Ds9701 Strain Mutagenesis And The Enzymatic Properties

Poly(3-hydroxybutyrate)(PHB) are attracting much commercial and academic interest as substitute for non-degradable petrochemically derived plastics because of their similar material properties to conventional plastics and complete biodegradability under natural environment.The ability to degrade PHB is widely distributed among bacteria and fungi and depends on the secretion of specific extracellular PHB depolymerase. The aim of this work is to find out the mutant, which can be able to degrade PHB efficiently and to investigate the characteristics of PHB depolymerase. The main results obtained from this work are as follows:1. The Penicillium.sp DS9701, a strain of degrading PHB, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, the best four were obtained with high-yield of stable PHB depolymerase, named as 02, 04, 09 and 14. The enzyme activity of the 09 mutant was increased to 5.4 u/ml as compared with 2.3 u/ml of the original.2. Comparative study among the crude extracts of 02, 04, 09 and 14 has been done. The optimum activity of 02 crude extract was observed at the temperature 45-50 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 4.0 to 5.0.The optimum activity of 04 crude extract was observed at the temperature 50 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 5.0 to 7.0.The optimum activity of 09 crude extract was observed at the temperature 50-60 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 5.0 to 7.0.The optimum activity of 14 crude extract was observed at the temperature 40 and at pH 5.8. The range of temperature stability isbelow 40 , the range of pH stability is from 5.0 to 8.0.3. The extracellular PHB depolymerase was purified from 09 by using hydrophobic column chromatography and gel filtration technique in sephadex G-100. The specific activity of the purified enzyme was increased by 37.9 folds over crude extract, and the recovery yield was 8.9%. Its molecular weight was determined running in SDS-PAGE and was found to be 41.5 KDa. The optimum activity of enzyme was observed at the temperature 50 and at pH 5.0. The range of temperature stability is below 50 , the range of pH stability is from 5.0 to 6.0. Different metal ions caused inhibition on the PHB depolymerase activity. Comparative study with DS9701 PHB depolymerase also had been done.Moreover, the mass spectrum analysis of the hydrolyzed water soluble products of PHB powder after treatment of enzyme was performed and the main product was identified to be dimers.