Formation of Nitrile Hydratase CLEAs in Mesoporous Silica

Formation of Nitrile Hydratase Cross-Linked Enzyme Aggregates in Mesoporous Onion-like Silica: Preparation and Catalytic Properties

Jing Gao, Qi Wang, Yanjun Jiang*, Junkai Gao, Zhihua Liu, Liya Zhou, and Yufei Zhang,

Ind. Eng. Chem. Res., 2015, 54 (1), pp 83–90

Nitrile hydratase CLEAs) were formed in mesoporous onion-like silica (NHase-CLEAs@MOS) by using macromolecular dextran polyaldehyde as a cross-linker through the carrier-bound CLEAs method. Effect of pH, thermal and storage stability, and kinetic parameters of NHase-CLEAs@MOS were also studied. The maximum amount of NHase absorbed in MOS was 535 mg/g. Under optimized conditions, the maximum activity recovery of NHase-CLEAs@MOS was 48.2%. The stabilities of NHase-CLEAs@MOS were improved significantly compared to the NHase@MOS prepared by physical adsorption and free NHase.

Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids using a hydroxynitrile lyase, nitrile-active enzymes and an amidase

This review (Journal of Molecular Catalysis B: Enzymatic, 114, 2015, 25–30) by van Rantwijk and Stolz covers the bienzymatic conversion of aldehydes into enantiomerically pure hydroxycarboxylic acids and amides via an enzymatic cascade of hydrocyanation and nitrile hydration/hydrolysis. It compares results obtained via cross-linked enzyme aggregates (CLEAs) as well as whole-cell Escherichia coli expressing two enzymes. It highlights these methods’ potential for yielding near-quantitative yield and ee at synthetically relevant concentrations.

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.