A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production

Abstract

A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37 °C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25 °C, which preserved 95% of activity after 120 h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100 mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050 mM 3-cyanopyridine was hydrolyzed completely in 20.8 h with eight substrate feedings, yielding 129.2 g/L production of nicotinic acid and thus showing a potential for industrial application.

Purification and Characterization of Nitphym, a Robust Thermostable Nitrilase From Paraburkholderia phymatum

“…For example, the well described nitrilase from Pseudomonas fluorescens strain EBC191 has similar temperature profile (optima temperature: 45–55°C) but is much more unstable than Nit phym at these temperatures with activity decreasing within 30 min above 40°C ( Table 2 ; Kiziak et al, 2005 ). More than 50% of activity was lost for the ones from Ralstonia eutropha ( Fan et al, 2017b ) or Pseudomonas psychrotolerans ( Sun et al, 2016 ) after storage of less than 10 h at 37–40°C, whereas they displayed broad temperature optima of 30–50°C and 30–60°C, respectively ( Table 2 ). The stability of Nit phym is impressive, with a half-live of 18 h at 60°C, compared to these nitrilases and the one from Pantoea sp.…”

“…In addition to its preference for trans -cinnamonitrile, cyanopyridines, and 3-phenylpropionitrile and its inactivity or very low activity toward arylacetonitriles, Nit phym harbored a substrate spectrum similar to those of PaNIT and REH16. These two enzymes share 71 and 81% protein sequence identity with Nit phym , respectively ( Fan et al, 2017b ; Zhang et al, 2018 ). Thus, Nit Phym is an attractive alternative to these efficient biocatalysts for the hydrolysis of such nitriles.…”

“…The activity reduced only by half in the presence of 20% (vol/vol) DMSO has to be noticed, this solvent being often harmful for enzymes at this ratio ( Egelkamp et al, 2020 ). However, Nit phym was strongly affected by even 5% (vol/vol) of THF, like REH16 from Ralstonia eutropha , yet highly tolerant to many solvents even at 25% (vol/vol) ( Fan et al, 2017b ). The effect of substrate loading was tested on 3-phenylpropionitrile, one of the top substrate of various reported nitrilases mainly showing major activities toward arylacetonitriles, such as Nit09 (0.8 U⋅mg –1 ) ( Egelkamp et al, 2020 ), nitrilase from P. fluorescens EBC191 (3.90 U⋅mg –1 ) ( Kiziak et al, 2005 ), BGC4 ( Fan et al, 2017a ), REH16 (61% of the activity of 3-cyanopyridine) ( Fan et al, 2017b ) and nitrilases from Cupriavidus necator , Janthinobacterium sp., and Acetobacter pasteurianus ( Sunder et al, 2020 ).…”

“…Such high productivity can be expected with its better substrates trans -cinnamonitrile and terephthalonitrile. Further amelioration including biotransformation with resting cells and fed batch process, as done with REH16 (1,050 mM of 3-cyanopyridine hydrolyzed in total from 13 batches of 50–100 mM in 20 h) ( Fan et al, 2017b ), can be considered to reach productivity at least such as the one of 26 g/(L h) obtained using PaNIT with terephthalonitrile ( Zhang et al, 2018 ). It is worthy to note that no amide was detected by UHPLC-UV analyses whatever the conditions tested in this study, demonstrating the absence of nitrile hydratase activity of this enzyme.…”

“…They are “easy” enzymes that do not require any cofactor or metal ions; thus, biocatalytical hydrolysis carried out by nitrilases tends to be preferred over chemical methods due to their efficiency and relative ecological friendliness ( Singh et al, 2006 ; Gong et al, 2012 ; Nigam et al, 2017 ; Bhalla et al, 2018 ). This last decade, they have been successfully developed to facilitate the production of chemicals ( Shen et al, 2021 ), such as acrylic acid ( Nagasawa et al, 1990 ) and ( R )-(-) mandelic acid ( Fan et al, 2017b ). Moreover, nitrilases have also found application in the surface modification of acrylic fibers in the textile industry ( Matama et al, 2007 ) and in industrial bioremediation processes, such as degradation of the highly toxic 2,6-dichloro-benzonitrile and bromoxynil (3,5-dibromo-4 hydroxybenzonitrile) present in wastewater ( Mueller et al, 2006 ; Nigam et al, 2017 ; Park et al, 2017 ).…”