Geoffrey W. Haywood

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126☆

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.

A survey of the accumulation of novel polyhydroxyalkanoates by bacteria

SummaryA wide range of bacterial strains were examined for their ability to accumulate polyhydroxyalkanoates (PHA) from various carbon sources. Strains were selected from those reported to accumulate poly-3-hydroxybutyrate (PHB), related organisms and laboratory stocks. Other strains known to utilize n-alkanes, n-alcohols or n-acids were chosen to investigate their ability to produce long-chain PHAs. Five strains accumulated only PHB, 13 accumulated PHAs containing only C4 and C5 units and 7 accumulated PHAs containing 3-hydroxyacid units in the range C5 to C10.

Biosynthesis and composition of bacterial poly(hydroxyalkanoates)

It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism. The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discussed and it is proposed that the substrate specificity of the polymerizing enzyme restricts the range of monomer units incorporated into PHA. Various other bacteria produce similar copolymers from propionic acid and/or valeric acid. A number of Pseudomonas species accumulate PHAs containing longer-chain monomer units from linear alkanoic acids, alkanes and alcohols.

4-acetamidobutyrate deacetylase in the yeast Candida boidinii grown on putrescine or spermidine as sole nitrogen source and its probable role in polyamine catabolism

SUMMARY: The yeast Candida boidinii (CBS 5777, ATCC 56897) when grown on spermidine, diaminopropane, putrescine, diaminopentane, diaminohexane, acetylputrescine or 4-acetamidobutyrate as sole nitrogen source contained a deacetylase (EC 3.5.1.-) catalysing the removal of the acetyl group from N-acetyl-β-alanine, 4-acetamidobutyrate and 5-acetamidopentanoate. The enzyme was synthesized early in the exponential growth phase when C. boidinii that had been grown in medium containing glucose and ammonium was transferred to medium in which putrescine replaced ammonium. The 4-acetamidobutyrate deacetylase was partially purified 250-fold. The stoicheiometry of the reaction was established using 4-acetamidobutyrate as substrate. The enzyme had a subunit relative molecular mass (M R) of 78 500 and a M R in the range 122000 to 143000. The pH optimum was 8·0. The K m for 4-acetamidobutyrate was 0·29 mm. The enzyme was found in a number of other yeast species and was usually associated with high levels of diamine acetyltransferase and acetylputrescine oxidase. The role of this enzyme in the catabolism of di- and polyamines (including those organisms able to use these amines as carbon source) is discussed.

The Production of Polyhydroxyalkanoates from Unrelated Carbon Sources

Several bacteria have been found to accumulate polyhydroxyalkanoates (PHAs) containing 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) monomers from glucose and other carbon sources. These bacteria are members of the taxonomically related genera Rhodococcus, Nocardia and Corynebacterium. The proportion of 3HV and 3HB monomers present in PHA is dependent on the carbon source but 3HV is generally the major 3-hydroxyacid.

Putrescine breakdown in the yeast Candida boidinii: subcellular location of some of the enzymes involved and properties of two acetamidoaldehyde dehydrogenases

SUMMARY: Two acetamidoaldehyde dehydrogenases were identified in Candida boidinii grown on putrescine as sole nitrogen source with glucose as carbon source. One of them, enzyme A, although present when cells were grown on ammonium or l-lysine, increased in activity when cells were grown on putrescine or spermidine. The other, enzyme B, was absent when the putrescine was replaced by l-lysine or ammonium, but was present if the nitrogen source was spermidine or acetylputrescine. Both dehydrogenases were active with NAD+ or NADP+ as electron acceptor. Apparent K m values for 3-acetamidopropionaldehyde and 4-acetamidobutyr-aldehyde were respectively 0·83 mM and 0·041 mM for enzyme A and 0·077 mM and 0·015 mM for enzyme B. Enzyme A was competitively inhibited by chloral hydrate with a K i of 0·6 mM, whiile enzyme B was unaffected. Both enzymes were slightly (20%) stimulated by 50 mM-KCl. Although both enzymes catalysed the oxidation of a range of aldehyde substrates, and are thus both general aldehyde dehydrogenases, it is suggested that acetamidoaldehyde dehydrogenase B is more probably specifically involved in putrescine degradation. Subcellular fractionation of spheroplast lysates showed that enzyme B was cytosolic, remaining unsedimented at 100000 g, while enzyme A co-sedimented with mitochondrial marker enzymes in a sucrose density gradient. It was also shown that acetamidoalkanoate deacetylase and acetylputrescine oxidase activities, two other key enzymes in the breakdown of putrescine and spermidine, were respectively cytosolic and peroxisomal in their location in the cell.

More Than One Amine Oxidase is Involved in the Metabolism by Yeasts of Primary Amines Supplied as Nitrogen Source

Summary: Twenty-seven yeast species were tested for growth on methylamine and n-butylamine as sole nitrogen sources. Five species (including Saccharomyces cerevisiae) failed to grow on either amine, and a further five grew only on n-butylamine. The remainder grew on both amines. Amine oxidase activity was detected in extracts of all the strains which could grow on amines, but was generally absent from cells grown on ammonia or nitrate. From measurements in cell-free extracts of oxidase activity for a number of different amines, it is concluded that most such strains have at least two amine oxidases of different substrate specificity, and that the substrate specificity also varies between different yeast species. Based on these observations, four different groups of yeasts could be distinguished. Formaldehyde dehydrogenase activity was elevated in cells grown on amines or nitrate compared with those grown on ammonia, and activity was generally higher in cells grown on methylamine than in those grown on n-butylamine or nitrate.

Partial Purification of a Peroxisomal Polyamine Oxidase from Candida boidinii and its Role in Growth on Spermidine as Sole Nitrogen Source

Candida boidinii grows well on spermidine as sole nitrogen source, but poorly on spermine. Cells grown on spermidine, cadaverine, putrescine and 1,3-diaminopropane contained a polyamine oxidase which attacks spermine and spermidine at the secondary amino groups, forming putrescine and a product thought to be 3-aminopropionaldehyde. The enzyme was synthesized before growth began when C. boidinii that had been grown in medium containing glucose + ammonium was transferred to medium in which spermidine replaced ammonium. Other enzymes increasing in specific activity during this adaptation were catalase, benzylamine oxidase and N AD-dependent glutamate dehydrogenase. The polyamine oxidase was purified to 50% homogeneity, but was too unstable to obtain completely pure. It had a pH optimum of 10.0, and could be stabilized by addition of inert protein. It oxidized spermine, spermidine, N 1- acetylspermidine, N-n-butylpropylamine, di-n-butylamine and di-n-hexylamine. It did not oxidize di-n-propylamine, diethylamine or N 1,N 8-diacetylspermidine. Apparent Km values were determined for the active substrates. The enzyme was potently inhibited by quinacrine and by divalent cations. The stoicheiometry of the enzyme reaction was established using di-n-butylamine as substrate. The enzyme has a molecular weight in the range 80000 to 110000. Putrescine (the oxidation product of spermidine) was not oxidized by cell-free extracts, but evidence of aminotransferase activity was found. The oxidation/transamination product of putrescine, 4-aminobutyraldehyde (1-pyrroline), was oxidized by extracts and a scheme is presented by which spermidine could be catabolized. Polyamine oxidase was shown to co-sediment with NAD-dependent glycerol 3-phosphate dehydrogenase and catalase in sucrose gradients after mechanical breakage of spheroplasts, and is thus a peroxisomal enzyme. Polyamine oxidase was present in some other yeasts when grown on spermidine, C. nagoyaensis, Hansenula polymorpha and Trichosporon melibiosaceum, but absent from C. steatolytica, Pichia pastoris and Sporopachydermia cereana. These latter yeasts probably contained an enzyme resembling benzylamine/putrescine oxidase which attacks the primary amino groups of spermidine.