Biocatalytic carbon nitrogen double bond reduction

Abstract

Within this research project the bioreduction of imines and oximes has been investigated as possible promiscuous activity of two different reductases families, the enoate reductase family (EC 1.3.1.31) and the carbonyl reductase family (EC: 1.1.1.1). The decision of targeting these enzyme classes has been based on the existing literature and on substrate analogy for the enoate reductases and based on in silico docking for the carbonyl reductase. As a carbonyl reductase was already available in our research group, an activity based screening for the isolation of enoate reductases was performed within this research project. As genetic material the DNA of Acetobacterium woodii and metagenomic DNA have been used. The DNA of Acetobacterium woodii has been cloned into E.coli and the obtained library was screened in High Throughput screening format. The same procedure, although with a different vector, has been followed for the metagenomic DNA. As no positive hits were identified within both the screened libraries, the conclusion that the adopted strategy was not successful for the isolation of enoate reductase was drawn. In order to overcome the problems that arise with an activity based screening (low protein expression and uncorrect folding), a sequence based screening was performed, designing degenerate primers based on the alignment of the already published enoate reductases. This strategy showed to be successful and led us to the isolation of a new enoate reductase from the metagenome. The obtained enoate reductase retrieved from the metagenome, together with the cloned enoate reductase from Clostridium acetobutylicum and the carbonyl reductase from Candida parapsilosis, have been tried in the bioreduction of imines and oximes, under a very diverse reaction conditions. The formation of the desired product, however, has never been detected