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.

Making an approximate 3D model of an enzyme

Sometimes we want to check out how an amino acid sequence from a database might translate into a 3D dimensional structure. This is straightforward enough if there exists an x-ray crystal structure for that exact enzyme in the Protein Data Bank or, like often happens in the nitrile hydratase class, there is very limited variation within sequences so overlaying a short portion of the sequence "by eye" on an existing enzyme is possible.
We are currently working on nitrilases, and despite increasing interest in their use as biocatalysts, the number of nitrilases (i.e. enzymes that convert nitriles to carboxylic acids not the other looser biochemical definition) which exist as structures in the PDB s precisely one. It is from Pyrococcus abyssi and is pretty much restricted in substrate tolerance to small aliphatic nitriles like fumaronitrile. It should not be a surprise that it isnt a great model for many other nitrilases.
If the PDB has come up short we tend to use a link out of the Uniprot database to give a prediction of 3D structure. So if you want to see an estimate of what the nitrilase from Aurantimonas manganoxydans looks like, than the link labelled "ModBase" under the subtitle "3D structure databases" leads you to a page of predictions and further tools. For this specific enzyme it is suggested that a mouse nitrilase superfamily structure contains the best 3D match.

A nitrile hydratase for cyanopyridines... and it's a bit more stable than usual.

There is a paper in Process Biochemistry which describes a new NHase from Aurantimonas manganoxydans which shows improved stability than you can normally expect from a NHase. It is entitled “Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy”, and it is by Xiaolin Peia, Hongyu Zhang, Lijun Meng, Gang Xu, Lirong Yang and Jianping Wu. This NHase is four times more rapid at converting 3-cyanopyridine to its corresponding amide as valeronitrile, and the authors emphasize their enzyme's stability though in the world of NHases where nothing is what you might describe as thermophilic, please don't get too expectant! They have a great table of NHase thermostability which I reproduce with their enzyme's data inserted.

 

E. coli expression of active nitrile hydratase from Aurantimonas manganoxydans needs cobalt ions

This paper in Biotechnology Letters from Pei and co-workers gives details of a cobalt centred nitrile hydratase from Aurantimonas manganoxydans, and the importance of getting the concentration right for good level of expression and activity. (DOI 10.1007/s10529-013-1215-5)