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Isolation, Identification and Characterization of a Novel Elastase from Chryseobacterium Indologenes

Author: ZhaoPeiPei
Tutor: WuZuo
School: Sichuan Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Chryseobacterium indologenes Elastase Purification Enzymatic properties Proteolytic characteristics
CLC: Q78
Type: Master's thesis
Year: 2012
Downloads: 25
Quote: 0
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Elastase is a kind of protease that can specially degrade elastin. It has a promising application prospect in the food industry, medicine, daily chemical industry, environmental protection and biological control, which shows the high commercial value of elastase. In the study, a microbial strain with the high elastic proteolytic activity was identified from the cowhells powder plate with contamination bacteria. And the elastase produced by the strain was purified. Furthermore, its enzymatic properties and the hydrolysis characteristics on different proteins were studied. The main results are as follows:(1) A contamination bacterium named StrainWZE87was obtained from laboratory due to its strong hydrolysis ability for cowhells powder. Its original enzyme activity was high up to71.06U/mL. Seen from the morphological identification, the physio-biochemical characteristics, and the16S rDNA phylogenetic analysis, the StrainWZE87was identified as Chryseobacterium indologenes.(2) By using (NH4)2SO4fractional precipitatation, Q sepharose fast flow chromatography, Sephadex G-75chromatography, the elastase was purified from C. indologenes WZE87. SDS-PAGE showed that the molecular weight of the electrophoresis pure elastase was about26KDa. The resutls indicated the overall purification was8.3-fold, the recovery of activity was5.8%, and the specific activity was up to2376.5U/mg.(3) Enzymatic characterizations of the purified elastase were measured. The results revealed that its optimal temperature was37℃. After incubation at50℃for30min, it maintained85%of the relative activity, which meant the elastase showed a some degree of thermostability. However, the relative activity was sharply dropped or eventually lost when the elastase was treated above60℃for30min. The optimal pH of the elastase was pH8.0or pH7.5in boric acid buffer or in Tris-HCl buffer, respectively. After treated in the pH5.5-6.5or pH10-11buffers for24h, the elastase remained over60%of its relative activity. Then, the elastase activities were inhabited partly and left with67%to96%relative activity in5mmol/L Li+, Na+, Ca2+, K+, EDTA and0.01mmol/L mercaptoethanol. Furthermore, Mn2+and SDS resulted in a reduction of the elastase activities more than50%. Particularly, Zn2+, Fe2+and Fe3+showed the obvious inhibition effect on the elastase activities, and the residual activities decreased to7%,0%and0%, respectively. However, Mg2+showed a weak activation to enhance the elastase activity up to103%.(4) The elastase from C. indologenesWZE87was prior to degrade elastin in the mixture of elastin and other proteins due to its specific adsorption for elastin. The results found the elastase also possessed the strong hydrolysis abilities for elastin, casein, gelatin, and isolated soy protein.

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