| Poly(β-Hydroxybutyrate-co-β-Hydroxyvalerate) (PHBV) are biodegradable polysters that are synthesized and accumulated intracellularly during unbalanced growth by some bacteria. It has attracted much commercial and academic interest as a kind of new biodegradable material. The ability to d egrade PHBV is widely distributed among b acteria and fungi, and depends o n the secretion of specific extracellular PHBV depolymerase which can hydrolyse the polymer to water-soluble products. The aim of this work is to select the mutant with the treatment of UV, which can degrade PHBV efficiently, to purify PHBV depolymerase and to study the fundamental characteristics of PHBV depolymerase. The main results obtained from this work are as follows: 1. The pinicillium sp. DS9702-D10, a strain of degrading PHBV, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, best five ones were obtained with high –yield of stable PHBV depolymerase, named as 05,06,11,19 and H2. The enzyme activity of the06 and H2 were 5.6 and 5.38 times of the original one. 2. The enzyme activity and the protein of the five muntants were surveyed with their crude enzyme, the highest enzyme yielding of them were found in 72h of 11, 19 ; 96h of H2 and 120h of 05,06. The activity of 11,19,H2,05,06 were 2.75;1.55;5.38;4.37;5.6 times of the original one. 3. Comparative study among the crude extracts of 06,H2 mutants and the original one。The optimum temperature of 06 was 20℃ higher and H2 was 10℃ higher than the original one, the range of temperature stability of 06 was from 40℃ to 60℃ and that of H2 was from 30℃ to 60℃, but the original one was from 30℃ to 50℃.The optimum pH of 06 was 7.38 and that of H2 was 6.98 and 8.04. It was different from the original one which was 7.0. Moreover, the range of pH stability of 06 and H2 was also much better. 4. The extracellular PHBV depolymerase was selected and purified from 06 and H2 mutants by using filtration,C2H5OH precipitation,gel filtration technique in Sephrouse CL-6B.The activity of the purified enzyme was increased by 33.81 folds and the recovery yield was 13.57% of 06. The molecular mass of 06 was 28KD and the optimum activity of enzyme was observed at the temperature 60℃ and at pH 7.38. Its range of temperature stability was 40℃~50℃ at pH 6.24~6.64; The activity of the purified enzyme was increased by 39.28 folds and the recovery yield was 3.27% of H2. The molecular mass of H2 was 44KD and the optimum activity of enzyme was observed at the temperature 50℃ and at pH 6.98 or 8.04. Its range of temperature stability was 40℃~50℃ at pH 7.6-8.4. some metal ions could activate or inhibit the two PHBV depolymerase activity and the hydroxyl-products with PHBV depolymerase were both monomer 3-hydroxybutyrate acid, analyzed by using mass spectrometer. |
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