Oxynitrilases overexpression

For the production of (5)-m-phenoxybenzaldehyde cyanohydrin (47) DSM established an enzymatic hydrocyanation process on an industrial scale (Scheme 31). An efficient (S)-oxynitrilase biocatalyst has been developed. This enzyme is derived from the plant Hevea brasiliensis, and has been cloned and overexpressed in a microbial host organism [117]. In the presence of this biocatalyst the desired product 47 has been obtained with high enantioselec-tivity. 

Among the 11 oxynitrilases isolated from six plant families and purified to homogeneity to date (Table 1), only four attained preparative interest due to their sufficient availability from natural plant sources or successful functional overexpression. 

Sorghum bicolor oxynitrilase was purified and characterised, but a functional overexpression has failed to date. The core protein was expressed correctly, but the posttranslational processing (glycosylation) could not be performed in the host organisms used. Since plants differ in the manner of glycosylation compared to simple eucaryotic organisms, functional overexpression of the oxynitrilase from Sorghum bicolor seems not to be possible in the foreseeable future [67]. 

Progress has also been made in the overexpression of the oxynitrilase from Manihot esculenta (cassava) [67,70] in Escherichia coli. As mentioned previously, this oxynitrilase is very similar to the Hevea enzyme [16,32], because both plants belong to the same plant family, the Euphorbiaceae. A fermentation on the 40 1 scale gave, after simple purification, a total amount of about 40,000 lU of oxynitrilase activity and allowed the exploration of its ability to catalyse the formation of (S)-cyanohydrins [67]. 

Good progress was also made for the (S)-oxynitrilase from Manihot esculenta [16, 62, 70]. Overexpression in E. coli gave sufficient amounts of enzyme to characterise its properties [67] and it was found that this oxynitrilase catalyses the formation of ahphatic, aromatic and hetero aromatic aldehydes and ketones [67]. The substrate range is very similar compared to that of the aforementioned oxynitrilase from Hevea brasiliensis [151-153] which is not surprising since these two enzymes belong to the same plant family and resemble each other in secondary and three dimensional structure (see Sect. 2.4). 

---