Purification And Characterization Of PCL Depolymerase

Polycaprolactone (PCL) is a synthetic polyester by ring opening polymerizationof ε-caprolactone, with excellent properties and biodegradability, recently has beenapplied in multiple areas. So far, information for the biodegrading of PCL was little，therefore, research on the bacteria and depolymerizing enzymes with high efficientdegradability appeared particularly important and urgent. Pseudomonas mendocinastrain DS1001with PCL biodegrability was selected from sludge in our laboratory.The conditions of enzyme production, degradation of PCL film, purification andcharacteration of PCL depolymerase, gene clone and expression, protein structure andfunction were studied in this paper, and conclusions are as follows:(1) The optimum fermentation conditions for the production of PCLdepolymerase by strain DS1001were investigated by response surface methodologybased on a four-variable, three-level Box-Behnken experimental design, obtained thehighest PCL depolymerase activity under the conditions of medium initial pH9.0,temperature at31.27℃, carbon content0.05%（w/v）and fermentation time for48h.Enzyme activity increased by40%after optimization.(2) PCL depolymerase was purified by DEAE sepharose Fast Flow, with relativemolecular mass of about28.4KDa. The optimum reaction temperature and pH of theenzyme were60℃and9.5,respectively; PCL depolymerase could keep stabileacticity within temperature4-50℃and pH5-12;enzyme activities were promoted byCa2+or Mg2+strongly, but inhibited by EDTA,PMSF; enzyme in organic solventssuch as methanol was very stabile, but not resistant to tween-80and Triton X-100.The degradation products of the PCL depolymerase mainly were hydroxyl caproicacid dimmers, and some monomers, trimers, tetramers via mass spectrometrydetection. The purified enzyme did not show interfacial activation, and had cutinbiodegradability, indicating that it is a cutinase.(3) The gene coding PCL depolymerase was cloned and heterologouslyexpressed in E.coli BL21successfully, then the recombinant protein with relativemolecular mass of29KDa was obtained by Ni+affinity chromatography. The N endof the enzyme contained a signal peptide sequence composed of22amino acids.Through sequence alignment and verified by site-directed mutation, PCLdepolymerase had a Ser148, Asp198, His228catalytic triplet and a Thr80-Gln149oxygen ions hole, either amino acid mutation would cause the loss of enzyme activity, but did not influence the expression of mutants.(4) Amorphous PCL film could be degraded completely in5days by strainDS1001, the whole degradation process can be divided into three phases: slow-fast-slow, and degradation was occurred from outer to inner step by step. The degradationmechanisms of PCL were discussed in this paper, the second ester bond was cutedpriority by PCL depolymerase, the catalytic mechanism of PCL depolymerase wassame with the traditional lipase. PCL was degraded due to identification in error bysome natural enzymes with broad substrate specificity, because of the structuresimilary of PCL with some natural substrates.