Preparation of CLEAs of a recombinant NHase ES-NHT-118

Preparation of Cross-linked Enzyme Aggregates of Nitrile Hydratase ES-NHT-118 from E. coli by Macromolecular Cross-linking Agent

Liya Zhou, Haixia Mou, Jing Gao, Li Ma, Ying He and Yanjun Jiang

Chinese Journal of Chemical Engineering (in press)

http://dx.doi.org/10.1016/j.cjche.2016.08.027

Cross-linked enzyme aggregates (CLEAs) of nitrile hydratase (NHase) ES-NHT-118 from E. coli were prepared by using ammonium sulfate as precipitating agent followed by cross-linking with dextran polyaldehyde for the first time. In this process, egg white was added as an amine source to aid formation of CLEAs. The optimal conditions of the immobilization process were determined. Michaelis constants (K_m) of free NHase and NHase CLEAs were also determined. The NHase CLEAs exhibited increased stability at varied pH and temperature conditions compared to its free counterpart. When exposed to high concentrations of acrylamide, NHase CLEAs also exhibited effective catalytic activity.

 

The cysteinesulfenic Acid in NHase as catalytic nucleophile

Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile.

Yamanaka, Y., Kato, Y., Hashimoto, K., Iida, K., Nagasawa, K., Nakayama, H., Dohmae, N., Noguchi, K., Noguchi, T., Yohda, M. and Odaka, M.

Angew. Chem. Int. Ed., (2015), 54: 10763–10767.

doi:10.1002/anie.201502731

The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2–1.3 Å). In combination with FTIR analyses of NHase following hydration in H218O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO−) atom of αCys114-SO−, followed by nucleophilic attack of the S(SO−) atom by a βArg56-activated water molecule to release the product amide and regenerate αCys114-SO−.

Active Site investigations on the Fe-centred NHase from Comamonas testosteroni Ni1

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu , Richard C. Holz

JBIC Journal of Biological Inorganic Chemistry (2015), 20/5, 885-894

A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k _cat = 10 ± 2 s⁻¹) accounts for less than 1 % of the wild-type activity (k _cat = 1100 ± 30 s⁻¹) while the K _m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k _cat values of 220 ± 40 and 77 ± 13 s⁻¹, respectively, and K _m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k _cat value of 132 ± 3 s⁻¹ and a K _m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys¹⁰⁴-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

3D content at http://proteopedia.org/w/Journal:JBIC:32

BASF opens new bio-acrylamide plant in Bradford, UK

From

http://www.worldofchemicals.com/media/basf-opens-new-bio-acrylamide-plant-in-bradford-uk/9710.html

“BRADFORD, UK: BASF said it has opened a new world-scale bio-acrylamide (BioACM) production facility at its site in Bradford, a major investment that will help ensure long-term future of one of the UK’s largest chemical manufacturing facilities, which employs around 600 people.”

“The development of a biocatalytic manufacturing process for acrylamide started in Bradford with collaboration with Huddersfield University. Subsequent work by scientists from Britain, Germany, South Africa and US, made significant improvements to the performance of the biocatalyzed conversion technology.”

Image from BASF.com

A poster on Production of 2,6-difluorobenzamide using the NHase from Aurantimonas manganoxydans

Production of 2, 6- difluorobenzamide via whole-cell biocatalysis by nitrile hydratase from Aurantimonas manganoxydans

Lirong Yang

AGM Society for Industrial Microbiology & Biotechnology

Nitrile hydratases (NHases) are enzymes which catalyze the hydration of nitriles, converting them into their corresponding amides. Amides are an important intermediates for pharmaceutical and pesticide industry. For example, 2, 6 – difluorobenzamide is used for the synthesis of fluorinated benzoyl urea pesticide.

Four NHase genes from Aurantimonas manganoxydans ATCC BAA-1229, Klebsiella oxytoca KCTC 1686, Pseudomonas putida NRRL-18668, Comamonas testosteroni 5-MGAM-4D were cloned and functionally expressed in Escherichia coli BL21 (DE3). All of the recombinant NHases can catalyze the hydration of 2, 6-difluorobenzonitrile to produce 2, 6-difluorobenzamide. Among them, the NHase from Aurantimonas manganoxydans ATCC BAA-1229 showed the highest activity.

Assisting soluble expression of recombinant and active Co-centred NHase

Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature

Xiaolin Pei, Qiuyan Wang, Lijun Meng, Jing Li, Zhengfen Yang, Xiaopu Yin, Lirong Yang, Shaoyun Chen and Jianping Wu

Journal of Biotechnology, Volume 203, 10 June 2015, Pages 9–16

doi:10.1016/j.jbiotec.2015.03.004

Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase from Aurantimonas manganoxydans has been functionally expressed in E. coli, and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.

NHase hydration of alicyclic 𝜶,𝝎-dinitrile 1-cyanocyclohexaneacetonitrile to 1-cyanocyclohexaneacetamide

Highly regioselective and efficient production of 1-cyanocyclohexaneacetamide by Rhodococcus aetherivorans ZJB1208 nitrile hydratase

Ren-Chao Zheng, Xin-Jian Yina and Yu-Guo Zheng

J Chem Technol Biotechnol 2016; 91: 1314–1319

DOI 10.1002/jctb.4724

A newly isolated NHase producing strain, Rhodococcus aetherivorans ZJB1208, was successfully used for hydration of1-CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed-batch biotransformation was performed in non-buffered water system with the continuous precipitation of 1-cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L⁻¹ (6.0 mol L⁻¹), giving a product concentration of 966.7 g L⁻¹ and biocatalyst yield (g product/g cat) of 204.2.

Clues towards the role of amide carbonyl groups in the active site of a cobalt centred NHase

Role of the Amide Carbonyl Groups in the Nitrile Hydratase Active Site for Nitrile Coordination Using Co(III) Complex with N2S3-type Ligand

Takuma Yano,  Tomohiro Ikeda, Tomonori Shibayama,  Tomohiko Inomata, Yasuhiro Funahashi,2 Tomohiro Ozawa,* and Hideki Masuda*

Chemistry Letters Vol. 44 (2015) No. 6 P 761-763

The role of the amide carbonyl oxygens in the nitrile hydratase (NHase) active site for the nitrile coordination to the metal center was studied using a distorted square-pyramidal N2S3-type Co(III) complex at room temperature in the presence of a folded-sheet mesoporous material (FSM) or sodium cation.

A proposed catalytic mechanism for NHase via a cyclic intermediate assessed by QM/MM

Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study

Megumi Kayanuma, Mitsuo Shoji, Masafumi Yohda, Masafumi Odaka, and Yasuteru Shigeta

J. Phys. Chem. B, Article ASAP

DOI: 10.1021/acs.jpcb.5b11363

The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from βTyr72 to the substrate via αSer113, and cleavage of the S–O bond of αCys114–SO^– and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of βArg56K, which is one of the essential amino residues in the enzyme, we analyzed a βR56K mutant in which βArg56 was replaced by Lys. The results suggest that βArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S–O bond via a hydrogen bond with the oxygen atom of αCys114–SO^–.

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.

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.

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.

PVA-chitosan based immobilization of NHase for dynamic kinetic resolution of rac-mandelonitrile

A recently published paper by Pawar and Yadav in Industrial and Engineering Chemistry Research entitled “Enantioselective Enzymatic Hydrolysis ofrac-Mandelonitrile to R-Mandelamide by Nitrile Hydratase Immobilized onPoly(vinyl alcohol)/Chitosan–Glutaraldehyde Support” describes the improvement in performance the nitrile hydratase from Rhodococcus rhodocrous ATCC BAA-870 exhibits when immobilized on PVA/chitosan. The reaction being tested is the DKR of rac-mandelonitrile.

A mechanism model for NHase using a cyclic and a disulfide intermediate.

There is a very interesting paper proposing a new mechanism for the hydration reaction of iron-centred NHases published in Inorganic Chemistry by Kathrin Hopmann. The paper, entitled "Full Reaction Mechanism of Nitrile Hydratase: A Cyclic Intermediate and an Unexpected Disulfide Switch" sides with the opinion that the nitrile ligates to the iron centre. Hopmann suggests that the oxygen of the cysteine post-translationally modified to a sulfenic acid then acts as the nucleophile attached the C-N bond.

 

This oxygen is the one that becomes the oxygen in the nascent amide carbonyl, with the two cysteine sulfurs combining to form a disulfide link.

After loss of the amide from the iron centre, the cysteine is oxidized again using an oxygen from water. My suspicion with all these mechanistic investigation is that it may be that a lot of different mechanisms may operate with the dominant one being very tied to the specific sequence/space properties of each enzyme. I have been a fan of nitrile-bound mechanisms solely on the basis that they seem much more likely to render chiral selectivity which we know is a possibility for some enzymes and some substrates.

I also wonder how the numbers from the calculations reported in this paper change for cobalt centre NHases.

A nitrile hydratase for cyanopyridines... and it's a bit more stable than usual.

There is a paper in Process Biochemistry which describes a new NHase from Aurantimonas manganoxydans which shows improved stability than you can normally expect from a NHase. It is entitled “Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy”, and it is by Xiaolin Peia, Hongyu Zhang, Lijun Meng, Gang Xu, Lirong Yang and Jianping Wu. This NHase is four times more rapid at converting 3-cyanopyridine to its corresponding amide as valeronitrile, and the authors emphasize their enzyme's stability though in the world of NHases where nothing is what you might describe as thermophilic, please don't get too expectant! They have a great table of NHase thermostability which I reproduce with their enzyme's data inserted.

 

A nitrile hydratase for cyanopyridines... (but it prefers aliphatic nitriles)

There has been a recent paper in Journal of Molecular Catalysis B on a nitrilase that converted cyanopyridines. This enzyme came from a strain of Pseudomonas putida. There is also a recent paper in the same journal entitled “Discovery of a new Fe-type nitrile hydratase efficiently hydrating aliphatic and aromatic nitriles by genome mining” by Xiaolin Peia, Lirong Yang, Gang Xu, Qiuyan Wang and Jianping Wu.which describes a nitrile hydratase, this time, from a Pseudomonas putida strain (F1) which they have shown to be able to turn over 3-cyanopyridine in a 1L fed batch reactor. Their activity data suggests it actually prefers aliphatic nitriles: acrylonitrile rates as 941 U/mg and valeronitrile as 535 U/mg as compared to 3-cyanopyridine at 26 U/mg. They describe how it was cloned into E. coli, needing the inclusion of an activator protein to get activity. They also include a nice phylogenetic tree showing the spread of known iron type NHases... plenty of examples in the Rhodococcus but spreading outwards into Pseudomonas.

But which is more popular nitrilase or nitrile hydratase?

Google have a rather entertaining Google Labs product called the NGram viewer where they allow you to check the ranking of the number of times that books published in a specific year use words which you define. Their examples are like "nursery school" versus "kindergarten". I thought it might be rather more relevant for this blog to try "nitrilase" against "nitrile hydratase".

NGram viewer- nitrilase v nitrile hydratase

Basically nitrilase has been more popular apart from a burst of enthusiasm for NHase between 1988 and 1994, and a photofinish victory in 2003.