Molecular Modification Strategies of Nitrilase for Its Potential Application in Agriculture.

Some feed source plants will produce secondary metabolites such as cyanogenic glycosides during metabolism, which will produce some poisonous nitrile compounds after hydrolysis and remain in plant tissues. The consumption of feed-source plants without proper treatment affect the health of the animals' bodies. Nitrilases can convert nitriles and have been used in industry as green biocatalysts. However, due to their bottleneck problems, their application in agriculture is still facing challenges. Acid-resistant nitrilase preparations, high-temperature resistance, antiprotease activity, strong activity, and strict reaction specificity urgently need to be developed. In this paper, the application potential of nitrilase in agriculture, especially in feed processing industry was explored, the source properties and catalytic mechanism of nitrilase were reviewed, and modification strategies for nitrilase application in agriculture were proposed to provide references for future research and application of nitrilase in agricultural and especially in the biological feed scene.

Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis.

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.

Genomic Insights into Cyanide Biodegradation in the Pseudomonas Genus.

Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In some Pseudomonas strains, the assimilation of cyanide has been linked to NitC, such as the cyanotrophic model strain Pseudomonas pseudoalcaligenes CECT 5344, which has been recently reclassified as Pseudomonas oleovorans CECT 5344. In this work, a phylogenomic approach established a more precise taxonomic position of the strain CECT 5344 within the species P. oleovorans. Furthermore, a pan-genomic analysis of P. oleovorans and other species with cyanotrophic strains, such as P. fluorescens and P. monteilii, allowed for the comparison and identification of the cioAB and mqoAB genes involved in cyanide resistance, and the nitC and cynS genes required for the assimilation of cyanide or cyanate, respectively. While cyanide resistance genes presented a high frequency among the analyzed genomes, genes responsible for cyanide or cyanate assimilation were identified in a considerably lower proportion. According to the results obtained in this work, an in silico approach based on a comparative genomic approach can be considered as an agile strategy for the bioprospection of putative cyanotrophic bacteria and for the identification of new genes putatively involved in cyanide biodegradation.

Astrobiological implications of the organic and inorganic cyanide utilization by a novel Antarctic hyperthermophilic Pyrococcus strain.

Organic and inorganic cyanides are widely distributed in nature, yet not much is known about the ability of microorganisms to use these compounds as a source of nitrogen and/or carbon at high temperatures (>80 °C). Here we studied the capacity of organic and inorganic cyanides to support growth of an hyperthermophilic Pyrococcus strain isolated from Deception Island, Antarctica. This microorganism was capable of growing with aromatic nitriles, aliphatic nitriles, heterocyclic nitriles, amino aromatic nitriles and inorganic cyanides as nitrogen and/or carbon source. This is the first report of an hyperthermophilic microorganism able to incorporate these compounds in its nitrogen and carbon metabolism. Based on enzymatic activity and genomic information, it is possibly that cells of this Pyrococcus strain growing with nitriles or cyanide, might use the carboxylic acid and/or the ammonia generated through the nitrilase enzymatic activity, as a carbon and/or nitrogen source respectively. This work expands the temperature range at which microorganisms can use organic and inorganic cyanides to growth, having important implications to understand microbial metabolisms that can support life on Earth and the possibility to support life elsewhere.

Modification of nitrilase based on computer screening and efficient biosynthesis of 4-cyanobenzoic acid.

4-cyanobenzoic acid serves as a crucial intermediate for the synthesis of various high-value organic compounds. The enzymatic hydrolysis of terephthalonitrile to produce 4-cyanobenzoic acid using nitrilase offers the advantages of a simple reaction pathway, environmental friendliness, and easy product separation. In order to efficiently develop nitrilases that meet industrial production requirements, the virtual screening method used in the study is established and mature. From a total of 371 amino acids in the nitrilase AfNIT, which exhibits activity in terephthalonitrile hydrolysis, three candidate sites (F168, S192, and T201) were identified, and a "small and accurate" mutant library was constructed. The triple mutant F168V/T201N/S192F was screened from this small mutant library with a specific activity of 227.3 U mg-1 , which was 3.8 times higher than that of the wild-type AfNIT. Using the whole-cell biocatalyst containing the mutant F168V/T201N/S192F, terephthalonitrile was successfully hydrolyzed at a concentration of 150 g L-1 to produce 4-cyanobenzoic acid with a final yield of 170.3 g L-1 and a conversion rate of 98.7%. The obtained nitrilase mutant F168V/T201N/S192F in this study can be effectively applied in the biomanufacturing of 4-cyanobenzoic acid using terephthalonitrile as a substrate. Furthermore, the results also demonstrate the significant improvement in predictive accuracy achieved through the latest AI-assisted computer simulation methods. This approach represents a promising and feasible new technological pathway for assisting enzyme engineering research, laying a theoretical foundation for other related studies.

Identification and evolution of non-traditional nitrilase from Spirosoma linguale DSM 74 with high hydration activity.

Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.

Plasmodiophora brassicae affects host gene expression by secreting the transcription factor-type effector PbZFE1.

The protist pathogen Plasmodiophora brassicae hijacks the metabolism and development of host cruciferous plants and induces clubroot formation, but little is known about its regulatory mechanisms. Previously, the Pnit2int2 sequence, a sequence around the second intron of the nitrilase gene (BrNIT2) involved in auxin biosynthesis in Brassica rapa ssp. pekinensis, was identified as a specific promoter activated during clubroot formation. In this study, we hypothesized that analysis of the transcriptional regulation of Pnit2int2 could reveal how P. brassicae affects the host gene regulatory system during clubroot development. By yeast one-hybrid screening, the pathogen zinc finger protein PbZFE1 was identified to specifically bind to Pnit2int2. Specific binding of PbZFE1 to Pnit2int2 was also confirmed by electrophoretic mobility shift assay. The binding site of PbZFE1 is essential for promoter activity of Pnit2int2 in clubbed roots of transgenic Arabidopsis thaliana (Pnit2int2-2::GUS), indicating that PbZFE1 is secreted from P. brassicae and functions within plant cells. Ectopic expression of PbZEF1 in A. thaliana delayed growth and flowering time, suggesting that PbZFE1 has significant impacts on host development and metabolic systems. Thus, P. brassicae appears to secrete PbZFE1 into host cells as a transcription factor-type effector during pathogenesis.

NIT24 and NIT29-mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice.

Auxin plays a pivotal role in the co-evolution of plants and microorganisms. Xanthomonas oryzae pv. oryzicola (Xoc) stands as a significant factor that affects rice yield and quality. However, the current understanding of Xoc's capability for indole 3-acetic acid (IAA) synthesis and its mechanistic implications remains elusive. In this study, we performed a comprehensive genomic analysis of Xoc strain RS105, leading to the identification of two nitrilase enzyme family (NIT) genes, designated as AKO15524.1 and AKO15829.1, subsequently named NIT24 and NIT29, respectively. Our investigation unveiled that the deletion of NIT24 and NIT29 resulted in a notable reduction in IAA synthesis capacity within RS105, thereby impacting extracellular polysaccharide production. This deficiency was partially ameliorated through exogenous IAA supplementation. The study further substantiated that NIT24 and NIT29 have nitrilase activity and the ability to catalyse IAA production in vitro. The lesion length and bacterial population statistics experiments confirmed that NIT24 and NIT29 positively regulated the pathogenicity of RS105, suggesting that NIT24 and NIT29 may regulate Xoc invasion by affecting IAA synthesis. Furthermore, our analysis corroborated mutant strains, RS105_ΔNIT24 and RS105_ΔNIT29, which elicited the outbreak of reactive oxygen species, the deposition of callose and the upregulation of defence-related gene expression in rice. IAA exerted a significant dampening effect on the immune responses incited by these mutant strains in rice. In addition, the absence of NIT24 and NIT29 affected the growth-promoting effect of Xoc on rice. This implies that Xoc may promote rice growth by secreting IAA, thus providing a more suitable microenvironment for its own colonization. In summary, our study provides compelling evidence for the existence of a nitrilase-dependent IAA biosynthesis pathway in Xoc. IAA synthesis-related genes promote Xoc colonization by inhibiting rice immune defence response and affecting rice growth by increasing IAA content in Xoc.

Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation.

Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. Fusarium oxysporum has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of nitrilase gene from F. oxysporum genomic DNA was optimized for cloning and expression in E. coli. The pUC57 containing synthetic optimized nitrilase gene was transferred into E. coli DH5α strain. This nitrilase gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in E. coli BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in E. coli proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The Km and Vm values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.

Arylacetonitrilases: Potential Biocatalysts for Green Chemistry.

Nitrilases are the enzymes that catalyze the hydrolysis of nitriles to corresponding carboxylic acid and ammonia. They are broadly categorized into aromatic, aliphatic, and arylacetonitrilases based on their substrate specificity. Most of the studies pertaining to these enzymes in the literature have focused on aromatic and aliphatic nitrilases. However, arylacetonitrilases have attracted the attention of academia and industry in the last several years due to their aryl specificity and enantioselectivity. They have emerged as interesting biocatalytic tools in green chemistry to synthesize useful aryl acids such as mandelic acid and derivatives of phenylacetic acid. The aim of the present review is to collate information on the arylacetonitrilases and their catalytic properties including enantioselectivity and potential applications in organic synthesis.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Nitrilase mediated mild hydrolysis of a carbon-14 nitrile for the radiosynthesis of 4-(7-hydroxycarbamoyl-[1-14 C-heptanoyl]-oxy)-benzoic acid methyl ester, [14 C]-SHP-141: A novel class I/II histone deacetylase (HDAC) inhibitor.

A strategy has been developed for the carbon-14 radiosynthesis of [14 C]-SHP-141, a 4-(7-hydroxycarbamoyl-heptanoyloxy)-benzoic acid methyl ester derivative containing a terminal hydroxamic acid. The synthesis involved four radiochemical transformations. The key step in the radiosynthesis was the conversion of the 7-[14 C]-cyano-heptanoic acid benzyloxyamide [14 C]-4 directly into the carboxylic acid derivative, 7-benzyloxycarbamoyl-[14 C]-heptanoic acid [14 C]-8 using nitrilase-113 biocatalyst. The final step involved deprotection of the benzyloxy group using catalytic hydrogenation to facilitate the release of the hydroxamic acid without cleaving the phenoxy ester. [14 C]-SHP-141 was isolated with a radiochemical purity of 90% and a specific activity of 190 µCi/mg from four radiochemical steps starting from potassium [14 C]-cyanide in a radiochemical yield of 45%.

Functional metagenomics uncovers nitrile-hydrolysing enzymes in a coal metagenome.

Introduction: Nitriles are the most toxic compounds that can lead to serious human illness through inhalation and consumption due to environmental pollution. Nitrilases can highly degrade nitriles isolated from the natural ecosystem. In the current study, we focused on the discovery of novel nitrilases from a coal metagenome using in silico mining. Methods: Coal metagenomic DNA was isolated and sequenced on the Illumina platform. Quality reads were assembled using MEGAHIT, and statistics were checked using QUAST. Annotation was performed using the automated tool SqueezeMeta. The annotated amino acid sequences were mined for nitrilase from the unclassified organism. Sequence alignment and phylogenetic analyses were carried out using ClustalW and MEGA11. Conserved regions of the amino acid sequences were identified using InterProScan and NCBI-CDD servers. The physicochemical properties of the amino acids were measured using ExPASy's ProtParam. Furthermore, NetSurfP was used for 2D structure prediction, while AlphaFold2 in Chimera X 1.4 was used for 3D structure prediction. To check the solvation of the predicted protein, a dynamic simulation was conducted on the WebGRO server. Ligands were extracted from the Protein Data Bank (PDB) for molecular docking upon active site prediction using the CASTp server. Results and discussion: In silico mining of annotated metagenomic data revealed nitrilase from unclassified Alphaproteobacteria. By using the artificial intelligence program AlphaFold2, the 3D structure was predicted with a per-residue confidence statistic score of about 95.8%, and the stability of the predicted model was verified with molecular dynamics for a 100-ns simulation. Molecular docking analysis determined the binding affinity of a novel nitrilase with nitriles. The binding scores produced by the novel nitrilase were approximately similar to those of the other prokaryotic nitrilase crystal structures, with a deviation of ±0.5.

A Proterozoic microbial origin of extant cyanide-hydrolyzing enzyme diversity.

In addition to its role as a toxic environmental contaminant, cyanide has been hypothesized to play a key role in prebiotic chemistry and early biogeochemical evolution. While cyanide-hydrolyzing enzymes have been studied and engineered for bioremediation, the extant diversity of these enzymes remains underexplored. Additionally, the age and evolution of microbial cyanide metabolisms is poorly constrained. Here we provide comprehensive phylogenetic and molecular clock analyses of the distribution and evolution of the Class I nitrilases, thiocyanate hydrolases, and nitrile hydratases. Molecular clock analyses indicate that bacterial cyanide-reducing nitrilases were present by the Paleo- to Mesoproterozoic, and were subsequently horizontally transferred into eukaryotes. These results present a broad diversity of microbial enzymes that could be optimized for cyanide bioremediation.

Biotransformation of 3-cyanopyridine to nicotinic acid using whole-cell nitrilase of Gordonia terrae mutant MN12.

In the present study, the Gordonia terrae was subjected to chemical mutagenesis using ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 5-bromouracil (5-BU) and hydroxylamine with the aim of improving the catalytic efficiency of its nitrilase for conversion of 3-cyanopyridine to nicotinic acid. A mutant MN12 generated with MNNG exhibited increase in nitrilase activity from 0.5 U/mg dcw (dry cell weight) (in the wild G. terrae) to 1.33 U/mg dcw. Further optimizations of culture conditions using response surface methodology enhanced the enzyme production to 1.2-fold. Whole-cell catalysis was adopted for bench-scale synthesis of nicotinic acid, and 100% conversion of 100 mM 3-cyanopyridine was achieved in potassium phosphate buffer (0.1 M, pH 8.0) at 40 °C in 15 min. The whole-cell nitrilase of the mutant MN12 exhibited higher rate of product formation and volumetric productivity, i.e., 24.56 g/h/g dcw and 221 g/L as compared to 8.95 g/h/g dcw and 196.8 g/L of the wild G. terrae. The recovered product was confirmed by HPLC, FTIR and NMR analysis with high purity (> 99.9%). These results indicated that the mutant MN12 of G. terrae as whole-cell nitrilase is a very promising biocatalyst for the large-scale synthesis of nicotinic acid.

Green synthesis aspects of (R)-(-)-mandelic acid; a potent pharmaceutically active agent and its future prospects.

(R)-(-)-mandelic acid is an important carboxylic acid known for its numerous potential applications in the pharmaceutical industry as it is an ideal starting material for the synthesis of antibiotics, antiobesity drugs and antitumor agents. In past few decades, the synthesis of (R)-(-)-mandelic acid has been undertaken mainly through the chemical route. However, chemical synthesis of optically pure (R)-(-)-mandelic acid is difficult to achieve at an industrial scale. Therefore, its microbe mediated production has gained considerable attention as it exhibits many merits over the chemical approaches. The present review focuses on various biotechnological strategies for the production of (R)-(-)-mandelic acid through microbial biotransformation and enzymatic catalysis; in particular, an analysis and comparison of the synthetic methods and different enzymes. The wild type as well as recombinant microbial strains for the production of (R)-(-)-mandelic acid have been elucidated. In addition, different microbial strategies used for maximum bioconversion of mandelonitrile into (R)-(-)-mandelic acid are discussed in detail with regard to higher substrate tolerance and maximum bioconversion.HighlightsMandelonitrile, mandelamide and o-chloromandelonitrile can be used as substrates to produce (R)-(-)-mandelic acid by enzymes.Three enzymes (nitrilase, nitrile hydratase and amidase) are systematically introduced for production of (R)-(-)-mandelic acid.Microbial transformation is able to produce optically pure (R)-(-)-mandelic acid with 100% productive yield.

An improved process for synthesis of nicotinic acid using hyper induced whole cell nitrilase of Gordonia terrae MTCC8139.

Nitrilase mediated synthesis of nicotinic acid (vitamin B3) from 3-cyanopyridine has emerged as promising viable alternative to its chemical synthesis. In the present investigation, the nitrilase production in Gordonia terrae MTCC8139 has been increased by two fold with inducer feeding approach [i.e. the addition of 0.5% (v/v) isobutyronitrile as inducer at 0, 16 and 24 h of incubation of the culture]. The use of hyper induced whole cell nitrilase of G. terrae as biocatalyst (10 U per ml reaction) to synthesize nicotinic acid from 3-cyanopyridine in a fed batch reaction at one litre scale resulted in accumulation of 1.65 M (202 g) nicotinic acid in 330 min. The catalytic productivity of hyper induced whole cell nitrilase was increased from 8.95 to 15.3 g/h/g dcw and the reaction time was reduced to half. This is the highest productivity of nicotinic acid in a nitrilase mediated process so far reported. The achievements of the present investigation will lead to mitigate the cost of nitrilase vis-a-vis nicotinic acid production at large scale.

Screening and characterization of a nitrilase with significant nitrile hydratase activity.

PURPOSE: We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. METHODS: A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases. RESULTS: Based on the previously reported "amide-formation hotspots," we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. CONCLUSIONS: NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.

Preparation, characterization and stability of cross linked nitrilase aggregates (nitrilase-CLEAs) for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid.

Nitrilases capable of performing hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid were screened, and ES-NIT-102 was the best nitrilase for said biotransformation. Nitrilase was immobilized as cross linked enzyme aggregates (nitrilase-CLEAs) by fractional precipitation with iso-propanol, and cross linked with glutaraldehyde. The nitrilase-CLEAs prepared with optimized 35 mM glutaraldehyde for 120 min cross linking time had 82.36 ± 4.45% residual activity, and displayed type-II structural CLEAs formation as confirmed by particle size, SEM, FTIR, and SDS-PAGE analysis. Nitrilase-CLEAs had superior pH and temperature stability, showed a shift in optimal temperature by 5 °C, and retained nearly 1.5 to 1.7 folds activity over free nitrilase at 50 °C and 55 °C after more than 9 h incubation. Nitrilase-CLEAs showed reduced affinity and decreased conversion of substrate as indicated by slightly higher Km values by 5.19% and reduced Vmax by 17%. Furthermore, these nitrilase-CLEAs showed 98% conversion, 94.72 g/L product formation, and 83.30% recovery after 24 h when used for hydroxylation of 2-chloroisonicotinonitrile to 2-chloroisonicotinic acid. Nitrilase-CLEAs were catalytically active for 3 cycles showcasing 81% conversion, 75.53 g/L product formation and 66.42% yield. The recovered product was confirmed by HPLC, FTIR, LC-MS, and 1H NMR, and displayed > 99% purity.

Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3  to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.