Purification And Properties Of Extracellular PHBV Depolymerase From Pseudomonas Mendocina

Polyhydroxyalkanoates (PHAs) are the most common examples of biodegradable polyesters. PHAs and conventional thermoplastic have similar properties of chemistry and physics, in addition, PHAs have such characters as good biodegradability biocompatibility and regeneration.PHBV(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) is one kind of PHAs,which has excellent properties of chemistry and physics and processing characteristics, possesses good commercial foreground and attractiveness.Hoever,there are few researches on the biodegradation of PHBV, The aim of this paper is to study the biodegradation of PHBV by the strain of Pseudomonas mendocina ,to isolate and purify extracellular PHBV depolymerases,and to syudy the fundamental charcteristics of the extracellular PHBV depolymerases. The main results obtained from this work are as follows:1. The biodegradation of PHBV.The strain of Pseudomonas mendocina was cultivated on solid agar media that contain the polymer as a sole source of carbon, and then appeared transparent clearing zones around the colonies.two days later,the diameter of the clearing zones was 2 centimeters,four days later,the diameter was 3 centimeters.The strain of Pseudomonas mendocina was cultivated in liquid media that contain the polymer as a sole source of carbon,when cultivated 24 hours the powders completely disappeared. The biodegradation of PHBV films indicated that on the 6th day the PHBV films were thoroughly degradated under the flask culture in the laboratory.2. The purification of extracellular PHBV depolymerase. This experiment was optimized separation and purification steps, after the centrifuge, ultrafiltration, lyophilization and gel chromatography,PHBV pure PHBV depolymerase was got.The purification of multiple enzymes was 109.06, the activity recovery was 4%.3. The determination of PHBV depolymerase molecular weight.The purified depolymerase which had been disposed by TCA precipitation were For SDS polyacrylamide gel electrophoresis (SDS-PAGE).The molecular weight of the purified depolymerase was 30.3kDa.4. Some characteristics about PHBV depolymerase.The enzymology characters of coarse enzymes and pure enzyme had no evident differences. The optimal reaction temperature is 50℃,when in 10~50 degrees Celsius temperature range was stable. The optimal reaction pH is 8.6, when PH valued for 8-9.5 the stability of the enzyme was good. Different kinds of metal ions,and different concentration had different effects on enzyme activity.Ca2+,Mn2+,Co2+,K+ increased the enzyme activity,but the enzyme was inhibited by Cu2+,Fe3+,Zn2+, Mg2+,Na+ didn't have apparent effect on the enzyme. When the concentration of metal ions was from 1mM to 5mM, the corresponding solutions had further strengthening on the enzyme. 0.1%(w/v)SDS and 4mol/L Urea increased the enzyme activity,whereas 0.1%(v/v)Tween-20,0.5(w/v)EDTA,0.5%(v/v) mercaptoethyl alcohol almost had no influence on it.5. The determination of degradation products. The mass spectrum analysis showed that the degraded products of enzymes was 3-hydroxybutyrate.