Enhanced Expression and Purification of Fungal Galactose Oxidase in Escherichia coli and Use for Analysis of a Saturation Mutagenesis Library

Abstract

Galactose oxidase (GO) displays broad primary alcohol substrate specificity and so offers potential for engineering new substrate specificity by directed evolution. Producing variant libraries of sufficient complexity ideally requires expression of functional protein in a host such as Escherichia coli. Wild-type GO is produced by the fungus Fusarium graminiarum and is expressed poorly in E. coli. We introduced silent mutations within codons 2-7 of the mature GO coding sequence to enhance GO translation and have combined these with other expression-enhancing mutations. We selected the best E. coli host strain, autoinduction medium, induction temperature, harvest time and cell lysis procedure to produce active recombinant GO. Although normally secreted by the fungus, we have expressed GO in the cytoplasm of E. coli and have used a C-terminal Streptag II sequences for single-step affinity purification. This resulted in purification of 240 mg of functional GO per litre of shake flask culture. We have generated a saturation mutagenesis library at residue Cys383, known to influence substrate binding, and have used the optimised expression conditions to purify and characterise the resulting enzymes.

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

“…The additional silent mutations, along with optimized cultivation conditions, increased GAO productivity over 20 times to 240 mg L -1 [92]. Systematic comparison of GAO expression in E. coli and P. pastoris confirmed that the modifications to codon sequence reported by Sun et al (2001) [24] increased GAO expression in E. coli, but did not improve GAO expression in P. pastoris [13].…”

“…GAO production in E. coli was further increased by introducing silent mutations in codons two to seven [92], in addition to the six substitutions reported by Sun et al (2001) (Table 5) [24].…”

Computational Enzyme Design

“…[17,18] Examples of its application include improved themostability, [88] tolerance to organic solvents, [89] strengthened proteinprotein interactions, [90] altered substrate promiscuity/specificity, [91,92] enhanced enzymatic activity, [93] and inversion of enantioselectivity. [94,95] In the directed evolution of catalytic function, a starting gene is mutagenized to create a library of variants, which is screened for enzymes with an improvement of the sought-after property (stability, substrate specificity, activity, etc.).…”

Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization

“…Expression has been increased by using directed evolution and site directed mutagenesis [21]–[23]. Higher yields were obtained by expression of the wild type enzyme in P. pastoris [15], [24]–[27] and A. nidulans [28].…”