Purification And Characterization Of A PCL Depolymerase From Pseudomonas Sp.DS1001

Poly(?-caprolactone)(PCL)is a kind of high molecular polyester material polymerized by ?-caprolactone,it can be completely degraded by the action of the enzyme secreted by microorganisms in the natural world.Our laboratory screened a strain Pseudomonas sp.DS1001 with high efficient degradation for PCL in prophase,the strain was able to produce a variety of PCL degrading enzymes.A PCL depolymerase with molecular weight of 28.4kDa was obtained.In this study,the fermentation broth was separated and purified,the purification process of PCL depolymerase I was optimized,and the PCL depolymerase ? with obvious difference between PCL depolymerase I was obtained,the enzymatic properties were tested,the gene was cloned and analyzed,at the same time,the degradation process of PCL was discussed,in order to better explain the degradation mechanism of PCL.Specific research contents and results are as follows.(1)By ion exchange chromatography and exclusion chromatography,PCL depolymerase I and PCL depolymerase ? were obtained respectively,the purification efficiency and the recovery rate of PCL depolymerase I were 5.02 and 18.69%,the purification efficiency and the recovery rate of PCL depolymerase ? were 4.01 and12.93%.SDS-PAGE of PCL depolymerase ? showed that its molecular weight was about 32.79 kDa.(2)The enzymatic properties of PCL depolymerase ? showed that the optimal reaction temperature was 40?,and the temperature stability was good in the temperature range of 20-60?;the optimum reaction pH was obtained in the Gly-NaOH buffer system of pH10.0,and the pH stability test showed that the enzyme was stable in the range of pH6.0-12.0;When the metal ion concentration is 1mM,the activity of the enzyme was affected in varying degrees,Zn2+ions inhibit enzyme activity,Mg2+?Na+?Ca2+?Co2+ increased enzyme activity in different degree;EDTA and PMSF had obvious inhibitory effect on the enzyme activity.The enzyme was well tolerated by common organic solvents such as methanol,ethanol and glycerol.Triton X-100 and Tween-80 inhibit the enzyme activity.(3)Study on the substrate specificity of PCL depolymerase ? results showed that PCL depolymerase ? have degradation activity to the substrates of aliphatic polyesters PBS,PHB,tribuyrin and different length of carbon chain of pNP esters;ithas no degradation activity to the substrates of PLA and apple peel crude cutin;it showed interfacial activation effect when tribuyrin and pNPC6 as substrates respectively.(4)The gene of PCL depolymerase ? was cloned and heterologous expression achieved in E.coli BL21,but there was a low expression,which needs to be studied in the future.The physical and chemical properties and structure of Pclase ? were predicted by online prediction softwares.The results showed that the Pclase ? was composed of 317 amino acids,with a theoretical molecular weight of 33.1994 kDa and a theoretical isoelectric point of 6.59;the N terminal may contain a signal peptide sequence consisted of 25 amino acid residues,30-316 amino acids residues have a conserved sequence of ?/? hydrolase family;the proteins with high amino acid sequence similarity to Pclase ? is derived from Pseudomonas.sp lipase;In the secondary structure,the irregular curl accounted for 53.94%,and the ?-helix and?-sheet accounted for 36.90% and 9.14%,respectively;Pseudomonas aeruginosa PAO1 lipase PAL(1ex9.1.A)was used as a template for three-dimensional modeling,and it was speculated that there is a movable ?-helix cap structure related to the interface activation effect.(5)Study on the degradation mechanism of PCL depolymerase ?.The experimental results show that products of the PCL emulsion substrate degradation in complete degradation stage are caprolactone monomer,dimer,trimer and four dimer,incomplete degradation stage,there are still five dimers and six dimers;the degradation rate of PCL film appears the trend of soon after the first slow;SEM results showed that with the enzyme preferentially degraded the non crystalline region.The spherical crystalline spherulites are degraded starting from the center,and expanding outward gradually.