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The resolution of the bicyclic synthon ( rac )-γ-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) is an important step in the synthesis of a group of chemotherapuetic agents known as carbocyclic nucleosides. The archaeon Sulfolobus solfataricus MT4 produces a thermostable γ-lactamase that has a high sequence homology to the signature amidase family of enzymes. It shows similar inhibition patterns of amidases towards benzonitrile, phenylmethylsulfonyl fluoride and heavy metals such as Hg 2+ , and is activated by thiol reagents. The enzyme selectively cleaves the (+)-enantiomer from a racemic mix of γ-lactam. It also exhibits general amidase activity by cleaving linear and branched aliphatic and aromatic amides. The enzyme catalyses the synthesis of benzoic hydrazide from benzamide preferentially to benzamide cleavage in the presence of excess hydrazine. This enzyme has potential for use in industrial biotransformations in the production of both carbocyclic nucleosides and hydrazides.

A thermostable archaeal l -aminoacylase from Thermococcus litoralis has been used in immobilisation trials to optimise its application in industrial biotransformation reactions. Immobilisation techniques used included direct adsorption and crosslinking of the enzyme onto solid supports, bioencapsulation, and covalent bonding onto a variety of activated matrices. The most successful immobilisation methods were covalent binding of the enzyme onto glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per gram of support (wet weight for glyoxyl-Sepharose), respectively, with nearly 80% activity recovery in both cases. Enzyme immobilised onto glyoxyl-agarose was stabilised 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant l -aminoacylase immobilised onto Sepharose beads was constructed with the substrate, N -acetyl- dl -Trp, continuously flowing at 60...

Silica gel treated with AlEt 2 Cl or TiCl 4 efficiently promotes the reaction of cyclopentadiene with ( Z )-2-phenyl-4-benzylidene-5(4H)-oxazolone ( 1a ) with very little Z/E isomerization. Silica gel treated with TiCl 4 and ZnCl 2 supported on silica gel leads to very high yields for the reaction of cyclopentadiene with ( E )-2-phenyl-4-benzylidene-5(4H)-oxazolone ( 1b ). Furthermore, E/Z isomerization is almost suppressed, which is not achieved with homogeneous Lewis acids. Whereas for the reaction of 1a with cyclopentadiene the exo cycloadduct ( 3a ) is always the major product, for the reaction of cyclopentadiene wth 1b the endo ( 2b )/ exo ( 3b ) selectivity is reversed as a function of the catalyst. Thus, in the reactions catalyzed by silica gel, alumina and exchanged clays, endo ( 2b ) is the major product However, the exo cycloadduct ( 3b ) is preferably obtained for reactions catalyzed by silica gel and alumina treated with TiCl 4 and ZnCl 2 supported on silica gel.

Biocatalysis is a useful tool in the provision of chiral technology and extremophilic enzymes are just one component in that toolbox. Their role is not always attributable to their extremophilic properties; as with any biocatalyst certain other criteria should be satisfied. Those requirements for a useful biocatalyst will be discussed including issues of selectivity, volume efficiency, security of supply, technology integration, intellectual property and regulatory compliance. Here we discuss the discovery and commercialization of an l-aminoacylase from Thermococcus litoralis, the product of a LINK project between Chirotech Technology and the University of Exeter. The enzyme was cloned into Escherichia coli to aid production via established mesophilic fermentation protocols. A simple downstream process was then developed to assist in the production of the enzyme as a genetically modified-organism-free reagent. The fermentation and downstream processes are operated at the 500 litre scale. Characterization of the enzyme demonstrated a substrate preference for N-benzoyl groups over N-acetyl groups. The operational parameters have been defined in part by substrate-concentration tolerances and also thermostabilty. Several examples of commercial biotransformations will be discussed including a process that is successful by virtue of the enzyme's thermotolerance.

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.

Providing sufficient biocatalyst to support the demands of multi tonne product supply can be problematical. Here we describe how screening for and cloning a gamma-lactamase overcame biocatalyst supply issues, and greatly improved the actual biocatalytic process. The isolation of an expressing gamma-lactamase clone from a gene library necessitated a combination of classical molecular biology techniques together with innovative screening methods to identify a functional clone. Once isolated the enzyme was characterised with regard to its process performance and proved to be active at 500 g L(-1) substrate. Further development of the recombinant fermentation and downstream processing has resulted in the ability to produce sufficient biocatalyst from one 5001 fermentation to resolve 5 metric tonnes of (+/-)-lactam, whilst simplifying the process chemistry greatly.

Silica gel treated with AlEt2Cl or TiCl4 efficiently promotes the reaction of cyclopentadiene with (Z)-2-phenyl-4-benzylidene-5(4H)-oxazolone (1a) with very little Z/E isomerization. Silica gel treated with TiCl4 and ZnCl2 supported on silica gel leads to very high yields for the reaction of cyclopentadiene with (E)-2-phenyl-4-benzylidene-5(4H)-oxazolone (1b). Furthermore, E/Z isomerization is almost suppressed, which is not achieved with homogeneous Lewis acids. Whereas for the reaction of 1a with cyclopentadiene the exo cycloadduct (3a) is always the major product, for the reaction of cyclopentadiene wth 1b the endo(2b)/exo(3b) selectivity is reversed as a function of the catalyst. Thus, in the reactions catalyzed by silica gel, alumina and exchanged clays, endo (2b) is the major product However, the exo cycloadduct (3b) is preferably obtained for reactions catalyzed by silica gel and alumina treated with TiCl4 and ZnCl2 supported on silica gel., This work was made possible by the generous financial support of the Comisión Interministerial de Ciencia y Tecnología (project MAT93-0224) and of the European Union (contract CHRX CT93-0276). One of us (E.P.) thanks the Diputación General de Aragón for a grant.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.