NHase hydration of alicyclic 𝜶,𝝎-dinitrile 1-cyanocyclohexaneacetonitrile to 1-cyanocyclohexaneacetamide

Highly regioselective and efficient production of 1-cyanocyclohexaneacetamide by Rhodococcus aetherivorans ZJB1208 nitrile hydratase

Ren-Chao Zheng, Xin-Jian Yina and Yu-Guo Zheng

J Chem Technol Biotechnol 2016; 91: 1314–1319

DOI 10.1002/jctb.4724

A newly isolated NHase producing strain, Rhodococcus aetherivorans ZJB1208, was successfully used for hydration of1-CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed-batch biotransformation was performed in non-buffered water system with the continuous precipitation of 1-cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L⁻¹ (6.0 mol L⁻¹), giving a product concentration of 966.7 g L⁻¹ and biocatalyst yield (g product/g cat) of 204.2.

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.

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.