California Institute of Technology

Basf Aktiengesellschaft

Basf Se Corporation

Universitat Basel

Rwth Aachen

Ulrich Schwaneberg

Frances H Arnold

Edgardo Farinas

Anton Glieder

Bernhard Hauer

Rolf Schmid

Juergen Pleiss

Jutta Schmitt

Markus Fischer

Qing-shan Li

Michael Breuer

Sabine Lutz-Wahl

Anton Blieder

Daniel Appel

WolfGang Meier

Tuck Seng Wong

Mathias Winterhalter

Nucleic acids encoding cytochrome P450 variants are provided. The cytochrome P450 variants of have a higher alkane-oxidation capability, alkene-oxidation capability, and/or a higher organic-solvent resistance than the corresponding wild-type or parent cytochrome P450 enzyme. A preferred wild-type cytochrome P450 is cytochrome P450 BM-3. Preferred cytochrome P450 variants include those having an improved capability to hydroxylate alkanes and epoxidate alkenes comprising less than 8 carbons, and have amino acid substitutions corresponding to V78A, H236Q, and E252G of cytochrome P450 BM-3. Preferred cytochrome P450 variants also include those having an improved hydroxylation activity in solutions comprising co-solvents such as DMSO and THF, and have amino acid substitutions corresponding to T235A, R471A, E494K, and S1024E of cytochrome P450 BM-3.

Cytochrome P450 oxygenases

The invention relates to novel proteins having esterase activity, to mutants thereof, to nucleic acid sequences coding therefor, to expression cassettes, vectors and recombinant microorganisms; to methods for preparing said proteins and to the use thereof for enzymic, in particular enantioselective enzymic, ester hydrolysis or transesterification of organic esters.

Mutants of a pseudomonas glumae butynol esterase

The present invention relates to modified cytochrome P450 monooxygenases with an altered substrate profile, to nucleic acid sequences coding therefor, to expression constructs and vectors, to recombinant microorganisms which comprise these vectors, and to processes for the microbiological production of terminally or subterminally hydroxylated aliphatic carboxylic acids.

Modified cytochrome P450 monooxygenases

The invention relates to novel cytochrome P450 monooxygenases comprising a modified substrate specificity, to nucleotide sequences which code therefor, to expression constructs and vectors containing these sequences, and to microorganisms transformed therewith. The invention also relates to methods for microbiologically oxidizing different organic substrates, such as methods for producing indigo and indirubin.

Cytochrome p450 monooxygenases and their use for oxidizing organic compounds

A process for the mutagenesis of a double-stranded polynucleotide sequence (master sequence) of n base-pairs having a (+)-strand and a complementary (&#x2212;)-strand comprising the steps (i) creation of a collection of single-stranded fragments of the (+)-strand of the master sequence wherein all members of the collection have the same 5'-terminus and have a deletion in the 3-terminus such that the collection represents (+)-strands with a length of n&#x2212;1, n&#x2212;2, n&#x2212;3, . . . nucleotides; (ii) introduction of at least one universal or degenerate nucleotide at the 3&#x2032;-terminus of the (+) strand produced in step (i); (iii) elongation of the (+)-strand produced in step (ii) to the full length of the master sequence using the (&#x2212;)-strand or fragments thereof as a template strand for the elongation; (iv) synthesis of a (&#x2212;)-strand by using the (+)-strand produced in step (iii) as a template strand thereby effecting mutations in the (&#x2212;)-strand at the positions of the previous universal or degenerate nucleotides compared to the master sequence.

Process for sequence saturation mutagenesis (SeSaM)

Modified cytochrome p450 monooxygenases

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Ritterhude, Germany

Ulrich Schwaneberg