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Studies On Recombinant Expression And Enzymatical Characteristics Of Three Imorotant Enzymes For Analytical Applications

Posted on:2013-12-19Degree:MasterType:Thesis
Country:ChinaCandidate:Y P LiuFull Text:PDF
GTID:2250330401969867Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Alkaline phosphatase, glutamate oxidase and alcohol oxidase have important application in food industry and medical test. The purpose of the present work is to increase the expression level of target proteins both in E. coli and Pichia pastoris expression system. Alkaline phosphatase (Apase) and glutamate oxidase (GOX) were chosen as the target proteins and suitable strategies were employed to improve their soluble productivity in recombinant E. coli. Then recombinant alcohol oxidase (AOX) was expressed in Pichia pastoris, and its expression level was increased greatly, especially for the secretive expression of recombinant AOX by optimizing the culture and induction situation. Exploring the features will provide necessary functional proteins for the development of simple and efficient analytical methods.Firstly, the expression levels of APase were compared in four different E. coli hosts (BL21(DE3), Rosetta (DE3), C41(DE3) and C43(DE3)), and the highest productivity of soluble APase protein was achieved in E. coli Rosetta (DE3). The recombiannt APase showed some resistance to detergents and proteases. In addition, the metal ions Ca2+and Mg2+could enhance the activity of recombinant APase.As to GOX, the constructed vector pET28a-GOX was transformed into E. coli Rosetta (DE3) for the expression and the volumetric productivity of soluble GOX reached as much as0.234g/L with a total activity of6660U/L. We succeeded in selecting out the right proteases (alkaline protease and protease K) correctly digest the GOX precursor to the mature one. It was observed that the mature GOX was more active and stable than the precursor one. Further study showed the molecular weight of GOX precursor was150kDa but the mature GOX was142kDa.Finally, the AOX was expressed in Pichia pastoris and about30%of total recombinant AOX was secreted to the culture supernatant by optimizing the culture and induction conditions. The affinity chromatography was employed to purify the recombinant AOX, and the recovery of the target protein was68.9%with a purity of93.2%. The results showed that the thermal stability of recombinant AOX was better than native one from Pichia pastoris. The recombinant AOX was tolerant to certain organic solvents. H2O2could competitively inhibit the catalytic reaction of alcohol with the Ki value1.28mM. Our studies here provides some base for the commercial and industrial application of these detective enzymes.
Keywords/Search Tags:Alkaline phosphatase, glutamate oxidase, alcohol oxidase, Pichia pastorisexpression system, Characterization
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