Purification And Characterization Of PBS Depolymerase

Polybutylene succinate(PBS)is currently recognized as the world's best comprehensive biodegradable plastics.Compared to other biodegradable materials,PBS has low cost and good performance.PBS can be very effective and other materials for effective polymerization.PBS performance between the polyethylene and polypropylene,so its industrial application prospects are very broad,with good market and economic value.In this paper,the fermentation conditions of the strain were optimized by single factor experiment and orthogonal experiment using Pseudomonas sp.DS1001 strain,which was able to degrade PBS by laboratory.At the same time,the depolymerase secreted by Pseudomonas sp.DS1001 strain was isolated and purified,and the enzymatic and enzymatic properties of the proteolytic enzyme were studied.Finally,the following conclusions were obtained.(1)The results of single factor optimization were as follows: carbon content was 0.2%,liquid volume was 150 m L/250 m L,culture time was 72 h,culture medium initial p H was 9.0;According to the single factor optimization results,a four-factor three-level orthogonal test was designed.The optimal conditions for the final strain were as follows: temperature was 37?,medium was 9.0,carbon content was 0.25% For 150 m L/250 m L.Orthogonal experiment was 45% higher than single factor experiment.(2)The fermented crude enzyme was separated and purified by ultrafiltration and DEAE anion exchange chromatography.Finally,a pure enzyme was obtained from Pseudomonas sp.DS1001 strain with a molecular weight of about 50 k Da.The optimum reaction temperature of PBS depolymerization enzyme is 50?,and it has good thermal stability at 4 ~ 50?.The optimum reaction p H was 8.0(Na2HPO4-Na H2PO4 buffer),and the remaining enzyme activity of PBS depolymerase was above 50% in the range of p H 4.0~10.0.Metal ions have an important effect on PBS depolymerization.In addition,EDTA,PMSF can strongly inhibit the activity of PBS depolymerase;PBS depolymerase on organic solvents have a certain tolerance,But Triton X-100,whether low or high concentrations can inhibit the PBS depolymerase.(3)It was found from peptide mass spectrometry that the PBS depolymerase purified from Pseudomonas sp.DS1001 strain was a lipase.Purified PBS depolymerization enzyme has certain degradation activity to aliphatic polyester such as PBS,PHB and PCL,and can not degrade PLA.The catalytic effect of PBS on the p-NPC4 was the best.The results showed that the surface of the PBS membrane was relatively smooth before the action of the PBS depolymerase,but under the action of PBS depolymerization,the surface of the PBS membrane showed obvious honeycomb pores,While with the extension of the degradation time of the PBS membrane surface pores become larger and deeper.Mass spectrometry analysis of the degradation products of PBS depolymerization products were succinic acid,succinic-acid-butanediol,but anediol-butanediol-succinic acid,succinic-acid-butanediol-succinic acid-butanediol.