The Study On The Degradation Of Polyethylene By Streptomyces Albogriseolus And Degradation Enzyme Gene Cloning

The rapid development of the industry in recent years has brought serious "white pollution" problem and affect our production and life. Among the many ways to alleviate the "white pollution" and deal with waste plastics, microbial degradation of plastics has become one of the hottest research areas. Polyethylene microorganism have been reported at home and abroad are mainly composed of bacteria and fungi, actinomycetes as polyethylene degradation strains were isolated is rarely reported.This experiment firstly screend and isolated a strain of actinomycetes to degrade polyethylene from the soil, by morphological observation, physiological and biochemical identification(gelatin liquefaction test, milk coagulation and peptone experiment, experiment of starch hydrolysis, tyrosine hydrolysis experiments, Hydrogen sulfide is produced experiment and with the different carbon source utilization experiment) and 16 S rDNA Sequence Cloning and sequencing, identification of the Streptomyces albogriseolus.Study on the degradation effect of the Streptomyces albogriseus on polyethylene next:(1) to study the relative molecular weight of polyethylene powder were 2000, 5000, 10 W and 40 W. The results showed that different molecular weight polyethylene powder can be Streptomyces albogriseus degradation, and the degradation degree from large to small is as follows: 2000 > 5000 > 10 W > 40W;(2) On the relative molecular weight > 100 W polyethylene film were studied. The results showed that Streptomyces albogriseolus attachment on polyethylene film growth and in the naked eye and scanning electron microscope observation were found there is any breakage and holes on the surface of membrane. This leads to the conclusion that in polyethylene film can also be degraded by Streptomyces albogriseolus. So known Streptomyces albogriseolus belongs not only to the low molecular weight polyethylene degrading bacteria, also belongs to the high molecular weight even ultra high molecular weight degradation bacteria.Once again explore the key enzyme of Streptomyces albogriseolus degradation of polyethylene: firstly with laccase and HBT(1-hydroxy benzene benzotriazole) constructs an intermediate or mediating, in 30 ℃ environment degradation relative molecular mass for 2000 of polyethylene powder after 3 days can be seen in the experimental group of the polyethylene powder particles become small, the solution was light yellow. According to relevant literature and experimental results show that the laccase is a key enzyme in the degradation of polyethylene.Using the ABTS assay for the detection of Streptomyces albogriseolus fermentation broth containing laccase protein and its enzyme activity was determined. Comparison in NCBI gene database published Streptomyces laccase gene sequence, primers were designed K-F2, K-R2, genomic DNA of Streptomyces albogriseolus laccase gene was amplified by polymerase chain reaction(PCR) in vitro and of blast and analysis. Laccase genes will be connected with the pEASY-T1 cloning plasmid Cloning Vector constructed recombinant pEASY-T1 cloning plasmid Cloning Vector / laccase, imported into Trans1-T1 Phage Resistant Chemically Competent Cell, and a series of positive clones verifying, such as blue-white selection, identification of PCR methods, restriction enzyme analysis and sequencing. The restriction enzyme XhoⅠ and BamHⅠon recombinant plasmid cloning pEASY-T1 Cloning Vector / laccase and prokaryotic expression vector pET-32a-c(+) Vector dual-enzyme, exposing sticky ends, using T4 DNA Ligase connects into BL21(DE3) Chemically Competent Cell, and a series of positive clones verifying, Such as blue-white selection, PCR identification, restriction enzyme analysis and sequencing.Finally, carrying the recombinant expression plasmid of Escherichia coli BL21(DE3) test:(1) be vaccinated in the polyethylene as the sole carbon source and the underlying carbon-free source of energy for growth on solid media, shows the colony grows, so that Escherichia coli BL21(DE3) polyethylene powder can be used;(2) will carry the pET-32a-c(+) Vector/laccase of Escherichia coli BL21(DE3) liquid broth made with ammonium sulfate precipitation enzyme, SDS-polyacrylamide gel electrophoresis of enzyme protein present. Results show that the presence of laccase protein and about 66.4 KDa in size.Through a series of gene cloning technology, the laccase genes in Streptomyces albogriseolus imported into Escherichia coli and success. Description key enzyme laccase is degradation of polyethylene, which will explore the mechanism of microbial degradation of polyethylene for the future, laid the foundation for screening laccase-high producing strain.