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.

 

ChromSoc nitrilase flow chemistry project 5

We have looked at the conversion of 4-cyanopyridine to isonicotinic acid using our mesoscale flow chemistry apparatus containing a nitrilase enzyme immobilized in alginate. Here is an example of our results.

Rob flowed the reaction medium through the device, and took a measurement of the amount of ammonia (using our colorimetric nitrilase assay) available after each cycle. Each cycle took about 20 minutes. It has not reached completion but seems to be a fairly robust system as far as it goes.

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−.

High concentration synthesis of 3-hydroxypropionic acid

Enzymatic synthesis of 3-hydroxypropionic acid at high productivity by using free or immobilized cells of recombinant Escherichia coli

 Shanshan Yua, Peiyuan Yao, Jianjiong Li, Jie Ren, Jing Yuan, Jinhui Feng, Min Wang, Qiaqing Wu,  Dunming Zhu,

Journal of Molecular Catalysis B: Enzymatic, Volume 129, July 2016, Pages 37-42

doi:10.1016/j.molcatb.2016.03.011

3-Hydroxypropionic acid (3-HP) is an important platform chemical for organic synthesis and high performance polymers. This paper describes an effective enzymatic method for the synthesis of 3-HP was achieved by using free or immobilized recombinant Escherichia coli BL21(DE3) cells harboring a nitrilase gene from environmental sample (NIT190). Under the optimal conditions (100 mmol/L Tris-HCl buffer, pH 8.0, 30 °C), the maximum substrate concentration which led to 100% hydrolysis by using free cells within 24 h was 4.5 mol/L (319.5 g/L). Furthermore, immobilization of the whole cells enhanced their substrate tolerance (up to 7.0 mol/L), stability, and reusability. The immobilized cells could be reused for up to 30 batches, and 70% of enzyme activity was retained after 74 batches in distilled water. A productivity (36.9 g/(L h)) was obtained after isolation and purification of 3-HP from the first 30 batches.

Figure shows free cell substrate tolerance (a) compared to three immobilized cell methods (b-d).

Simultaneous KRED and NHase/amidase activity

Developing a Biocascade Process: Concurrent Ketone Reduction-Nitrile Hydrolysis of 2-Oxocycloalkanecarbonitriles

Elisa Liardo, Nicolás Ríos-Lombardía, Francisco Morís, Javier González-Sabín, and Francisca Rebolledo

Org. Lett., 2016, 18 (14), pp 3366–3369

DOI: 10.1021/acs.orglett.6b01510

 

 A stereoselective bioreduction of 2-oxocycloalkanecarbonitriles was concurrently coupled to a whole cell-catalyzed nitrile hydrolysis in one-pot. The first step, mediated by ketoreductases, involved a dynamic reductive kinetic resolution, which led to 2-hydroxycycloalkanenitriles in very high enantio- and diastereomeric ratios. Then, the simultaneous exposure to nitrile hydratase and amidase from whole cells of Rhodococcus rhodochrous provided the corresponding 2-hydroxycycloalkanecarboxylic acids with excellent overall yield and optical purity for the all-enzymatic cascade.

ChromSoc nitrilase flow chemistry project 4

Once you have the track filled with immobilized enzyme and the two halves stuck together, you just need to condition it and check there are no leaks.

Then it is just a case of getting the reaction going using a water bath to get the appropriate temperature. We tend to set it up so that we have a separate starting and receiving flask so that we can track aliquots through the enzyme bed, but you can just the two pipe operating out of /into the same flask obviously.

ChromSoc nitrilase flow chemistry project 3

Rob has shown made a supply of  the plates that go together to make a flow cell for flow biocatalysis. The 3D printed master copy (the one with the wall around the shape) has provided another silicone mould which has then be used to make polyurethane casts.

They can then be stuck together with the track filled with immobilized enzymes. Alongside these reactions we are also running comparable batch reactions in glassware to see how they compare.

ChromSoc nitrilase flow chemistry project 2

We have a range of nitrilases which we use as starting points for all our projects. Most of them are listed in this ChemComm. We've used them both as cell free extract and with many different types of immobilization. A good place to start we have found is in simple alginate beads which are easy to make, and give a consistent performance under our standard reaction conditions. Their synthesis using a powered syringe dropping into a stirred beaker has a somewhat hypnotic quality.

ChromSoc nitrilase flow chemistry project 1

I have an undergraduate student, Rob, working with us this summer on nitrilase reactions. He is kindly sponsored by the Chromatographic Society to work on a project using HPLC and GC to compare batch processes run on immobilized enzyme in a flask with those run on the same enzyme preparation using our in-house mesoscale flow reactor system. The flow reactor system is something we have been working on for a while and its genesis was part of a project based around using 3D printing to make bespoke laboratory equipment which is described in the Tumblr blog here, with a video of the system in operation doing a nitro reduction here. My intention is to relate here a real time log of progress with this project.

Two copies of the track fit together, and the solution

is flowed through. The track contains enzyme immobilized

on beads.

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

Creating bioactive molecules from online data

From a genome sequence to that protein in your hand is a well –established process

Until I started collaborating closely with biologists ten years ago, my impression of how easy it was to get a sample of a complex biomolecule like a protein was based on popular science stories describing days/months of tedious purification of buckets of biomass (sea sponges, exotic fungi, cow bile, etc) to end up with a single vial containing so little it was invisible to the naked eye.

Follow the link to see the schematic explained.

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.

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.