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.

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.

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.

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.

Nitrilase and nitrile hydratase from Pseudomonas sp. UW4

Having just finished a project where we looked at a range of nitrilases and what their preferred substrates are, it is always interesting to ponder what the bacterium actually wanted the enzyme for (as compared to the host of xenobiotics you threw at it). We have often had the situation where we have an enzyme which we reckon ought to be active but doesn't seem interested in any of the forty or so compounds we have in our simple screen.
There is a recent paper in Applied and Environmental Microbiology by Duca, Rose and Glick which is concerned with investigating the biosynthesis of indoleacetic acid (IAA), which is a plant growth hormone. This compound comes from indoleacetonitrile (IAN) and there are two obvious pathways to get from there to IAA- via the NHase and via a nitrilase. These workers cloned both enzymes in to E. coli, and then looked at their level of interest in IAN. Interestingly the nitrilase had a habit of producing a proportion of amide as well as the usual acid. Also of interest is that the enzymes have different pH and temperature optima (Nase like 50 degrees C and pH6, the iron-centred NHase likes 4 degrees C and pH7.5), though I wonder if the lower temperature for the NHase is due to the fundamental lack of stability of iron NHases rather than an adaption. Additionally, the authors use some bioinformatics to confirm their experimental findings that this is an aromatic nitrile active system.

Nitrilase from sequence to 50 litre scale

We have been working on developing a specific nitrilase reaction in a consortium with Chemoxy and Biocatalysts Ltd with funding assistance from the UK Technology Strategy Board. In nine months, we have gone from the selection of amino acid sequences to expressed active proteins to assessment of substrate preference using a novel greener assay method which works with cell-free extracts to chemistry on the one litre scale, and finally at the end of last month to doing the reaction in the 50 litre plant at CPI's National Industrial Biotechnology Facility. Just to finish the boasting, the outcome of that reaction was over a kilo of product with great conversion.

We are now going into a TSB-funded collaborative research and development project with the same partners to really scale this process and bring it nearer to market by optimizing the enzyme, its use and reuse. It's going to be fun.

Selectivity in nitrilases

One of the things we are interested in looking at with our increasingly wide panel of nitrilases is how substrate selectivity works with classic Group 1 nitrilases.
 Our standard panel of substrates we are assaying our nitrilases against has fifty substrates so we span quite a range of different structural types and traits. Here is an excerpt from our posters' table of data.

Colour coding mirrors the response that we see with our assay and carries handy semi-quantitative values as well which we get from UV/vis spectroscopy. What we have here is a table with different substrates along the top and different nitrilases down the side.
The top row here shows a nitrilase which hydrolyzes arylaliphatic substrates with great gusto (showing a 10 against 2-phenylacetonitrile) but has not a lot of interest in alkyl or aryl nitriles generally. The fifth row shows a nitrilase which is an enthusiast for aromatic nitriles peaking at para-substituted aromatics but with some interest in almost everything else!
To some extent the other patterns of activties are also interesting... we see some nitrilases which show activities which are active against only a few compounds which are quite structurally diverse. This may be a low expression level only letting us see the most active members of wider groupings (because this data table comes out of a screening programme of cell-free extracts), and this is something we will be investigating. It would be nice to hope that there was something a bit deeper going on though.
Finally we have some enzymes like one in the fourth line here which appear to be oblivious to everything. These show up a difference of response within our team. I, as the chemist, say "perhaps it just isn't active at all"; our molecular biologists say "nonsense- with that sequence it's just got to be a nitrilase, you haven't found the right substrate yet"!
We'll keep looking... for new nitrilases and new substrates.

How specific are nitrilases for their substrates?

As part of our ongoing nitrilase discovery project, we are trying a panel of about 50 substrates of various types against an ever-growing panel of nitrilases. The first round panel has 20 enzymes, and using a chromogenic assay we are seeing the rather nice selectivity that the literature indicates these enzymes show. Below, an example of two nitrilases (with each repeated) and their response to 4 simple nitriles- the depth of colour scales nicely with the amount of ammonia released by the nitrile hydrolysis.

There are similar levels of diversity of response for aryl over alkyl nitriles, and short chain over long chain nitriles. After the colour screening we are moving onto GCMS for confirmatory results, as well as doing a bit of NMR for that final structural evidence.

125 Nitrilase activities all in the one place.

Imagine you are working on a panel of enzymes and their activities against a library of substrates looking to try to link substrate specificity to microbial origin, and then someone comes along and publishes a very similar thing…. Argh! Let me recommend to you Nitrilase Activity Screening on Structurally Diverse Substrates Providing Biocatalytic Tools for Organic Synthesis by fourteen authors  with corresponding author Anne Zaparucha, which is in Advanced Synthesis and Catalysis at DOI:10.1002/adsc.201201098.
It is good to see that full information is given on the nature of the nitrilases (Uniprot codes are given for each NIT reference) and they have a neat assay for nitrilase activity. Obviously a working nitrilase kicks out ammonia stoichiometrically so that's the best thing to be looking for in an activity assay. There are several ways to do this (for example pH change has been advocated e.g. Banerjee's bromothymol blue screen in J Biomol Screen at DOI: 10.1177/1087057103256910 but we have found it not to work for cell free extract based enzymes- too many other endogenous sources of pH change I'd guess) but this paper describes a nice coupled assay which uses the evolved ammonia as a substrate for a glutamate dehydrogenase with readout through the level of NADH/NAD+.

We are currently assaying nitrilase sequence space (as the jargon that got us the grant says!) as well. Our first round involves twenty enzymes and fifty substrates... we will be adding to the number of enzymes but we think 50 substrates sounds about right to assess selectivity. We have our own assay methodology which seems to give about the level of activity readout we want for a semiquantitative read on a plate reader... here's an example- top row shows a colour chart of increasing activity, the rest of the wells are substrates with a single enzyme.

 

NHase numbers to May 2013, and now nitrilase numbers too

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 4782 hits (+ 14% since last November), of which 2440 (+ 50% since August) were RefSeq data. It appears that there has been a lot of RefSeq going on with this enzyme class.

There are 24848 sequences labelled as "nitrilase" (not sure how robust that is currently), of which 12037 are pegged as RefSeq data.

Slight pivot

I have been doing this blog for 2 years and just over 100 posts, and over that time I have focussed very narrowly on nitrile hydratases. Whilst they are lovely enzymes and I will continue to work on/with them, a growing interest of mine has become their cousins, the nitrilases. I now have more research effort going into nitrilases than nitrile hydratases, so from henceforth I intend to write about NHases and nitrilases. I don't think I am going to stretch to amidases, even if they are on the same operon (or whatever molecular biologists call it!). Just to add a bit of contemporary experimental data to the usual reports, I intend to report some interesting and as yet unreported results here (obviously without compromising my ability to publish them eventually) on an ongoing nitrilase discovery project as they happen.