Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea) II. Effect of freezing,inorganic ions,and urate on O2 binding in vitro

Summary The effect of variations in [K], [Ca], [Mg], [NaCl], and [urate] on the in vitro O₂ binding properties of haemocyanin (Hc) from three talitroidean species, viz. the aquatic Apohyale pugettensis, the semi-terrestrial Megalorchestia californiana, and the semi-/euterrestrial Traskorchestia traskiana were studied. Freezing altered the cooperativity of Hc from A. pugettensis and M. californiana but not T. traskiana. Variations in [NaCl], [K], and [Mg] had no effect on cither O₂ affinity or cooperativity of the Hc except for A. pugettensis Hc where an increase in [Mg] resulted in an increase in both O₂ affinity and cooperativity. Increasing [Ca] or [urate] increased O₂ affinity of both A. pugettensis and M. californiana but not T. traskiana Hc. These effects were most marked in A. pugettensis. The results suggest a negative correlation between sensitivity to Hc effectors and the degree of terrestrial adaptation of a particular amphipod species.Abbreviations Hc haemocyanin - STR Stock traskorchestia ringer     

Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea)

Summary Haemolymph PO₂ and pH of two amphipod species, Apohyale pugettensis (aquatic) and Megalorchestia californiana (semi-terrestrial) in vivo were examined during immersion and emersion at 15 and 25°C, and also after activity in air at 15°C. For M. californiana arterial O₂ tensions were higher in air than in water. This situation was reversed in A. pugettensis, although all O₂ tensions measured for both species were comparatively high. No arterial-venous PO₂ difference was apparent in the haemolymph of quiescent M. californiana. Haemocyanin (Hc) was 100% saturated in vivo only in the following; A. pugettensis in water (15 and 25°C) and air (15°C), and M. californiana in air (15°C). The Hc of both species becomes important in O₂ transport during activity; under such circumstances the haemolymph of M. californiana delivered more O₂ to the tissues than did that of A. pugettensis, despite the greater O₂ content of the latter. The animals studied here may exhibit a stage (size class?) where cutaneous gas exchange is sufficient for resting aerobic metabolism while specialized respiratory carriers (and respiratory structures) are important in meeting the increased aerobic demands of activity or environmental stress.Abbreviations Hc haemocyanin - PO₂ partial pressure of oxygen     

Chemical and biochemical studies for the differentiation of coagulase-positive staphylococci

The cell wall composition, the configuration of lactic acid produced from glucose under anaerobic conditions, the occurrence of fructose-1,6-diphosphate (FDP) activatedl-lactate dehydrogenase (l-LDH), and the esterase pattern were determined from more than 80 strains of coagulase-positive staphylococci isolated from man and animal. Strains isolated from man, swine, bovines and hares form a rather homogencous group. They exhibit a similar cell wall composition, produce predominantlyd,l-lactate and have a characteristic and simple esterase pattern. Coagulasepositive staphylococci isolated from dogs, horses, minks and pigeons are quite distinct from typicalStaphylococcus aureus strains. They exhibit a different cell wall composition, produce onlyl-lactate, possess anl-LDH which is specifically activated by FDP, and have a quite complex esterase pattern.List of Abbreviations BBP bromphenol blue - FDP fructose-1,6-diphosphate - d-LDH d-lactate dehydrogenase - l-LDH l-lactate dehydrogenase - NAD nicotinamide adenine dinucleotide     

l-lactate or pyruvate stimulated growth of novikoff rat hepatoma cells

Summary Novikoff rat hepatoma cells (subline N1S1-67) grew when 30mm l-lactate or pyruvate was substituted ford-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lackingl-lactate, pyruvate ord-glucose, cell growth of 0.42 generation was obtained. Growth withl-lactate was dependent on thel-lactate concentration up to 30mm at which the greatest increase in cell number occurred. Significant growth did not occur whend-lactate, glycerol, acetate, α-ketoglutarate, succinate or malate, each at 30mm, was substituted ford-glucose. Growth in the medium containingl-lactate was not due to the utilization ofd-glucose or some other substrate carried into the culture with the inoculum. Medium contamination byd-glucose was insufficient to explain the growth obtained in the medium containingl-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration ofl-lactate in the medium remained unchanged. The increase in cell number cannot be explained byl-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoproteins in the medium.l-Lactate does not serve as a significant carbon or energy source in the growth of these cells. This investigation was supported by grants from the National Institute of Allergy and Infectious Disease, the National Science Foundation, and the United States Public Health Service.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Oxygen and carbon dioxide transport by the haemocyanin of an amphibious crab,Holthuisana transversa

Summary The oxygen and carbon dioxide transporting properties of the haemolymph from an amphibious Australian crab,Holthuisana transversa were investigated. Within the temperature range 15 to 35°C increasing temperature markedly decreased oxygen affinity (H=–54 kJ·mol^–1). The Bohr effect was small at all temperatures with a mean value of –0.13. Over the temperature range 15–35°C there was a significant increase in the cooperativity of oxygen binding. Changing the concentration of Ca,l-lactate or haemocyanin in the haemolymph could elicit no significant change in either O₂ affinity or cooperativity of O₂ binding. There was no evidence in support of a specific effect of CO₂ on oxygen affinity of either non-dialysed or dialysed haemolymph.The amount of CO₂ that could be carried byH. transversa haemolymph was significantly reduced by increased temperature (approx. 14 to 12.5 mmol·l^–1 CO₂). Comparisons of oxygenated and deoxygenated haemolymph at a fixed pH were unable to demonstrate the presence of a significant Haldane effect. Combining data from oxygenated and deoxygenated haemolymph the buffer value was calculated to be in the range –6.2 to –8.5 mmol·l^–1 HCO ₃ ^– ·pH unit^–1.The insensitivity ofH. transversa haemocyanin function to all modulating influences except temperature is discussed with respect to the ecology of this crab.     

Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation

Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 M phosphate two new products were formed in addition to ethanol, acetate, H₂ and CO₂: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides. The formation of 1,2-propanediol from methylglyoxal proceeded via lactaldehyde. The enzyme methylgloxal synthase was inhibited by phosphate. Clostridium glycolicum, C. nexile, C. cellobioparum, C. oroticum and C. indolis did not produce propanediol under the condition of phosphate limitation. The latter two species, however, formed d(-)-lactate.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acid-base and electrolyte regulation,and haemolymph gas transport in crayfish,Astacus astacus,exposed to soft,acid water with and without aluminium

Summary Intermoult crayfish (Astacus astacus) were exposed to acid (pH 4), soft water ([Ca⁺⁺]=100 mol·l^–1) in the absence and presence of aluminium (25 mol·l^–1) for variable time periods (up to 21 days) in order to assess the consequences for acid-base and electrolyte balance and haemolymph gas transport. Haemolymph osmolality and concentration of major ions decreased drastically and to a similar extent in acid and acid-aluminium water. Muscle tissue ion concentrations were, however, regulated at an almost constant level. A severe metabolic acidosis was gradually developed, attaining a haemolymph metabolic acid load of 6–7 mequiv·l^–1 after 12–21 days. The acidosis was partially compensated by ventilatory means, with the postbranchial haemolymph PCO₂ decreasing earlier in acidaluminium-exposed than in acid-exposed specimens. Hyperventilation seemed to be a direct acid-base regulatory response, since the rise in postbranchial PCO₂ had only minimal influence on haemolymph O₂ transport. The Bohr effect of Astacus astacus haemocyanin was low (log P₅₀/GdpH=-0.24), and the mean P₅₀ only increased from 15 to 19 mmHg after 21 days of acid exposure. The decrease in O₂ affinity with decreasing pH was accompanied by a decrease in the cooperativity of O₂ binding. The haemolymph haemocyanin concentration was not affected by acid and acid-aluminium exposure, but decreased after 21 days due to starvation. Muscle tissue aluminium concentrations were unaffected, whereas gill tissue concentrations increased in acid-aluminium exposed crayfish, most likely due to accumulation of aluminium on the gill surface. Mortality was low, and an internal hypoxia and lactacidosis was not developed in either of the experimental groups. This suggests that the gas transfer qualities of the chitincovered gills of crayfish are much less sensitive to acid and acid-aluminium stress than the gills of teleost fish.Abbreviations Hc haemocyanin - SO₂ saturation of Hc with O₂ - P ₅₀ oxygen tension of haemolymph at 50% SO₂ - n ₅₀ Hills coelficient around 50% SO₂     

Aerobic electron transport in Propionibacterium shermanii effects of cyanide

Cyanide inhibited d- and l-lactate and NADH oxidase activities of membrane particles from Propionibacterium shermanii but only at relatively high concentrations. Inhibition occurred at two different sites in the electron transport pathway. One site, with a half-maximal inhibition concentration (I _0.5) of 2 to 3 mM KCN, is located at the terminal oxidase involved in cytochrome b oxidation; the evidence is consistent with cytochrome d being the major oxidase involved. At high concentrations, cyanide inhibited reduction of cytochrome b by d-lactate (I _0.5 value 20–25 mM cyanide). A proportion of the oxygen-uptake remained uninhibited even by 100 mM cyanide; this proportion was about 80% for succinate, 30% for l-lactate, 15% for d-lactate and 10% for NADH. The oxygen uptake per mol of substrate oxidised increased with increasing cyanide concentration and was accompanied by the formation of hydrogen peroxide as a product of a cyanide-insensitive oxidase system.Abbreviations PMS Phenazine methosulphate     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Oxidation of short-chain fatty acids by sulfate-reducing bacteria in freshwater and in marine sediments

Colony counts of acetate-, propionate- and l-lactate-oxidizing sulfate-reducing bacteria in marine sediments were made. The vertical distribution of these organisms were equal for the three types considered. The highest numbers were found just beneath the border of aerobic and anaerobic layers.Anaerobic mineralization of acetate, propionate and l-lactate was studied in the presence and in the absence of sulfate. In freshwater and in marine sediments, acetate and propionate were oxidized completely with concomitant reduction of sulfate. l-Lactate was always fermented. Lactate-oxidizing, sulfate-reducing bacteria, belonging to the species Desulfovibrio desulfuricans, and lactate-fermenting bacteria were found in approximately equal amounts in the sediments. Acetate-oxidizing, sulfate-reducing bacteria could only be isolated from marine sediments, they belonged to the genus Desulfobacter and oxidized only acetate and ethanol by sulfate reduction. Propionate-oxidizing, sulfate-reducing bacteria belonged to the genus Desulfobulbus. They were isolated from freshwater as well as from marine sediments and showed a relatively large range of usable substrates: hydrogen, formate, propionate, l-lactate and ethanol were oxidized with concomitant sulfate reduction. l-Lactate and pyruvate could be fermented by most of the isolated strains.     

Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A novel transglycosylation reaction from sucrose to l-ascorbic acid by a recombinant sucrose phosphorylase from Bifidobacterium longum was used to produce a stable l-ascorbic acid derivative. The major product was detected by HPLC, and confirmed to be 2-O-α-d-glucopyranosyl-l-ascorbic acid by LC-MS/MS analysis.     

l-Galactose replaces l-fucose in the pectic polysaccharide rhamnogalacturonan II synthesized by the l-fucose-deficient mur1 Arabidopsis mutant

Arabidopsis thaliana mur1 is a dwarf mutant with altered cell-wall properties, in which l-fucose is partially replaced by l-galactose in the xyloglucan and glycoproteins. We found that the mur1 mutation also affects the primary structure of the pectic polysaccharide rhamnogalacturonan II (RG-II). In mur1 RG-II a non-reducing terminal 2-O-methyl l-galactosyl residue and a 3,4-linked l-galactosyl residue replace the non-reducing terminal 2-O-methyl l-fucosyl residue and the 3,4-linked l-fucosyl residue, respectively, that are present in wild-type RG-II. Furthermore, we found that a terminal non-reducing l-galactosyl residue, rather than the previously reported d-galactosyl residue, is present on the 2-O-methyl xylose-containing side chain of RG-II in both wild type and mur1 plants. Approximately 95% of the RG-II from wild type and mur1 plants is solubilized as a high-molecular-weight (>100 kDa) complex, by treating walls with aqueous potassium phosphate. The molecular mass of RG-II in this complex was reduced to 5–10 kDa by treatment with endopolygalacturonase, providing additional evidence that RG-II is covalently linked to homogalacturonan. The results of this study provide additional information on the structure of RG-II and the role of this pectic polysaccharide in the plant cell wall.Abbreviations AIR Alcohol-insoluble residue - d-Gal d-Galactosyl - EPG Endopolygalacturonase - ESI–MS Electrospray ionization mass spectrometry - GC–MS Gas chromatography–mass spectrometry - ¹H-NMR Proton nuclear magnetic resonance spectroscopy - l-Fuc l-Fucosyl - l-Gal l-Galactosyl - 2-O-MeFuc 2-O-Methyl l-fucosyl - 2-O-MeGal 2-O-Methyl l-galactosyl - 2-O-MeXyl 2-O-Methyl d-xylosyl - MWCO Molecular weight cut-off - RG-II Rhamnogalacturonan II - ppm Parts per million - RI Refractive index - SEC Size-exclusion chromatography - TFA Trifluoroacetic acid - WT Wild type     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Hydroxyurea,a natural metabolite and an intermediate in d-cycloserine biosynthesis in streptomyces garyphalus

It was found that hydroxyurea, l-arginine and l-citrulline respectively significantly stimulated the formation of d-cycloserine in Streptomyces garyphalus. The formation of [¹⁴C]-hydroxyurea by washed cells was demonstrated after incubation with l-[guanido-¹⁴C]-arginine and l-[ureido-¹⁴C]-citrulline. The ¹⁵N of H₂NCO¹⁵NHOH was incorporated to 40% in d-cycloserine. The mass spectrum as well as the ¹⁵N NMR spectrum of labelled N,2-dicarbobenzyloxy-d-cycloserine derived from [¹⁵N]-hydroxyurea showed that hydroxyurea was the source of the heterocyclic nitrogen in the biosynthesis of d-cycloserine.     

The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Production of l-lactic acid by Rhizopus species

Of 19 Rhizopus spp. only four produced l-lactic acid in shake-flask culture. Aerobically and in the presence of a neutralizing agent, Rhizopus oryzae NRRL 395 produced the highest concentration of l-lactic acid (65 g/l) but with O₂-limited growth ethanol was produced instead.C.R. Soccol and V.I. Stonoga are with the Laboratório de Processos Biotecnológicos, Departmento de Engenharia Química da Universidade Federal do Paraná, 81531-970 Curitiba, PR, Brazil; M. Raimbault is with the Centre ORSTOM, Laboratoire de Biotecnologie, Physiologie et Métabolisme Cellulaires, 911, Avenue Agropolis, 34032 Montpellier, Cedex 1, France.