Process optimization of a continuous airlift tower-loop reactor

Based on the experimental investigations with H. polymorpha and Methylomonas M 15 in bench-scale airlift tower-loop reactors, a general distributed parameter model was developed and used to simulate to cultivation process in a 40-m-high production reactor. This general model was simplified with regard to the gas phase and loop balances and was employed to optimize cell productivity and/or profit in a 20-m-high pilot-plant airlift tower-loop reactor. Maximum cell productivity always occurs in the oxygen-transfer-limited growth range. In case of a high "penalty factor" for nonconsumed substrate, maximum profit is attained at the boundary between substrate and oxygen-transfer-limited growth. Oxygen-transfer limitation exists in the lower half of the tower, whereas in the upper half, substrate limitation prevails. The longitudinal dissolved oxygen concentration passes a minimum in this case as has been determined experimentally in the bench-scale column. The simulation results agree fairly well with the data measured in the pilot plant.     

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.     

Comparison of the solution conformations of human [Zn7]-metallothionein-2 and [Cd7]-metallothionein-2 using nuclear magnetic resonance spectroscopy.

The solution structure of native human [Zn7]-metallothionein-2 has been compared with the previously determined structure of human [Cd7]-metallothionein-2. The comparison was based on complete sequence-specific 1H nuclear magnetic resonance assignments for human [Zn7]-metallothionein-2 obtained using the sequential assignment method. The secondary structure was found to be very similar in the [Zn7]- and [Cd7]- forms of the protein. Only seven amide protons in [Zn7]- metallothionein-2 were found to have exchange rates lower than approximately 0.2 min-1 at pH 7.0 and 10 degrees C, which corresponds closely to the results of amide proton exchange studies with the [Cd7]- form of the protein. Finally, the 1H-1H distance constraints determined from nuclear Overhauser enhancement spectroscopy for human [Zn7]-metallothionein-2 were checked for compatibility with the [Cd7]-metallothionein-2 structure. Overall, although no direct method is available for identifying the metal-polypeptide co-ordinative bonds in the Zn(2+)-containing protein, these measurements provided several independent lines of evidence showing that the [Zn7]- and [Cd7]- forms of human metallothionein-2 have the same molecular architecture.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of melatonin treatment on human circadian rhythmicity before and after a simulated 9-hr time shift.

The hormone melatonin is currently proposed by some investigators to be an efficient means for decreasing the impairing effects of jet lag. Eight healthy male subjects, aged 20 to 32, underwent a 9-hr advance shift in the isolation facility of our institute during two periods each of 15 days' duration. In a double-blind, crossover design, subjects took either melatonin or placebo at 1800 hr local time for 3 days before the time shift and at 1400 hr for 4 days afterwards. The time shift was simulated on days 7 and 8 by shortening the sleep period by 6 hr and the following wake period by 3 hr. Body temperature was recorded every 90 min, and urine was collected at 3-hr intervals all day and night. Melatonin treatment enhanced the resynchronization speed of some, but not all, hormone and electrolyte excretion rates for several days after the time shift. The adaptation speed of the temperature rhythm significantly increased during one postshift day. In addition, the circadian temperature rhythm had a significantly higher amplitude under melatonin treatment than under placebo after the time displacement. For the placebo group, the rhythm of 6-hydroxymelatoninsulfate excretion exhibited an advance shift in five subjects, whereas the other three showed a delay shift, and adjustment did not achieve more than one-half of the expected value within 8 days. A significantly different adjustment could be observed in the melatonin-treated group: Seven subjects underwent an advance shift of the expected 9 hr within an average of 8 days. The results suggest that melatonin treatment can accelerate resynchronization of the melatonin excretion rhythm after eastward time zone transitions. The improvement is not, however, sufficiently great that we can recommend melatonin for the alleviation of jet lag.     

The fine structure of calcified Mandl's corpuscles in teleost fish scales

Calcified Mandl's corpuscles present in the internal layer (or fibrillary plate) of the teleost fish scale were studied by transmission and scanning electron microscopy for a better understanding of this special type of mineralization process. The corpuscles show a great variability in their structure, form and surface features depending on the arrangement of the collagen fibrils in the internal layer of the different fish species studied, on the localization of the corpuscles in the scale and on the technical treatment to which the scale is subjected.     

Ribonucleotide reductase: A structural study of the dimeric iron site

The iron-containing B2 subunit of ribonucleotide reductase from Escherichia coli has been investigated by Raman spectroscopy. Both the tyrosyl radical-containing native protein and the radical-free protein exhibit a resonance-enhanced Raman band at 500 cm^?1. This band is assigned to an Fe-O vibrational mode arising from an oxygen-containing ligand. The failure to observe any tyrosinate ring modes makes it unlikely that ribonucleotide reductase is an iron-tyrosinate protein and rules out tyrosinate oxygen as a ligand. It is proposed that the 500 cm^?1 band in ribonucleotide reductase is analogous to the 510 cm^?1 Fe-O vibrational mode of methemerythrin and arises from an oxo- or carboxylate-bridge between the antiferromagnetically-coupled Fe(III) ions.     

Investigation of the structure of two-phase flow fermentation model media in bubble-column bioreactors

Summary Electrical conductivity microprobes have been used to estimate the transverse variation of bubble size, local gas holdup and local specific gas/liquid interfacial area in bench scale bubble column bioreactors containing fermentation model media. Inserted O₂-electrodes and plane parallel windows alter the structure of the two phase flow. Even slight tilting of the column strongly influences the transverse profiles of the bubble size and local gas holdup. The larger bubbles are collected at the wall, where they can be redispersed. These observations open up new possibilities for the construction of bubble column bioreactors.