Screening for lipases performing chemo-enzymatic epoxidation

TÖRNVALL, ULRIKA ; ADLERCREUTZ, DIETLIND ; MARTINEZ, M. ALEJANDRA; HATTI-KAUL, RAJNI

Venice

Simposio; Summer School on Green Chemistry 7th Edition. Venice, Italy; 2004

CORDIS, the Community Research and Development Information Service

Screening for lipases performing chemo-enzymatic epoxidation ULRIKA TÖRNVALL, DIETLIND ADLERCREUTZ, ALEJANDRA MARTINEZ, RAJNI HATTI-KAUL   Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden   Epoxidized unsaturated fatty acids and their esters are valuable products for use as building blocks in the synthesis of a vast amount of industrially important products such as PVC-stabilizers, plasticizers, lubricating oils and as additives to surface coatings. The industrial process for the manufacture of these epoxides involves the use of a strong acid catalyst. The replacement of this procedure by a chemo-enzymatic epoxidation using lipases is expected to result in both a more environmentally benign and a more specific process. The lipase catalyses the conversion of unsaturated fatty acids, in the presence of hydrogen peroxide, to unsaturated peracids. The peracids subsequently undergo spontaneous self-epoxidation. The stability of the enzyme towards the peroxide and the peracids is an important parameter for an efficient process and therefore methods for rapid screening of lipases exhibiting peroxidation activity are being investigated.          We are using both activity-based and gene-based approaches for screening of lipases suitable for epoxidation reactions. Selectivity of the screening method is important for obtaining lipase candidates with desired activity and stability features. The activity-based method makes use of the ability of the lipase to generate peracid from a fatty acid in the presence of hydrogen peroxide. The peracid formed is then used for selective oxidation of the substance 2,2´-Azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS) to its intensively green radical cation. The ABTS assay has been used for screening of lipase activity of a few commercial enzymes and isolates from extreme environments. Commercially available immobilised lipases from Candida antarctica and Rhizomucor miehei have shown the highest activity.          Based on this knowledge, lipases related to C. antactica and R. miehei will be isolated and cloned using the genome-sequence based approach. The use of PCR-fishing with highly degenerated primers designed towards consensus regions of these lipases, such as the oxy-anion hole and the active serine regions, will be used to search for novel sequences from environmental isolates and samples. The results are planned to be used for directed evolution, hopefully making it possible to create an enzyme more stable against hydrogen peroxide and peracids.