Epoxides are cyclic ethers that readily react with various nucleophilic compounds. Consequently, epoxides can be used in many chemical synthesis reactions. Two enantiomeric forms of an epoxide are possible if one of the carbon atoms is chiral. This means that the epoxide is actually a racemic mixture of its two enantiomers. Due to the universal presence of chirality in nature it is important to use the proper epoxide enantiomer in the synthesis of compounds such as pharmaceuticals and agrochemicals, which should affect biological processes. Therefore, enantiopure epoxides are valuable intermediates in the synthesis of biologically active compounds by the pharmaceutical and agrochemical industries.
Epoxide hydrolases (EHs) catalyze the hydrolysis of an epoxide into its corresponding diol. Moreover, EHs can hydrolyze racemic epoxide mixtures in an enantioselective manner. This results in the hydrolysis of one enantiomer, while the other remains unaffected and thus enantiopure. Therefore, EHs might be valuable tools to obtain enantiopure epoxides from racemic mixtures.
EHs from yeast species, Rhodotorula glutinis in particular, have been usedto hydrolyze various epoxides with high activity and enantioselectivity. Consequently, the EHs from yeast species are promising biocatalysts that can be used in the production of enantiopure epoxides.
In chapter 1 the work that is presented in this thesis is introduced. The aims of the research project were to gain important fundamental knowledge on EHs from yeasts and to develop biotechnological processes based on the use of these enzymes for the production of enantiopure epoxides. To introduce EHs in general, a broad overview is given in chapter 2 dealing with the molecular biology, biochemistry and potential application of these enzymes.
In order to determine the relationship of these yeasts EHs to other known EHs, the EH-encoding genes and cDNA sequences from the yeast strains Xanthophyllomyces dendrorhous and R. glutinis were isolated (chapters 3 and 4). The genes were denominated EPH1 . Whereas the X . dendrorhous EPH1 open reading frame (ORF) of 1236 bp was interrupted by 8 introns, the 1230 bp-large R. glutinis EPH1 ORF was interrupted by 9 introns. The genes encoded polypeptides of 411 and 409 amino acids respectively, with corresponding calculated molecular masses of 46 kDa. The deduced amino acid sequences were similar to that of mammalian microsomal epoxide hydrolases. These enzymes belong to thea/bhydrolase fold family of enzymes, which have similar enzymatic structures and mechanisms. The EPH1 cDNA sequences were expressed in Escherichia coli to demonstrate their function. The epoxides, 1,2-epoxyhexane and 1-methyl-cyclohexene oxide, were hydrolyzed in an enantioselective manner. The inactivation of the EPH1 gene of X. dendrorhous showed that it was not essential for growth in rich medium under laboratory conditions.
The epoxide hydrolase of R. glutinis was overproduced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system (chapter 5). A strong improvement in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis , despite the formation of inactive Eph1 inclusion bodies. Co-expression of genes encoding molecular chaperones (DnaK-DnaJ-GrpE, GroEL-GroES, and trigger factor) decreased the amount of Eph1 inclusion bodies. However, there was no equivalent increase in active soluble Eph1. An increase in the level of soluble Eph1 was demonstrated by lowering the cultivation temperature from 37ºC to 21ºC and by using a fermenter for cultivation. Compared to R. glutinis the total increase in Eph1 activity for the recombinant E . coli towards 1,2-epoxyhexane was over 200 times, without loss of enantioselectivity. The utility of this Eph1 overproduction system was demonstrated by the hydrolysis of 1-oxa-spiro[2.5]octane-2-carbonitrile, which is a new R. glutinis Eph1 substrate and a versatile building block in organic synthesis. Whereas the recombinant E. coli , expressing R. glutinisEPH1 , could be used to hydrolyze 1-oxa-spiro[2.5]octane-2-carbonitrile with high Eph1 activity in an enantioselective manner. This was not possible using R. glutinis itself.
To explore the biological diversity and potential industrial use of EHs from yeasts the Eph1-encoding cDNA sequences were also isolated from the carotenoid producing yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031. These cDNA sequences encoded polypeptides of 409, 409, and 410 amino acids large respectively with molecular masses of 46 kDa. The deduced amino acid sequences were similar to that of the epoxide hydrolases from R. glutinis , X. dendrorhous and Aspergillus niger , which all correspond to the microsomal epoxide hydrolase sequence. Consequently, these cloned Eph1s probably belong to thea/bhydrolase fold family of enzymes. The epoxide hydrolase encoding cDNAs of the Rhodosporidium and Rhodotorula species were expressed in Escherichia coli BL21(DE3). The recombinant strains were able to hydrolyze trans -1-phenyl-1,2-epoxypropane in an enantioselective manner. The recombinant counterpart of Rhodosporidium toruloides CBS 14 was found to be a highly active and enantioselective biocatalyst for this substrate, despite the low activity and enantioselectivity of Rhodosporidium toruloides CBS 14 itself.
|Qualification||Doctor of Philosophy|
|Award date||1 Mar 2002|
|Place of Publication||S.l.|
|Publication status||Published - 2002|
- microsomal epoxide hydrolase
- gene expression