Publications by authors named "Ian N Taylor"

4 Publications

  • Page 1 of 1

An improved racemase/acylase biotransformation for the preparation of enantiomerically pure amino acids.

J Am Chem Soc 2012 Nov 15;134(47):19310-3. Epub 2012 Nov 15.

The EastChem School of Chemistry, Joseph Black Building, The University of Edinburgh, Edinburgh, EH9 3JJ, UK.

Using directed evolution, a variant N-acetyl amino acid racemase (NAAAR G291D/F323Y) has been developed with up to 6-fold higher activity than the wild-type on a range of N-acetylated amino acids. The variant has been coupled with an enantiospecific acylase to give a preparative scale dynamic kinetic resolution which allows 98% conversion of N-acetyl-DL-allylglycine into D-allylglycine in 18 h at high substrate concentrations (50 g L(-1)). This is the first example of NAAAR operating under conditions which would allow it to be successfully used on an industrial scale for the production of enantiomerically pure α-amino acids. X-ray crystal analysis of the improved NAAAR variant allowed a comparison with the wild-type enzyme. We postulate that a network of novel interactions that result from the introduction of the two side chains is the source of improved catalytic performance.
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http://dx.doi.org/10.1021/ja305438yDOI Listing
November 2012

Directed evolution of a thermostable l-aminoacylase biocatalyst.

J Biotechnol 2011 Oct 30;155(4):396-405. Epub 2011 Jul 30.

The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, UK.

Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65°C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k(cat) values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.
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http://dx.doi.org/10.1016/j.jbiotec.2011.07.029DOI Listing
October 2011

Managing the risk of glyphosate resistance in Australian glyphosate- resistant cotton production systems.

Pest Manag Sci 2008 Apr;64(4):417-21

Leslie Research Centre, Queensland Department of Primary Industries and Fisheries, Toowoomba, Australia.

Background: Glyphosate-resistant cotton varieties are an important tool for weed control in Australian cotton production systems. To increase the sustainability of this technology and to minimise the likelihood of resistance evolving through its use, weed scientists, together with herbicide regulators, industry representatives and the technology owners, have developed a framework that guides the use of the technology. Central to this framework is a crop management plan (CMP) and grower accreditation course. A simulation model that takes into account the characteristics of the weed species, initial gene frequencies and any associated fitness penalties was developed to ensure that the CMP was sufficiently robust to minimise resistance risks.

Results: The simulations showed that, when a combination of weed control options was employed in addition to glyphosate, resistance did not evolve over the 30 year period of the simulation.

Conclusion: These simulations underline the importance of maintaining an integrated system for weed management to prevent the evolution of glyphosate resistance, prolonging the use of glyphosate-resistant cotton.
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http://dx.doi.org/10.1002/ps.1508DOI Listing
April 2008

A thermostable L-aminoacylase from Thermococcus litoralis: cloning, overexpression, characterization, and applications in biotransformations.

Extremophiles 2002 Apr;6(2):111-22

School of Chemistry, University of Exeter, UK.

A thermostable L-aminoacylase from Thermococcus litoralis was cloned, sequenced, and overexpressed in Escherichia coli. The enzyme is a homotetramer of 43 kDa monomers and has an 82% sequence identity to an aminoacylase from Pyrococcus horikoshii and 45% sequence identity to a carboxypeptidase from Sulfolobus solfataricus. It contains one cysteine residue that is highly conserved among aminoacylases. Cell-free extracts of the recombinant enzyme were characterized and were found to have optimal activity at 85 degrees C in Tris-HCl at pH 8.0. The recombinant enzyme is thermostable, with a half-life of 25 h at 70 degrees C. Aminoacylase inhibitors, such as mono-tert-butyl malonate, had only a slight effect on activity. The enzyme was partially inhibited by EDTA and p-hydroxymercuribenzoate, suggesting that the cysteine residue and a metal ion are important, but not essential, for activity. Addition of Zn2+ and Co2+ to the apoenzyme increased the enzyme activity, whereas Sn4+ and Cu2+ almost completely abolished enzyme activity. The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids. preferring substrates containing hydrophobic, uncharged, or weakly charged amino acids such as phenylalanine, methionine, and cysteine.
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http://dx.doi.org/10.1007/s007920100230DOI Listing
April 2002