Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS [g of lactose](-1)) and 2.88 (0.0288 g of EPS [g of glucose](-1)). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase). These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.     

Competition between oxygen and nitrate respirations in continuous culture of Pseudomonas aeruginosa performing aerobic denitrification

Continuous culture of P. aeruginosa was conducted with nitrate-containing media under the dilution rates (D) of 0.026, 0.06, and 0.13/h and the dissolved oxygen concentrations (DO) of 0-2.2 mg/L. The bacterium performed simultaneous O(2) and nitrate respiration in all of the systems studied. For each D, the (apparent) cell yield from glucose (Y(X/S)) was lower at zero DO, but did not change substantially with non-zero DO. In non-zero DO systems, Y(X/S) increased with increasing D, and when fit with a model considering cell death, gave the following parameters: maximum cell yield Y(X/S) (m) = 0.49, maintenance coefficient M(S) = 0.029 (/h), and cell decay constant k(d) = 0.014/h. The same model failed to describe the behaviors of zero-DO systems, where neither glucose nor nitrate was limiting and the limiting factor(s) remained unknown. The cell yield from accepted electron (Y(X/e)) was however relatively constant in all systems, and the energy yield per electron accepted via denitrification was estimated at approximately 69% of that via O(2) respiration. A closer examination revealed that increasing DO enhanced O(2) respiration only at extremely low DO ( <0.05 mg/L), beyond which the increasing DO only slightly increased its weak inhibition on denitrification. While O(2) was the preferred electron acceptor, the fraction of electrons accepted via denitrification increased with increasing D.     

Factors affecting microbial sulfate reduction by Desulfovibrio desulfuricans in continuous culture: limiting nutrients and sulfide concentration

The effects of sulfate and nitrogen concentrations of the rate and stoichiometry of microbial sulfate reduction were investigated for Desulfovibrio desulfuricans grown on lactate and sulfate in a chemostat at pH 7.0. Maximum specific growth rates (mu(max)), half-saturation coefficients (K(sul)), and cell yield (Y(c/Lac)) of 0.344 +/- 0.007 and 0.352 +/- 0.003 h (-1), 1.8 +/- 0.3 and 1.0 +/- 0.2 mg/L, and 0.020 +/- 0.003 and 0.017 +/- 0.003 g cell/g lactate, respectively, were obtained under sulfate-limiting conditions at 35 degrees C and 43 degrees C. Maintenance energy requirements for D. desulfuricans were significant under sulfate-limiting conditions. The extent of extracellular polymeric substance (EPS) produced was related to the carbon: nitrogen ratio in the medium. EPS production rate increased with decreased nitrogen loading rate. Nitrogen starvation also resulted in decreased cell size of D. desulfuricans. The limiting C : N ratio (w/w) for D. desulfuricans was in the range of 45 : 1 to 120 : 1. Effects of sulfide on microbial sulfate reduction, cell size, and biomass production were also ivestigated at pH 7.0. Fifty percent inhibition of lactate utilization occurred at a total sulfide concentration of approximately 500 mg/L. The cell size of D. desulfuricans decreased with increasing total sulfide concentration. Sulfide inhibition of D. desulfuricans was observed to be a reversible process. (c) 1992 John Wiley & Sons, Inc.     

Activity of Pseudomonas aeruginosa in biofilms: Steady state

Aerobic glucose metabolism by Pseudomonas aeruginosa in steady-state biofilms at various substrate loading rates and reactor dilution rates was investigated. Variables monitored were substrate (glucose), biofilm cellular density, biofilm extracellular polymeric substance (EPS) density, and suspended cellular and EPS concentrations. A mathematical model developed to describe the system was compared to experimental data. Intrinsic yield and rate coefficients included in the model were obtained from suspended continuous culture studies of glucose metabolism by P. aeruginosa. Experimental data compared well with the mathematical model, suggesting that P. aeruginosa does not behave differently in steady-state biofilm cultures, where diffusional resistance is negligible, than in suspended cultures. This implies that kinetic and stoichiometric coefficients for P. aeruginosa derived in suspended continuous culture can be used to describe steady-state biofilm processes.     

Effects of temperature and phosphorous concentration on microbial sulfate reduction by Desulfovibrio desulfuricans

The effects of temperature and phosphorous concentration on the rate and the extent of microbial sulfate reduction with lactate as carbon and energy source were investigated for Desulfovibrio desulfuricans. The continuous culture experiments (chemostat) were conducted at pH 7.0 from 12 to 48 degrees C. The maximum specific growth rate (mu(max)) was relatively constant in the range 25 degrees C-43 degrees C and dramatically decreased outside this temperature range. The half-saturation coefficient was minimum at 25 degrees C. Cell yield was highest in the optimum temperature range (35 degrees C-43 degrees C) for growth. Maintenance energy requirements for D. desulfuricans were not significant. Two moles of lactate is consumed for every mole of sulfate reduced, and this stoichiometric ratio is not temperature dependent. Steady state rate and stoichiometric coefficients accurately predicted transient behavior during temperature shifts. The extent of extracellular polymeric substance (EPS) is related to the concentration of phosphorous in the medium. EPS production rate increased with decreased phosphorous loading rate. Failure to discriminate between cell and EPS formation by D. desulfuricans leads to significant overestimates of the cell yield. The limiting C:P ratio for D. desulfuricans was in the range of 400:1 to 800:1.     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

Formation of compound I and compound II ferryl species in the reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

The formation of ferryl heme (Fe(IV) = O) species, i.e., compound I and compound II, has been identified as the main intermediates in heme protein peroxidative reactions. We report stopped-flow kinetic measurements which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce ferryl intermediates compound I and compound II. Compound I appears relatively stable displaying an absorption at 648 nm. The rate constant value (k'(2)) for the conversion of compound I to compound II is 3.0 x 10(-2) s(-1), more than 100 times smaller than that reported for myoglobin. The rate constant value for the oxidation of the ferric heme (k'(12) + k'(13)) is 2.0 x 10(2) M(-1) s(-1). These values suggest an alternate route for the formation of compound II (by k'(13)) avoiding the step from compound I to compound II (k'(2)). In HbI from L. pectinata the stabilization of compound I is attribute to the unusual collection of amino acids residues (Q64, F29, F43, F68) in the heme pocket active site of the protein.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

Biochemical basis of glucokinase activation and the regulation by glucokinase regulatory protein in naturally occurring mutations

Glucokinase (GK) has several known polymorphic activating mutations that increase the enzyme activity by enhancing glucose binding affinity and/or by alleviating the inhibition of glucokinase regulatory protein (GKRP), a key regulator of GK activity in the liver. Kinetic studies were undertaken to better understand the effect of these mutations on the enzyme mechanism of GK activation and GKRP regulation and to relate the enzyme properties to the associated clinical phenotype of hypoglycemia. Similar to wild type GK, the transient kinetics of glucose binding for activating mutations follows a general two-step mechanism, the formation of an enzyme-glucose complex followed by an enzyme conformational change. However, the kinetics for each step differed from wild type GK and could be grouped into specific types of kinetic changes. Mutations T65I, Y214C, and A456V accelerate glucose binding to the apoenzyme form, whereas W99R, Y214C, and V455M facilitate enzyme isomerization to the active form. Mutations that significantly enhance the glucose binding to the apoenzyme also disrupt the protein-protein interaction with GKRP to a large extent, suggesting these mutations may adopt a more compact conformation in the apoenzyme favorable for glucose binding. Y214C is the most active mutation (11-fold increase in k(cat)/K(0.5)(h)) and exhibits the most severe clinical effects of hypoglycemia. In contrast, moderate activating mutation A456V nearly abolishes the GKRP inhibition (76-fold increase in K(i)) but causes only mild hypoglycemia. This suggests that the alteration in GK enzyme activity may have a more profound biological impact than the alleviation of GKRP inhibition.     

Metabolite profiles and growth characteristics of Rhizobium meliloti cultivated at different specific growth rates

Rhizobium meliloti (ATCC 55340) was grown at different specific growth rates in a chemostat apparatus. Metabolic products, relating to the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle, were measured and quantified to probe the influence of specific growth rate on the distribution of important metabolites. The detection of propionate in the fermentation broth implies that the imbalance of reducing equivalents of FADH(2) and NADH + H(+) resulted in a partially reductive operation of the TCA cycle. Additionally, experimental results show that the specific growth rate plays an essential role in modulating the biomass concentration, the specific substrate uptake rate, the cell length, the specific exopolysaccharide (EPS) production rate, the distribution of EPS molecular weight, and the profiles of carbohydrate and organic acid. The specific EPS production rate (varying from 13.3 to 111 mg EPS/g-DW/h) follows a growth-associated pattern at the specific growth rate ranging from 0.06 to 0.20 h(-1) and switches into non-growth-associated mode when the specific growth rate is over 0.20 h(-1).     

The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin

A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway.     

Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas

A thermophilic strain isolated from sea sand at Maronti, near Sant' Angelo (Ischia), is described. The organism grows well at an optimal temperature of 60 °C at pH 7.0. The thermophilic bacterium, named strain 4004, produces an exocellular polysaccharide (EPS) in yields of 90 mg/l. The EPS fraction was produced with all substrates tested, although a higher yield was obtained with sucrose or trehalose as sole carbon source. During growth, the EPS content was proportional to the biomass. Three fractions (EPS1, EPS2, EPS3) were obtained after purification. Quantitative monosaccharide analysis of the EPSs revealed the presence of mannose:glucose:galactose in a relative ratio of 0.5:1.0:0.3 in EPS1, mannose:glucose:galactose in a relative ratio of 1.0:0.3:trace in EPS2, and galactose:mannose:glucosamine:arabinose in a relative ratio of 1.0:0.8:0.4:0.2 in EPS3. The average molecular mass of EPS3 was determined to be 1×10⁶ Da. From comparison of the chemical shift values in ¹H and ¹³C spectra, we conclude that EPS3 presents a pentasaccharide repeating unit. Electronic Publication     

Kinetics of batch ethanol fermentation of cheese-whey powder (CWP) solution as function of substrate and yeast concentrations

A lactose utilizing yeast strain, Kluyveromyces marxianus DSMZ-7239 was used for ethanol formation from cheese-whey powder (CWP) solution in batch experiments. Effects of initial substrate (CWP) and yeast concentrations on the rate and extent of ethanol formation were investigated. The initial pH and oxidation-reduction potential (ORP) was kept at 5 and -250 mV, respectively. The rate and extent of ethanol formation increased with increasing CWP concentration up to 156 g l(-1) (75 g l(-1) sugar) and then decreased for larger CWP concentrations due to substrate inhibition at high sugar concentrations. The ethanol yield coefficient was also maximum (0.54 g EtOH/g sugar) and equal to the theoretical yield at CWP concentration of 156 g l(-1). The growth yield coefficient was found to be Y(x/s)=1.2+/-0.1g biomass g sugar(-1). The rate of sugar utilization and ethanol formation also increased linearly with increasing initial biomass concentrations. A kinetic model describing the rate of sugar utilization and substrate inhibition as function of the initial substrate and the biomass concentrations was developed. The kinetic constants were determined using the experimental data. Model predictions of sugar utilization rates were in good agreement with the experimental data. The results indicated that the initial sugar concentration should be below 75 g l(-1) (CWP<156 g l(-1)) and the initial biomass should be above 850 mg l(-1) to obtain high rates and yields of ethanol formation and to avoid substrate inhibition.     

Cultivation of E. coli in single- and ten-stage tower-loop reactors

E. Coli was cultivated in batch and continuous operations in the presence of an antifoam agent in stirred-tank and in single- and ten-stage airlift tower reactors with an outer loop. The maximum specific growth rate, mu(m), the substrate yield coefficient, Y(x/s), the respiratory quotient, RQ, substrate conversion, U(s), the volumetric mass transfer coefficient, K(L)a, the specific interfacial area, a, and the specific power input, P/V(L), were measured and compared. If a medium is used with a concentration of complex substrates (extracts) 2.5 times higher than that of glucose, a spectrum of C sources is available and cell regulation influences reactor performance. Both mu(m) and Y(X/S), which were evaluated in batch reactors, cannot be used for continuous reactors or, when measured in stirred-tank reactors, cannot be employed for tower-loop reactors: mu(m) is higher in the stirred-tank batch than in the tower-loop batch reactor, mu(m) and Y(x/s) are higher in the continuous reactor than in the batch single-stage tower-loop reactor. The performance of the single-stage is better than that of the ten-stage reactor due to the inefficient trays employed. A reduction of the medium recirculation rate reduces OTR, U(s), Pr, and Y(X/S) and causes cell sedimentation and flocculation. The volumetric mass transfer coefficient is reduced with increasing cultivation time; the Sauter bubble diameter, d(s), remains constant and does not depend on operational conditions. An increase in the medium recirculation rate reduces k(L)a. The specific power input, P/V(L), for the single-stage tower loop is much lower with the same k(L)a value than for a stirred tank. The relationship k(L)a vs. P/V(L) evaluated for model media in stirred tanks, can also be used for cultivations in these reactors.     

Activity of Pseudomonas aeruginosa in biofilms: effect of calcium

Aerobic glucose metabolism by Pseudomonas aeruginosa biofilms at various calcium loading rates was investigated. The influence of calcium on specific growth rate, extracellular polymeric substance (EPS) formation rate, biofilm detachment rate, and biofilm calcium concentrations was determined. Calcium accumulated in the biofilm in proportion to the liquid phase concentration. Increasing calcium concentration increased the cohesiveness of the biofilm as indicated by a lower relative detachment rate. Specific activity in the biofilm was the same as that measured in a chemostat and was not influenced by changing calcium concentration. EPS formation rate in the biofilm was unaffected by calcium concentration but was higher than that observed in a chemostat.     

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l(-1)) and at 72 h (751 mg l(-1)) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30 degrees C and 100 rpm. It reached 8373 mg l(-1) when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.     

Batch and continuous culture kinetics for production of carotenoids by beta-ionone-resistant mutant of Xanthophyllomyces dendrorhous

A beta-ionone-resistant mutant strain isolated from the red yeast Xanthophyllomyces dendrorhous KCTC 7704 was used for batch and continuous fermentation kinetic studies with glucose media in a 2.5-1 jar fermentor at 22 degrees C and pH 4.5. The kinetic pattern of growth and carotenoid concentration in the batch fermentations exhibited a so-called mixed-growth-associated product formation, possibly due to the fact that the content of intracellular carotenoids depends on the degree of physical maturation toward adulthood. To determine the maximum specific growth rate constant (microm) and Monod constant (k(s)) for the mutant, glucose-limited continuous culture studies were performed at different dilution rates within a range of 0.02-0.10 h(-1). A reciprocal plot of the steady-state data (viz., reciprocal of glucose concentration versus residence time) obtained from continuous culture experiments was used to estimate a microm of 0.15 h(-1) and k(s) of 1.19 g/l. The carotenoid content related to the residence time appeared to assume a typical form of saturation kinetics. The maximum carotenoid content (Xm) for the mutant was estimated to be 1.04 microg/mg dry cell weight, and the Lee constant (k(m)), which was tentatively defined in this work, was found to be 3.0 h.     

Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S(P)- and R(P)-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R(P)-enantiomer of the sarin analogue (k(cat)=2600 s(-1)) was the preferred substrate for the wild-type PTE relative to the corresponding S(P)-enantiomer (k(cat)=290 s(-1)). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k(cat) values for the R(P)- and S(P)-enantiomers were 410 and 4200 s(-1), respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R(P)R(C)-stereoisomer of the soman analogue was the preferred substrate for PTE. The k(cat) values for the soman analogues were measured as follows: R(P)R(C,) 48 s(-1); R(P)S(C), 4.8 s(-1); S(P)R(C), 0.3 s(-1), and S(P)S(C), 0.04 s(-1). With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k(cat) values for the soman analogues were found to be as follows: R(P)R(C,) 0.3 s(-1); R(P)S(C), 0.3 s(-1); S(P)R(C), 11s(-1), and S(P)S(C), 2.1 s(-1). Prior investigations have demonstrated that the S(P)-enantiomers of sarin and soman are significantly more toxic than the R(P)-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.