A more extreme than usual NHase

There is a cobalt centred nitrile hydratase from Rhodococcus opacus which has been the subject of work by Yasukawa and patents and commercial usage by Degussa. If you BLASTp the alpha chain of this, as NCBI titles it, "unnamed protein product" against their RefSeq collection, you find there are a lot of similar NHases. One that is relatively new comes from the newly released draft genome sequence of the bacterium Caldalkalibacillus thermarum TA2.A1. There is a good clue in its name to the fact that this bacterium grows optimally 65-70 degrees C and pH 9.5. That is pretty hot for a NHase, and with those pH conditions, no surprise it isnt an iron-centred version!

Microbial production of acrylamide by nitrile hydratase

I have just obtained a copy of the "Acrylamide, Microbial Production by Nitrile Hydratase" by R-C Zheng, Y-G Zheng and Y-C Shen from the Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation and Cell Technology, published in 2010; DOI: 10.1002/9780470054581.eib004. It is new, interesting resource for how much large scale use of NHases there is going on, and includes some information I havent seen anywhere else before.

• It gives a worldwide estimate for the biocatalytic production of acrylamide as 400,000 t/a
• There are "more than 10 plants" in China running microbial acrylamide production using a Nocardia strain to yield a total production capacity which "has exceeded 200,000 t/a", with plans to increase this to 300,000 t/a
• Degussa have a plant in Russia with a production capacity of 24,000 t/a using a R. rhodocrous M8 strain.
• Mitsui Chemical have a JV acrylamide plant in Pusan, Korea.
• SNF Floerger built a 20,000 t/a plant using the Nitto technology in 1999 (but it doesnt say where), and have plans for a further 5 of the same size.

More on the industrial exploitation of Nitrile Hydratases

I have just been pointed in the direction of a rather nice summary ("Nitrile hydratases in synthesis") of the state of play for nitrile hydratase commercial exploitation from the May/June 2008 issue of Chemistry Today from Sander van Pelt. It includes more details of the Mitsubishi Rayon, Lonza, and DuPont usage, and shows how SNF Floerger were looking to introduce biocatalytic acrylamide production to Europe.

The length of the beta unit in a nitrile hydratase

I have also had a quick look at the length of the beta chain in NHases using much the same method as I did for the alpha chains. I have to say that this time, I did do a bit of manual pruning because there was loads of inappropriate sequences in the list I downloaded. Anywhere here is the answer- about 200 again.

Salpingoeca

There is another marine eukaryote which has single subunit NHase- it is Salpingoeca. If you CLUSTALW it against Monosiga, it looks pretty similar (B subunit comes before A subunit, plenty of stars and colons but not the big section of histidines on the link.)

A better estimate of the number of NHases.

I have spent some time devising a spreadsheet into which you can download a list of FASTA data, and it will scan through looking for the "CTLCSC" fragment which indicates cobalt-centred and "CSLCSC" fragment which indicates iron centring, and total up how many hits there are of each in the list. It is all a bit more definite than just relying on "nitrile hydratase alpha" to give you accurate numbers!
Using the term "nitrile hydratase alpha" and then selecting the RefSeq selections, you get 121 cobalt centred NHases and 18 iron centred NHases. If on the other hand, you use all those sequences which are tagged as "bacteria", you get 278 cobalt-centred NHases and 131 iron-centred NHases. There are a few things to mention- these numbers don't include those sequences which have X in the tag to indicate that the cysteine is oxidized (these tend to be the PDB linked sequences) but do include some sequences which don't start with M and would make a molecular biologist wince, and the list hasn't been checked for duplication.

Double length equals a single subunit?

Following on from the last post... it seems reasonable to check that if you get sequences thrown up by a search which are about the double the length of one subunit in a two subunit enzyme that you don't have a situation where the two subunits are actually linked. This is the situation for the nitrile hydratase from Monosiga brevicollis which can be seen to the right of the histogram in the last post. For this reason, it seems like a good idea to check out the two sequences in the n=360 and n=380 boxes. Well the one in the n=380 box is titled "nitrile hydratase regulator [Agrobacterium radiobacter K84]" and doesn't look right at all. Scratch that one. The other one is simply called GK12401 and is from Drosophila willistoni... a eukaryote like Monosiga. Looking at the GenPept file it is easy to see how the thing was given the NHase alpha coding (I have put a green line around the bit that looks like the metal binding bit of a NHase) though I am not convinced it is what it actually is or does (based on trying to align it to Monosiga NHase or an example alpha subunit from AJ270).

The length of the alpha chain in a nitrile hydratase

If you look at number of amino acids in the alpha chain of a nitrile hydratase, I think it is safe to say that you get suspicious of its attribution as NHase if the number was much different from 200. I was wondering if this is actually a reasonable hunch to have so here is a graph.

I got this in the usual not terribly robust way of using a text search for "nitrile hydratase alpha", and then downloading only those which were RefSeq. Today that gives you 174 sequences of which some are definitely bobbins, but I feel bad at hand-pruning datasets if your initial search is a bit of a rough design to start with. The mean length I calculated to be 199, and as you can see the mode in the histogram above is for sequences between 200 and 210. Nice to see that for once, a hunch is backed by some data! Other things of note are that the Monsiga brevicollis NHase is found by this search and is the tiny block to the far right, and a quick scan through the sequences with n=60-140 indicates these are non-NHase waifs and strays picked up by my rough search terms.

Industrial Exploitation of Nitrile Hydratases

Nitrile hydratases are one of the big successes of industrial biocatalysis. An enzymatic way to convert a nitrile to an amide is obviously attractive for its controlability and selective hydration.
There are several companies which use NHase-based nitrile hydration. The original company I think was Nitto Chemical Industries (now Mitsubishi Rayon) who use have used Rhodococcus and Pseudomonas to convert acrylonitrile to acrylamide. Wolfgang Aehle in his 2007 book "Enzymes in Industry: Production and Applications" indicates that this process runs (p281/2 available via a Google Books search) at 30,000 t/a. There are indications behind a paywall that this business may no longer be owned by Mitsubishi but by SNF Floerger Group of France but I don't want to know enough to pay up.
Lonza are another company often quoted as industrial users of NHases but in the conversion of 3-cyanopyridine to nicotinamide. They have a 3000 t/a plant in Guangzhou and it has been reported that they use the Nitto technology for this conversion.
A more recent entrant (2008) into the acrylonitrile to acrylamide bioconversion is Senmin in South Africa who have used Ciba-BASF technology to do the hydration. I dont know the scale of the operation but apparently (slides 37 onwards) they could make 20,000 tons of polyacrylamide polymer from its output.
It is not easy finding verifiable information on industrial usage of biocatalysis- I am going back to reading the academic literature!