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).

Keywords::

• Arthroderma benhamiae
• Arylacetonitrilase
• Nectria haematococca
• phenylacetonitrile
• (R,S)-mandelonitrile

Acknowledgements

The authors wish to thank Dr. A. Stolz (University of Stuttgart) for his useful advice on the sequential analysis.

Declaration of interest: The authors declare no conflict of interests. Financial support via projects P504/11/0394, TA01021368 (Technology Agency of the Czech Republic), 305/09/H008 (Czech Science Foundation), OC09046 (Ministry of Education of the Czech Republic), internal project RVO61388971 (Institute of Microbiology) and the Erasmus fellowship to P.W. is gratefully acknowledged.