Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits.

To test the potential usefulness of transgenic rabbits as production systems for human proteins of pharmaceutical value, we cloned the rabbit beta-casein promoter and fused it to the genomic sequence of the human interleukin-2 (hIL2) gene. Four transgenic female rabbits were tested for expression and biological activity of the foreign protein in their milk. The milk of all four females proved to contain biologically active hIL2. The results show that transgenic rabbits may represent a convenient and economic system for the rapid production of biologically active protein in milk.


Microbial glycolipid production under nitrogen limitation and resting cell conditions.

Rhodococcus erythropolis is able to synthesize an anionic trehalose-2,2',3,4-tetraester during cultivation on n-alkanes. Preconditions for an overproduction are nitrogen limitation, temperature- and pH-shift. The optimum carbon source was technical grade n-C-10, which led to 0.35 g g-1 of glycolipid per n-alkane. Electron microscopical observations showed that n-C-14,15 (technical grade) grown cells contained numerous lipid inclusions in contrast to n-C-10 (technical grade) grown cells. Nocardia corynebacteroides synthesizes a novel pentasaccharide lipid and as size products small amounts of trehalose-corynomycolates. Optimum precursors for overproduction are n-alkanes from n-tetradecane to n-hexadecane with yields in the range of 0.17 g g-1 of glycolipid per carbon source.


Immobilization of enzymes with polyaziridines.

A novel method of enzyme immobilization using a low molecular weight prepolymer of tri-functional aziridines which can immobilize enzymes both by covalent attachment and entrapment within a gel matrix is described. The enzymes are immobilized on a solid support and exhibit an excellent retention of enzymatic activity. The immobilization procedure is essentially a single step process which can be easily performed at room temperature or 4 degrees C in either aqueous solution or in an inert organic solvent. The polyaziridines used in the immobilization are nontoxic, available in bulk at low cost and completely miscible with water and many organic solvents, thus providing one of the most satisfactory methods of immobilization available.


Continuous itaconic acid production by immobilized biocatalysts.

The continuous itaconic acid production from sucrose with Aspergillus terreus TKK 200-5-3 mycelium immobilized on polyurethane foam cubes was optimized in column bioreactors using statistical experimental design and empirical modelling. The highest itaconic acid product concentration calculated on the basis of the obtained model was 15.8 g l-1 in the investigated experimental area, when sucrose concentration was 13.5%, aeration rate 150 ml min-1 and residence time 178 h. From sucrose with immobilized A. terreus TKK 200-5-3 mycelium itaconic acid production was stable for at least 4.5 months in continuous column bioreactors. In comparison, using glucose as substrate and immobilized A. terreus TKK 200-5-1 mycelium as biocatalyst similar stability was obtained with higher product concentration. The omission of copper sulphate from the production medium gave the highest itaconic acid product concentration (26 g l-1) from 9% glucose with 0.25% ammonium nitrate and 0.095% magnesium sulphate.


Trimethyl lead degradation by free and immobilized cells of an Arthrobacter sp. and by the wood decay fungus Phaeolus schweinitzii.

The continuing production of leaded petrol generates liquid wastes containing recalcitrant trialkyl lead, for which no suitable chemical treatment has been formulated. This investigation explores the feasibility of using microorganisms to catalyse the rate-limiting step of trimethyl lead degradation to dialkyl lead; this disproportionates chemically to give, ultimately, Pb2+ which is treatable by classical methods. An Arthrobacter sp. and a wood decay macrofungus, Phaeolus schweinitzii provide novel evidence for metabolic trimethyl lead (Me3Pb+) degradation. The retention of this activity in immobilized cell column reactors challenged with Me3Pb(+)-supplemented flows suggests that a future biotreatment process may be possible.


Thermodynamic evidence of trophic microniches in methanogenic granular sludge-bed reactors.

The Gibbs free energy changes in methanogenic granular biomass from sludge-bed reactors were evaluated using the in situ concentrations and partial pressures of metabolites during the metabolism of acetate, hydrogen, formate and propionate. Based on mass balance calculations it appeared that the degradation of propionate into acetate, hydrogen and bicarbonate was endergonic, even if propionate was effectively degraded. On the other hand, the methane-producing reactions, both from acetate and from hydrogen plus bicarbonate, were found to be exergonic and the free energy change was sufficient for the formation of ATP. Formate was detected in only one of the two reactors. When formate, instead of hydrogen, was considered as the electron carrier between propionate-degrading and methanogenic bacteria, similar thermodynamic results were obtained. The existence of trophic microniches in the granular biomass is suggested to explain propionate degradation even though the Gibbs free energy change in the liquid surrounding the granules was positive. Hence, to make propionate degradation exergonic the dissolved hydrogen concentration surrounding the propionate-degrading bacteria would have to be about 30 times lower than in the free liquid.


Hyaluronic acid--a versatile biopolymer.

Since its discovery from bovine synovial fluid, hyaluronic acid has found application in several diverse areas. It is now extensively used in ophthalmic surgery, in the treatment of lameness in racehorses and as an ingredient in cosmetics. It can also be used in drug delivery, orthopedics, cardiovascular aids and in wound healing. Even so, its potential as a therapeutic agent is yet to be fulfilled. New improved products, formed by crosslinking hyaluronic acid with itself or other chemicals, are being produced. Hyaluronic acid is clearly a most valuable therapeutic biopolymer. This article reviews the properties of hyaluronic acid, its biosynthetic pathway and its methods of commercial production, and provides an estimation of its current and future world demand. Experiments designed to improve production economics have been performed in our laboratory. These involved control of glucose and yeast extract concentrations and fermentation pH. Hyaluronic acid production by Streptococcus zooepidemicus was found to be lower at pH 6.5 than at pH 7.1. A new method of extending the production of hyaluronic acid by S. zooepidemicus into the stationary phase has also been achieved.


How ANUTECH transfers technology.

In summary, we would draw your attention to the things that are important, when transferring technology from a higher education institution. 1. The institute should have a separate department to deal with these matters. 2. We see advantages in this being a private company. 3. The type of agreements should be flexible so that most projects can be accommodated and having specialized staff helps.


A TAFE strategy for biotechnology skills training.

The outline of a strategy for training technicians who will undertake biotechnology-oriented tasks as employees is the focus of this review. Technical and Further Education (TAFE) in Western Australia has recently restructured electives offered in certificate and diploma courses of Applied Science. The aim of restructuring was to allow a broader choice catering for specialized streams. With a few additions it would be possible to list a set of biotechnology options for technicians specializing in this area. After identifying employers in Western Australia, data on technician employment characteristics and training needs was gathered through a survey and used to formulate a future development strategy. In the past little attention has been given anywhere in Australia to the training needs of this group. The availability of such appropriately trained employees will increasingly be recognized as a vital link in the success of any biotechnology enterprise.


Characterization of peptidyl-nucleoside antifungal antibiotics from fermentation broth.

Characterization of sinefungin related antifungal antibiotics from fermentation broth was accomplished by coupling photodiode array (PDA) detection to high performance liquid chromatography (HPLC). From the combined HPLC-PDA evaluation of broth filtrate, we detected five sinefungin related components. Fast atom bombardment (FAB) mass spectroscopic evaluations, mass-analysed ion kinetic energy spectra (MIKES) and collision activated (CA) MIKES of these components confirmed their respective identities. Our findings from the combination of HPLC photodiode array acquisition and FAB-mass spectrometry suggest we have detected the presence of a previously unreported sinefungin analogue.


Environmental factors influencing methanogenesis in a shallow anoxic aquifer: a field and laboratory study.

The environmental factors influencing methanogenesis in a shallow anoxic aquifer were probed in a combined field and laboratory study. Field data collected over a year revealed that 'in situ' rates of methane production were depressed in winter and elevated in summer. Over the same period, ground water pH values ranged from 6.0 to 7.8 while temperatures varied from 7-22 degrees C. 'In situ' methanogenesis was severely inhibited at temperatures less than 13 degrees C or by pH values less than 7. The influence of these factors on microbial methane formation from both endogenous and exogenous substrates were tested in aquifer slurries adjusted to pH 5-9 and incubated at temperatures ranging from 5-45 degrees C. Temperature optima for methane production from endogenous substrates varied as a function of pH, but the pH optimum was 8 at all temperatures. Optimal conditions for acetoclastic methanogenesis were found at pH 8 and 35 degrees C. An analysis of variance revealed that pH, temperature, and a pH-temperature interaction are all significant variables influencing aquifer methanogenesis. In addition transient sulfate accumulations were also found to limit methane production in some areas. A comparison of field and laboratory methane production patterns suggest that pH, temperature, and sulfate accumulations are important, but not the only environmental variables influencing the mineralization of organic matter in shallow aquifers.


Characterization of the requirements and substrates for reductive dehalogenation by strain DCB-1.

An obligately anaerobic bacterium known as strain DCB-1 was grown under a variety of conditions to determine the requirements for dehalogenation as well as factors which stimulated or inhibited the process. Dechlorination was obligately anaerobic since introduction of O2 immediately inhibited the reaction. Sulfuroxy anions, which also serve as electron acceptors for DCB-1, inhibited dechlorination but NO3- and fumarate did not. The optimum growth medium for dechlorination was 0.2% Na pyruvate and 20% rumen fluid in basal salts. Media with either pyruvate or rumen fluid alone did not support dechlorination. DCB-1 also consumed H2 but typical substrate concentrations of H2 (80 kPa) delayed dechlorination. Once the H2 concentration was reduced to less than 20 microM (2.67 kPa), dechlorination resumed. Dehalogenation by DCB-1 was restricted to the meta substituted benzoates as halogens in other positions and chloroaromatic compounds with other functional groups were not dechlorinated.


Antibiotic sensitivity testing of bacteria by microcolony inhibition and image analysis.

A rapid method for determining the sensitivity of Staphylococcus aureus to beta-lactams is described. The method involves the culture of microcolonies of the bacterium on the surface of antibiotic-containing agar medium on microscope slides. The areas of the microcolonies formed are measured by means of a microscope and image analyzer. The sensitivity of the bacterium can be estimated in 4 h by this technique and the results are comparable to those obtained by overnight incubation.


Enumerating low densities of genetically engineered Erwinia carotovora in soil.

An inexpensive, quantitative, and sensitive technique was developed for detection of genetically engineered Erwinia carotovora in soil samples. Enrichment media, antibiotic resistance, and most probable number (MPN) analysis were used to enumerate as few as 1 to 10 target cells/10 g soil. The MPN technique recovered significantly higher cell densities than plating; however, densities estimated by the two techniques were strongly correlated. After inoculation of soil microcosms with genetically engineered E. carotovora, a decline rate of 1.2 log units/g soil/10 days and then subsequent disappearance was observed using the MPN technique.


Oxygen derepresses deacetoxycephalosporin C synthase and increases the conversion of penicillin N to cephamycin C in Streptomyces clavuligerus.

When dissolved oxygen (DO) was maintained at saturation level during batch fermentations of Streptomyces clavuligerus (NRRL 3585), the accumulation of the intermediate penicillin N was lowered while formation of the end product cephamycin C was increased relative to fermentations without DO control. The specific activity of the penicillin ring-expansion enzyme deacetoxycephalosporin C synthase (DAOCS) was increased 2.3-fold under oxygen saturated conditions, whereas the penicillin ring-cyclizing enzyme isopenicillin N synthase (IPNS) showed only a 1.3-fold increase. Thus oxygen derepression of DAOCS appears to be an important regulatory mechanism in the conversion of penicillin N to cephamycin C in S. clavuligerus. IPNS, an early acting enzyme in cephamycin C biosynthesis, and DAOCS, which acts late in the pathway, both disappeared from cell extracts at 60 h, just prior to cessation of cephamycin production.


Ultrafiltration membranes as carriers for lipase immobilization.

The feasibility of directly incorporating lipase from Rhizopus in the interior of poly(vinyl chloride) ultrafiltration membranes during the phase inversion process for their manufacturing has been demonstrated. The obtained membranes used for plant oil hydrolysis have shown better time stability as compared with those of lipase immobilized by adsorption. The specific activity of entrapped lipase achieves values higher than those of the soluble one. The activity of immobilized lipase is strongly affected by the rate of removing fatty acids from the interior of the membrane.


Papain-catalysed synthesis of dipeptides: a novel approach using free amino acids as nucleophiles.

For the first time, papain-catalysed synthesis of peptide bonds was successfully carried out using free amino acids as nucleophiles. In kinetically controlled experiments employing pH-Stat-mode, the ester substrates Z-Ala-OMe and Z-Gly-OMe were coupled with alanine, glutamine, and Cys(Acm)-OH, respectively. Under optimized reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 10 mumol substrate mg-1 papain), the peptide yields ranged from 17% to 79%, depending on the structure of the amino and/or carboxyl component. The peptides formed were not hydrolysed under the chosen reaction conditions. With Z-Gly-OMe as the ester substrate, formation of the dipeptide was both rapid and high yielding. Papain-catalysed formation of peptide bonds applying free amino acids as nucleophiles might serve as an economic and easily manageable approach for the synthesis of short-chain peptides to be used in clinical nutrition.


Enhancement of cyclosporin production in a Tolypocladium inflatum strain after epichlorohydrin treatment.

Following treatment of conidia of the cyclosporin producer fungus, Tolypocladium inflatum, with 0.15 M epichlorohydrin, strain M6 was isolated. The new strain exhibited a similar growth rate to the parent organism but more extensive conidiation and several-fold higher overall cyclosporin production. Strain M6 reached titres of 318 mg l-1 cyclosporin A in agar cultures, whereas in liquid medium it produced 140 mg l-1 cyclosporin A and 68 mg l-1 cyclosporin C. It also maintained a steady volumetric productivity of 0.48 mg l-1 h-1 cyclosporin A over 2 weeks of submerged cultivation in maltose-based semisynthetic medium. The new strain holds potential for improved cyclosporin production due to the superior titres and demonstrated capacity to sustain elevated production of cyclosporin for periods greater than the wild type.


Synthesis and biological activity of some new 2-chlorophenothiazine derivatives.

10-(Hetero/arylthio)acetyl-2-chlorophenothiazines II have been synthesized via interaction of 10-chloroacetyl-2-chlorophenothiazine I with heterocyclic and/or aromatic potassium mercaptide in ethanol. Oxidation of II using H2O2/CH3COOH mixture was studied. Structures of the products were verified by elemental and spectral analyses. Some compounds were screened in vitro for their antibacterial activities.


Landfill co-disposal of phenol-bearing wastewaters: organic load considerations.

A multi-stage model, operated with single elution, was used to investigate the effects of organic loadings on the attenuation of a model phenolic wastewater in domestic refuse. Although 100% dissimilation of influent phenol (2-5 mmol dm-3) was recorded at a dilution rate of 0.007 h-1, partial inhibition of both phenol degradation and species competing with methanogens for a common electron donor(s) was apparent at concentrations greater than or equal to 4 mmol dm-3. On extended perfusion with 8 mmol phenol dm-3, the progressive inhibition of phenol dissimilation was not obviated by nutrient supplementation. Simultaneous degradation of the catabolic intermediate, hexanoic acid, and elevated methane release rates suggested that the transformation of phenol to hexanoate was rate limiting.


Mass spectral characterization of acyloxymethyl phosphates.

A series of organophosphates having one or two acyloxymethyl groups have been characterized by methane chemical ionization mass spectrometry: twelve are model compounds with phenyl and benzyl groups while four are derived from pyrimidine-3'-deoxyriboside-5'-monophosphates. Some are protonated cyclohexyl-ammonium salts. In addition the phenyl and benzyl phosphate spectra exhibit an abundance of fragmentation and rearrangement ions, the majority of which were products of three processes--expulsion of formaldehyde, loss of the acyloxymethyl group with transfer of hydrogen to the phosphate, and fission of the methylene-acyloxy bond. The pyrimidine-3'-deoxyriboside-5'-monophosphate esters give less fragmentation under similar circumstances; prominent ions in their spectra are the protonated molecular ions and losses of one and two molecules of formaldehyde from them and the protonated bases.


Production, thermal stability and immobilisation of inulinase from Fusarium oxysporum.

Fusarium oxysporum produced maximum extracellular inulinase after 9 days of its growth at 25 degrees C on a medium (pH 5.5) containing 3% fructan and 0.2% sodium nitrate. The level of this enzyme decreased on the addition of either glucose, fructose, galactose or sucrose to F. oxysporum already growing on a fructan-containing medium. A significant increase in invertase production which resulted in an increase of the invertase/inulinase (S/I) ratio, was observed on addition of inulin to this fungus growing on other carbon sources. Glycerol (10%) gave better protection to inulinase against thermal denaturation at 50 degrees C compared to ethylene glycol and sorbitol. Inulinase immobilised in polyacrylamide gel retained 45% of its original activity. The immobilised enzyme showed a higher optimum temperature (45 degrees C) compared to free enzyme (37 degrees C). The immobilised enzyme after storage at 25 degrees C for 96 h showed 58% activity. Thermal stability of entrapped inulinase increased in the presence of inulin.


Comparison of oxygen supply methods for cultures of shear-stress sensitive organisms including animal cell culture.

In animal cell culture, oxygen supply sometimes limits cell growth. Therefore, four oxygen supply methods (free surface aeration, porous Teflon tubing, perfluorocarbon addition and external aerator) were compared in terms of oxygen transfer rate for suspension culture of animal cells. Oxygen transfer rate with the free surface aeration was dependent on the vertical position of the impeller and the stirred Reynolds number for h/HL greater than 0.35, but no effect of the impeller position was observed for h/HL less than 0.35. The relationship between the Reynolds and Sherwood numbers could be expressed by simple correlations. These correlations could also be applied to oxygen transfer using porous Teflon tubing. When oxygen was supplied with perfluorocarbon saturated with air, the overall mass transfer coefficient KL was estimated to be about 9.0 X 10(-3) cm s-1, which was five-fold greater than that of the surface aeration. Maximum cell densities which would be supported by four methods were calculated from values of KLa for different sizes of fermentor assuming that oxygen supply would be the rate-limiting factor in animal cell culture.


Biotransformation of alkyl and aryl carbonates. Microbial degradation.

An enriched mixed culture was successfully grown on model alkyl and aryl carbonates. These compounds were degraded by microorganisms at different rates. P-Chlorophenyl-2-octyl carbonate and p-nitrobenzyl-2-octyl carbonate were metabolized through the formation of p-chlorophenol and p-nitrobenzyl alcohol respectively. A strain of Acinetobacter calcoaceticus isolated from the mixed culture utilized phenyl-2-octyl carbonate by an intracellular hydrolase to phenol and 2-octanol which were further metabolized.


Genetic complementation of Streptomyces tendae deficient in nikkomycin production.

Streptomyces tendae Tü 901 produces the nucleoside peptide antibiotic nikkomycin. In shot-gun cloning experiments using pIJ699 as vector we isolated a 9.4-kb DNA fragment from S. tendae which complemented the nikkomycin nonproducing mutant NP9 to the formation of nikkomycin C/Cx and Kx. Nikkomycins were identified by HPLC analyses and their characteristic UV spectra. In Southern hybridization experiments the cloned DNA exclusively reacted with S. tendae DNA sequences. As shown by Northern dot blotting, transcripts of the isolated DNA fragment were only detected during stationary growth and correlated with the extent of nikkomycin production. When the recombinant plasmid pNP113 containing the 9.4-kb DNA fragment was transferred into the over-producing mutant Tü901/S2566, transformants exhibited a significantly decreased capacity for forming nikkomycin. Southern analysis of genomic DNA of these transformants revealed that severe rearrangements occurred in DNA sequences homologous to the 9.4-kb insert of pNP113.


Optimum conditions for efficient transformation of Streptomyces venezuelae protoplasts.

Optimum conditions for protoplast regeneration and transformation of Streptomyces venezuelae ETH14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10(7) transformants/micrograms DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30 degrees -32 degrees C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10(5) regenerating protoplasts per plate. Higher protoplasts densities considerably decreased the regeneration frequency of the transformants.


Screening of basidiomycetes for lignin peroxidase genes using a DNA probe.

Basidiomycetes were screened for lignin peroxidase (LPO) genes using a DNA probe prepared from the LPO restriction fragment of Phanerochaete chrysosporium. Southern blot analysis showed restriction fragments of chromosomal DNA of Bjerkandera adusta and Coriolus consors hybridized with the probe. Bjerkandera adusta produced LPO in a glucose-peptone medium. Ion-exchange chromatography showed that this fungus produced multiple molecular forms of LPO. One of the enzymes, LPO-2, was purified and characterized. The molecular weight of LPO-2 was 41,000 with a pI of 4.2. Spectral analysis demonstrated that LPO-2 is a haem protein. The enzyme cleaved lignin model dimers mainly at the C alpha-C beta position of the side chain. The LPO-2 exhibited close similarity to LPOs of P. chrysosporium with respect to their basic properties.


Cloning of a DNA fragment from Corynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase.

The Corynebacterium glutamicum/Escherichia coli shuttle vector plasmid pZ1 was used to clone the S-(2-aminoethyl)-D,L-cysteine (AEC)-resistance gene from a lysine-excreting, AEC-resistant strain of C. glutamicum, the aspartokinase activity of which was released from feedback inhibition by mixtures of lysine and threonine or AEC and threonine respectively. A recombinant plasmid designated pCS2 carrying a 9.9-kb chromosomal insert that conferred AEC resistance and the ability to excrete lysine to its host was isolated. The aspartokinase activity of the pCS2-carrying strain was resistant towards inhibition by mixtures of lysine and threonine or AEC and threonine respectively. By deletion analysis the DNA region conferring AEC resistance to the host and feedback resistance to its aspartokinase activity could be confined to a 1.2-kb DNA fragment.


Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate.

Actinorhodin production in Streptomyces coelicolor A3(2) was relatively insensitive to the carbon source concentration but was elicited by nitrogen or phosphate depletion, or by a decline in the growth rate. In starch-glutamate media with nitrogen limitation, increasing the nitrogen supply delayed the onset of antibiotic synthesis and, at concentrations above 30 mM, decreased its rate. In a similar medium with phosphate limitation, increasing the initial phosphate concentration delayed actinorhodin formation and, above 2.5 mM, reduced the rate of synthesis. Experiments in which actinorhodin synthesis was elicited by phosphate depletion at various nitrogen concentrations demonstrated strong suppression by residual glutamate. Cultures in which actinorhodin biosynthesis was initiated by nitrogen depletion were not similarly suppressed by increasing amounts of residual phosphate. The results suggest that actinorhodin production in S. coelicolor A3(2) responds to interacting physiological controls, notable among which is nitrogen catabolite regulation.


Effect of the addition of microbial surfactants on hydrocarbon degradation in a soil population in a stirred reactor.

The hydrocarbon degradation rate could be doubled by the addition of sophorose lipids as biosurfactants in a model system containing 10% soil and a 1.35% hydrocarbon mixture of tetradecane, pentadecane, hexadecene, 1,2,4-trimethylcyclohexane, pristane (2,6,10,14-tetramethylpentadecane) phenyldecane and naphthalene suspended in mineral salts medium. The adaptation phases for two degradation phases were shortened, and the extent of degradation and final biomass were increased. The added biosurfactants were degraded after they had facilitated degradation of all hydrocarbon components.


Degradation of phenanthrene, fluorene and fluoranthene by pure bacterial cultures.

Bacterial mixed cultures able to degrade the polycyclic aromatic hydrocarbons (PAH) phenanthrene, fluorene and fluoranthene, were obtained from soil using conventional enrichment techniques. From these mixed cultures three pure strains were isolated: Pseudomonas paucimobilis degrading phenanthrene; P. vesicularis degrading fluorene and Alcaligenes denitrificans degrading fluoranthene. The maximum rates of PAH degradation ranged from 1.0 mg phenanthrene/ml per day to 0.3 mg fluoranthene/ml per day at doubling times of 12 h to 35 h for growth on PAH as sole carbon source. The protein yield during PAH degradation was about 0.25 mg/mg C for all strains. Maximum PAH oxidation rates and optimum specific bacterial growth were obtained near pH 7.0 and 30 degrees C. After growth entered the stationary phase, no dead end-products of PAH degradation could be detected in the culture fluid.


Production of correctly processed human serum albumin in transgenic plants.

We have used a modified CaMV 35S promoter to direct the expression of chimaeric genes encoding human serum albumin (HSA) in transgenic potato and tobacco plants. To secrete the protein, either the human prepro-sequence or the signal sequence from the extracellular tobacco protein PR-S was used. We demonstrate secretion of HSA with both types of signal sequences in transgenic leaf tissue and in suspension cultures. HSA produced in transgenic potato plants was purified to chromatographic homogeneity. N-terminal amino acid sequence analysis revealed that the processing of the precursor protein was dependent on the type of signal sequence. Expression of the human preproHSA gene lead to partial processing of the precursor and secretion of proHSA. Fusion of HSA to the plant PR-S presequence resulted in cleavage of the presequence at its natural site and secretion of correctly processed HSA that is indistinguishable from the authentic human protein.


Regulation of isopenicillin N synthetase (IPNS) gene expression in Acremonium chrysogenum.

Total RNA was extracted daily from the beta-lactam antibiotic producing fungus A. chrysogenum strain CO728 during a 7 day cephalosporin C fermentation. IPNS mRNA species, with a size of about 1.5 kb, were detected by Northern blotting at high levels between days 2 and 4. The rapid appearance of IPNS mRNA in mycelial extracts up to day 2 suggests that IPNS is regulated at the transcriptional level. Primer extension and S1 endonuclease mapping studies indicate the existence of two major and at least two minor transcription initiation start sites. There was no change in the relative levels of the four transcripts during the period they could be detected. A region upstream of the IPNS structural gene (pcbC) has been sequenced and the transcription initiation sites appear as major and minor pairs on either side of one of the pyrimidine-rich blocks that punctuate the promoter sequence.


An investigation of engineering parameters for the use of immobilized biomass particles in biosorption.

Immobilized, inactive mycelia of Rhizopus arrhizus are preferential to native biomass for use in the biosorption of metal ions. Refinement of a proprietary immobilization technique previously developed at McMaster University enabled production of particles of immobilized Rhizopus arrhizus biomass having a 12-23% wt of polymer additive. The effects of production stage parameters on the intrinsic uptake capacity of the immobilized biomass were examined. Kinetic experiments showed the following trends: a decrease in the weight percent of the added polymer leads to an increase in the apparent uranium uptake capacity of the immobilized biomass particles for a given contact time. A decrease in the particle size improved the kinetics of metal uptake and led to an increase in the apparent uranium uptake capacity for the same contact time. An increase in the initial concentration of the uranium solution caused equilibrium conditions to be attained faster.


Biomass production by a thermotolerant yeast: Hansenula polymorpha.

Biomass production at high temperature by Hansenula polymorpha as part of a lignocellulosic utilizing process was studied. Compromise growth conditions (45 degrees C and pH = 4.8) with an eventual saccharification step were established. The effects of stirring rate and initial glucose concentration on biomass yield coefficient, volumetric productivity and maximal cell density were determined. Process optimization led to a fed-batch fermentation process: high yield (0.63 g dry cell g-1 glucose), volumetric productivity (1.3 g dry cell dm-3 h-1) and cell concentration (60 g dry cell dm-3) were obtained. At these conditions, significant arabitol excretion (18 g dm-3) as a unique by-product associated with cell mass production was obtained, making more interesting a high temperature operating process.


Effect of diffusional resistances on the action pattern of immobilized alpha-amylase.

Alpha-amylase from Aspergillus oryzae has been immobilized onto corn grits and porous silica (specific areas 180 and 440 m2 g-1). Kinetic parameters of immobilized enzyme have been determined. Immobilization of alpha-amylase results in the formation of less polymerized products resulting in an apparent decrease in the number of transglycosylation reactions, for both maltotetraose and starch as substrates, when compared with free enzyme. Diffusional limitations for substrate and products have been quantified in the case of the three supports used. External diffusional resistances were important in all cases for the reaction products, whilst they became negligible for the substrate in the case of silica supports. Moreover, internal transfer limitations were identified with silica 180 m2 g-1 support. It was demonstrated that diffusional resistances were in direct relation to the apparent modification of the enzyme action pattern after immobilization.


Factors influencing the activity and thermostability of immobilized porcine pancreatic lipase.

Lipase from porcine pancreas was immobilized on cellulose beads having various degrees of hydrophobicity, by covalent linking and by hydrophobic adsorption. Lipolytic activity was measured in heterogeneous organic-aqueous systems of various hydrophobicities using olive oil as a substrate. The main factors influencing lipase activity were hydrophobicity of the reaction mixture and of the carrier. Carriers with increased hydrophobicity enhanced lipase activity more than less hydrophobic ones. Lipase immobilized covalently on cellulose beads was less active than that adsorbed onto tritylcellulose but was considerably more thermostable.


Metabolic activation in isolated rat hepatocytes.

Hepatocytes were exposed in vitro to the hepatocarcinogen N-hydroxy-2-aminofluorene (N-OH-AAF) in order to determine the nature and repair of DNA adducts formed. N-OH-AAF formed 3 DNA adducts, N-(deoxyguanosin-8yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF). The removal of these adducts was measured up to 38 h following cessation of exposure to N-OH-AAF. The dG-C8-AAF adduct was removed with a half-life of about 10 h, while the other two remained relatively constant throughout the incubation period. The dG-C8-AAF adduct is probably responsible for the induction of unscheduled DNA synthesis (UDS) in this model in vitro system.


Natural transformation in Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus is a metabolically versatile microorganism that is naturally competent for DNA uptake and incorporation. We have exploited the natural state of competency for studies involving the cloning, organization and expression of genes encoding catabolic enzymes. A. calcoaceticus is able to take up, at high efficiency, genetically engineered DNA, incorporate the DNA and stably maintain and express the DNA. Sequence analysis of cloned A. calcoaceticus DNA reveals a great deal of internal repetition and secondary structure, but no specific sequences associated with uptake appear to be present. Uptake and transformation occurs in solid and liquid medium, at a wide range of DNA concentrations and with little restriction barrier to the source of the transforming DNA.


The recombinant human parvoviruses for gene therapy of hemoglobinopathies.

Towards a goal of using adeno-associated viruses (AAV), the human parvovirus, as the gene transfer vector for gene therapy of hemoglobinopathies, the human beta-globin (h beta G) cDNA was ligated downstream of the P40 promoter of AAV type 2 (AAV2) genome. Transfection via electroporation of the construct into human 293 cells (embryonal kidney cell line) resulted in expression of the cloned h beta G cDNA, as evidenced by the synthesis of transcripts hybridizable to h beta G probe. The transfection led to the recombinant genome to be excised out of the plasmid and replicate in the cell, followed by production of the recombinant AAV that harbors h beta G cDNA.


Intramolecular thiolester linkages in apolipoprotein B.

Intramolecular thiolester bonds in apolipoprotein B (ApoB) were studied using [14C]methylamine (MA) to cleave the thiolester and [3H]- or [14C]iodoacetate (IA) to titrate the newly generated sulfhydryls. Covalent incorporation of [14C]MA and [3H]carboxylmethyl group into the previously carboxymethylated LDL or the reduced and carboxymethylated ApoB was observed and both radioactivities coincided with ApoB-100 band on SDS-polyacrylamide gel electrophoresis. The [14C]MA-labeled ApoB was completely trypsinized and cross-linked to the activated thiol Sepharose 4B beads. The peptides were eluted with DTT and the free -SH groups blocked with IA then separated on FPLC. Two fractions contained [14C]MA. Sequence analyses showed that these labeled peptides contained Cys-51 and Cys-3734, respectively. Evidence suggests that the thiolester is formed between Cys-51 and gamma-Glu-54 for one, and Cys-3734 and beta-Asp-3737 for the other, with Lys and a hydrophobic amino acid, Val/Leu, in between. This is the first evidence for the presence of intramolecular thiolester linkages in ApoB. The presence of high energy, labile thiolester bonds may explain many of the unusual properties of ApoB and LDL.


Expression of chimeric human transferrin genes in transfected human tumor cell lines.

The iron-binding plasma protein transferrin (TF) is essential for supplying iron to cells and the prevention of iron toxicity. Our laboratory has cloned and characterized the human TF gene. Comparison of promoter regions of TF genes from human, chicken, and mouse reveals a strong nucleotide sequence conservation. This study demonstrates that 5' flanking regions of the TF gene are sufficient for directing expression of a heterologous gene in transgenic mice and transfected cells. For cell-specific expression, more than 150 base pairs appear to be required.


Determination of oxygen profiles in biocatalyst particles by means of a combined polarographic oxygen microsensor.

When studying the effect of immobilization of enzymes or whole cells on the conversion of substrate, more information is gained if measurements of substrate inside the biocatalyst particles are possible. With the methods used until now, only measurements outside the particle can be performed. In this article a method for measuring oxygen profiles in a biocatalyst particle under steady state conditions is described. The biocatalyst particle was made of agarose and contained the enzyme L-lactate 2-monooxygenase. This enzyme decarboxylates lactic acid to acetic acid in the presence of oxygen. The experiments were carried out in a flow chamber with the use of a micromanipulator and a stereomicroscope. The data were sampled by means of a computer. Four different profiles were measured using four different enzyme concentrations. The measured oxygen profiles were reproducible and the signal was very stable. It was also possible to measure the boundary layer around the particle. With the use of the oxygen microsensor, measurements in a biocatalyst particle could be performed accurately, giving way for model validation.


Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.


New monoclonal antibodies to 3 beta-hydroxy-gibberellins.

Two new monoclonal antibodies to 3 beta-hydroxy-gibberellins are described. Monoclonal antibodies GA1-1 and GA1-2 were derived from immunizations with the hapten-protein conjugate gibberellin A1-17-KLH. Cross-reactivities with a panel of 43 gibberellins and gibberellin derivatives are compared with those of other anti-gibberellin monoclonal antibodies.


Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.


Composition of the gum from Combretum paniculatum and four other gums which are not permitted food additives.

Only three gum exudates are permitted for pharmaceutical and food use by international regulatory authorities, viz. gum tragacanth (Asiatic Astragalus spp.), gum karaya (Sterculia spp.) and gum arabic [Acacia senegal (L.) Willd.], but a wide range of other tree exudates is used for a variety of uses in their countries of origin. This paper presents analytical data for the gum exudates from Atalaya hemiglauca, Cassine aethiopica, Combretum paniculatum, Sclerocarya birrea, and Pseudocedrela kotschyi. These gums may have local technological applications, but are not recommended for addition to foodstuffs.


Phlinosides A, B and C, three phenylpropanoid glycosides from Phlomis linearis.

Three new phenylpropanoid glycosides, phlinosides A, B and C were isolated from a methanolic extract of the aerial parts of Phlomis linearis. On the basis of chemical and spectral evidence their structures were determined as 3,4-dihydroxy-beta-phenylethoxy-O-beta-D-glucopyranosyl-(1----2)-a lpha-L- rhamnopyranosyl-(1----3)-4-O-caffeoyl-beta-D-glucopyranoside, 3,4 dihydroxy-beta-phenylethoxy-O-beta-D-xylopyranosyl-(1----2)-alpha- L- rhamnopyranosyl-(1----3)-4-O-caffeoyl-beta-D-glucopyranoside and 3,4-dihydroxy-beta-phenylethoxy-O-alpha-L-rhamnopyranosyl-(1----2) -alpha- L-rhamnopyranosyl-(1----3)-4-O-caffeoyl-beta-D-glucopyranoside, respectively.


Two flavonol glycosides from the underground parts of Vancouveria hexandra.

Two new flavonol glycosides were isolated from the roots and rhizomes of Vancouveria hexandra. The glycosides were determined by chemical and spectral means to be anhydroicaritin 3-galactosyl(1----3) rhamnoside-7-glucoside and anhydroicaritin 3-[6-O-acetyl-galactosyl(1----3)rhamnoside]-7-glucoside.


Flavonol triglycosides from Blackstonia peroliata.

Three new flavonol triglycosides quercetin, kaempferol and isorhamnetin 3-rhamnosyl(1----2)galactoside-7-glucosides have been isolated from leaves and stems of Blackstonia perfoliata. This species together with three other genera of the tribe Gentianeae, subtribe Chlorae: Centaurium, Coutoubea and Eustoma, is unusual in producing flavonol glycosides instead of C-glycosyl flavones, the more characteristic flavonoid constituents of the Gentianaceae.


Flavonol diglycosides from Blackstonia perfoliata.

Two unusual diglycosides, quercetin and kaempferol 3-rhamnosyl(1----2)galactosides and the new isorhamnetin 3-rhamnosyl(1----2)galactoside have been isolated from the aerial parts of Blackstonia perfoliata. Instead of C-glycosylflavones, the occurrence of flavonol glycosides in this species as well as in three other genera of the Gentianaceae: Centaurium, Coutoubea and Eustoma, is in agreement with the grouping of these four genera in the subtribe Chlorae of the Gentianeae.


Comparative studies on the fermentative production of lantibiotics by staphylococci.

The production of the lanthionine-containing polypeptide antibiotics gallidermin from Staphylococcus gallinarum TU 3928 and pep 5 from S. epidermidis 5 is investigated with respect to regulation and stimulation of productivity by media components, optimization of both the media used and the fermentation process and is compared to the production of the lantibiotic epidermin from S. epidermidis TU 3298. Efficient methods for rapid quantification of lantibiotics, optimization of the media and a primary enrichment by adsorption chromatography are reported.


Purification and characterization of acid urease from Lactobacillus fermentum.

Acid urease was purified to an electrophoretically homogeneous state, and the molecular weight was estimated to be 220,000. The enzyme consisted of three kinds of subunits, designated alpha, beta and gamma, with molecular weights of 67,000, 16,800 and 8600, respectively, in a (alpha 1 beta 2 gamma 1)2 structure. The isoelectric point of the enzyme was 4.8. The nickel content was found to be 1.9 atoms of nickel per alpha 1 beta 2 gamma 1 unit. The amino acid profile was different from those of known bacterial neutral ureases. The enzyme was most active at pH 2 and around 65 degrees C. It was stable between pH 3 and 9, and below 50 degrees C. The Km for urea was 2.7 mM at pH 2. The enzyme activity was inhibited by Ag+, Hg2+, Cu2+, p-chloromercuribenzoate and acetohydroxamate. The enzyme was separated into three subunits by reverse phase HPLC. The amino terminal amino acid sequences of the subunits alpha, beta and gamma were Ser-Phe-Asp-Met-, Met-Val-Pro-Gly- and Met-Arg-Leu-Thr-, respectively.


Chitosan-poly(acrylic acid): mechanism of complex formation and potential industrial applications.

The food industry is interested in polyelectrolytic coagulants of natural origin for the clarification of food beverages and the recovery of colloidal and dispersed particles from processing waste streams. This paper discusses potential industrial applications of recent findings on polymer complex formation obtained with a chitosan-poly(acrylic acid) model system. Process recommendations could be made on the basis of the ionic strength, pH, and charged group concentration of the fluid to be treated. Ionic strength does not affect the complex formation process. The amount of chitosan in the complex formed is controlled by the solution pH. The mechanism of complex formation indicates that pH measurements could be used to monitor the coagulation process.


Mechanism of the stimulatory effect of cyclodextrins on lankacidin-producing Streptomyces.

Gel-filtration analysis of a mixture of cyclodextrin (CyD) and lankacidin C showed that beta-CyD had strong, gamma-CyD weak and alpha-CyD no affinity for lankacidin C. Lankacidin C production activity, which was assayed by measuring the incorporation of L-[methyl-14C-]methionine into the lankacidin molecule, was the greatest with cells grown in the presence of beta-CyD, less with gamma-CyD and the least with alpha-CyD. Lankamycin and T-2636M, which are by-products in lankacidin C fermentation, were not included by beta-CyD and their production was not stimulated by beta-CyD. It was apparent that the stimulatory effect of CyD was closely related to the formation of an inclusion complex between CyD and the antibiotic. Lankacidin C biosynthesis was repressed by preincubating cells with lankacidin C, while the repressive effect of lankacidin C was abrogated by the inclusion by beta-CyD. Thus, abrogation of feed-back repression seems to be a main mechanism of the effect of CyD. However, alpha-CyD, which had no affinity for lankacidin C, stimulated the production to the least extent and exhibited a complementary effect on the stimulation by beta-CyD or gamma-CyD, alpha-CyD also caused a change in cell morphology and cell-surface hydrophobicity. It was assumed that the modification of the cell surface is a secondary mechanism of the effect of CyD.


Experimental collision efficiencies of polymer-flocculated animal cells.

The determination of particle collision kinetics is useful to decouple the effects of process parameters on individual events in flocculation. This paper discusses the effects of flocculation conditions on the collision efficiency of ATCC strain CRL 1606 hybridomas flocculated with poly-L-histidine. Experimental determinations of the collision efficiency of cells in Couette flow are presented over a range of experimental conditions. The collision efficiency correlates with the cell zeta potential to the -2.4 power at high surface coverage, consistent with literature results in latex systems. At low coverage, accounting for the distribution of polymer on the cells corrects for deviation from the high-coverage behavior. Collision is dependent on the hydrodynamic environment as well. At high surface coverage, collision efficiency is weakly dependent on hydrodynamic conditions and follows a dependency on the shear rate and viscosity to the -0.32 power. This is consistent with ionic coagulation theory. At low surface coverage, the collision efficiency is strongly dependent on the viscous fluid forces. The results versus both dose and shear rate over the entire range of surface coverages are consistent with weak intercell bonding. Collision kinetics in the presence of high molecular weight dextrans show steric hindrance to cell collision.


Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585.

We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various beta-lactam-producing Streptomyces spp. but absent in species which are not known to produce this class of antibiotics. Furthermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicillin N synthase gene of the cephalosphorin pathway.


Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.


Highly sensitive and fast detection of Phoma tracheiphila by polymerase chain reaction.

A new method for the diagnosis of the plant pathogenic fungus Phoma tracheiphila has been developed. The method takes advantage of the enzymatic amplification of a specific 102 bp-long target sequence of fungal DNA by the polymerase chain reaction (PCR) using Thermus aquaticus DNA polymerase. The amplified DNA was characterized by agarose-gel electrophoresis, molecular hybridization using a synthetic oligonucleotide probe and direct sequencing. The application of the new method makes possible fast and direct detection of the pathogen in lignified plant tissues, a goal not previously achieved when a cloned probe and a dot-blot test were employed. In addition the PCR test can be used to advantage as a particularly simple and fast way of typing fungal isolates. This is achieved by submitting to DNA amplification crude homogenates of fungal mycelium and analysing the amplified DNA on an agarose mini-gel.


Two-barrel bile-acids-sensitive microelectrodes based on liquid ion exchanger.

Several liquid membrane microelectrodes sensitive to bile acids (two barrel, tip diameter about 0.5 micron) are described. The results of different liquid ion exchangers such as Aliquat 336/decanol, trioctylmethylammonium/decanol, hexadecyltrimethylammonium/decanol, benzyldimethylhexadecylammonium/decanol, hexadecyltributylammonium/5% hexachlorobenzene + 0.5% bromoacetanilide in o-dichlorobenzene are compared with each other, and the better one among them is the mixture of benzyldimethylhexadecylammonium cholate/decanol with hexadecyltributylammonium taurocholate/5% hexachlorobenzene + 0.5% bromoacetanilide in o-dichlorobenzene because of its quicker response time and low drift. The calibration curves, slopes, test limits, selective coefficients, drifts, and response times of the various bile-acids-sensitive microelectrodes in different calibration solutions were demonstrated and compared with each other.


Bacterial metabolism of side-chain-fluorinated aromatics: unproductive meta-cleavage of 3-trifluoromethylcatechol.

Sixteen bacterial strains capable of degrading alkylbenzenes and alkylphenols were directly isolated from soil and water. The degradation pathways are discussed. Alkylcatechols are almost exclusively cleaved via meta-ring fission. Meta-cleavage of 3-trifluoromethyl-(TFM)-catechol was observed with all strains at different rates although the reaction rates compared to catechol as a substrate varied considerably. All 2-hydroxy-6-oxohepta-2,4-dienoic acid hydrolases investigated showed strong binding of 7,7,7-trifluoro-2-hydroxy-6-oxohepta-2,4-dienoic acid, the ring fission product of 3-TFM-catechol. Turnover rates, however, were negligible indicating this compound to be a general dead-end metabolite during metabolism of TFM-substituted compounds via meta-cleavage pathways.


Correlation of biocatalytic activity in an organic-aqueous two-liquid phase system with solvent concentration in the cell membrane.

Results presented here show that loss of progesterone 11 alpha-hydroxylase activity in Rhizopus nigricans in aqueous-organic two-liquid phase and cosolvent systems correlates well with the concentration of solvent in the cell membranes. Rhizopus nigricans is shown to retain full 11 alpha-hydroxylase activity at saturating aqueous phase concentrations of hexane and the higher primary alcohols. This reflects their inability to attain a critical concentration in the cell membranes. The relationship between our own findings and the previously described correlation of the logarithm of the partition coefficient with activity retention is explained and design parameters are proposed that may be used to select solvents for future biocatalytic systems.


Simultaneous determination of glucose, ethanol and lactate in alcoholic beverages and serum by amperometric flow injection analysis with immobilized enzyme reactors.

Glucose, ethanol and lactate were determined simultaneously in a flow injection system by using a parallel configuration of immobilized enzyme reactors. Hydrogen peroxide produced was monitored amperometrically at the potential of +0.65 V vs. Ag/AgCl. Linear relations between sensor responses and each species were observed in the ranges of 0.02-10 mM (glucose), 5 x 10(-4)-0.1% (v/v) (ethanol) and 0.005-1 mM (lactate) with correlation coefficients larger than 0.999 for each species. The relative standard deviations for 10 successive injections were 1.4, 0.5 and 1.1% for glucose (1 mM), ethanol (5 x 10(-3)% (v/v] and lactate (0.05 mM), respectively. Analysis of serum samples was performed with urate-eliminating reactors which were set just before each immobilized enzyme reactor. Interference of ascorbate in a serum sample was completely eliminated by using an ascorbate-eliminating reactor which was set before the sample injection valve. Application of the system to alcoholic beverages and control serum was described and the results were compared with those of free enzymatic, spectrophotometric analysis (F-kit or C-test method).


A flow injection analysis system involving immobilized NADH oxidase in column form for clinical analysis.

A highly sensitive FIA system for chemiluminometric determination of reduced coenzyme, NADH, was developed, using immobilized NADH oxidase from Brevibacterium ammoniagenes. The enzyme catalyzed the oxidation of NADH generating hydrogen peroxide which emitted chemiluminescence when mixed with luminol and potassium ferricyanide. The immobilized enzyme reactor was a mini-column, measuring 1 or 2 mm in inner diameter and 20 mm in length, and the sample volume was only 1 microliter per assay, with a feeding speed of one sample per min and a lowest detection limit of 10 pmol NADH. A FIA system was also developed for the determination of magnesium in human serum, using an enzyme column reactor with simultaneously coimmobilized hexokinase, D-glucose-6-phosphate dehydrogenase, and NADH oxidase. The performance of the system was as satisfactory as a routine colorimetric assay, but with much higher sensitivity.


Determination of alanine aminotransferase in human serum in an open-closed flow injection configuration.

Open-closed flow injection systems allow the development of methods based on multidetection by using conventional detectors. The methods proposed here for the determination of alanine aminotransferase (ALT) utilize a photometric detector included in the closed circuit, which allows one to perform fixed-time and reaction-rate measurements whose sensitivity can be increased by using the sum of the analytical signals. The methods, with determination ranges between 1 and 500 U l-1 of ALT, feature sampling frequencies up to 60 h-1. Their application to serum samples provided excellent results, with recoveries between 95 and 106% and averaging at 99.9%.


Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference.


Prerequisites for the on-line control of microbial processes by flow injection analysis.

Problems associated with the use of biosensors in process control, e.g. difficulties of sterilization and sensor fouling, are shortly displayed, and possibilities to overcome them are outlined. The advantages of flow injection analysis (FIA) are demonstrated and examples for efficient sampling systems connected with this method are reviewed. Special emphasis is given to problem-orientated sample pretreatments, preventing fast inactivation of immobilized enzymes in the analysis system. Examples of problem-orientated sample pretreatment units are given. A proposal for a computer-controlled self-calibrating FIA system is given.


An automated spectrophotometric flow-injection procedure for the determination of cellulase activity in bioreactor preparations.

A spectrophotometric procedure for the determination of cellulase activity in precipitated bioreactor preparations and culture filtrates is described. It is based on the determination of reducing sugar produced by the action of the enzyme on carboxymethylcellulose. The reducing sugar is derivatized with p-aminobenzoyl-hydrazide and permits a limit of detection of 0.1 U ml-1 cellulase in the presence of background sugar, with a sampling rate of 5 h-1. The method can readily be applied to the determination of any carbohydrase acting on soluble substrates and producing reducing sugars, e.g. amylase, dextranase, xylanase, glucanase and polygalacturonase.


Monitoring of enzymes during chromatographic separations.

An on-line enzyme assay is presented based on flow injection techniques combined with fluorimetric detection. It allows to monitor NAD-dependent oxidoreductases during the purification of microbial crude extracts or partially purified enzymes by fast protein liquid chromatography (FPLC) in a near real-time mode. The arrangement is simple and can be easily integrated in the chromatographic system avoiding dead volumes. A high measuring frequency (up to 180 samples h-1) and a short response time (10-30 s) are achieved. The method has a low limit of detection (approximately 0.01 U ml-1), and a good reproducibility (1-4%), the injected sample volume is only 2 microliters.


Urease immobilized on chitosan membrane: preparation and properties.

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65 degrees C for the free urease and 75 degrees C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.


Growth and hyoscyamine production of 'hairy root' cultures of Datura stramonium in a modified stirred tank reactor.

The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg.g-1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g-1 wet weight). Growth at 30 degrees C was initially faster than at either 25 degrees C or 35 degrees C and by day 12, the drained weight of roots in the fermentor at 30 degrees C was about fourfold greater than at 25 degrees C and twice that at 35 degrees C. The ultimate hyoscyamine levels attained (approximately 0.5 mg.g-1 wet weight) were similar at both 25 degrees C and 30 degrees C but some 40% lower at 35 degrees C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25 degrees C and the highest production rate of 8.2 mg hyoscyamine 1(-1) per day was obtained for roots grown at 30 degrees C. In continuous fermentation at 25 degrees C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30 degrees C or 35 degrees C.


A study of N- and P-dependence of nikkomycin production in continuous culture with immobilized cells.

The influence of nitrogen and phosphate on the biosynthesis of nikkomycin was studied in chemically defined medium. Cells of Streptomyces tendae were immobilized on porous glass particles in a fluidized-bed reactor for continuous production of nikkomycin. Phosphate had no significant influence on the biosynthesis of nikkomycin. However, even a very low concentration of phosphate in the production medium (0.0125 mmol/l) resulted in microbial growth on the particles. The concentration of nitrogen was highly effective in the regulation of the biosynthesis of nikkomycin. A high level of antibiotic production (maximum 3.05 mg/g dry cell weight per hour) was maintained for a period of about 200 h in a medium that contained nitrogen at a concentration of 0.2 g NH4NO3/l.


The use of free and immobilised Arthrobacter simplex in organic solvent/aqueous two-liquid-phase reactors.

The use of free and immobilised Arthrobacter simplex (NCIB 8929) for steroid delta 1-dehydrogenation in two-liquid-phase, stirred-tank reactors has been compared. Product formation is related to the logarithm of the water-octanol partition coefficient (log P) of the organic solvent employed, but the relationship is different for the two forms of the biocatalyst. No reaction was seen with either biocatalyst in media containing solvents of log P less than or equal to 2.5. For free bacteria, product formation rose linearly with log P thereafter to a maximum at a value of 9.8. With immobilised bacteria, product formation reached a maximum with a solvent of log P = 4.0 and remained constant with solvents of higher log P value. Consequently extended reactor operation was possible with immobilised bacteria, and the production of high quality (greater than 95% purity) steroid product was demonstrated.


Transformation of microcrystalline hydrocortisone by free and immobilized cells of Arthrobacter simplex.

The transformation of microcrystalline hydrocortisone by free and immobilized cells of Arthrobacter simplex resulted in formation of prednisolone. The effect of medium composition, non-ionogenic surfactants and exogenous electron acceptors on the delta 1-dehydrogenase activity of free and immobilized cells is described.


Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium.

The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp. Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h-1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methylphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions. 2,4-DP-Grown cells (10(8] of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h. Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol.


Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1.

The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55 degrees C, and optimum pH was 6.5-7.5, giving a minimum generation time of 12.6-13.9 h (mu max = 0.050-0.055 h-1) and a maximum value of the specific acetate consumption rate (qmaxs) of 14-20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55 degrees C and at constant pH 7 resulted in a Km value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively.


Quantitative flow measurements in bioreactors by nuclear magnetic resonance imaging.

We have developed nuclear magnetic resonance (NMR) flow imaging techniques to measure fluid flow in a cell-free hollow fiber bioreactor (HFBR). Using 1H NMR we track the motion of protons and obtain velocity distributions as a function of position and time. These measurements enable the visualization of flow patterns needed for module design and for establishing desired operating conditions. Uneven flow in the cell-containing region of an HFBR can result in concentration gradients and uneven cell distribution that may lead to reduced cell viability. Results from this non-invasive method could be used to design more efficient cell bioreactors or membrane separation devices.


Expression and secretion of a functional scorpion insecticidal toxin in cultured mouse cells.

We have expressed a synthetic gene encoding the insecticidal neurotoxin of scorpion Androctonus australis (AaIT) in NIH/3T3 mouse fibroblast cells under the transcriptional control of a murine retroviral long terminal repeat. The secretion of the toxin into the culture medium was directed by the signal peptide of human interleukin-2. The recombinant AaIT produced was selectively toxic to yellow-fever mosquito larvae and harmless to mice.


A new method for protein crystallization using high pressure.

Crystallization methods for proteins have been the subject of decades of development yet protein crystallization remains the limiting step in structural studies. We present here a new method for protein crystallization--based on the use of high pressure--that enabled us to accelerate dramatically the growth of glucose isomerase crystals. We think this method may have a more general utility.


Long-term continuous synthesis of aspartame precursor in a column reactor with an immobilized thermolysin.

N-(Benzyloxycarbonyl)-L-asparty-L-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25 degrees C with the substrates (N-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl2 with or without the substrate at 25 degrees C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h-1.


Investigations on cephalosporin C adsorption kinetics and equilibria.

The kinetics and equilibria of cephalosporin C adsorption on different commercial adsorbents were investigated. Adsorption isotherms could be analysed according to the Brunauer, Emmett and Teller theory. For the interpretation of adsorption kinetics it was necessary to develop a more complex model comprising both a rapid and a slower step. An integrated approach combining kinetics and equilibria allowed for simulation of the experimental data and can be used as a basis for predicting technical approaches such as fluidized-bed technology.


Chromone glycosides from Schumanniophyton magnificum.

Two new chromone glycosides, schumanniofiosides A and B have been isolated from the root bark of Schumanniophyton magnificum and their structures shown to be 2-methyl-5,7-dihydroxychromone 5-O-beta-D-glucopyranoside and 2-methyl-5,7-dihydroxychromone 7-O-beta-D-glucopyranosyl-(1----2)-apiofuranoside, respectively. The structures were elucidated by a combination of spectral data and chemical degradation.


Biotransformation of digitoxigenin by ginseng hairy root cultures.

Five new compounds (three esters and two glycosides) and seven previously reported compounds were isolated as biotransformation products of digitoxigenin by ginseng hairy root cultures. The new esters and glycosides were elucidated as digitoxigenin stearate, digitoxigenin palmitate, digitoxigenin myristate, 3-epidigitoxigenin beta-D-gentiobioside and digitoxigenin beta-D-sophoroside using 1H and 13CNMR and FAB mass spectral data. Biotransformations involving esterification (of stearic acid, palmitic acid, myristic acid and lauric acid) and glycosylation (of gentiobiose and sophorose) of digitoxigenin have been demonstrated for the first time in the plant cell and tissue cultures. The hairy roots showed high glycosylation ability to the digitoxigenin molecule.


A galactomannan from Crotalaria medicaginea seeds.

A polysaccharide isolated from the seeds of Crotalaria medicaginea is composed of D-galactose and D-mannose in the molar ratio of 10:31. Structural studies were performed by methylation analysis, partial acid hydrolysis, chromic oxide oxidation, mild hydrolysis with dilute oxalic acid and 13CNMR analysis of the polymer.


Steroidal glycosides from Agave cantala.

Two new steroidal glycosides, agaveside A and B, isolated from the fruits of Agave cantala were characterized as 3 beta-O-[beta-D-xylopyranosyl-(1----2),beta-D-xylopyranosyl-(1----3), beta-D-glucopyranosyl-(1----3)-[beta-D-xylopyranosyl-(1----3)-beta-D- galactopyranosyl-(1----2)]-beta-D-glucopyranosyl]-(25R)-5 alpha-spirostane and 3 beta-O-[beta-D-xylopyranosyl-(1----2), beta-D-xylopyranosyl-(1----3)-beta-D-glucopyranosyl-(1----3)- [beta-D-galactopyranosyl-(1----2)]-beta-D-glucopyranosyl]-(25R)-5 alpha-spirostane. The structures were elucidated by a combination of 13CNMR spectroscopy, chemical degradation and fast atom bombardment mass spectrometry.


Affinity immobilization of a genetically engineered bifunctional hybrid protein.

Protein A from Staphylococcus aureus (SpA) is a receptor for the Fc domain of several classes of antibodies including immunoglobin G (IgG). A hybrid protein consisting of protein A and the enzyme beta-lactamase has been constructed using recombinant DNA techniques. The functional characteristics of the hybrid protein adsorbed on IgG-coated Sepharose matrices were studied in detail and compared to those of (i) the hybrid protein in solution and (ii) beta-lactamase covalently immobilized on CNBr-activated Sepharose. Protein A--beta-lactamase bound tightly and specifically to IgG-Sepharose and could be stored for at least 4 weeks without dissociation. The rate of penicillin G hydrolysis by the beta-lactamase domain of the immobilized hybrid protein was found to depend on the amount of IgG covalently coupled to the support. For all IgG loads, higher specific activities and lower Km values relative to covalently immobilized beta-lactamase were obtained. Adsorption of the hybrid protein on the support resulted in increased stability to thermal deactivation. These results indicate that bifunctional hybrid proteins can be useful for the affinity immobilization of enzymes.


Conceptual design of a cellular filter to remove trace viral contaminants in blood.

Various process alternatives and designs of using a filter containing cellular adsorbents to remove trace viral contaminants from blood and other protein solutions have been studied. Sterilization charts have been developed that can be used to estimate the filter size required to achieve a desired sterilization criterion. A parametric study was carried out to identify various process parameters that may affect this physical trace removal process. It has been demonstrated that the adsorption rate constant is a critical parameter in the design of an efficient cellular filter for viral contaminant removal. This constant is characteristic of the virus-cell system under consideration and is shown to be particularly sensitive to the cell surface receptor density, adsorbent diameter, and fluid flow rate. Higher log titer reduction in virus concentrations can be achieved with low flow rates and no recycle. Preliminary analyses indicate the feasibility of using a magnetically stabilized fluidized filter (MSFF) reactor design for effective virus removal from these complex solutions.


Protein separation using membrane-encapsulated soluble ligand conjugates.

A new approach for isolating and recovering biological macromolecules using membrane-encapsulated soluble ligand conjugates was investigated. Membrane-encapsulated solid adsorbents have been successfully developed and employed in our laboratory to isolate and purify proteins and enzymes directly from culture broths. This new concept also makes it possible to use soluble ligand conjugates instead of solid adsorbents inside membrane capsules. In this work, model membrane-encapsulated soluble and insoluble ligands comprising Blue Dextran and Blue Sepharose entrapped within calcium alginate membranes were studied to compare adsorption characteristics such as capacities and rates. Experimental results suggest that membrane-encapsulated soluble ligands may be expected to result in higher overall adsorption capacity compared to membrane-encapsulated solid adsorbents with comparable adsorption rates.


Chemical decomposition of glutamine in cell culture media: effect of media type, pH, and serum concentration.

The chemical decomposition of glutamine to ammonia and pyrrolidonecarboxylic acid was studied at 37 degrees C in a pH range of 6.8-7.8 in different media preparations containing various amounts of fetal bovine serum. The media type influenced the decomposition rate, and the first-order rate constants increased with increasing pH values. The serum concentration had little or no effect on the decomposition rate. The importance of chemical decomposition of glutamine on the analysis of glutamine and ammonia metabolism was illustrated by an example of batch cultivation of a hybridoma cell line. The difference between the apparent uptake rate of glutamine and the actual uptake rate (which is corrected for the chemical decomposition) is shown to be as high as 200%. Similar discrepancy between the apparent and actual ammonia production rate is observed. Mathematical analysis was carried out to develop the relationship between the apparent and actual glutamine uptake and ammonia production rates. The analysis reveals that there are three important dimensionless parameter ratios that govern the difference between the apparent and actual glutamine uptake and ammonia production rates.


Multimolecular process in a packed-bed immobilized enzyme reactor: numerical simulation and back-mixing effects.

In a previous report, we presented a new analytical model describing the performance of a packed-bed catalytic unit, where the reaction between two cosubstrates is catalyzed by an enzyme immobilized on a porous carrier. The model explicitly takes into account the changes in concentrations of both cosubstrates along the reactor, as well as the hydrodynamic regimen (i.e., back-mixing) prevailing in the packed bed. In the present report, and on the basis of the procedures developed, we present a detailed analysis of the performance of the reactor. With numerical simulations, the effects of internal diffusion limitations, the depth of the pores, the substrates' concentration in the feed, and kinetic parameters are evaluated. Particular attention is also given here to the back-mixing effects prevailing in the reactor. An experimental procedure for assessing their extent is described.


Patents and technology transfer.

Although the discovery and transfer of technology from universities to industry has been taking place for many years, the surge of activity in areas related to biotechnology, over the past fifteen years, has been remarkable. As the very essence of university research requires rapid publication of results, it is particularly important that timely patenting activity take place if an orderly and profitable transfer of technology is to occur.


HIV-1 testing: product development strategies.

Strategies for the development of diagnostic products for acquired immune deficiency syndrome (AIDS) are inextricably linked to the status of our knowledge of the human immunodeficiency virus (HIV) and the events associated with the pathogenesis of AIDS. This review traces product development strategies from 1984 to the present day. Product development activities in the HIV-1 antibody screening test market were a response to the need to remove contaminated units from the blood supply. With the successes in screening blood and blood products, there has been a shift towards product development for personal health care and applicant suitability. Identification of markers for disease progression and the need to monitor therapeutic efficacy is now leading to tests for patient disease staging, monitoring and prognosis.


Approaches to HIV vaccine design.

Contemporary vaccines are relying increasingly on modern biotechnology and a vaccine against the AIDS virus is expected to depend even more on new technological advances. Four basic areas of vaccine development are discussed in this context: (1) selection and preparation of candidate immunogens; (2) presentation of immunogens to the immune system; (3) pre-clinical testing of vaccine candidates; and (4) monitoring of vaccine clinical trials.


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.


Protein-based medical adhesives.

There are many naturally occurring adhesive proteins which have potential for application in medicine and dentistry. Cloning and expression of their genes enables the modes of action of these proteins to be better understood and increases their availability for practical applications. This article concentrates on the adhesive protein from the blue mussel Mytilus edulis but also describes medical adhesives based on fibrin isolated from human blood.


Alginate as immobilization matrix for cells.

In recent years, entrapment of cells within spheres of Ca2+ alginate has become the most widely used technique for immobilizing living cells. This versatile method includes applications ranging from immobilization of living or dead cells in bioreactors, immobilization of plant protoplasts for micropropagation and immobilization of hybridoma cells for production of monoclonal antibodies, to entrapment of animal cells for implantation of artificial organs. This review evaluates the potential of this method on the basis of the current knowledge of structural and functional relationships in alginate gels.


Supercritical fluid extractions in biotechnology.

Over the past decade, supercritical fluid (SCF) extraction has been shown to deserve consideration as an alternative to liquid-liquid extraction or distillation. Most current commercial applications of SCF extraction involve biologically produced materials; the technique may be particularly relevant to extraction of biological compounds in cases where there is a requirement for low-temperature processing, high mass-transfer rates and negligible carry over of solvent into the final product. New advances, in which extraction is combined with reaction or crystallization steps, may further increase the attractiveness of SCFs in the bioprocessing industries.


High efficiency transformation of intact yeast cells by electric field pulses.

We have developed an efficient method that electrically introduces DNA into intact yeast cells. Saccharomyces cerevisiae was used as a model in order to optimize the transformation protocol. Transformation efficiencies of 10(7) transformants/micrograms of plasmid DNA were obtained with a square wave electric pulse of 2.7 kV/cm during 15 milliseconds. The technique is simple and rapid. Even small quantities of DNA (100 pg) can be used to transform 10(8) cells. Important parameters are the pulse field strength and duration. Pretreatment of the yeast cells in the early phase of exponential growth with dithiothreitol increases transformation efficiency. The method has been successfully applied to various strains of S. cerevisiae as well as to other types of yeast.


Immunoaffinity adsorption: process-scale isolation of therapeutic-grade biochemicals.

This review describes immunoaffinity purification as a tool for the process-scale isolation of high-value, therapeutic-grade biochemicals from complex media. To this end, practicalities of activation, antigen coupling and operation in anti-human IgG monoclonal antibody recovery have been studied using cyanogen-bromide activated Sepharose CL-4B and composite Macrosorb K4AX immobilised with polyclonal human IgG as a model for general immunoaffinity systems. The characteristics required of solid phases are discussed in the context of the efficient application of bioselective processes early in the purification sequences. The importance of the technique for isolating biologically active biomolecules for in-vivo administration has been discussed in the light of current, stringent regulatory requirements. Finally, the prospect of scale-up and automation of the technique has been evaluated.


Cooperative interactions in transcriptional regulation.

Cooperative interactions between regulatory proteins and RNA polymerase are a common feature of transcriptional systems. We have developed a method, based on the electrophoresis mobility shift assay, for the measurement of cooperative effects in the binding of proteins to DNA restriction fragments. Using this approach we have identified a hitherto unknown interaction between the E. coli lactose repressor and CAP proteins. We suggest that this interaction plays a role in the control of the lactose operon that is not predicted by current regulatory models.


Gene construction and mutagenesis for site specific modification of protein with carbohydrate.

We reported the construction of the structural gene for trans-activator (Tat) protein of human immunodeficiency virus. While maintaining the same amino acid sequence as the viral protein, the corresponding nucleotide sequence was modified to create additional recognition sites for restriction endonucleases and to prevent basepair mismatch during gene assembly. The oligonucleotides were synthesized chemically, purified and assembled into five gene blocks. The gene blocks were cloned into plasmid vectors and later reassembled into a complete gene. The use of gene blocks facilitated in vitro mutagenesis by the cassette mutagenesis method.


Molecular cloning of the xylL-xylE region from the P. putida TOL plasmid, pDK1.

A 5.2 kilobase EcoRI restriction fragment from the Pseudomonas putida HS1 TOL plasmid pDK1, encoding a portion of the lower toluene degradation pathway, was cloned into the E. coli plasmid pBR325. A detailed map of the restriction endonuclease sites was constructed and the nucleotide sequence of three contiguous XhoI fragments, with a combined total length of approximately 3.9 kilobases, has been investigated. This region was determined to contain a total of four separate open reading frames, each preceded by an identical putative ribosome-binding site (nucleotide sequence of 5'-GAGGTG-3'). These open reading frames have been tentatively identified as encoding the lower pathway enzymes catechol 2,3-dioxygenase (C23O) and 1,2-dihydroxycyclohexa-3,5-diene carboxylate dehydrogenase (DHCDH) and a subunit of the toluate 1,2-dioxygenase complex (TO).


Tumor cell drug resistance and its reversal.

Tumors that formerly were uniformly fatal can now be cured by cancer chemotherapy. However, successful anticancer therapy is faced by many obstacles, such as excessive normal tissue toxicity and drug resistance. Tumor drug resistance may be either intrinsic or acquired. The multidrug resistance (MDR) is a unique phenomenon and is characterized by tumor resistance to various structurally unrelated drugs. Known mechanisms for MDR include overexpression of a membrane P-glycoprotein 170 and elevated cellular levels of reducing agents, such as glutathione (GSH). Currently available strategies for overcoming drug resistance include competitive inhibitors of the P-glycoprotein 170, inhibitors of GSH synthesis, and adjuvant therapy with hyperthermia. Development of drug resistance is analogous to a physiological detoxification mechanism and may continue to limit the effectiveness of cancer chemotherapy in the near future.


The tryptophan synthase multienzyme complex: exploring structure-function relationships with X-ray crystallography and mutagenesis.

The bifunctional tryptophan synthase alpha 2 beta 2 complex that catalyzes the final two reactions in tryptophan biosynthesis is a classic example of a multienzyme complex that "channels" a metabolic intermediate (indole) between two active sites. The three-dimensional structure of the alpha 2 beta 2 complex from Salmonella typhimurium reveals that the four polypeptide subunits are arranged in an extended alpha beta beta alpha order forming a complex 150 A long. The active sites of the neighboring alpha and beta subunits are separated by about 30 A and appear to be connected by a tunnel, which may facilitate the intramolecular transfer of indole. The active site of the alpha subunit, which is centrally located near one end of an eight-fold alpha/beta barrel structure, contains the sites of most of the missense mutations which were identified as key residues by Yanofsky and colleagues in early genetic studies. Site-directed mutagenesis is being used to replace residues found in the active sites of the alpha and beta subunits in order to probe the mechanism of catalysis. Recombinant DNA technology should also be useful in analyzing protein-protein interaction, protein folding and the channeling phenomenon.


The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences.

We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae. Studies with five KEX2 processed leader sequences, namely the S. cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described. We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product. In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product. Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.


Use of the Escherichia coli SSB gene to prevent bioreactor takeover by plasmidless cells.

Reactor takeover by plasmidless cells is a major problem encountered when producing proteins from plasmid-borne genes in genetically engineered bacteria. We have approached this problem by deleting the essential ssb gene from the Escherichia coli chromosome and placing it on a plasmid. Plasmidless cells do not accumulate even after growing such strains under non-selective continuous culture conditions for extended periods of time. Other ssb-containing plasmids can be readily introduced into this E. coli strain by a plasmid-displacement technique. Using this system, we have achieved very high levels of beta-lactamase production in continuous culture without selective pressure.


Development of a piezoelectric immunosensor for the detection of Salmonella typhimurium.

A piezoelectric biosensor has been developed for the detection of Salmonella typhimurium. The antibody to Salmonella was immobilized on the crystal by various immobilization procedures. The best result was obtained when antibody was immobilized on the crystal precoated with a thin layer of polyethyleneimine. The response of the coated crystal for S. typhimurium in a microbial suspension was in the range of 10(5) to 10(9) cells ml-1. The time required for a complete interaction between the crystal and the cells appeared to depend upon the cell concentration of the analyzed sample. The antibody-bound crystal lost no activity over 4 days at 4 degrees C and it could be reused for 6-8 consecutive assays.


Hydrolysis of concentrated sucrose syrups by invertase immobilized on anion exchanger waste cotton thread.

Yeast invertase was immobilized on polyethyleneimine-coated cotton thread by adsorption followed by crosslinking with glutaraldehyde. The thread-bound invertase was used as an easily retrievable system for the hydrolysis of 80% w/v commercial sucrose syrups. The immobilized enzyme was stable for over 90 days to a temperature of 50 degrees C, only when stored in 80% sucrose solution. Above this temperature, inactivation of enzyme was observed. The cotton threads were used in a batch reactor for hydrolysis of sucrose in about 30 batches carried out over a period of 50 days without loss in activity. The threads could also be used in a packed bed reactor (1.51) for 97% hydrolysis of 80% sucrose syrups at 50 degrees C at a rate of about 360 kg per month for a period of 3 months.


Gene expression and the development of live enteric vaccines.

Rationally attenuated live Salmonella vaccines provide good protection against homologous challenge and can act as carriers of heterologous antigens. However, the optimum expression system for each heterologous antigen will need to be established individually. This will ensure that the antigen in question is produced at appropriate levels, in the correctly folded conformation, within or at the surface of the carrier cell, and can therefore elicit the optimal immune response.


Biomaterials aspects of porous microcarriers for animal cell culture.

Porous microcarriers are new support materials with important advantages in both industrial cell-culture processes and the culture of cells of medical importance. Porous microcarriers are now commercially available with internal architecture and surface chemistry suitable for culture of both anchorage-dependent and anchorage-independent animal cells.


Oriented immobilization of bacterial photosynthetic membrane.

We have examined a method for oriented immobilization of photosynthetic membrane fragments on a solid surface by specific avidin-biotin interaction. Photosynthetic membrane fragments from the purple non-sulphur photosynthetic bacterium Rhodopseudomonas viridis, of which the H-subunit of the photosynthetic reaction centre was biotinylated, was immobilized on an avidin-adsorbed plate. Orientation of the immobilized membrane on the plastic plate was checked by an antisera binding assay that could react to the respective sides of the membrane: the H-subunit side was selectively adsorbed on the plate. Light-induced potential and current responses could be measured when the membrane immobilized on the SnO2-coated glass plate was dried and sandwiched with a counter electrode of Hg. The electrical response in the immobilized membrane was much improved in comparison with the control (membranes were simply adsorbed on the plate), supporting the idea that the membranes have an orientation on solid surfaces.


Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.