belongs to the Aspergillus genera according to its morpha characters of colonies and filamentous.3. After successive purification by (NH4)2SO4 ethanol, DE52, Sephadex G-100, one band was obtained by SDS-PAGE of the purified enzyme. Molecular weight of the enzyme was about 15,942D.4. The optimal carboxypeptidase's reaction pH was at 5.0, optimal reaction temperature were at 30 ℃ for Cbz-gly-tyr. Metal ions such as Mg2+, Fe2+,Mn2+,Ca2+, Ba2+ can increase the activity of carboxypeptidase. But its activity was inhibited by EDTA, Cu2+, so it should be a metaloenzyme.Studies on stability of the carboxypeptidase under different pH and against heat treatment shows that the enzyme was stable in the pH range of 6~7, not stable against heat, more than 72% of the activity of the enzyme was lost at 50℃ after 30min.