Biomedical subjects

G Eggink

Publications and source records attributed to G Eggink.

At least 19 recordsLinked to original sources

PubMed2001

Production of microbial polyesters: fermentation and downstream processes.

Poly(3-hydroxyalkanoates) (PHAs) constitute a large and versatile family of polyesters produced by various bacteria. PHAs are receiving considerable attention because of their potential as renewable and biodegradable plastics, and as a source of chiral synthons since the monomers are chiral. Industrial PHA production processes have been developed for poly(3-hydroxybutyrate) (poly(3HB)) and poly(3-hydroxybutyrate-co-3-valerate) (poly(3HB-co-3HV). More than 100 other poly(3HAMCL)s, characterized by monomers of medium chain length, have been identified in the past two decades. These monomers typically contain 6-14 carbon atoms, are usually linked via-3-hydroxy ester linkages, but can occasionally also exhibit 2-, 4-, 5-, or 6-hydroxy ester linkages. Such polyesters are collectively referred to as medium chain length PHAs poly(3HAMCL)s. The vast majority of these interesting biopolyesters have been studied and produced only on the laboratory scale. However, there have been several attempts to develop pilot scale processes, and these provide some insight into the production economics of poly(3HAMCL)s other than poly(3HB) and poly(3HB-co-3HV). These processes utilize diverse fermentation strategies to control the monomer composition of the polymer, enabling the tailoring of polymer material properties to some extent. The best studied of these is poly(3-hydroxyoctanoate) (poly(3HO)), which contains about 90% 3-hydroxyoctanoate. This biopolyester has been produced on the pilot scale and is now being used in several experimental applications.

Chemical Engineering↗

PubMed2001

Properties, modifications and applications of biopolyesters.

Poly(hydroxyalkanoates) (PHAs), of which poly(hydroxybutyrate) (PHB) is the most common, can be accumulated by a large number of bacteria as energy and carbon reserve. Due to their biodegradability and biocompatibility these optically active biopolyesters may find industrial applications. A general overview of the physical and material properties of PHAs, alongside with accomplished applications and new developments in this field is presented in this chapter. The properties of PHAs are dependent on their monomer composition and therefore it is of great interest that recent research has revealed that, in addition to PHB, a large variety of PHAs can be synthesized microbially. The monomer composition of PHAs depends on the nature of the carbon source and microorganism used. PHB is a typical highly crystalline thermoplastic whereas medium chain length PHAs are elastomers with low melting points and a relatively lower degree of crystallinity. By (chemical) modification of the PHAs, the ultimate properties of the materials can be adjusted even further, when necessary. Applications that have been developed from PHB and related materials (e.g. Biopol) can be found in very different application areas and cover packaging, hygienic, agricultural and biomedical products. Recent application developments based on medium chain length PHAs range from high solid alkyd-like paints to pressure sensitive adhesives, biodegradable cheese coatings and biodegradable rubbers. Technically, the prospects for PHAs are very promising. When the price of these materials can be further reduced, application of biopolyesters will also become economically very attractive.

Bacteria↗

PubMed2001

Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor.

Pseudomonas putida GS1 is able to convert limonene to perillic acid (up to 64 mM,(11 g/l) when the bacteria is cultivated in fed-batch culture with non-limiting amounts of glycerol. ammonium, and limonene. P. putida GS1 can use p-cymene as a single source of carbon and energy, and the enzymes that are responsible for the conversion of limonene to perillic acid belong to the degradation pathway of p-cymene. The p-cymene pathway of P putida GS1 is very similar, if not identical, to the cym pathway of P. putida F1. The latter strain, and a recombinant Escherichia coli strain that carried the genes of the cym pathway of P. putida Fl, also converted limonene to perillic acid. However, the final concentrations that were obtained in batch cultures with these two strains were lower than those obtained with P. putida GS1.

Bioreactors↗

PubMed2001

Characterisation of extracellular polysaccharides produced by Crypthecodinium cohnii.

The valuable polyunsaturated fatty acid, docosahexaenoic acid, can be produced by cultivation of the heterotrophic microalga, Crypthecodinium cohnii. During batch growth of C. cohnii on glucose, sea salt and yeast extract for 5 days, so far unreported extracellular polysaccharides were produced. These caused an increased viscosity and a strong drop in the maximum oxygen transfer. The viscosity increased most markedly as cells entered the stationary phase. The polysaccharides varied in size (from 6 kDa to >1,660 kDa) and monomer distribution. A high molecular mass fraction (from 100 kDa to >1,660 kDa) and a medium molecular mass fraction (6-48 kDa) were prepared. The high molecular mass fraction contained (on a molar basis) 71.7% glucose, 13.1% galactose and 3.8% mannose, whereas the medium molecular mass fraction contained 37.7% glucose, 19.8% galactose and 28.1% mannose. Other monomers present in both fractions were fucose, uronic acid and xylose. Monomers were coupled mainly via alpha-(1-3) links. Increased viscosity due to polysaccharide production complicates the development of commercial, high cell-density processes for the production of docosahexaenoic acid.

Animals↗

PubMed2001

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12.

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.

1-Octanol↗

PubMed2001

Spontaneous formation of a mannitol-producing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose.

We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides. The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain. It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars. A possible explanation of these observations is discussed.

Fructose↗

PubMed2000

Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate) films: effects of epoxidation and curing.

Medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers derived from linseed oil (PHA-L) have a relatively small molar mass and contain a high concentration of unsaturated side-chains. As such, these polymers are amorphous and take on the consistency of a viscous liquid at room temperature. In order to increase the application potential of this material, the side-chain olefinic groups of PHA-L were converted to epoxy derivatives (PHA-LE) using m-chloroperoxybenzoic acid (m-CPBA). Epoxidation resulted in a 37% conversion of olefinic to epoxy groups. The epoxy groups enhanced the PHA-LE film susceptibility to crosslinking upon exposure to air. PHA-LE films began to crosslink and stiffen in less than 25 days, whereas PHA-L films began to crosslink between days 50 and 75. The PHA-LE films showed an increase in tensile strength (TS, from 4.8 to 20.7 MPa) and Young's modulus (YM, from 12.9 to 510.6 MPa) between 25 and 100 days. In contrast, PHA-L had a TS of 25.0 MPa and YM of 767.8 MPa after 100 days. Epoxidation helped induce crosslink formation; however, aging for 100 days ultimately resulted in crosslinked films from both PHA-L and PHA-LE with higher strength and durability than the original materials.

Biomechanical Phenomena↗

PubMed1997

Cloning and expression of the delta 9 fatty acid desaturase gene from Cryptococcus curvatus ATCC 20509 containing histidine boxes and a cytochrome b5 domain.

To allow genetic modification of the fatty acid biosynthesis routes in the lipid-accumulating yeast Cryptococcus curvatus the delta 9 fatty acid desaturase gene was cloned and characterized. The 1668-bp gene encodes a protein of 556 amino acids with a calculated molecular mass of 62 kDa. The gene shows strong homology to previously cloned delta 9 fatty acid desaturase genes from yeast and rat. Homology includes three histidine boxes characteristic for membrane-bound desaturases and a cytochrome b5 domain responsible for electron transport. The delta 9 desaturase gene has a high G+C content of 61% and displays a codon usage different from that of Saccharomyces cerevisiae, but similar to that of the basidiomycete Schizophyllum commune. Expression of the delta 9 desaturase gene of C. curvatus ATCC 20509 was studied in the presence of different fatty acids in the growth medium. Repression of desaturase mRNA signals was found if fatty acids with a double bond at the delta 9 position were present. Fatty acids with a double bond at another position (delta 10 or delta 6) or saturated fatty acids had no effect on the transcription of the cloned gene.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of a delta-9 fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570.

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the delta-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned delta-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the delta-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.

Amino Acid Sequence↗

PubMed1995

Formation of novel poly(hydroxyalkanoates) from long-chain fatty acids.

Poly(hydroxyalkanoates) (PHAs) were isolated from Pseudomonas aeruginosa 44T1 cultivated on euphorbia oil and castor oil. With the aid of 2-D proton NMR spectra and proton-detected multiple bond coherence NMR spectra the structures of the PHAs were determined. In addition to the usual PHA constituents (C6-C14 3-hydroxy fatty acids), PHAs formed from euphorbia oil contained delta 8,9-epoxy-3-hydroxy-5c-tetradecenoate, and probably delta 6,7-epoxy-3-hydroxydodecanoate and delta 4,5-epoxy-3-hydroxydecanoate. These novel constituents account for approximately 15% of the total amount of monomers and are clearly generated via beta-oxidation of vernolic acid (delta 12,13-epoxy-9c-octadecenoic acid), the main component of euphorbia oil. In PHAs formed from castor oil, 7% of the monomers found were derived from ricinoleic acid (12-hydroxy-9c-octadecenoic acid). The presence of 3,8-dihydroxy-5c-tetradecenoate was clearly demonstrated. Furthermore, NMR analysis strongly suggested the presence of 3,6-dihydroxydodecanoate, 6-hydroxy-3c-dodecenoate, and 4-hydroxydecanoate.

Castor Oil↗

PubMed1994

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.

Acyltransferases↗

PubMed1993

Heteronuclear NMR analysis of unsaturated fatty acids in poly(3-hydroxyalkanoates). Study of beta-oxidation in Pseudomonas putida.

Poly(3-hydroxyalkanoates) (PHAs) were isolated from Pseudomonas putida KT2442 cultivated on petroselenic acid, oleic acid, and linoleic acid to study beta-oxidation of unsaturated fatty acids. Both saturated and unsaturated medium chain length 3-hydroxy fatty acids were found to be constituents of these polymers. With the aid of proton-detected multiple quantum coherence and proton-detected multiple bond coherence NMR spectra the structures of the unsaturated monomers were identified as 3-hydroxy-5-cis-tetradecanoate for PHA produced on oleic acid, and 3-hydroxy-6-cis-dodecanoate and 3-hydroxy-5-cis-8-cis-tetradecadienoate for PHA produced on linoleic acid. The identified structures, which are derived from fatty acid degradation intermediates, indicate a degradation of oleic acid via the enoyl-CoA isomerase-dependent route and a degradation of linoleic acid via the dienoyl-CoA reductase-dependent route.

Carbon Isotopes↗

PubMed1992

DNA sequence determination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans.

The alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides. The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane-bound and converts aliphatic medium-chain-length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants. AlkK is homologous to a range of proteins which act by an ATP-dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long-chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl-CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm. AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk-genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown. The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT-plasmid and the P. putida chromosome.

Alcohol Dehydrogenase↗

PubMed1992

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers.

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas putida KT2442 during growth on carbohydrates was studied. PHAs isolated from P. putida cultivated on glucose, fructose, and glycerol were found to have a very similar monomer composition. In addition to the major constituent 3-hydroxydecanoate, six other monomers were found to be present: 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate. The identity of all seven 3-hydroxy fatty acids was established by gas chromatography-mass spectrometry, one-dimensional 1H-nuclear magnetic resonance, and two-dimensional double-quantum filtered correlation spectroscopy 1H-nuclear magnetic resonance. The chemical structures of the monomer units are identical to the structure of the acyl moiety of the 3-hydroxyacyl-acyl carrier protein intermediates of de novo fatty acid biosynthesis. Furthermore, the degree of unsaturation of PHA and membrane lipids is similarly influenced by shifts in the cultivation temperature. These results strongly indicate that, during growth on nonrelated substrates, PHA monomers are derived from intermediates of de novo fatty acid biosynthesis. Analysis of a P. putida pha mutant and complementation of this mutant with the cloned pha locus revealed that the PHA polymerase genes necessary for PHA synthesis from octanoate are also responsible for PHA formation from glucose.

Acyltransferases↗

PubMed1990

Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints.

The oxidation of alkanes to alkanols by Pseudomonas oleovorans involves a three-component enzyme system: alkane hydroxylase, rubredoxin and rubredoxin reductase. Alkane hydroxylase and rubredoxin are encoded by the alkBFGHJKL operon, while previous studies indicated that rubredoxin reductase is most likely encoded on the second alk cluster: the alkST operon. In this study we show that alkT encodes the 41 x 10(3) Mr rubredoxin reductase, on the basis of a comparison of the expected amino acid composition of AlkT and the previously established amino acid composition of the purified rubredoxin reductase. The alkT sequence revealed significant similarities between AlkT and several NAD(P)H and FAD-containing reductases and dehydrogenases. All of these enzymes contain two ADP binding sites, which can be recognized by a common beta alpha beta-fold or fingerprint, derived from known structures of cofactor binding enzymes. By means of this amino acid fingerprint we were able to determine that one ADP binding site in rubredoxin reductase (AlkT) is located at the N terminus and is involved in FAD binding, while the second site is located in the middle of the sequence and is involved in the binding of NAD or NADP. In addition, we derived from the sequences of FAD binding reductases a second amino acid fingerprint for FAD binding, and we used this fingerprint to identify a third amino acid sequence in AlkT near the carboxy terminus for binding of the flavin moiety of FAD. On the basis of the known architecture and relative spatial orientations of the NAD and FAD binding sites in related dehydrogenases, a model for part of the tertiary structure of AlkT was developed.

Amino Acid Sequence↗

PubMed1990

Bioconversions of aliphatic compounds by Pseudomonas oleovorans in multiphase bioreactors: background and economic potential.

Pseudomonas oleovorans can grow on linear alkanes and alkenes in the hexane to dodecane range by virtue of enzymes encoded by the alk genes. By introducing selected alk genes into Pseudomonas strains and by supplying alkanes in the growth medium as a bulk liquid phase, specific alkane oxidation products can be accumulated in the alkane phase. We review the genetics and enzymology of the alk system and the potential of bioconversions in two-liquid-phase bioreactors, and suggest that such systems might eventually allow the biotechnological production of intermediate value compounds.

Alkanes↗

PubMed1989

Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads.

The fluorescent pseudomonads are classified as a group, one characteristic of which is that they do not accumulate poly-3-hydroxybutyrate (PHB) during nutrient starvation in the presence of excess carbon source. In this paper we show that prototype strains from this subclass, such as Pseudomonas aeruginosa, Pseudomonas putida, and Pseudomonas fluorescens, do accumulate poly-3-hydroxyalkanoates (PHA) when grown on fatty acids. These PHAs are composed of medium-chain-length (C6 to C12) 3-hydroxy fatty acids. The ability to form these polyesters does not depend on the presence of plasmids. A specificity profile of the enzymes involved in the biosynthesis of PHA was determined by growing Pseudomonas oleovorans on fatty acids ranging from C4 to C18. In all cases, PHAs were formed which contained C6 to C12 3-hydroxy fatty acids, with a strong preference for 3-hydroxyoctanoate when Ceven fatty acids were supplied and 3-hydroxynonanoate when Codd fatty acids were the substrate. These results indicate that the formation of PHAs depends on a specific enzyme system which is distinct from that responsible for the synthesis of PHB. While the fluorescent pseudomonads are characterized by their inability to make PHB, they appear to share the capacity to produce PHAs. This characteristic may be helpful in classifying pseudomonads. It may also be useful in the optimization of PHA production for biopolymer applications.

Fatty Acids↗

PubMed1988

Alkane utilization in Pseudomonas oleovorans. Structure and function of the regulatory locus alkR.

The OCT plasmid-localized alkBAC operon encodes enzymes for alkane hydroxylation and alkanol dehydrogenation. The positively controlled expression of the operon is very efficient in both Pseudomonas putida and Escherichia coli. Two regulatory functions have been ascribed to the regulatory locus alkR: inducer recognition and transcriptional activation of the operon. We have cloned and localized the alkR locus on a 4.9-kilobase pair SalI fragment. The alkR region was analyzed for translation productions in E. coli minicells. Two proteins were identified: a 99- and a 48-kDa peptide. The positions of the cistrons encoding these proteins were established. Both cistrons were shown to be essential for an Alk phenotype. The first cistron (alkS), which encodes the 99-kDa protein, complemented alkR mutations affecting inducer specificity. Furthermore, we found that alkS is responsible for activation of expression of the alkBAC operon since it is required for the induction of the alkB gene product alkane hydroxylase. The second cistron (alkT), which encodes the 48-kDa protein, is required for reconstitution of an Alk phenotype but has no function in regulation of alkBAC expression. Thus, the expression of the alkBAC operon is regulated by a 99-kDa protein, whereas the 48-kDa protein is probably a component of the alkane hydroxylase complex.

Bacterial Proteins↗