New patent on NHase from Mitsubishi Rayon

Mitsubishi Rayon have published a new US patent (20140120588) protecting a specific set of primary sequences of NHase.

Enhancement of NHase stability with self assembling peptides

There is an in-press paper available online entitled “Enhancement ofthermo-stability and product tolerance of Pseudomonasputida nitrile hydratase by fusing with self-assembling peptide” in the Journal of Bioscience and Bioengineering by Zhemin Zhou and co-workers. They describe how they have used some self-assembling peptide based tags appended to the beta subunit to enhance thermal stability and substrate tolerance.

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.

Two publications on the nitrilase from Sphingomonas wittichii RW1

Sphingomonas wittichii RW1 is a bacterium which has been shown to have the ability to degrade polychlorinated dioxins, and hence has had its genome fully sequenced. It has two different nitrilases in its genome.

YP_001264656.1

YP_001261492.1

Two papers just released with the corresponding authors Er-Zheng Su and Dong-Zhi Wei report on the nitrilase with the accession code YP_001264656. These authors and enzyme have previously been mentioned on this blog- I complained there was no information on the enzyme discovery... here it is!

The first entitled “Cloning, Overexpression, andCharacterization of a High Enantioselective Nitrilase from Sphingomonaswittichii RW1 for Asymmetric Synthesis of (R)-Phenylglycine” and published in Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology describes the construction of a mini-library of nitrilase enzymes chosen by the similarity to a nitrilase from Pseudomonas fluorescens EBC191 (GenBank accession no. AAW79573) which has been shown to be highly activity toward phenylglycinonitrile. The nitrilase from Sphingomonas wittichii RW1 was shown to be most promising nitrilase to achieve chiral synthesis of R-phenylglycine in a kinetic resolution mode.

The second entitled “Efficient asymmetric synthesis ofD-N-formyl-phenylglycine via cross-linked nitrilase aggregates catalyzeddynamic kinetic resolution” and published in Catalysis Communications describes the preparation of cross-linked enzyme aggregates (CLEAs) of nitrilase from Sphingomonas wittichii RW1, and then its use to perform dynamic kinetic resolution to make D-N-formyl-phenylglycine. The paper also demonstrates that the CLEAs have improved stability over non-immobilized enzyme and are reuseable up to six times whilst retaining 70% initial activity.

NHase expression in Corynebacterium glutamicum

Industrial scale biocatalytic production of acrylamide relies on the use of engineered strains of Rhodococcus. There is a newly accepted manuscript in Applied Microbiology and Biotechnology from three Korean groups lead by J-H Lee and H-S Kim where the NHase from a Rhodococcus strain is expressed in Corynebacterium glutamicum (a cell factory already used commercially in amino acid biosynthesis) and tested for its ability to hydrate acrylonitrile. Whilst it didnt have the same activity as the NHase in the homologous system, the advantage the authors propose is that the rate of growth and hence enzyme production is higher with C. glutamicum.

 

Chiral intermediate for cilastatin by nitrilase hydratase/amidase combo

There is a new accepted manuscript in the Journal of Molecular Catalysis B from Yu-Guo Zheng and co-workers entitled "Industrial production of chiral intermediate of cilastatin by nitrile hydratase and amidase catalyzed one-pot, two-step biotransformation" which describes their research into using a chiral nitrile to carboxylic acid conversion which results from a cascade of NHase from (Rhodococcus boritolerans FW815) then amidase (from Delftia tsuruhatensis ZJB-05174). The compound they are aiming to hydrolyze is rac 2,2-dimethylcyclopropanecarbonitrile to the S-2,2-dimethylcyclopropanecarboxamide which is an intermediate in the synthesis of cilastatin (a booster component to some antibiotics which prolongs their activity by blocking the kidney’s dehydropeptidase activity). This is achieved using a NHase which is notably active but not chirally selective, and an amidase which is only avid for the R enantiomer of the amide. The R acid and S amide are separated using macroporous resin adsorption chromatography and the R acid is converted to the acid chloride. This can than have its chirality scrambled with a bit of heat such that its conversion to amide by addition of ammonia gives a fresh racemic batch of amide to challenge the R selective amidase with. The conditions described are to get it to work on the 100kg scale. Considering that this is a report on a process which has been undergoing continuous optimization since 2005, I guess the choice of amidase to do the chiral resolution was “baked in” before there was literature evidence of significantly stereoselective nitrile hydratases which might have prompted an enzyme discovery effort in that direction. Having said that racemization using the wrong acid chloride and heat rather than on the wrong isomer of the nitrile is a neat touch.

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.