Kurzbeschreibung
(Englisch)
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The project is focussed on the construction of a library of well-expressed oxygenases (alkane hydroxylases and Baeyer-Villiger monooxygenasees), on the identification of mutations in oxygenase genes or host genes that allow high-level functional expression of these oxygenases.
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Partner und Internationale Organisationen
(Englisch)
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AT, BE, CH, CZ, DE, DK, ES, FI, FR, GR, HU, IT, LT, LV, NL, NO, PlL, PT, SE, SI, SK, UK
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Abstract
(Englisch)
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CYP153 P450 enzymes are useful biocatalysts for the production of a wide range of (terminal) alcohols and epoxides from linear, alicyclic and aromatic compounds. Eleven CYP153 genes were cloned from various organisms such as Mycobacterium sp. HXN1500, Sphingomonas sp. HXN200, and Acinetobacter sp. EB104, and eight of these could be functionally expressed using pCom12 in Pseudomonas putida and/or Pseudomonas fluorescens. To further improve functional expression, the CYP153s are being tested with their cognate ferredoxins and ferredoxin reductase genes. Further characterisation of the CYP153s resulted in: i. protein crystals of two CYP153 enzymes; ii. production of large amounts of all recombinants; iii. characterization of specificy and activity towards a range of substrates including linear alkanes and alkenes, styrene, and limonene; iv. development of selection and screening methodologies for directed evolution experiments; v. improvement of the activity of CYP153A1 through forced selection experiments. Some of the recombinants are being tested by COST-partners in Graz. A Class 1 Baeyer-Villiger monooxygenase (BVMO) cloned from Xanthobacter flavus ZL5 was expressed in E. coli, and has been characterized by our partners in Vienna. This new biocatalyst is able to convert a wide range of ketones with different structural constraints in excellent enantioselectivity like other BVMO enzymes. Sterically-demanding substrates not transformed by other BVMOs were successfully oxidized by the X. flavus ZL5 BVMO.
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