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 .

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

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

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 .

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 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.

molecular evolution • Random mutagenesis of the gene encoding the biocatalyst (e.Protein engineering Directed evolution • Evolutive biotechnology. by error-prone PCR) • DNA shuffling: recombination of gene fragments (staggered extension process or random priming recombination) .g.

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 Directed evolution strategy • • • • • • • • Random mutagenesis Library of mutated genes Transformation in E.

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 .

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

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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