Protein engineering

1. 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

2. 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

3. 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.

4. 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

5. 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 wild-type enzyme necessary

6. 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

7. 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)

8. 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.

9. Protein engineering Selection parameters • Substrate range • Stability in organic solvent • Stability towards reaction conditions • Thermal stability • High-throughput product analysis • Robot technology

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

11. 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

12. 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

13. 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