Browsing by Author "Hauer, Bernhard (Prof.Dr.)"

Now showing 1 - 1 of 1

Open Access
Development of highly efficient CYP153A-catalysed terminal hydroxylation of fatty acids
(2016) Notonier, Sandra; Hauer, Bernhard (Prof.Dr.)
Terminally hydroxylated fatty acids (omega-OHFA) are of great interest to industry: in the area of high end polymers, in fine chemicals, in the cosmetic and fragrance industry. The activation and oxidation of C-H bonds, is however still chemically challenging. It requires rough conditions, high pressure, high temperature and the processes still suffer from poor selectivity. Alternatively, the functionalization of carbon atoms can be achieved by using versatile cytochrome P450 monooxygenases with high regio- and stereoselectivity (P450s or CYPs). These heme containing proteins can be found in many organisms, they are involved in a diverse range of reactions and they are able to catalyse the conversion of a large panel of substrates. CYP153A from Marinobacter aquaeolei (CYP153AM.aq.) constitutes a promising catalyst for the oxidation of non-activated carbon atoms due to its high regioselectivity in the hydroxylation of different small to medium chain alkanes, fatty acids and primary alcohols as well as its efficient expression in Escherichia. coli at high yields. For bacterial whole-cell applications, the enzyme was further engineered as a protein chimera by fusing the reductase domain of P450BM3, from Bacillus megaterium, to the heme domain of CYP153AM.aq. (CYP153AM.aq.-CPRBM3). The mutant G307A (position located in the binding pocket) was further identified as improved variant towards medium chain-length fatty acids regarding activity, regioselectivity and coupling efficiency. To perform efficient whole-cell biotransformations and therefore to increase the yield of bioconversion of fatty acids into omega-OHFA, the system requires the identification of present bottlenecks. The approaches to characterize and to optimise the up-scaling process involved testing different feeding strategies, evaluation of substrate/product inhibition, transport limitations estimation, cofactor availability evaluation and biocatalyst stability investigations. Such limitations often associated to the P450 biocatalysts and/or related to whole-cell biotransformations were evaluated in this study, using the fusion constructs CYP153AM.aq.-CPRBM3 towards the model substrate dodecanoic acid (C12:0) in vitro and in vivo. Strategies to overcome existing bottlenecks regarding low activity involved the generation of mutant libraries to screen and to characterize improved versions of the current chimera biocatalyst. In parallel to the mutant libraries generation, a whole-cell colorimetric assay was also established.