Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis: impact on titer, analogue levels, and the identification of new analogues of pneumocandin B0

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the “serine analogues” of pneumocandins B₀ and B₅ and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B₀ and E₀, whereas pneumocandins C₀ and D₀ decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit α-ketoglutarate-dependent dioxygenases, affected the pneumocandin B₀ titer and altered the levels of pneumocandins B₁, B₂, B₅, B₆, and E₀, analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns. Journal of Industrial Microbiology & Biotechnology (2001) 26, 216–221. Received 05 November 2000/ Accepted in revised form 27 January 2001     

Energy cost and running mechanics during a treadmill run to voluntary exhaustion in humans

The aim of the present study was to examine the physiological and mechanical factors which may be concerned in the increase in energy cost during running in a fatigued state. A group of 15 trained triathletes ran on a treadmill at velocities corresponding to their personal records over 3000m [mean 4.53 (SD 0.28) m · s⁻¹] until they felt exhausted. The energy cost of running (C _R) was quantified from the net O₂ uptake and the elevation of blood lactate concentration. Gas exchange was measured over 1 min firstly during the 3rd–4th min and secondly during the last minute of the run. Blood samples were collected before and after the completion of the run. Mechanical changes of the centre of mass were quantified using a kinematic arm. A significant mean increase [6.9 (SD 3.5)%, P < 0.001] in C _R from a mean of 4.4 (SD 0.4) J · kg⁻¹ · m⁻¹ to a mean of 4.7 (SD 0.4) J · kg⁻¹ · m⁻¹ was observed. The increase in the O₂ demand of the respiratory muscles estimated from the increase in ventilation accounted for a considerable proportion [mean 25.2 (SD 10.4)%] of the increase in C_R. A mean increase [17.0 (SD 26.0)%, P < 0.05] in the mechanical cost (C _M) from a mean of 2.36 (SD 0.23) J · kg⁻¹ · m⁻¹ to a mean of 2.74 (SD 0.55) J · kg⁻¹ · m⁻¹ was also noted. A significant correlation was found between C _R and C _M in the non-fatigued state (r = 0.68, P < 0.01), but not in the fatigued state (r = 0.25, NS). Furthermore, no correlations were found between the changes (from non-fatigued to fatigued state) in C _R and the changes in C _M suggesting that the increase in C _R is not solely dependent on the external work done per unit of distance. Since step frequency decreased slightly in the fatigued state, the internal work would have tended to decrease slightly which would not be compatible with an increase in C _R. A stepwise regressions showed that the changes in C _R were linked (r = 0.77, P < 0.01) to the changes in the variability of step frequency and in the variability of potential cost suggesting that a large proportion of the increase in C _R was due to an increase in the step variability. The underlying mechanisms of the relationship between C _R and step variability remains unclear. Accepted: 15 September 1997     

Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the CC double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3–4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candidaantarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid. Received: 29 October 1998 / Received revision: 18 December 1998 / Accepted: 21 December 1998     

Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up

A combination of microbial strain improvement and statistical optimization is investigated to maximize pneumocandin B₀ production from Glarea lozoyensis ATCC 74030. Atmospheric and room temperature plasma (ARTP) was used to enhance G. lozoyensis ATCC 74030 in pneumocandin B₀ yield. Mutant strain G. lozoyensis Q1 exhibited 1.39-fold increase in pneumocandin B₀ production to 1134 mg/L when compared with the parent strain (810 mg/L). Further, the optimized medium provided another 1.65-fold in final pneumocandin B₀ concentration to 1873 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively pneumocandin B₀ production from G. lozoyensis and could be a tool to improve other secondary metabolites production by fungal strains.     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

Efficient mediator-independent hydrogenation of carbon-carbon double bonds,using the obligate anaerobe,Acetobacterium woodii,with fructose as an electron donor

Fructose and H₂ were compared as electron donors for hydrogenation of carbon-carbon double bonds using Acetobacterium woodii. Caffeate was used as a model substrate. An electron donor was required and both fructose and H₂ were suitable. With fructose as the donor, the K _s for caffeate was 0.5 mM and the V _max was 678 mmol kg_dry weight ⁻¹ h⁻¹.␣Fructose oxidation was coupled very efficiently to caffeate reduction by an alteration in the fructose fermentation so that acetate was no longer produced. Received: 24 June 1996 / Accepted: 1 July 1996     

Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Trans-pyridine tetraammine complexes of RuII and RuIII with N7-coordninated purine nucleosides

 The synthesis, spectroscopic, and electrochemical properties of trans-[L(Pyr)(NH₃)₄Ru^II/III] (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, or 4-bnpy; L=H₂O, Guo, dGuo, 1MeGuo, Gua, Ino, or G⁷-DNA) are reported. As expected, the Pyr ligand slows DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ relative to [(H₂O)(NH₃)₅Ru^II]²⁺ and favors reduction of Ru^III by about 150 mV. The pyridine ligand also promotes the disproportionation of Ru^III to afford the corresponding complexes of Ru^II and, presumably, Ru^IV. For L=Ino, disproportionation follows the rate law: d[Ru^II]/dt=k ₀[Ru^III]+k ₁[OH^–][Ru^III], k ₀=(2.7±0.7)×10^–4s^–1 and k ₁=70±1 M^–1s^–1. Received: 11 May 1998 / Accepted: 3 March 1999     

Adenosine 5′-triphosphate-mediated activation of sucrose-phosphate synthase in bundle sheath cells of C4 plants

We report the ATP-mediated activation of sucrose-phosphate synthase in bundle sheath cells prepared from C₄ species. Sucrose synthesis was followed by measuring the incorporation of [¹⁴C]fructose 6-phosphate into sucrose in bundle sheath cells also provided with uridine 5′-diphosphoglucose (UDPGlc). Studies with Panicum miliaceum L. cells showed that activation was largely due to an increase in the affinity for UDPGlc and was therefore only evident at limiting UDPGlc concentrations. The apparent K _m UDPGlc for sucrose synthesis by cells pretreated and assayed with ATP was about 0.7 mM compared with 7–8 mM for control cells without ATP. The γ-thio derivative of ATP had a similar effect to ATP. The effect was also evident when ATP was rapidly removed from cells prior to assay. Sucrose-phosphate synthase activity in extracts from cells pretreated with or without ATP showed similar differences in K _m UDPGlc. These observations support the view that ATP is inducing a covalent modification of the enzyme. However, several protein kinase inhibitors did not prevent activation. Changes of more than threefold were observed for the K _m UDPGlc with sucrose-phosphate synthase extracted from bundle sheath cells rapidly isolated from attached leaves that were subjected to dark/light treatments. The possible relationship between these changes and those induced by ATP with isolated cells is discussed. Received: 22 October 1996 / Accepted: 7 January 1997     

Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1

The synthesis of polyhydroxyalkanoates (PHA) by Pseudomonas putida PGA1, using saponified palm kernel oil (SPKO), was investigated. The PHA produced from SPKO was compared with those produced by the major free fatty acids found in the palm kernel oil. Owing to the absence of lipase activity in P.␣putida, palm kernel oil did not support cell growth. However, SPKO could support cell growth and produced relatively high yield of both dry cells and PHA. The polyester produced was similar in properties to those derived from lauric (C_12:0) and myristic (C_14:0) acids, while oleic acid (C_18:1) gave rise to PHA that was sticky and of broader molecular mass distribution. Nuclear magnetic resonance and gas chromatography showed that these PHA were copolymers consisting mainly of n-alkanoate monomers ranging from C₆ to C₁₄, with C₈ as the predominant component. PHA derived from SPKO and oleic acid also contained a small amount of unsaturated monomers. Received: 25 March 1996 / Received last revision: 30 September 1996 / Accepted: 18 October 1996     

Carbon:nitrogen ratio interacts with initial concentration of total solids on insecticidal crystal protein and spore production in Bacillus thuringiensis HD-73

A response-surface methodology was used to study the effect of carbon:nitrogen ratio (C:N) and initial concentration of total solids (C _TS) on insecticidal crystal protein production and final spore count. Bacillus thuringiensis var. kurstaki HD-73 was grown in a stirred-tank reactor using soybean meal, glucose, yeast extract, corn steep solids and mineral salts. Soybean meal and glucose were added according to a central composite experimental design to test C:N ratios ranging from 3:1 to 11:1 and C _TS levels from 60␣g/l to 150 g/l. Cry production was quantified using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The response-surface model, adjusted to the data, indicated that media with a C:N of 7:1 yielded the highest relative Cry production at each C _TS. The spore count was higher at low C:N ratio (4:1) and high C _TS (near 150 g/l). Specific Cry production varied from 0.6 to 2.2 g Cry/10¹⁰ spores. A 2.5-fold increase in C _TS resulted in a six-fold increase of protoxin production at a 7:1 C:N ratio. It is concluded that the best production conditions for Cry and for spores are different and optimization of B. thuringiensis processes should not be done on a spore-count basis but on the amount of Cry synthesized. Received: 5 September 1997 / Received revision: 22 December 1997 / Accepted: 2 January 1998     

Energetics of swimming at maximal speeds in humans

The energy cost per unit of distance (C _s, kilojoules per metre) of the front-crawl, back, breast and butterfly strokes was assessed in 20 elite swimmers. At sub-maximal speeds (v), C _s was measured dividing steady-state oxygen consumption (V˙O₂) by the speed (v, metres per second). At supra-maximal v, C _s was calculated by dividing the total metabolic energy (E, kilojoules) spent in covering 45.7, 91.4 and 182.9 m by the distance. E was obtained as: E = E _an+V˙O_2max t _p−V˙O_2max(1−e^{−( t p/)}), where E _an was the amount of energy (kilojoules) derived from anaerobic sources, V˙O_2max litres per second was the maximal oxygen uptake, α (=20.9 kJ · l O₂ ⁻¹) was the energy equivalent of O₂, τ (24 s) was the time constant assumed for the attainment of V˙O_2max at muscle level at the onset of exercise, and t _p (seconds) was the performance time. The lactic acid component was assumed to increase exponentially with t _p to an asymptotic value of 0.418 kJ · kg⁻¹ of body mass for t _p ≥ 120 s. The lactic acid component of E _an was obtained from the net increase of lactate concentration after exercise (Δ[La]_b) assuming that, when Δ[La]_b = 1 mmol · l⁻¹ the net amount of metabolic energy released by lactate formation was 0.069 kJ · kg⁻¹. Over the entire range of v, front crawl was the least costly stroke. For example at 1 m · s⁻¹, C _s amounted, on average, to 0.70, 0.84, 0.82 and 0.124 kJ · m⁻¹ in front crawl, backstroke, butterfly and breaststroke, respectively; at 1.5 m · s⁻¹, C _s was 1.23, 1.47, 1.55 and 1.87 kJ · m⁻¹ in the four strokes, respectively. The C _s was a continuous function of the speed in all of the four strokes. It increased exponentially in crawl and backstroke, whereas in butterfly C _s attained a minimum at the two lowest v to increase exponentially at higher v. The C _s in breaststroke was a linear function of the v, probably because of the considerable amount of energy spent in this stroke for accelerating the body during the pushing phase so as to compensate for the loss of v occurring in the non-propulsive phase. Accepted: 14 April 1998     

Kinetics and mechanism for reduction of anticancer-active tetrachloroam(m)ine platinum(IV) compounds by glutathione

trans -[PtCl₄(NH₃)(thiazole)] (1), trans-[PtCl₄(cha)(NH₃)] (2), cis-[PtCl₄(cha)(NH₃)] (3) (cha =cyclohexylamine), and cis-[PtCl₄(NH₃)₂] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]_tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of these protolytic equilibria. The thiolate species GS⁻ is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS⁻ are (1.43±0.01)×10⁷, (3.86±0.03)×10⁶, (1.83±0.01)×10⁶, and (1.18±0.01)×10⁶M⁻¹s⁻¹, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter to increase anticancer activity, at least for this class of compounds. Received: 8 December 1999 / Accepted: 15 February 2000     

Inheritance of high palmitic acid content in the seed oil of sunflower mutant CAS-5

 Sunflower genotypes with increased levels of palmitic acid (C16 : 0) in the seed oil could be useful for food and industrial applications. The objective of the present study was to determine the inheritance of the high C16 : 0 content in the sunflower mutant line CAS-5 (>25% of the total oil fatty acids). This mutant was reciprocally crossed with the lines HA-89 (5.7% C16 : 0) and BSD-2-691 (5.4% C16 : 0), the latter being the parental line from which CAS-5 was isolated. No maternal effect for the C16 : 0 content was observed from the analysis of F₁ seeds in any of the crosses. The inheritance study of the C16 : 0 content in F₁, F₂ and BC₁F₁ seeds from the crosses of CAS-5 with its parental line BSD-2-691 indicated that the segregation fitted a model of two alleles at one locus with partial dominance for the low content. The analysis of the fatty acid composition in the F₂ populations from the crosses with HA-89 revealed a segregation fitting a ratio 19 : 38 : 7 for low (<7.5%), middle (7.5–15%), and high (>25%) C16 : 0 content, respectively. This segregation was explained on the basis of three loci (P1, P2, P3) each having two alleles showing partial dominance for low content. The genotypes with a high C16 : 0 content were homozygous for the recessive allele p1 and for at least one of the other two recessive alleles, p2 or p3. This model was further confirmed with the analysis of the F₃ and the BC₁F₁ generations. It was concluded that both the recessive alleles p2 and p3 were already present in the BSD-2-691 line, the allele p1 being the result of a mutation from P1. This genetic study will facilitate breeding strategies associated with the incorporation of the high C16 : 0 trait into agronomically acceptable sunflower hybrids. Received: 30 March 1998 / Accepted: 13 August 1998     

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats

Anaerobic tetrachloroethene(C₂Cl₄)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C₂Cl₄ and trichloroethene (C₂HCl₃) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C₂H₂Cl₂), whereas, with l-lactate or formate, complete dechlorination of C₂Cl₄ via C₂HCl₃, cis-C₂H₂Cl₂ and chloroethene (C₂H₃Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C₂H₂Cl₂ was obtained, whereas C₂HCl₃ was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C₂Cl₄ was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h⁻¹) and influent C₂Cl₄-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C₂Cl₄ was transformed at high rates (above␣140 μmol l⁻¹ h⁻¹, corresponding to 145 nmol Cl⁻ min⁻¹ mg protein⁻¹) into cis-C₂H₂Cl₂ and C₂H₃Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C₂Cl₄) suggest that C₂Cl₄-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C₂H₂Cl₂ and C₂H₃Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l⁻¹ h⁻¹ (20 nmol min⁻¹ mg protein⁻¹), and that of the combined anoxic → oxic reactor system was 43.4 μmol l⁻¹ h⁻¹. This is significantly higher than reported thus far. Received: 17 April 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His₆-tag fusions and metal chelate affinity chromatography. In vivo His₆-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg⁻¹ and 0.035 U mg⁻¹ protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg⁻¹ when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a K _i of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 10⁴ g mol⁻¹, and which occurred independently of pre-existing PHA granules. Received: 3 December 1999 / Revision received: 10 January 2000 / Accepted: 14 January 2000     

The molecular structure of a curl-shaped retinal isomer

Computational studies of retinal protonated Schiff base (PSB) isomers show that a twisted curl-shaped conformation of the retinyl chain is a new low-lying minimum on the ground-state potential energy surface. The curl-shaped isomer has a twisted structure in the vicinity of the C₁₁=C₁₂ double bond where the 11-cis retinal PSB isomerizes in the rhodopsin photoreaction. The twisted configuration is a trapped structure between the 11-cis and all-trans isomers. Rotation around the C₁₀–C₁₁ single bond towards the 11-cis structure is prevented by steric interactions of the two methyl groups on the retinyl chain and by the torsion barrier of the C₁₀–C₁₁ bond in the other direction. Calculations of spectroscopic properties of the 11-cis, all-trans, and curl-shaped isomers provide useful data for future identification of the new retinal PSB isomer. Circular dichroism (CD) spectroscopy might be used to distinguish between the retinal PSB isomers. The potential energy surface for the orientation of the β-ionone ring of the 11-cis retinal PSB reveals three minima depending on the torsion angle of the β-ionone ring. Two of the minima correspond to 6-s-cis configurations and one has the β-ionone ring in 6-s-trans position. The calculated CD spectra for the two 6-s-cis configurations differ significantly indicating that the sign of the β-ionone ring torsion angle could be determined using CD spectroscopy. Calculations of the CD spectra suggest that a flip of the β-ionone ring might occur during the first 1 ps of the photoreaction. Rhodopsin has a negative torsion angle for the β-ionone ring, whereas the change in the sign of the first peak in the experimental CD spectrum for bathorhodopsin could suggest that it has a positive torsion angle for the β-ionone ring. Calculated nuclear magnetic resonance (NMR) shielding constants and infrared (IR) spectra are also reported for the retinal PSB isomers. Figure The figure shows the optimized molecular structure of the curl-shaped retinal isomer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induction of H2-Uptake and Nitrogenase Activities in the Cyanobacterium Anabaena variabilis ATCC 29413: Effects of Hydrogen and Organic Substrate

A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H₂-uptake [2–7 μmoles of H₂ · mg⁻¹ (Chl a) · h⁻¹] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H₂ on in vivo H₂-uptake was observed during the early stages of nitrogenase induction. However, exogenous H₂ does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth. Received: 22 November 1995 / Accepted: 22 December 1995     

Haemolymph oxygen transport and acid-base status in Glyptonotus antarcticus Eights

Haemolymph samples were withdrawn from routinely active male intermoult Glyptonotus held at 0 ± 0.5°C, and analysed for blood-gas and acid-base variables. In both the arterialised (a) and venous (v) haemolymph, over 50% of the oxygen was transported as dissolved oxygen at PaO₂ and PvO₂ levels of 12.0 ± 1.15 and 7.70 ± 1.89 kPa, respectively. The maximum oxygen-carrying capacity of the haemocyanin (C^maxHcO₂) was relatively low at 0.19 ± 0.05 mmol l⁻¹, accompanied by relatively low protein and [Cu²⁺] levels indicating low circulating haemocyanin concentrations. Arterialised haemolymph had a mean pH of 7.88 ± 0.02(6) at a PCO₂ of 0.12 ± 0.01(6) kPa and a bicarbonate level of 12.95 ± 0.80(6) mequiv l⁻¹ with small differences in PCO₂ and pH between arterial and venous haemolymph. The non-bicarbonate buffering capacity of Glyptonotus haemolymph was low at −2.0 mequiv l⁻¹ HCO₃ ⁻ pH unit⁻¹. Haemolymph [l-lactate] and [d-glucose] levels were similar at < 1 mmol l⁻¹ in animals held in the laboratory and those sampled in Antarctica. The blood-gas and acid-base status of Glyptonotus haemolymph may be a reflection of the low and stable temperatures experienced by this Antarctic crustacean. Received: 14 August 1996 / Accepted: 3 November 1996     

Determination of the kinetic parameters during continuous cultivation of the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil

A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 °C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l⁻¹ h⁻¹ at a dilution rate of 0.4 h⁻¹. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant K _S of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, μ_max, of 1.0 h⁻¹. Oxygen uptake rates of up to 2.9 g l⁻¹h⁻¹ were calculated and the yield coefficient, Y _X/O, was determined to be 0.29 g dry cell weight/g O₂. From an overall material balance the yield coefficient, Y _X/S, was estimated to be 0.60 g dry cell weight/g olive oil. Received: 8 January 1997 / Received revision: 30 April 1997 / Accepted: 4 May 1997