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Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene. The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon. This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries. In vivo overexpression consistently led to the synthesis of a 16-kDa protein. The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine. Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon. It was preceded by a cyclic AMP receptor protein motif centered at position -41.5. Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine. This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism. Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway. Expression of a caiF-lacZ operon fusion was subject to FNR regulator-mediated anaerobic induction and cyclic AMP receptor protein activation. The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors. Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.

The production of extracellular sophorose lipid by Candida apicola IMET 43747 was studied after growth on glucose as sole carbon source. In the presence of citrate and after 120 h of cultivation, the microcrystalline lactonic sophorose lipid started to appear in the medium. The lipid was shown to accumulate in the cell wall and only a minor part was liberated into the medium. By means of NMR studies it was shown that the cell wall-bound and the liberated lactonic sophorose lipid were identical and had a hydroxy Δ 8 -hexadecenoic acid backbone. Biotransformation of d -[1- 13 C]glucose revealed that in glucose-grown cells glucose was degraded to trioses which were rearranged and flow backward via the gluconeogenic pathway into the glycolipid as documented by scrambling of the 13 C label from the C-1 position into the d -[6- 13 C]glucose of sophorose. A further part of the trioses was used for de novo synthesis of the glycolipid fatty acid moiety via acetyl-CoA. By use of a mixed substrate, i.e., glucose and hexadecane, part of the glucose added was directly incorporated into the carbohydrate moiety of the lipid. These results are discussed in terms of the physiological significance of the sophorose lipid to act as extracellular carbon storage material.

Summary: The membrane-bound aldehyde dehydrogenase from ‘Acetobacter rancens’ CCM 1774, solubilized from the membrane fraction by treatment with surfactants and subsequently purified to homogeneity, was characterized with respect to the M r of the dimeric enzyme (145000), pH optimum (5·1), pI value (5·3), substrate specificity towards straight-chain aldehydes, substrate inhibition and the effects of various inhibitors and ions. Methanol extracts of the membrane fraction and of the purified enzyme showed properties identical with pyrroloquinoline quinone as revealed by spectrophotometric methods and reactivity with apoquinoprotein glucose dehydrogenase. Pyrroloquinoline quinone could not be liberated by EDTA treatment. The enzyme activity of an apoenzyme could be partly restored by addition of membrane extracts containing pyrroloquinoline quinone adducts. The solubilized, purified quinoprotein aldehyde dehydrogenase and the membrane-bound enzyme differed in substrate binding, substrate inhibition, pH optimum and linkage to a c-type cytochrome which acts as electron acceptor in membrane fractions. Since double-reciprocal plots of initial reaction rates with various concentrations of aldehyde or electron acceptor showed intersecting lines, and different inhibition patterns were obtained for the two forms of the enzyme, a ping-pong kinetic behaviour with the two reactants was excluded. The kinetic mechanism was suggested to be changed after solubilization from a random-order type to a compulsory-order type, and is thus atypical for quinoproteins reported so far.

The yeast Torulopsis apicola produces a microcrystalline glycolipid mixture which major component was identified as the novel 16-([2′-O-s-D-glucopyranosyl-s-D-glucopyranosyl]-oxy)-hexadecanoic acid 1′,4″-lactone 6′,6″-diacetate 1 originating from the fatty acid ω-hydroxylation route. Full carbon and proton n.m.r. signal assignment and structure determination is possible using the peracetylated derivative 2 .

The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l−1 h−1). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used.

Similarly to the recently described methanol dehydrogenase (MDH) from Methylocystis sp. GB 25 (Grosse et al. 1997) MDH from Methylosinus sp. WI 14 is able to catalyse the oxidation of methanol to formate directly. The enzyme was purified about 9-fold to electrophoretic homogeneity and is localised in the soluble fraction. The relative molecular mass of the native enzyme has been determined to be 140 kDa. It is composed of two identical subunits of relative molecular mass 70 kDa. The amino terminal sequence shows a strong similarity (a match of 80% over the first 20 amino acids) to the MDH from Methylocystis sp. GB 25. PQQ could be detected as the prosthetic group of MDH in the purified enzyme fraction by using the apoenzyme of a membrane-bound glucose dehydrogenase from Pseudomonas aeruginosa. A PQQ ratio of 1.3 per mole MDH was estimated. The purified enzyme has an optimum activity at pH 9.0 and at 57 degrees C. MDH from Methylosinus sp. WI 14 oxidises only primary alcohols up to octanol and several aldehydes. The estimated K(m)-values vary between 0.18 mM for the sorbic alcohol and 6.3 mM for butanol and show no dependence upon the chain length.

L-Carnitine, a quaternary ammonium compound, plays an important role in beta-oxidation of fatty acids in mammals. The increasing demand for this compound in medicine has led to the development of numerous procedures for L-carnitine production. This review discusses the possibilities of microbial and enzymatical synthesis of L-carnitine and gives an overview on the pathways of L-carnitine metabolism and related enzymes in microorganisms.

The fine structure of the cell envelope of Acinetobacter has been determined from electron micrographs of thin sections, negatively-stained and freeze-etched preparations of whole cells, and of envelopes treated to remove the various layers (Thornley and Glauert, 1968; Thorne et al., 1973; Sleytr et al., 1974; Thornley and Sleytr, 1974; Aurich et al., 1977). These early studies revealed the presence of an outer membrane typical of Gram-negative bacteria. Furthermore, there are numerous reports of superficial material covering the outer surface of several Acinetobacter strains; such material includes crystalline surface layers of protein (S-layers) (Thornley et al., 1973, 1974), and polysaccharide capsules (Taylor and Juni, 1961; Pines et al., 1983). These additional surface layers are of tremendous importance in determining the surface properties of the cells, and the polysaccharide capsular material has proven to be a potent extracellular emulsifying agent (reviewed by Rosenberg and Kaplan, 1987).

The ability to oxidise alkanes is widely distributed among prokaryotic and eukaryotic microorganisms. Acinetobacter is among the bacterial genera most often found in petroleum-contaminated habitats and has been extensively used in studies of n-alkane oxidation (Einsele, 1983). Research with this bacterium has contributed to the exploration of many aspects of microbial n-alkane metabolism.

L(-)-Carnitine (R(-)-3-hydroxy-4-trimethylaminobutyrate) is a ubiquitously-occurring substance which is essential for the β-oxidation of long-chain fatty acids in mitochondria. Bacteria are able to metabolise this trimethylammonium compound in different ways. Species of the genus Pseudomonas can utilise L-carnitine as sole source of carbon and nitrogen under aerobic conditions. After growth on L(-)-carnitine, the highly specific L(-)-carnitine dehydrogenase (EC 1.1.1.108) is induced in P. aeruginosa (Aurich et al., 1967) and P. putida (Kleber et al., 1978). A second group of carnitine-metabolising microorganisms comprising different Enterobacteriaceae, e.g., Escherichia coli, Salmonella typhimurium and Proteus vulgaris, can almost quantitatively convert L(-)-carnitine into ɣ-butyrobetaine under anaerobic conditions (Seim et al., 1980, 1982a,c,d). They do not assimilate the carbon and nitrogen skeleton. The metabolism of L(-)-carnitine by E. coli includes at least a two-step reduction of L(-)-carnitine to ɣ-butyrobetaine with crotonobetaine as intermediate (Seim et al., 1982d). The function of this reaction sequence is still unknown. Seim et al. (1982a,c,d) postulated that crotonobetaine serves as an external electron acceptor similar to (e.g.) nitrate (Haddock and Jones, 1977). One of the two enzymes involved, the carnitine dehydratase, was recently purified and characterised (Jung et al., 1989).

The n-alkane metabolizing strain Acetobacter rancens CCM 1774 possesses a dye-linked membrane-bound aldehyde dehydrogenase. The application of a sequential solubilization procedure at defined protein-detergent ratios allowed fast and effective purification without loss in enzyme activity. Both forms of aldehyde dehydrogenase—the membrane-bound and the solubilized enzyme— exhibited different properties, such as stability and electron transfer to cyto-chromes. By means of spectrophotometric investigations the presence of heme, FAD and other known groups in the purified enzyme could be excluded. Preliminary investigations with regard to the natural electron acceptor indicated the participation of PQQ in electron transfer. The fluorescence spectrum recorded for methanol extracts of the pure enzyme are comparable with those of adducts of PQQ. Inactivated aldehyde dehydrogenase could be reactivated by addition of these extracts, following saturation kinetics. Both enzyme forms catalyzed the oxidation of straigh...

A method for the separation of the outer membrane (OM) from the cytoplasmic membrane (CM) of Acinetobacter calcoaceticus 69/V grown on different carbon sources is described. The contamination of the OM with CM was less than 10%. Independent of the carbon source, five protein bands with apparent molecular weights of 47 000, 33000, 21 000, 19 000 and 12 000 were found by solubilization at 37°C and six bands at 100°C (apparent Mr 53 000, 47 000, 38 000, 26 000, 21000, 12000). Three proteins were modifiable by heat. With the periodic acid-Schiff procedure the bands with apparent Mr of 33 000 and 12 000 were made visible. After growth on d , l -carnitine an additional two non-heat-modifiable protein bands with apparent Mr between 40 000 and 45 000 were detected. By cultivation on acetate and peptone as carbon source one additional band (Mr 15 000) from OM of cells could be found.

Die Aufnahme von Pyruvat, α-Ketoglutarat, Succinat und L-Malat durch intakte nicht wachsende Zellen von Acinetobacter calcoaceticus wird in Abhangigkeit von der C-Quelle (Acetat, n-Alkane, Succinat, L-Malat, Hefeextrakt) untersucht, und einige kinetische Parameter der Aufnahme dieser organischen Anionen werden ermittelt. Der „scheinbare” KM-Wert fur die Aufnahme von Pyruvat betragt 1,8 × 10−3M, fur α-Ketoglutarat 7,6 × 10−4M, fur Succinat 3,3 × 10−4M und fur L-Malat 1,4 × 10−4M. Die Aufnahme von Pyruvat wird von α-Ketoglutarat und den untersuchten Dicarbonsauren nicht-kompetitiv gehemmt. Die Dicarbonsauren hemmen einander bei der Aufnahme kompetitiv. α-Ketoglutarat beeinflust den Transport von Succinat und L-Malat nicht. Umgekehrt dagegen wird die Aufnahme von α-Ketoglutarat durch Dicarbonsauren kompetitiv (durch Oxalacetat allerdings nicht-kompetitiv) gehemmt. Uranylacetat, Hemmstoffe der Atmungskette und Protonenleiter hemmen die Aufnahme von Pyruvat, α-Ketoglutarat, Succinat und L-Malat z. T. vollstandig.

The strain ‘Acetobacter rancens’ CCM1774 was investigated concerning the occurrence of aldehyde dehydrogenases after growth on different carbon sources. Two constitutive enzyme activities, a NADP+-dependent one and a pyridine nucleotide-independent one, have been detected. The catalytic properties of the latter membrane-bound aldehyde dehydrogenase suggest its participation in the degradation of long-chain n-alkanes.

Cell-free growth liquor of Acinetobacter calcoaceticus 69-V contains an extracellular lipase. Its activity depends on growth phase and carbon source. During growth on acetate or succinate the activity ist low or zero, respectively. Growth on alkanes causes an increase in the extracellular lipase activity. Activity reaches maximum values during the exponential phase of growth which significantly decrease in the stationary phase During the growth on alkanes some surfactants (Tauroglycocholate, Triton X-405) stimulate the excretion of the enzyme and some other (Tween, Brij, Triton X-100) inhibit the lipase and growth of cells, respectively Air-water and alkane-water interfaces inhibit the lipase activity. During starvation of the bacteria grown on alkanes lipase is excreted in the starvation medium.

The conditions for the production of extracellular glycolipid with Torulopsis apicola IMET 43747 have been investigated. Different culture conditions resulted in the production of either water-soluble or crude crystalline glycolipids. Growth was always accompanied by a strong decrease of the pH-value in the reaction medium. Cultivation at pH-value below 2 yielded the water-soluble product. The addition of either sodium citrate or sodium hydroxide to correct the pH-value to 3, resulted in the formation of large amounts of crystalline glycolipids. Depending on the kind of carbon source and its relative concentration, the product concentration was 5–90 g 1−1 with maximal yields of 0.46 g g−1 (product per substrate) after growth on a mixture of plant oils and glucose. The crude crystalline glycolipid mixture can be separated from the culture medium by filtration. It is composed of 80% of one major crystalline glycolipid and different minor compounds. Purification by liquid chromatography on silica gel yields the pure compound. This main product is a nonionic glycolipid with remarkable interfacial activities.

The levels of serum total cholesterol, LDL- and HDL-cholesterol, triglycerides and apolipoprotein B were determined in urban and rural persons of Ethiopia in dependency on age. Regarding total and LDL cholesterol a comparable age-dependency as in the population of other countries is observed. On the other hand higher values of these parameters were found in the serum of urban persons in comparison with rural subjects which may be a reflection of the different life style.