Purification And Research Of PCL Depolymerase From Pseudomonas Aeruginosa DS0801

More and more attention has been paid to the biodegradable plastics because of the serious white pollution caused by extensive use of traditional plastics. Polycaprolactone (PCL) is one of the biodegradable polyesters, which is obtained by ring-opening polymerization of s-caprolactone. This semi-crystalline polymer can be widely applied in many fields, such as the bio-medical area. Currently, the researches on the PCL depolymerases are not enough to provide the correct evaluation of bio-degradation mechanisms of PCL.The laboratory has screened a PCL degrading strain DS0801which is identified as Pseudomonas aeruginosa in the previous work. Firstly, this paper optimizes its fermentation conditions. Through single factor and orthogonal experiments, we get the optimum conditions for the production of the PCL depolymerase:the initial pH of culture medium is7.0, the incubation temperature is32℃, the liquid volume is50/250mL, the carbon content is0.15%(w/v), and the phosphorus content is1.904mg/mL. Under this condition, after incubating for48h, the activity of PCL depolymerase secreted from the strain increase about48.5%.Many steps are taken to purify the PCL depolymerase from the fermentation broth, such as centrifugation, ultrafiltration, lyophilization, DEAE Sepharose Fast Flow and Phenyl Sepharose6Fast Flow chromatography. Then we obtain a purified enzyme, the molecular weight of which is about30.4KDa. The optimum temperature is40℃, and the optimum pH is8.0. The depolymerase can keep good stability when temperature is below50℃or pH is between6-9. The enzyme activity can be promoted by K+、Fe2+、Mn2+at1mM and K+、Na+、Mn2+at10mM. Ca2+、Cu2+、 PMSF、EDTA and SDS can significantly inhibit enzyme activity. The enzyme can hydrolyse PCL effectively, which generates monomers, dimers and trimmers. We speculated that the depolymerase prefers in cutting the second ester bond of the PCL chain terminal because the dimers account for most of the proportion of the hydrolysis products.Through MALDI-TOF-PMF analysis, we find a99.99%similarity between the PCL depolymerase and an extracellular lipase from Pseudomonas aeruginosa. The enzyme can work on p-nitrophenyl (C2-C16) substrates, and the enzyme prefer the long-chain substrates. Thus, we identify the enzyme as a lipase and study its property. After incubating for6h in organic solvents, such as methanol and acetone,the residual activity maintains a high level. Lipases can catalysis some important synthesis reactions in organic solvents, so the PCL depolymerase has great potential in applications as organic solvent tolerant enzyme.