Uv Mutagenesis Of. Ds9713a Strain And Its Phb Depolymerase Enzyme Properties

Poly(3-hydroxybutyrate) (PHB) are biodegradable polysters that are synthesized and accumulated intracellularly during unbalanced growth by a large of bacteria .It has attracted much commercial and academic interest as a new biodegradable materials.The ability to degrade PHB is widely distributed among bacteria and fungil and depends on the secretion of specific extracellular PHB 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 PHB efficiently ,to purify PHB depolymerase and to study the fundamental characteristics of PHB depolymerase. The main results obtained from this work are as follows:1. The penicillium.sp DS9713a ,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 five were obtained with high-yield of stable PHB depolymerase ,named as 01 , 02 , 06 , 07 and 11.The enzyme activity of the 01 mutant was higher 97.42% than that of the original.2. The enzyme activity and the protein of five mutants was surveyed with their crude enzyme ,the highest enzyme yielding of them were found at 72h except 11 strain at 96h ,furthermore ,the enzyme activity of 02 , 06 , 07 , 11 were higher 48. 93%, 57. 51%, 48. 93%, 54. 51% respectively than that of the original.3. Comparative study among the crude extracts of 01 mutant and the original .The optimum temperature of 01 is higher 10 C than that ofthe original ,the range of temperature stability is from 20 C to 50 C,but the original is from 20 C to 40 C.The original with optimum pH at 6.4 and 8.6 was changed to only an optimum pH -8.6, under this condition the enzyme activity also had been apparently increased,morever,the range of pH stability of 01 is much better.4. The extracellular PHB depolymerase was selected and purified from 01 mutant by using filtration , (NH4) 2SO4 precipitation , gel filtration technique in SephroseCL-6B.The activity of the purified enzyme was increased by 38.44 folds over crude extract and the recovery yield was 11.59%. The molecular weight is 15.1 KD deterimed by SDS-PAGE. The optimum activity of enzyme was observed at the temperature 50 C and at pH 8.6.The range of temperature stability was below 37 C,the range of pH stability was from 8.0 to 9.2 .The PHB depolymerase activity was activated or inhibited by some metal ions. The hydroxy-products with PHB depolymerase was monomer 3-hydroxybutyrate acid, analyed by using mass spectrometer.