Stephen John Clifford Taylor

A novel pathway for the catabolism of 4-nitrotoluene by Pseudomonas

Eleven strains of Pseudomonas were isolated by selective enrichment on 4-nitrotoluene (4NT). They all utilized 4NT, 4-nitrobenzyl alcohol (4NBA) or 4-nitrobenzoate (4NBZate) as sole sources of carbon and nitrogen. One strain, TW3, was used for more detailed studies. 4NT-grown cells of TW3 take up O2 when incubated in the presence of 4NBA, 4-nitrobenzaldehyde (4NBZ) and 4NBZate. HPLC analysis of culture supernatants showed that 4NBZ and 4NBZate were formed when 4NT-grown cells wer incubated with 4NBA, whereas only 4NBZate was found when they were incubated with 4NBZ. Two dehydrogenases were detected in extracts of 4NT-grown cells. 4NBA dehydrogenase could be assayed by a dye-linked assay whereas 4NBZ dehydrogenase activity was linked to NAD+ reduction. No nitrite was detected in supernatants of 4NBZate-grown cells incubated with 4NBZate but the nitrogen appeared as ammonium. The only aromatic ring-cleavage dioxygenase that was induced during growth on the nitroaromatics was protocatechuate 3,4-dioxygenase. It is proposed that the pathway for 4NT catabolism proceeds via 4NBA, 4NBZ and 4NBZate and ultimately to protocatechuate with release of the nitro group as ammonium.

Novel screening methods--the key to cloning commercially successful biocatalysts.

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.

Biochemically generated chiral intermediates for organic synthesis: The absolute stereochemistry of 4-bromo-cis-2,3-dihydroxycyclohexa-4,6-diene-1-carboxylic acid formed from 4-bromobenzoic acid by a mutant of pseudomonas putida. ☆

Abstract The absolute stereochemistry of the 4-bromo- cis -2,3-dihydroxycyclohexa-4,6-diene-1-carboxylic acid, one of a series of diols produced by P. putida strain JT 107, was shown by X-ray crystal analysis to be (2-R, 3-R).

Enzyme-catalysed kinetic resolution of 4-endo-hydroxy-2-oxabicyclo[3.3.0]oct-7-en-3-one and employment of the pure enantiomers for the synthesis of anti-viral and hypocholestemic agents

The endo-hydroxylactone (+/-)-(1) was resolved by enantioselective acetylation using Candida cylindracea lipase or preferentially Pseudomonas fluorescens lipase (pfl). Alternatively the corresponding butyrate (+/-)-(3) was hydrolysed with pfl to give the ester (+)-(1S,4R,5S)-(3) and the alcohol (-)-(1R,4S,5R)-(1). The latter compound was converted into carbovir (-)-(1R,4S)-(12) while the ester (+)-(3) was transformed into the delta-lactone (+)-(3R,5S)-(18). The exo-hydroxylactone (+/-)-(2) was resolved less efficiently by a trans-esterification process employing pfl and vinyl acetate.

Immobilisation of the Thermostable l -aminoacylase from Thermococcus litoralis to Generate a Reusable Industrial Biocatalyst

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°C for 10 days. No loss of activity was detected over five days, with 32% activity remaining after 40 days at 60°C. These results show the potential of the use of immobilised l -aminoacylase in biotransformation reactions for the production of fine chemicals.

Submerged Fermentation of Penicillium paxilli Biosynthesizing Paxilline, a Process Inhibited by Calcium-induced Sporulation

SUMMARY: A submerged fermentation process for the production of the tremorgenic mycotoxin paxilline by Penicillium paxilli has been developed. The fungus did not sporulate and accumulated paxilline to 1·5% (w/w) in freeze-dried cells within 6 d in a 60 1 stirred fermenter. Induction of extensive differentiation of conidiophores and profuse sporulation by adding 2% (w/v) CaCl2. 2H2O to the medium at batching reduced paxilline yield by 97%. Paxilline biosynthesis occurred when the glucose in the medium had been exhausted, implying that carbon catabolite repression may be involved in the biosynthesis of this alkaloid, even when calcium-induced sporulation inhibits or delays the onset of paxilline biosynthesis. Sporulation-induced inhibition of indole-terpenoid alkaloid biosynthesis in P. paxilli contrasts with the situation in some other penicillia elaborating indole alkaloids and allows disassociation of aspects of secondary metabolite biosynthesis from growth-associated differentiation, which formerly seemed to be linked.

Unexpected stereoselectivity in the cis dihydroxylation of some 2-cyclopentene-1-carboxamides

While 4-substituted-2-cyclopentene-1-carboxylate esters gave no facial selectivity in the cis dihydroxylation of the olefin function with osmium tetroxide/N-methylmorpholine-N-oxide, the corresponding carboxamides unexpectedly gave high diastereoselectivity for the isomer useful for carbocyclic ribofuranosyl nucleosides.

A short, scaleable synthesis of both enantiomers of 2-benzoylsulfanyl-5-phthalimidopentanoic acid from ornithine

Abstract An efficient “one-pot” synthesis of (R)- and (S)-2-bromo-5-phthalimidopentanoic acid from ornithine is described. Subsequent reaction with potassium thiobenzoate affords a concise, scaleable route to (R)- and (S)-enantiomers of 2-benzoylsulfanyl-5-phthalimidopentanoic acid, an intermediate in the synthesis of MMP inhibitors.