The nitrile hydratase catalyzed hydration of 2-amino-2,3-dimethylbutyronitrile to 2-amino-2,3-dimethylbutyramide in various green solvent–aqueous reaction systems was investigated in this study.

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 ...

Nitrilase from Arthrobacter aurescens CYC705 can hydrolyze the iminodiacetonitrile to iminodiacetic acid (IDA) efficiently, and its high-level production in Escherichia coli has not been established. In the present work, the production of this nitrilase expressed in E. coli BL21(DE3) with a recombinant plasmid pET28a-cyc705 was optimized. Various culture conditions and process parameters including medium components and concentrations, inducer types and concentrations, inducing temperature and time were systematically examined in a shake flask. After optimization, the OD600 , nitrilase activity, and productivity were obviously improved and achieved to 40.91 ± 1.341, 98.12 ± 1.248 U/mL, and 2,230 ± 28.36 U L(-1)  H(-1) , respectively, about 2.1-, 30-, and 33-fold increases as compared with those in the primary medium. Furthermore, four different fermentation strategies were adopted to scale up cultivation of the recombinant E. coli BL21(DE3)/pET28a-cyc705 in a 3.7-L fermenter. Substituting the peanut powder with fish peptone and accompanying with 1.0% glycerol feeding could significantly reduce the bubble production and shorten the fermentation time, which resulted in a nitrilase productivity of 4,653 ± 38.16 U L(-1) H(-1) that was about two times higher than that in a shake flask. The high-level production of A. aurescens CYC705 nitrilase established in this study will meet the need of industrial biosynthesis of IDA.

In the synthesis of imidazolinone herbicides, 2-Amino-2,3-dimethylbutyramide (ADBA) is an important intermedium. In this study, the recombinant production of nitrile hydratase (NHase) in Escherichia coli for ADBA synthesis was explored. A local library containing recombinant NHases from various sources was screened using a colorimetric method. NHase from Pseudonocardia thermophila JCM3095 was selected, fused with a His-tag and one-step purified. The enzymatic properties of recombinant NHase were studied and indicated robust thermal stability and inhibition of cyanide ions due to substrate degradation. After systematic optimization of fermentation conditions, the OD600 (optical density at 600 nm), enzyme activity and specific activity of recombinant strain E. coli BL21(DE3)/pET-28a+NHase reached 19.4, 3.72 U/mL and 1.04 U/mg protein at 42 h, representing 5.86-, 26.6- and 4-fold increases, respectively. These results offered an efficient recombinant whole-cell biocatalyst for ADBA synthesis.