Sequence alignment of Nit6803 from Syechocystis sp. PCC6803 with other nitrilases

Secondary structure elements are drawn on the basis of structures of Nit6803 and shown at the top of the aligned sequences. b-Sheets are shown as arrows in yellow, whereas a-helices are shown as bars in red. Residues involved in enzymatic catalysis are indicated are highlighted in red rectangle, whereas the proposed key residue involved in substrate preference is highlighted in blue rectangle. Nit6803, PaNit, PH0642, DNCAase and RrNit indicate Syechocystis sp. PCC6803 nitrilase (GI: 16331918), hyperthermophilic nitrilase (GI: 14521598), Pyrococcus horikoshii hypothetical protein (GI: 14590532), N-carbamoyl-D-amino-acid amidohydrolase (GI: 34921541) and Rhodococcus rhodochrous ATCC 33278 nitrilase (GI: 417384).

 from this paper by Yuan, Wei and co-workers.

NCBI Sequence numbers for nitrile hydratase and nitrilase to 27/2.16

Looking at the bare search term "nitrile hydratase" amongst protein sequences (and remember, most aren’t but it’s a rough measure), today gives me 10224 hits, of which 4117 were RefSeq data. 

There are 80688 sequences labelled as "nitrilase" (not sure how robust that is currently), of which 20872 are pegged as RefSeq data.

A crystal structure of nitrilase Nit6803 from Syechocystis sp. PCC6803

PDB: 3WUY_A

>gi|742261201|pdb|3WUY|A Chain A, Crystal Structure Of Nit6803
GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP
PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT
PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME
VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL
DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH

PDB: 3WUY_B

>gi|742261202|pdb|3WUY|B Chain B, Crystal Structure Of Nit6803
GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP
PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT
PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME
VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL
DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH

A new thermophilic nitrilase from Pyrococcus sp. M24D13

A new thermophilic nitrilase from an Antarctic hyperthermophilic microorganism by Geraldine V. Dennett and Jenny M. Blamey

Front. Bioeng. Biotechnol.  doi: 10.3389/fbioe.2016.00005

Several environmental samples from Antarctica were collected and enriched to search for microorganisms with nitrilase activity. A new thermostable nitrilase from a novel hyperthermophilic archaea Pyrococcus sp. M24D13 was purified and characterized. The activity of this enzyme increased as the temperatures rise from 70 up to 85 °C. Its optimal activity occurred at 85 °C and pH 7.5. This new enzyme shows a remarkable resistance to thermal inactivation retaining more than 50% of its activity even after 8 h of incubation at 85 °C. In addition, this nitrilase is highly versatile demonstrating activity towards different substrates such as benzonitrile (60 mM, aromatic nitrile) and butyronitrile (60 mM, aliphatic nitrile). Moreover the enzyme NitM24D13 also presents cyanidase activity.

Mutagenesis of a fungal nitrilase from Gibberella intermedia for improved rate and different acid/amide

Engineering of a fungal nitrilase for improving catalytic activity and reducing by-product formation in the absence of structural information from Jin-Song Gong,   Heng Li,   Zhen-Ming Lu,   Xiao-Juan Zhang,   Qiang Zhang,   Jiang-Hong Yu,   Zhe-Min Zhou,   Jin-Song Shi and Zheng-Hong Xu

Catal. Sci. Technol., 2016, DOI: 10.1039/C5CY01535A

This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermedia was performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.

Immobilization of nitrilase for synthesis of 2-hydroxy-4-(methylthio) butanoic acid

Immobilization of nitrilase on bioinspired silica for efficient synthesis of 2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio) butanenitrile  from Li-Qun Jin, Dong-Jing Guo, Zong-Tong Li, Zhi-Qiang Liu, Yu-Guo Zheng

Journal of Industrial Microbiology & Biotechnology, DOI 10.1007/s10295-016-1747-5

This paper describes a simple and effective method to immobilize recombinant nitrilase, for efficient production of 2-hydroxy-4-(methylthio) butanoic acid from 2-hydroxy-4-(methylthio) butanenitrile. The immobilized enzyme displayed better thermal stability, pH stability and shelf life compared to free nitrilase. Moreover, it showed excellent reusability and could be recycled up to 16 batches without significant loss in activity. 200 mM 2-hydroxy-4-(methylthio) butanenitrile was completely converted by the immobilized enzyme within 30 min, and the accumulation amount of 2-hydroxy-4-(methylthio) butanoic acid reached 130 mmol/g of immobilized beads after 16 batches.

Characterization of the NHase from Ensifer meliloti CGMCC 7333

Characterization of a versatile nitrile hydratase of the neonicotinoid thiacloprid-degrading bacterium Ensifer meliloti CGMCC 7333

Shi-Lei Sun, Tian-Qi Lu, Wen-Long Yang, Jing-Jing Guo, Xue Rui, Shi-Yun Mao, Ling-Yan Zhou and Yi-Jun Dai - RSC Advances, 2016

The nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 and its nitrile hydratase (NHase) degrade the neonicotinoid insecticides, thiacloprid (THI) and acetamiprid (ACE), to their corresponding amide metabolites. The NHase gene cluster is composed of α-subunit and β-subunit genes and a hypothetical protein gene. The functionality of the hypothetical protein downstream of the NHase coding genes and the characteristics of CGMCC 7333 NHase were explored in this study. Co-expression of the hypothetical protein coding gene with NHase (α- and β-subunit genes) in Escherichia coli Rosetta enhanced NHase hydration of THI and ACE two- and four-fold, respectively, and also significantly improved NHase solubility compared with the absence of the hypothetical protein coding gene. The NHase displayed an optimal reaction temperature of 50 °C for THI hydration and was unstable when the incubation temperature exceeded 40 °C. The optimum reaction pH was 7.0 and the NHase activity was stable in the pH range of 6 to 9. The enzyme activity for THI hydration was slightly inhibited by copper, zinc, and iron, and decreased by 68.6%, 75.7%, and 70.3% when 2% ethanol, ethyl acetate, and acetone were added to the reaction mixture, respectively, whereas dichloromethane and trichloromethane had no effect. The K_m and k_cat values of CGMCC 7333 NHase for THI hydration were 12.39 mmol L⁻¹ and 131.36 s⁻¹, respectively. Substrate specificity analysis indicated that CGMCC 7333 NHase also transformed 3-cyanopyridine, benzonitrile, and indole-3-acetonitrile to the corresponding amide products, with maximum specific activities of 652.52, 255.32, and 263.93 U mg⁻¹ protein, respectively.

Hydration of a N-cyanoimine group to a N-carbamoylimine by NHase

Degradation of the Neonicotinoid Insecticide Acetamiprid via the N-Carbamoylimine Derivate (IM-1-2) Mediated by the Nitrile Hydratase of the Nitrogen-Fixing …

LY Zhou, LJ Zhang, SL Sun, F Ge, SY Mao, Y Ma, ZH Liu, YJ Dai, and S Yuan J. Agric. Food Chem., 2014, 62 (41), pp 9957–9964

The metabolism of the widely used neonicotinoid insecticide acetamiprid (ACE) has been extensively studied in plants, animals, soils, and microbes. However, hydration of the N-cyanoimine group in ACE to the N-carbamoylimine derivate (IM-1-2) by purified microbes, the enzyme responsible for this biotransformation, and further degradation of IM-1-2 have not been studied. The present study used liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy to determine that the nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 transforms ACE to IM-1-2. CGMCC 7333 cells degraded 65.1% of ACE in 96 h, with a half-life of 2.6 days. Escherichia coli Rosetta (DE3) overexpressing the nitrile hydratase (NHase) from CGMCC 7333 and purified NHase converted ACE to IM-1-2 with degradation ratios of 97.1% in 100 min and 93.9% in 120 min, respectively. Interestingly, IM-1-2 was not further degraded by CGMCC 7333, whereas it was spontaneously hydrolyzed at the N-carbamoylimine group to the derivate ACE-NH, which was further converted to the derivative ACE-NH2. Then, ACE-NH2 was cleaved to the major metabolite IM-1-4. IM-1-2 showed significantly lower insecticidal activity than ACE against the aphid Aphis craccivora Koch. The present findings will improve the understanding of the environmental fate of ACE and the corresponding enzymatic mechanisms of degradation.

Surface modification of polyacrylonitrile fibre by NHase

Surface Modification of Polyacrylonitrile Fibre by Nitrile Hydratase from Corynebacterium nitrilophilus

S Chen, H Gao, J Chen, J Wu - Applied biochemistry and biotechnology, 2014

Previously, nitrile hydratase (NHase) from Corynebacterium nitrilophilus was obtained and showed potential in polyacrylonitrile (PAN) fibre modification. In the present study, the modification conditions of C. nitrilophilus NHase on PAN were investigated. In the optimal conditions, the wettability and dyeability (anionic and reactive dyes) of PAN treated by C. nitrilophilus NHase reached a similar level of those treated by alkali. In addition, the chemical composition and microscopically observable were changed in the PAN surface after NHase treatment. Meanwhile, it revealed that cutinase combined with NHase facilitates the PAN hydrolysis slightly because of the ester existed in PAN as co-monomer was hydrolyzed. All these results demonstrated that C. nitrilophilus NHase can modify PAN efficiently without textile structure damage, and this study provides a foundation for the further application of C. nitrilophilus NHase in PAN modification industry.

NHase patent on stabilizing nitrile active enzymes in cells

Methods for preserving and/or storing cells having a nitrilase or nitrile hydratase activity

T Zelinski, M Keβeler, B Hauer - US Patent 8,815,569, 2014

The invention from BASF relates to a method for preserving and/or storing microorganisms which exhibit at least one nitrile hydratase or nitrilase enzyme activity, with the preservation and/or storage being effected in an aqueous medium which comprises at least one aldehyde, with the total aldehyde concentration being in a range from 0.1 to 100 mM/l.

Book chapter on sulfur oxygenation and functional models of NHase

Chapter 12 in the book “Bioinspired Catalysis”

Authors: Davinder Kumar and Craig A. Grapperhaus

This chapter highlights selected complexes with tetra- and penta-dentate chelates that provide key insights into the oxidized sulfur environment at the enzyme active site. Small-molecule mimics with variable S-oxidation levels provide an attractive method to address these interactions. It discusses a brief history of metal-thiolate sulfur-oxygenation is provided, followed by selected S-oxygenation studies relevant to nitrile hydratase (NHase). The NHase are divided by metal type and organized according to the donor atoms of the chelates. Several ruthenium catalysts have been reported as nitrile hydration catalysts. Ruthenium is also a logical choice for oxidation studies as the second-row transition metal maintains a consistent low spin, which was found to promote S-oxygenation.

A switch in a substrate tunnel for directing regioselectivity of nitrile hydratases towards α,ω-dinitriles

A switch in a substrate tunnel for directing regioselectivity of nitrile hydratases towards α,ω-dinitriles

Zhongyi Cheng, Wenjing Cui, Zhongmei Liu, Li Zhou, Min Wang, Michihiko Kobayashi and Zhemin Zhou 

The β37 residue of nitrile hydratase (NHase) from Pseudomonas putida and NHase from Comamonas testosteroni played a critical role in directing enzyme regioselectivity. Amino acid substitution in this site modulated or even inverted enzyme regioselectivity towards aliphatic α,ω-dinitriles.

Cartoon model of the substrate access tunnel of (a) wild-type PpNHase and its (b) L37F and (c) L37Y variants, and (d) wild-type CtNHase and its (e) F37L and (f) F37P variants. The protein structures of PpNHase and CtNHase are shown as the grey cartoon. The β37 residues of NHases are shown as blue sticks. The purple balls and sticks represent the catalytic site of NHase. The bottleneck-forming amino acids are shown as red sticks. The tunnels are shown as green spheres, and the tunnel bottlenecks are coloured in yellow. All the figures share the same size proportion.