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The Study on Prokaryotic Expression, Purification and Application of Key Enzymes Related to Formaldehyde Decomposition

Author: ZhangZuo
Tutor: ChenLiMei
School: Kunming University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Glutathione-dependet formaldehyde oxidation pathway Formaldehyde dehydrogenase S-formylglutathione hydrolase formate dehydrogenase Prokaryotic expression Formaldehyde contamination
CLC: Q814
Type: Master's thesis
Year: 2010
Downloads: 87
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Abstract

Formaldehyde (HCHO) is one of the main indoor air pollutants, which can be found or detected in many decorative materials. It is also a highly reactive compound that has a great harm to human health through its nonspecific reactivity with proteins, nucleic acid and lipid. Using physical adsorption or chemical elimination methods to remove formaldehyde usually has a little effect, and these methods also lead to a secondary pollution. Using plants or Enzymes to remediate formaldehyde pollution is an economic and environmental method and will not produce a secondary pollution.The glutathione (GSH)-dependent formaldehyde oxidation pathway is a most widespread system to repair formaldehyde damage in organisms. GSH-dependent formaldehyde dehydrogenase (ADH), S-formylglutathione hydrolase (FGH) and formate dehydrogenase (FDH) are the three key enzymes in this pathway. However, the formaldehyde dehydrogenase from Pseudomonas putida PADH is not dependent on GSH but requires NAD+as an electron acceptor.In this study, the genomic DNA of Escherichia coli, P. putida, Candida boidinii was used as the template, respectively, to amplify the E. coli ADH gene (Eadh) and FGH gene (fgh) as well as P. putida PADH gene (padh) and C. boidinii FDH gene (fdh). The ADH gene for Arabidopsis ADH was obtained from the TA-clone pMD18-T-Atadh (Song Master Thesis) by restriction enzyme digestion. These genes were subcloned into the prokaryotic expression vectors, pET32a or pET28a, respectively, to construct pET32a(+)-Eadh, pET28a(+)-padh, pET32a(+)-fgh, pET28a(+)-fdh and pET32a(+)-Atadh expression vectors.The expression vectors were introduced into E. coli BL21 or Rosseta, respectively, to induce the target protein expression. The optimal conditions for expression of each target protein were determined by adjusting the IPTG concentration, induction temperature and induction time. The expressed proteins accounted for 70% of the total proteins. The results from SDS-page showed that all the expressed recombinant proteins formed more or less inclusion bodies.80% of AtADH and E.coli FGH protein was expressed in inclusion bodies. Using gel purification method, the AtADH, FGH, FDH recombinant proteins were purified from the inclusion bodies. The pure proteins were used as antigens to produce their antibodys.The soluble recombinant proteins of PADH and FDH accounted for 40% of the total soluble proteins. By enlarge the culture scale, His-tag affinity chromatography and (NH)4SO4 precipitation, the soluble PADH and FDH proteins was purified to homogeneity. The Enzymatic activity analysis showed that both PADH and FDH had catalytic activity and thermostability. PADH protein had higher catalytic activity at 30℃and 40℃and the highest specific activity,1.95U/mg, was obtained at the optimal temperature of 50℃. It had 60% residual activity even incubated for 10 min at 65℃. The FDH enzymatic activity varied slightly at 30℃and 50℃. The highest specific activity,0.376U/mg, was achieved at 40℃. It still remained 51% residual activity even incubated at 65℃for 100min. FDH had a more extensive reaction temperature and high temperature tolerance compared with ADH. The denaturant resistance experiments revealed that the enzymatic activity of ADH and FDH was strongly influenced by guanidiniumhydrochloride treatment. When exposed to 0.5 mM guanidiniumhydrochloride for 13h, the residual activity of ADH and FDH decreased dramatically to zero.Furthermore, the ADH, FDH proteins and NAD+were fixed into some supporter. A preliminary investigation was performed to evaluate formaldehyde absorption efficiency of the fixed enzymes. The results showed that the fixed enzymes with ADH, FDH proteins and NAD+had considerable formaldehyde absorption capacity.The results of this study established valuable foundations for using enzymatic method to remediate indoor formaldehyde contamination. The AtADH, FGH, FDH antibodys also provided a strong support for detection of these proteins when expressed in transgenic plants generated by genetic engineering.

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