Genes from Pseudomonas sp. strain BS involved in the conversion of L-2-amino-Delta(2)-thiazolin-4-carbonic acid to L-cysteine

DL-2-amino-Delta(2)-thiazolin-4-carbonic acid (DL-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of L-ATC to L-cysteine from Pseudomonas sp. strain BS. The introduction of this DNA fragment into Escherichia coli cells enabled them to convert L-ATC to cysteine via N-carbamyl-L-cysteine (L-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has L-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the L-cysteine synthesis from DL-ATC. These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of E. coli cells carrying deletion derivatives of pTK10. The results indicate that atcB and atcC are involved in the conversion of L-ATC to L-NCC and the conversion of L-NCC to cysteine, respectively. atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with L-N-carbamoylases. Since both enzymes can only catalyze the L-specific conversion from L-ATC to L-NCC or L-NCC to L-cysteine, it is thought that atcB and atcC encode L-ATC hydrolase and N-carbamyl-L-cysteine amidohydrolase, respectively.     

Cloning, expression, and identification of genes involved in the conversion of DL-2-amino-Delta2-thiazoline-4-carboxylic acid to L-cysteine via S-carbamyl-L-cysteine pathway in Pseudomonas sp. TS1138

Two novel genes (tsB, tsC) involved in the conversion of DL-2-amino-Delta2-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine through S-carbamyl-L-cysteine (L-SCC) pathway were cloned from the genomic DNA library of Pseudomonas sp. TS1138. The recombinant proteins of these two genes were expressed in Escherichia coli BL21, and their enzymatic activity assays were performed in vitro. It was found that the tsB gene encoded an L-ATC hydrolase, which catalyzed the conversion of L-ATC to L-SCC, while the tsC gene encoded an L-SCC amidohydrolase, which showed the catalytic ability to convert L-SCC to L-cysteine. These results suggest that tsB and tsC play important roles in the L-SCC pathway and L-cysteine biosynthesis in Pseudomonas sp. TS1138, and that they have potential applications in the industrial production of L-cysteine.     

Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Summary A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.     

Induction of 2-amino-D2-thiazoline-4-carboxylic acid hydrolase and N-carbamoyl-l-cysteine amidohydrolase by S-compounds in Pseudomonas putida AJ3865

The induction of 2-amino-Delta(2)-thiazoline-4-carboxylic acid hydrolase (ATCase) and N-carbamoylcysteine amidohydrolase (NCCase), both of which are involved in the conversion step of 2-amino-Delta(2)-thiazoline carboxylic acid (ATC) to cysteine, was studied with Pseudomonas putida AJ3865. We found that L-ATC induced L-ATCase and L-NCCase, but that D-ATC induced only L-NCCase, whereas L- or D-NCC and thiazoline derivatives did not induce both enzymes. The bacterium showed neither D-ATCase nor D-NCCase activities, indicating that the role of L-ATC and D-ATC was different in the enzyme induction. We also found new inducers, d- and l-methionine, S-methyl-L-cysteine, cysteic acid, and 2-aminoethane sulfonic acid. However, the induction level of both enzymes by new inducers was much lower than those by L-ATC and D-ATC. Furthermore, the induction rate of both enzymes was synergistically increased only under a combination of D,L-ATC and new inducers. S-Compounds, however, such as new inducers except S-methyl-L-cysteine, inhibited both enzyme activities. This is the first report on the new inducers, synergistic induction, and the new inhibitors of L-ATCase and L-NCCase.     

NADP-Glutamate Dehydrogenase Activity Is Increased under Hyperosmotic Conditions in the Halotolerant Yeast <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis>

Glutamate plays an important role in osmoprotection in various bacteria. In these cases, increased intracellular glutamate pools are not attributable to the NADP-dependent glutamate dehydrogenase (NADP-GDH) or the glutamate synthase, which do not increase their activities under hyperosmotic conditions, but rather to changes in other enzymes involved in glutamate metabolism. We performed a study which indicates that, as opposed to what happens in bacteria, the activity of NADP-GDH is fivefold higher when the halotolerant yeast Debaryomyces hansenii is grown in the presence of 1 M NaCl, compared with growth in media with no added salt. Since purified NADP-GDH activity in vitro was not enhanced by the presence of salt and was more sensitive to ionic strength than the two isoenzymes from S. cerevisiae, increased enzyme synthesis is the most plausible mechanism to explain our results. We discuss the possibility that increased NADP-GDH activity in D. hansenii plays a role in counteracting the inhibitory effect of high ionic strength on the activity of this enzyme.     

Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C–C bond cleavage in symmetrical β‐diketones by 6‐oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β‐diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily. Biotechnol. Bioeng. 2011;108: 2815–2822. © 2011 Wiley Periodicals, Inc.     

Roles of gibberellins in increasing sink demand in Japanese pear fruit during rapid fruit growth

Our previous work demonstrated that exogenous gibberellins (GAs) applications during rapid fruit growth significantly increases sink demand and results in a larger fruit in Japanese pear. In an attempt to unravel the mechanism of increased sink demand by applied GAs, the histology, cell wall components of the flesh, and carbon accumulation in the fruit were assessed for Japanese pear (Pyrus pyrifolia, cultivar ‘Kousui’), as were the activities of sucrose- and sorbitol-cleaving enzymes. Our results show that most vascular tissues occurred in core tissue with very little vascular tissue in the flesh. Application of a mixture of GA₃ + GA₄ in lanolin paste significantly increased the amount of ethanol-insoluble solids, e.g., total pectins, hemicellulose, and cellulose in the cell walls. There was a significantly increased sink demand (assessed by ¹³C accumulation in the fruit) by the applied GAs, and this increased sink strength was closely related to increased activities of cell wall-bound invertase in the core, neutral invertase and NAD-dependent sorbitol dehydrogenase in the flesh during rapid fruit growth. As well, concentrations of sorbitol and sucrose in the flesh were decreased by GA application, while glucose concentration increased. Most importantly, the fact that sink activity can be increased by GA application implies that endogenous GAs are likely to be important modulators for sugar metabolism. Hence, selecting for genotypes with elevated GA production in the growing fruit and increased activities of key enzymes for sugar metabolism could result in increased fruit size.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media

The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (a(w) = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89 degrees C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72 degrees C). (c) 1995 John Wiley & Sons, Inc.     

Differential Effects of Ionic Strength,Divalent Cations and pH on the Pore-forming Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins

The combined effects of ionic strength, divalent cations, pH and toxin concentration on the pore-forming activity of Cry1Ac and Cry1Ca were studied using membrane potential measurements in isolated midguts of Manduca sexta and a brush border membrane vesicle osmotic swelling assay. The effects of ionic strength and divalent cations were more pronounced at pH 10.5 than at pH 7.5. At the higher pH, lowering ionic strength in isolated midguts enhanced Cry1Ac activity but decreased considerably that of Cry1Ca. In vesicles, Cry1Ac had a stronger pore-forming ability than Cry1Ca at a relatively low ionic strength. Increasing ionic strength, however, decreased the rate of pore formation of Cry1Ac relative to that of Cry1Ca. The activity of Cry1Ca, which was small at the higher pH, was greatly increased by adding calcium or by increasing ionic strength. EDTA inhibited Cry1Ac activity at pH 10.5, but not at pH 7.5, indicating that trace amounts of divalent cations are necessary for Cry1Ac activity at the higher pH. These results, which clearly demonstrate a strong effect of ionic strength, divalent cations and pH on the pore-forming activity of Cry1Ac and Cry1Ca, stress the importance of electrostatic interactions in the mechanism of pore formation by B. thuringiensis toxins.     

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

ABSTRACT: BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, wefirst tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coli L-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (cysI and cysJ) and the L-cysteine synthase gene (cysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (cysC or cysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coli L-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell . CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

Comparison of Three Ionic Liquid-Tolerant Cellulases by Molecular Dynamics

We have employed molecular dynamics to investigate the differences in ionic liquid tolerance among three distinct family 5 cellulases from Trichoderma viride, Thermogata maritima, and Pyrococcus horikoshii. Simulations of the three cellulases were conducted at a range of temperatures in various binary mixtures of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate with water. Our analysis demonstrates that the effects of ionic liquids on the enzymes vary in each individual case from local structural disturbances to loss of much of one of the enzyme’s secondary structure. Enzymes with more negatively charged surfaces tend to resist destabilization by ionic liquids. Specific and unique structural changes in the enzymes are induced by the presence of ionic liquids. Disruption of the secondary structure, changes in dynamical motion, and local changes in the binding pocket are observed in less tolerant enzymes. Ionic-liquid-induced denaturation of one of the enzymes is indicated over the 500 ns timescale. In contrast, the most tolerant cellulase behaves similarly in water and in ionic-liquid-containing mixtures. Unlike the heuristic approaches that attempt to predict enzyme stability using macroscopic properties, molecular dynamics allows us to predict specific atomic-level structural and dynamical changes in an enzyme’s behavior induced by ionic liquids and other mixed solvents. Using these insights, we propose specific experimentally testable hypotheses regarding the origin of activity loss for each of the systems investigated in this study.     

Efficient one-step production of (S)-1-phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli

Background

Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli.

Results

Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell.

Conclusions

In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions

We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.     

Isolation and Characterization of Thermophilic Bacteria from Gavmesh Goli Hot Spring in Sabalan Geothermal Field,Iran: Thermomonas hydrothermalis and Bacillus altitudinis Isolates as a Potential Source of Thermostable Protease

Abstract

Thermophilic bacteria have attracted great attention due to their ability to produce thermostable enzymes. The aim of this study was the isolation and characterization of thermophilic bacteria from Gavmesh Goli hot spring in Sareyn, North West of Iran. Of 10 water samples collected, 36 thermophilic bacteria were obtained. The thermophilic bacteria were tested for their ability to produce hydrolase enzymes. All the isolates were potentially protease producers. Lipase, DNase, and amylase activities were confirmed in 34 (94.4%), 8 (22.2%), and 3 (8.3%) isolates, respectively. Five isolates with higher levels of enzyme activity were selected for further studies. Morphological, biochemical, and molecular analysis by 16S rRNA gene sequencing revealed that four isolates (DH15, DH16, DH20, and DH29) could be identified as Thermomonas hydrothermalis and one (PA10) Bacillus altitudinis. The protease produced by these isolates was optimally active at 50–55?°C, pH 8–8.5, and 0–0.5?M NaCl. In this first time study, we isolated T. hydrothermalis and B. altitudinis from Iranian hot springs and demonstrated the characteristics of T. hydrothermalis protease. Accordingly, due to the valuable potential of these bacteria such as the production of protease with high temperature and pH stability, these isolates can be introduced as promising candidates for industrial applications.     

Catalytic performance of cross-linked enzyme aggregates of Penicillium expansum lipase and their use as catalyst for biodiesel production

Cross-linked enzyme aggregates (CLEAs) of lipase from Penicillium expansum (PEL) were prepared directly from fermentation broth, a more practical and economically viable procedure than the generally used methods that require purified or partially purified enzymes for CLEA preparation. A systematic study of the activity and stability of PEL-CLEAs was undertaken in aqueous solution, organic solvents, and ionic liquids (ILs). Immobilization of the enzyme resulted in a significantly enhanced stability in aqueous solution with regard to pH and temperature. PEL-CLEAs showed an improved activity in the IL [BMIm][PF₆] relative to that observed in hexane, both keeping increased with temperature (up to 90 °C in the IL and 60 °C in hexane). The effect of water content and water activity in these two nonaqueous media showed similar patterns as for the uncrosslinked enzyme. The half life of the CLEAs was higher in hydrophobic organic solvents (hexane and chloroform) than in aqueous solution, and presented a sigmoid relationship with the log P of the organic solvent tested. PEL-CLEAs catalyzed biodiesel production from microalgal oil in the IL [BMIm][PF₆] with a conversion of 85.7%, demonstrating that they can be taken as a promising catalyst for this application.     

Improvement of oxidoreductase stability of cethyltrimethylammoniumbromide permeabilized cells ofZymomonas mobilis through glutaraldehyde crosslinking

Summary Permeabilization ofZymomonas mobilis with CTAB(Cetyltrimethylammoniumbromide) was investigated in order to obtain a stable process for sorbitol production in the immobilized system. The optimum conditions for sorbitol formation were treating cells with 0.2% CTAB at 4°C for 10 min. For the immobilized system permeabilized cells were treated with glutaraldehyde to improve the system with cross-linking of enzymes. In this way, no significant loss of enzyme activity was apparent during 30 day operation in a continuous process. The productivity of the continuous process at a dilution rate 0.2 h^–1 was 6.51g/L-h for sorbitol. The CTAB-permeabilized cells could be used to produce sorbitol and gluconic acid simultaneously in the long term continuous process.     

Diverse associations in the ternary systems of β-cyclodextrin, simple carbohydrates and phenyl derivatives of inorganic oxoacids

Complex formation reactions of phenylboronic, phenylphosphonic, phenylarsonic and 4-aminophenyl arsonic acids with β-cyclodextrin (cycloheptaamylose, β-CD) and some simple carbohydrates (mannitol, sorbitol, glucose) have been studied using spectrophotometric, potentiometric methods and solubility measurements, supplemented with HPLC and IR analyses of the solid samples. Equilibrium constants have been determined at ionic strength of 0.2 M (NaCl) and 25 °C. β-CD forms the most stable complexes with the neutral, undissociated forms of the acids, the stability constants are as follows: phenylboronic acid: 320 ± 36, phenylphosphonic acid: 108 ± 25, phenylarsonic acid: 97 ± 4 and 4-aminophenyl arsonic acid: 107 ± 10. The stability constants for the β–CD-complexes of the ionic forms are much lower. Ternary complexes of low stability could be detected in the case of phenylphosphonic acid and sorbitol with the undissociated form and with glucose and the dianion. In more concentrated solutions phenylboronic acid forms insoluble complexes with mannitol, sorbitol and β-CD. The solid phases obtained in the ternary systems are predominantly mixtures of ester type 3:1 complexes with the carbohydrate and 1:1 inclusion complex with the β-CD. No significant interaction has been found with glucose. The phenomena can be explained by the differences in the structures of the components and by the changes in the H-bonding network of β-CD on the complex formation.     

Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH,and on Substrate Relative Concentration

The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I⁻ and SCN⁻ in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3‐fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength. ¹³C‐labelled SCN⁻ and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I₂SCN⁻), giving an intense signal at 49.7 ppm in ¹³C‐NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN⁻: I⁻ ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN⁻>I⁻. We demonstrated that the formation of the signal required the interaction between I₂ and SCN⁻, and MS showed the presence of I₂SCN⁻.     

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability

The use of Moloney murine leukaemia virus (MoMLV) derived retroviral vectors in gene therapy requires the production of high titer preparations. However, obtaining high titers of infective MoMLV retroviral vectors is difficult due to the vector inherent instability. In this work the effect of the cell culture medium osmotic pressure upon the virus stability was studied. The osmolality of standard medium was raised from 335 up to 500 mOsm/kg using either ionic (sodium chloride) or non-ionic osmotic agents (sorbitol and fructose). It was observed that, independently of the osmotic agent used, the infectious vector inactivation rate was inversely correlated with the osmolality used in the production media; therefore, the use of high medium osmolalities enhanced vector stability. For production purposes a balance must be struck between cell yield, cell productivity and retroviral stability. From the conditions tested herein sorbitol addition, ensuring osmolalities between 410 and 450 mOsm/kg, yields the best production conditions; NaCl hampered the viral infectious production while fructose originates lower cell yields. Lipid extractions were performed for cholesterol and phospholipid analyses showing that more stable viral vectors had a 10% reduction in the cholesterol content. A similar reduction in cholesterol was observed in the producer cells. A detailed analysis of the major phospholipids composition, type and fatty acid content, by mass spectrometry did not show significant changes, confirming the decrease in the cholesterol to phospholipids ratio in the viral membrane as the major reason for the increased vector stability.