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).

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.

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.

PVA-chitosan based immobilization of NHase for dynamic kinetic resolution of rac-mandelonitrile

A recently published paper by Pawar and Yadav in Industrial and Engineering Chemistry Research entitled “Enantioselective Enzymatic Hydrolysis ofrac-Mandelonitrile to R-Mandelamide by Nitrile Hydratase Immobilized onPoly(vinyl alcohol)/Chitosan–Glutaraldehyde Support” describes the improvement in performance the nitrile hydratase from Rhodococcus rhodocrous ATCC BAA-870 exhibits when immobilized on PVA/chitosan. The reaction being tested is the DKR of rac-mandelonitrile.

Supporting a nitrile hydratase for better performance

There is a new accepted manuscript for the Journal of Molecular Catalysis B: Enzymatic which adds to the literature on the support of a nitrile hydratase to increase this notoriously sensitive enzyme’s robustness.  PVA/Chitosan-glutaraldehyde cross-linkednitrile hydratase as reusable biocatalyst for conversion of nitriles to amides by SV Pawar and GD Yadav  describes how to prepare a cross linked assembly containing the NHase from Rhodococcus rhodochrous ATCC BAA-870. When they test this immobilized enzyme in the hydration of 3-cyanopyridine, they find it is able to withstand a higher temperature and more basic conditions. They also show that their preparation is a reusable format which retains 50% of activity after 8 batches.

The figure below from the paper shows relative activity of free and immobilized NHase over time at 45 degrees C in 0.1M phosphate buffer. The upper line is the immobilized form.

 

Methods of immobilization of NHase, and a new one.

There is a new paper on the immobilization of nitrile hydratase to give greater stability. There are a few previous examples of this topic including:

·         Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme from Sander van Pelt, Sandrine Quignard, David Kubac, Dimitry Y. Sorokin, Fred van Rantwijk and Roger A. Sheldon in Green Chemistry in 2008. (DOI: 10.1039/b714258g)

·         Production of Acrylamide using Alginate-Immobilized E. coli Expressing Comamonas testosteroni 5-MGAM-4D Nitrile Hydratase from Lawrence J. Mersinger, Eugenia C. Hann, Frederick B. Cooling, John E. Gavagan, Arie Ben-Bassat, Shijun Wu, Kelly L. Petrillo, Mark S. Payne, and Robert DiCosimo in Advanced Synthesis and Catalysis in 2005. (DOI: 10.1002/adsc.200505039)

·         Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats from David Kubáč, Alena Čejková, Jan Masák, Vladimír Jirků, Marielle Lemaire, Estelle Gallienne, Jean Bolte, Radek Stloukal, Ludmila Martínková in Journal of Molecular Catalysis B: Enzymatic in 2006. (doi:10.1016/j.molcatb.2006.01.004)

This one is Catalytic Properties of a Nitrile Hydratase Immobilized on Activated Chitosan by Yu. G. Maksimova, T. A. Rogozhnikova, G. V. Ovechkina, A. Yu. Maksimov, and V. A. Demakov in Applied Biochemistry and Microbiology (DOI: 10.1134/S0003683812030076).

They have used a nitrile hydratase isolated from a strain of Rhodococcus ruber gt1 and immobilized it on chitosan activated with 0.1% benzoquinone solution. They show that this immobilized enzyme can be used for 50 consecutive cycles of acrylonitrile transformation with activity holding up well.

They also found that their immobilized nitrile hydratases remain active at pH 3.0–4.0 which usefully extends its effective pH range.