Sun, Lianhong (2003) Engineering galactose oxidase to increase expression level in E. coli, enhance thermostability, and introduce novel activities. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10032002-101924
For the first time, we functionally expressed the fungal glycosylated copper-containing enzyme, galactose oxidase, in Escherichia coli. The generation of fully functional GOase confirms that the formation of the unusual thioether bond is an auto-catalytic process involving a self-processing mechanism. This process does not rely on the pro-peptide of native GOase and is not dependent on the auto-cleavage of the precursor peptide during maturation, a question which can not be illustrated with other expression systems. The kinetic parameters of recombinant GOase and native GOase are comparable, but the former is less stable than the latter, revealing that the small contents of carbohydrate in native GOase confer a more rigid structure, but not a beneficial effect on catalytic efficiency to the enzyme. We applied directed evolution to generate GOase variants with improved thermostability, increased catalytic efficiency, and enhanced expression level in Escherichia coli. The final mutants are comparable to fungal GOase in their thermostability, long-term stability and radical stability. We also applied saturation mutagenesis to endow the protein with a novel D-glucose 6-oxidase activity, an activity that has never been reported in nature or laboratory. The resultant mutant M-RQW, with three amino acid substitutions (R330K, Q406T/S and W290H), shows low but significant activity in the selective oxidation of D-glucose's 6-hydroxyl group and has decreased activity towards its native substrate, D-galactose. None of the mutations can be obtained by single nucleotide substitutions. The mutation on W290, a residue proposed to stabilize the radical of GOase, supports the conclusion that W290 restricts substrates from entering the active center of GOase. Moreover, kinetic characterization of the mutant indicates that W290 might facilitate access of D-galactose to the active center. It is also found that radical stability is not only affected by reduction potential, but also structural factors. The synergistic interactions between the R330K and Q406S/T substitutions improve D-glucose accessibility to the active center. Mutant M-RQW can also accept aliphatic secondary alcohols to generate corresponding ketones. This activity, compared with the bioinorganic mimics of GOase, illustrates the flexible, yet robust, active center configuration of GOase.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||directed evolution; expression; galactose oxidase; glucose 6-oxidase; radical enzymes; thermostability|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||23 September 2002|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||08 Oct 2002|
|Last Modified:||26 Dec 2012 03:03|
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