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Tuesday, 27 September 2016

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

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 (Km) 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.


Thursday, 1 September 2016

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.

Friday, 5 August 2016

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.

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

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


 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.

Wednesday, 3 August 2016

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.