Improving Functional Enzyme Expression and Secretion

Other reports show that intracellular expression of misfolded or unstable proteins can be dramatically improved by directed evolution. For example, directed evolution increased the expression of disulfide-containing antibody fragments in E. coli 50-fold, to reach a level of more than 0.5 g/L 212L The expression of a wide spectrum amidase of B. stearothermophilus in E. coli was improved 23-fold by two mutations 2101. And, in vivo fluorescence of the green fluorescent protein was improved 45-fold by increasing its solubility and native folding in E. co/t[1401. 

Secretion into the culture medium is preferred for some industrial enzymes because it can facilitate purification. This is especially true if the protein reaches high concentrations. Schellenberger s group at Genencor devised a method to select for improved subtilisin-secreting mutants in Bacillus1 7 1. Mutants secreting up to five times as much enzyme were found after one round of error-prone PCR and selection. 

Directed evolution can improve functional enzyme expression and even allow expression of enzymes that are otherwise difficult to produce in recombinant systems. Results obtained by directed evolution, however, will depend on the particular system, and high expression will undoubtedly be best achieved by a combined approach of molecular biology, fermentation optimization and directed evolution. 

Target enzyme Target function Change effected Approach Organism Reference 

Kanamycin nucleotidyltransferase Thermostability 200-fold increase in half-life at 60-65 °C Mutator strain + selection B. stearothermophilus (167) 

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