From sequence to function: a new workflow for nitrilase identification

Abstract

Nitrilases are industrially important biocatalysts due to their ability to degrade nitriles to carboxylic acids and ammonia. In this study, a workflow for simple and fast recovery of nitrilase candidates from metagenomes is presented. For identification of active enzymes, a NADH-coupled high-throughput assay was established. Purification of enzymes could be omitted as the assay is based on crude extract containing the expressed putative nitrilases. In addition, long incubation times were avoided by combining nitrile and NADH conversion in a single reaction. This allowed the direct measurement of nitrile degradation and provided not only insights into substrate spectrum and specificity but also in degradation efficiency. The novel assay was used for investigation of candidate nitrilase-encoding genes. Seventy putative nitrilase-encoding gene and the corresponding deduced protein sequences identified during sequence-based screens of metagenomes derived from nitrile-treated microbial communities were analyzed. Subsequently, the assay was applied to 13 selected candidate genes and proteins. Six of the generated corresponding Escherichia coli clones produced nitrilases that showed activity and one unusual nitrilase was purified and analyzed. The activity of the novel arylacetonitrilase Nit09 exhibited a broad pH range and a high long-term stability. The enzyme showed high activity for arylacetonitriles with a K M of 1.29 mM and a V max of 13.85 U/mg protein for phenylacetonitrile. In conclusion, we provided a setup for simple and rapid analysis of putative nitrilase-encoding genes from sequence to function. The suitability was demonstrated by identification, isolation, and characterization of the arylacetonitrilase. Key points • A simple and fast high-throughput nitrilase screening was developed. • A set of putative nitrilases was successfully screened with the assay. • A novel arylacetonitrilase was identified, purified, and characterized in detail.

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

“…Despite a decrease in activity of about a factor of 2 on the substrate 3-phenylpropionitrile compared to a purification by chromatography of affinity, this heat treatment can be a quick, easy, and economically interesting Nit phym purification method for large-scale applications. The molecular mass of Nit phym (37 kDa) is close to molecular masses of other known nitrilases such as Nit09 (36 kDa) (Egelkamp et al, 2020). Its high oligomericity of more than 18 subunits is in accordance with many described nitrilases, the preferred native forms of nitrilases seem to be large aggregates of 6-26 units (Banerjee et al, 2002).…”

“…Some nitrilases were reported to be stable in alcoholic cosolvent such as MeOH, EtOH, or iPrOH mainly in 1–10% (vol/vol) range, but a residual activity of more than 30% at 15–20% (vol/vol) in MeOH as observed for Nit phym seemed much more unusual ( Table 2 ). For example, the arylacetonitrilase Nit09 displayed less than 10% of residual activity at 20% (vol/vol) with all tested solvent, except glycerol ( Egelkamp et al, 2020 ). 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 ).…”

“…For example, the arylacetonitrilase Nit09 displayed less than 10% of residual activity at 20% (vol/vol) with all tested solvent, except glycerol ( Egelkamp et al, 2020 ). 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 ).…”

“…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 ). This nitrile was chosen for this study, despite it was not the best substrate of Nit phym ( Table 1 ), to attest even more its potential.…”

“…Furthermore, thermostable biocatalysts are often tolerant to high pressure and solvents, experimental conditions often used in industrial processes. Inter alia , nitrilases satisfying these criteria are searched among the (hyper)thermophilic biodiversity ( Atalah et al, 2019 ), through functional screenings ( Soares Bragança et al, 2017 ) or (meta)genome mining approaches ( Zhu et al, 2007 ; Seffernick et al, 2009 ; Vergne-Vaxelaire et al, 2013 ; Bordier et al, 2014 ; Vesela et al, 2016 ; Sharma et al, 2017 ; Egelkamp et al, 2020 ). Despite all the characterized nitrilases, no common feature of protein sequences was found that could explain their stability, only highly specific motifs were proposed as being important, thus making protein engineering complicated.…”