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Breeding of Bacillus sp. DSL09-60b to Degrade PLA and the Research of PLA Depolymerase

Author: LiuLingZuo
Tutor: ChenShan
School: Northeast Normal University
Course: Microbiology
Keywords: poly L-latic acid (PLA) strains identification PLA depolymerase bio-degradation determined the optimal enzyme production conditions
CLC: X172
Type: Master's thesis
Year: 2012
Downloads: 21
Quote: 0
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Polylactic Acid (PLA) is a newly emerging polymer, which polymerized bylactic acid. Because of its excellent physical and chemical properties,biocompatibilityand biodegradability, is considered to be the most potential green biological plastic.The experiment screened out a biodegradable PLA strains from the activatedsludge of Fushun petroleum three factory. And the strain’s mutational type has beenmutagenized through ultraviolet which is numbered for DSL09-60b. And the strainsof the conditions of fermentation has optimized, the PLA degrading enzymes has beenisolatied and purifyied, which properties has been preliminarily research. The mainresults are as follows:1. To PLA as the only carbon source, we screened out a biodegradable PLAstrains from the activated sludge of Fushun petroleum three factory which isnumbered for DSL09. Through the morphological observation, physiological andbiochemical characteristics of the determination and16S rDNA appraisal, we foundthat the strains close to Bacillus licheniformis, but the difference is the strains in55℃conditions can’t grow. So DSL09could belong to the new kind or the new subspeciesof Bacillus sp.2. In order to improve the DSL09degradation of the PLA, the ultraviolet raymutation processing that obtain a strains has strong vitality to degrade PLA andprotease, which is numbered DSL09-60b.3. The strains can degrade PLA film. PLA film is gradually from the transparentfilm state into white opaque, surface appear many holes, and will appeardisintegration phenomenon. By scanning electron microscope (SEM), surfacemorphology of the PLA degradation become very rough, has had the obvious erosion,further proof DSL09-60b strain of PLA has degradation ability.4. The strains DSL09-60b product the conditions of PLA depolymerase. Thesingle optimal conditions of production PLA: cultivate time for54h, culture mediumfor pH8, speed175r/min, inoculation amount of6%(V/V), the best inductor—thecasein of0.5%. At the same time design four factors of three levels orthogonalexperiment, determine the optimal fermentation conditions: pH8, speed175r/min,seed medium inoculated quantity5%(V/V), training time for72h.5. Some research has been done on the PLA depolymerase which is obtainedfrom DSL09-60b. The most suitable reaction temperature and pH of this PLAdepolymerase respectively is50℃and8.6. Between temperature20~50℃andbetween pH8.0~10.0, the depolymerase is stable. Respectively through Q SepharoseFast Flow and Sephacryl S-100gel chromatography column, get PLA depolymerase.Through SDS-PAGE electrophoresis show that the PLA depolymerase did notpurification, which needs further research.

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