125 Nitrilase activities all in the one place.

Imagine you are working on a panel of enzymes and their activities against a library of substrates looking to try to link substrate specificity to microbial origin, and then someone comes along and publishes a very similar thing…. Argh! Let me recommend to you Nitrilase Activity Screening on Structurally Diverse Substrates Providing Biocatalytic Tools for Organic Synthesis by fourteen authors  with corresponding author Anne Zaparucha, which is in Advanced Synthesis and Catalysis at DOI:10.1002/adsc.201201098.
It is good to see that full information is given on the nature of the nitrilases (Uniprot codes are given for each NIT reference) and they have a neat assay for nitrilase activity. Obviously a working nitrilase kicks out ammonia stoichiometrically so that's the best thing to be looking for in an activity assay. There are several ways to do this (for example pH change has been advocated e.g. Banerjee's bromothymol blue screen in J Biomol Screen at DOI: 10.1177/1087057103256910 but we have found it not to work for cell free extract based enzymes- too many other endogenous sources of pH change I'd guess) but this paper describes a nice coupled assay which uses the evolved ammonia as a substrate for a glutamate dehydrogenase with readout through the level of NADH/NAD+.

We are currently assaying nitrilase sequence space (as the jargon that got us the grant says!) as well. Our first round involves twenty enzymes and fifty substrates... we will be adding to the number of enzymes but we think 50 substrates sounds about right to assess selectivity. We have our own assay methodology which seems to give about the level of activity readout we want for a semiquantitative read on a plate reader... here's an example- top row shows a colour chart of increasing activity, the rest of the wells are substrates with a single enzyme.

 

Can genes be patented?

This article reports on the American Supreme Court ruling about the ability to patent DNA sequences from a genome. It can't be done now. That is sensible. Being able to patent genes on discovery can only stiffle innovation and fund legal process not research.

New nitrile hydratase papers

Enzyme–Substrate Binding Landscapes in the Process of Nitrile Biodegradation Mediated by Nitrile Hydratase and Amidase from Yu Zhang, Zhuotong Zeng, Guangming Zeng, Xuanming Liu, Ming Chen, Lifeng Liu, Zhifeng Liu & Gengxin Xie in Applied Biochemistry and Biotechnology describes molecular modelling experiments using the crystal structures 2QDY (AJ270 Fe based NHase) and 1IRE (Pseudonocardia thermophila Co based NHase).This is a docking study for these two enzymes and a downstream amidase. Available at DOI 10.1007/s12010-013-0276-1. Shown below is Fig1b which illustrates a binding mode between the P. thermophila Co based NHase and 3-cyanopyridine.

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli by Yi Liu, Wenjing Cui , Yueqin Fang, Yuechun Yu, Youtian Cui, Yuanyuan Xia, Michihiko Kobayashi and Zhemin Zhou adds to debate around the activator which is used for the maturation of the NHase with inclusion of the metal centre  (they say an activator is always needed, but is that true?). This paper is found in BMC Biotechnology at DOI:10.1186/1472-6750-13-48 and describes a methodology to ensure that the P14K activator is expressed successfully and in a more stable form.