Heterologous expression, purification and characterization of arylacetonitrilases from Nectria haematococca and Arthroderma benhamiae
Abstract
Two novel arylacetonitrilases were purified from Escherichia coli BL21-Gold (DE3) expressing nit genes from Nectria haematococca mpVI 77-13-4 (EEU45207; NitNh) and Arthroderma benhamiae CBS 112371 (EFE30690; NitAb). The nitrilases formed holoenzymes of 360 and 336 kDa through gel filtration, while their apparent subunit size in SDS-PAGE was approximately 36 and 37 kDa, respectively.
The preferred substrates of the purified enzymes were phenylacetonitrile, (R, S)-mandelonitrile, and 3-indolylacetonitrile. Both enzymes hydrolyzed (R)-mandelonitrile preferentially but with different degrees of selectivity, the e.e.s of the product (R)-mandelic acid being 63 and 89% in NitAb and NitNh, respectively, at pH 8.0.
NitAb exhibited a higher temperature and pH stability than NitNh. Significant amounts of amide (> 5% of total product) were produced only by NitNh (from 2-cyanopyridine, (R, S)-mandelonitrile and phenylacetonitrile).