Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid

Abstract

A nitrilase gene cyc705 from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid (IDA) was cloned. This gene contained a 930 bp ORF, which encoded a polypeptide of 310 amino acids. A recombinant Escherichia coli BL21(DE3)/ pET28a-cyc705 was constructed to achieve the heterologous expression of cyc705. This recombinant nitrilase was purified to homogeneity with a molecular weight of 36.7 kDa on SDS-PAGE and mass spectrometry, and characterized to be an oligomer of 14 subunits by gel permeation chromatography. Using iminodiacetonitrile (IDAN) as the substrate, the V max , K m , k cat and k cat / K m were 9.05 U mg 1 , 43.17 mM −1 , 94.1 min −1 and 2.18 10 3 min −1 M −1 , respectively. The optimum temperature and pH were 25 C and 5.8. The suitable substrates for the purified nitrilase were short-chain aliphatic dinitriles. High concentration of IDAN could be hydrolyzed to IDA in a shorter time.Key words: Arthrobacter aurescens; characterization; iminodiacetic acid; nitrilase Introduction Nitrilase (EC 3.5.5.1), as an important industrial enzyme, belongs to the nitrilase superfamily (Brenner, 2002). Nitrilases are widely derived from various organisms, including bacteria, fungi and plants. Initial investigations suggested nitrilases to be specific for aromatic nitriles. But now, depending on the substrate spectrum, nitrilases are reported from different sources (Banerjee et al., 2002), which can be active on aromatic or heterocyclic nitriles (Vejvoda et al., 2010) and aliphatic nitriles (Bayer et al., 2011) as well as arylacetonitriles (Sosedov et al., 2010). Nitrilases can convert nitriles directly into the corresponding carboxylic acids and ammonia. Compared with chemical methods of producing carboxylic acids, nitrilases are widely used due to the mild reaction conditions, regioselectivity (Wang et al., 2014) and enantioselectivity (Vejvoda et al., 2010). Therefore, they are used to catalyze the production of various valuable carboxylic acids.IDA is an important fine chemical intermediate. It has wide applications and is mainly used in producing glyphosate herbicides, chelating agents and surfactants (Ni et al., 2009). Currently, the production of IDA mainly uses chemical methods, including the hydrocyanic acid method (Rmon, 1991), diethanolamine method (Bornscheuer and Kazlauskas, 2006), and chloroacetic acid method (Farbwerke Hoechst, 1969). These methods produce a large amount of waste water and byproducts, which are unfriendly to the environment (Duan et al., 2007). Enzymatic production of IDA has some advantages, such as the mild reaction conditions, environmental friendliness and low cost. So far, only a few papers have reported nitrilase-catalyzed production of IDA. A nitrilase-produced strain named Alcaligenes faecalis ZJB-09133 was screened by using IDAN as the carbon source, and the whole cells were used to catalyze IDAN to IDA, which showed the conversion only reached 65.3% at 8 h after optimization of the reaction conditions (Liu et al., 2011). The Acidovorax facilis nitrilase was cloned ...

High‐level production of Arthrobacter aurescens CYC705 nitrilase in Escherichia coli for biosynthesis of iminodiacetic acid

“…Recombinant E. coli BL21(DE3)/pET28a‐cyc705 with the nitrilase gene (Genbank accession No. KC961317) from A. aurescens CYC705 was constructed for the expression of nitrilase protein in our previous work and used in the present study. The recombinant cells were maintained on TYGN agar plate containing kanamycin for short‐term storage.…”

“…Regarding the future industrial applications, it is necessary to achieve an available nitrilase with high enzyme activity on IDAN. Previously, a novel nitrilase from Arthrobacter aurescens CYC705 that shows high catalytic efficiency on IDAN had been screened, cloned, and overexpressed in Escherichia coli in our lab . The main purpose of this work will focus on how to enhance the production of the nitrilase for biosynthesis of IDA in a cost‐effective way.…”