Wednesday, 29 June 2016
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.
ChromSoc nitrilase flow chemistry project 1
I have an undergraduate student, Rob, working with us this summer on nitrilase reactions. He is kindly sponsored by the Chromatographic Society to work on a project using HPLC and GC to compare batch processes run on immobilized enzyme in a flask with those run on the same enzyme preparation using our in-house mesoscale flow reactor system. The flow reactor system is something we have been working on for a while and its genesis was part of a project based around using 3D printing to make bespoke laboratory equipment which is described in the Tumblr blog here, with a video of the system in operation doing a nitro reduction here. My intention is to relate here a real time log of progress with this project.
Two copies of the track fit together, and the solution
is flowed through. The track contains enzyme immobilized
on beads.
Friday, 22 April 2016
Active Site investigations on the Fe-centred NHase from Comamonas testosteroni Ni1
Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1
Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu , Richard C. Holz
A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k cat = 1100 ± 30 s−1) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s−1, respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s−1 and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.
BASF opens new bio-acrylamide plant in Bradford, UK
From
“BRADFORD, UK: BASF said it has opened a new world-scale
bio-acrylamide (BioACM) production facility at its site in Bradford, a major
investment that will help ensure long-term future of one of the UK’s largest
chemical manufacturing facilities, which employs around 600 people.”
“The development of a biocatalytic manufacturing process for
acrylamide started in Bradford with collaboration with Huddersfield University.
Subsequent work by scientists from Britain, Germany, South Africa and US, made
significant improvements to the performance of the biocatalyzed conversion
technology.”
Image from BASF.com
Tuesday, 19 April 2016
Creating bioactive molecules from online data
From a genome sequence to that protein in your hand is a
well –established process
Until I started collaborating closely with biologists ten
years ago, my impression of how easy it was to get a sample of a complex
biomolecule like a protein was based on popular science stories describing
days/months of tedious purification of buckets of biomass (sea sponges, exotic fungi,
cow bile, etc) to end up with a single vial containing so little it was
invisible to the naked eye.
Follow the link to see the schematic explained.
Monday, 18 April 2016
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
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
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.
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