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Protein engineering

Tailor-made biocatalysts
The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity Rational (re)design versus directed evolution

Protein engineering
Genetic manipulation techniques
Large-scale supply of enzymes at reasonable price Identification of new biocatalysts (screening) doesnot always yield suitable enzymes for a given synthetic problem Computer-aided site-directed mutagenesis Directed (molecular) evolution

Protein engineering
Site-directed mutagenesis
Requires structural information and knowledge about relationship between sequence, structure, function and mechanism Very information-intensive Rapid progress in NMR / X-ray methods Genome sequence information Molecular modeling, bioinformatics Prediction of selectivity, activity, stability etc.

Protein engineering
Rational redesign strategy
Protein structure Planning of mutants, SDM Vectors containing mutated genes Transformation in E. coli Protein expression and purification Mutant enzyme analysis Negative mutants Improved mutant enzymes

Protein engineering
Rational redesign
Amino acid substitutions often selected by sequence comparison with homologous sequences Results have to be carefully interpreted Minor changes by a single point mutation may cause significant structural disturbance Comparison of 3D-structure of mutant and wildtype enzyme necessary

Protein engineering
Inversion of stereospecificity of VAO
Current Opinion in Chemical Biology (2001)
A very nice study on alteration of enantioselectivity based on structural comparison of two members of structurally related FAD-dependent oxidoreductases, of which one is (R)-specific and the other (S)-specific Site-directed mutagenesis introduced (S)-selectivity in the (R)-selective wild-type enzyme Structural analysis of the mutant enzyme revealed that the mutations are really site-directed

Protein engineering
Directed evolution
Evolutive biotechnology, molecular evolution

Random mutagenesis of the gene encoding the biocatalyst (e.g. by error-prone PCR) DNA shuffling: recombination of gene fragments (staggered extension process or random priming recombination)

Protein engineering
Directed evolution strategy
Random mutagenesis Library of mutated genes Transformation in E. coli Mutant library > 10.000 clones Protein expression in microtiter plates Selection parameters Mutant enzyme and product analysis In vitro-recombination, transformation etc.

Protein engineering
Selection parameters
Substrate range Stability in organic solvent Stability towards reaction conditions Thermal stability

High-throughput product analysis


Robot technology

Protein engineering
Selection parameters
Hydrolysis of esters: agar-plate assay based on pH indicators Parallel assaying of replica-plated colonies with substrate analog Isotopically labeled substrates Capillary electrophoresis (7000 samples per day) Optimization with saturation mutagenesis

Protein engineering
Digital image screening
Naphthalene hydroxylation by P450cam Co-expression of horseradish peroxidase Fluorescent products amenable by digital screening

P450 hydroxylation of indole to indigo Inversion of enantioselectivity Increase of peroxidase specificity with guaiacol

Protein engineering
Improving thermostability
Cold-adapted proteases Combined screening for activity, thermostability, organic solvent tolerance and pH-profile Engineering of entire metabolic pathways Phytoene desaturase and lycopene cyclase shuffling for carotenoid biosynthesis Molecular breeding

Protein engineering
Biochemistry
Vanillyl-alcohol oxidase Production of natural vanillin 2-Hydroxybiphenyl monooxygenase Large-scale production of substituted catechols

Galactose oxidase Production of new oligosaccharides

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