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The Separation and Purification of AHL-Degrading Enzyme in Rhodosporidium Toruloides

Author: LuoLiLong
Tutor: SongShuiShan
School: Hebei University of Technology
Course: Biochemical Engineering
Keywords: quorum-quenching enzyme separation and purification gel recovery
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 44
Quote: 0
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Abstract


Quorum-Sensing(QS), which regulates gene expression depending on population density, is universal in bacteria. QS is involved in the regulation of virulent factor and pathogenic gene expression, and plays a key role in causing plant disease by plant bacterial pathogens. Hense, targeting the bacterial QS seem to be a promising approach for control the disease caused by QS-mediated bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key singnal in bacterial Quorum-Sensing. Several groups of AHL-degradation enzymes have recently been identified in a rang of living organisms including bacteria and eukaryotes. Expression of these genes encoding AHL-dagrading enzyme in AHL-dependent pathogens and plants efficiently quench the acceleration of QS signal and blocks pathogenic infection.Discovery of these novel quorum quenching enzymes has not only provided a promising means to control bacterial infections,but also presents new challenges to investigate their roles in host organisms and their potential impact on ecosystems. Qiu jian et al screened a yeast which can be able to degrade AHL and was identified as Rhodosporidium toruloides. The discovery riched the quorum quenching enzyme.In this paper we success in separation and purification of this enzyme. The R. toruloides was cultured in MacConkey medium and then we collected the bacterial by centrifugation. Through the ultrasonic we get the total proteins. The total proteins were concentrated by ammonium sulphate fractionation and then purified by anion exchange chromatography, hydrophobic interaction and gel recovery and finally showed a single band on the SDS-PAGE. Through experiment we studied the pH and the salt concentration on the impact of the AHL-degrading enzyme and the AHL-degrading enzyme substrate spectrum as well as the degradation activity. We get the molecular weight and the isoelectric point of the enzyme by two-dimensional electrophoresis.

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CLC: > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )
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