Influence of pH and malate-glucose ratio on the growth kinetics of Leuconostoc oenos

Summary Growth, substrate utilization and product formation were studied in batch cultures of a Leuconostoc oenos strain. The effect of various culture conditions, i.e. pH-control at different values and various initial concentrations of malate and glucose, on growth and metabolism were investigated. Addition of malate resulted in a marked stimulation of growth, with only a slight increase in final biomass but a high conversion yield of glucose. Under pH control this stimulation was much greater than could be accounted for from changes in pH profile resulting from malate utilization. The specific rate of malate utilization was maximal at pH 4.0 whereas the specific rate of glucose consumption was highest at pH 5.5. During co-metabolism of malic acid and glucose, substrate utilization and product formation agreed with the stoichiometric relationships of the malo-lactic reaction and the heterolactic fermentation of glucose. Offsprint requests to: A. Pareilleux     

Microbial modification of sugars as building blocks for chemicals

Summary Investigations on the microbial modification of sucrose to the corresponding 3-keto-derivative were carried out with resting cells of Agrobacterium tumefaciens NCPPB 396. This highly specific oxidation to yield the 3-keto-derivative has been analysed kinetically with varying substrate and cell mass concentrations. The formation of the corresponding 3-keto-derivative depended strongly on the reaction time and the aeration rate, and was maximal at aeration rates up to 11.5 volume air/cultivation volume per minute with resting cells. The product formation increased with increasing substrate concentrations. However, the product yield was maximal at substrate concentrations below 20 g/l. Data pertaining to the production of active cell mass as well as for maximal 3-keto-derivative formation are presented in this paper. Also included are some applications for these derivatives.     

Alkylamine-dependent oxidation and oxygenation of alpha-monoamino acids by lysine monooxygenase.

Lysine monooxygenase, a pseudomonad flavoprotein, was almost inactive with an α-monoamino acid as substrate, but the addition of an alkylamine, a counterpart of the fragmented lysine molecule, caused marked oxygen consumption. The rate of oxygen consumption was examined and compared with various combinations of α-monoamino acids and alkylamines. Hydrogen peroxide was formed and the corresponding α-keto acid was produced from each amino acid. In most cases, the hydrogen peroxide formation was nearly equal to the oxygen consumption. In some other cases, however, the oxygen consumption exceeded the hydrogen peroxide formation, and the production of an acid amide in addition to the α-keto acid was qualitatively demonstrated. Thus, both an oxidative deamination and an oxygenative decarboxylation of the same substrate occurred concomitantly, although the ratio of the two types of reaction varied with different combinations of amino acids and alkylamines. Alkylamines at higher concentrations competitively inhibited the lysine oxygenation, whereas lower concentrations of alkylamines stimulated the lysine oxygenation by decreasing the sigmoidicity of the saturation curve for lysine. These results suggest a dual function of the alkylamine; i.e., the interaction with the active site as a fragment of substrate and the effect on the regulatory property of the enzyme.     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.     

Improved synthesis of 3-keto, 4-ene-3-keto, and 4,6-diene-3-keto bile acids

Cholic and deoxycholic acids can be converted into 3-keto derivatives in 75-80% yield, by a four-step synthesis consisting of formylation, selective deformylation of the 3-formoxyl group, oxidation, then deformylation of the remaining formoxyl groups. The intermediate 3-keto formoxyl acids in this sequence were shown to be suitable starting compounds for the synthesis of 4-ene-3-keto acids, in 55-60% yield, via bromination, dehydrobromination, and deformylation. By extending the dehydrobromination reaction, the 7 alpha-formoxyl group of the intermediate 4-ene-3-keto-7 alpha,12 alpha-diformoxyl acid is also lost, hence providing a useful synthetic route to 4,6-diene-3-keto bile acids.     

Effect of glycerol,polyethylene glycol and glutaraldehyde on stability of phenylalanine ammonia-lyase activity in yeast

Summary The polyhydric alcohols, glycerol and sorbitol, significantly increased the rate ofl-phenylalanine production from trans-cinnamic acid using whole cells ofRhodotorula rubra. Chloride ions and oxygen prevented the stimulatory effect of the polyhydric alcohols. Furthermore, the severe inhibition, of the biotransformation by high trans-cinnamic acid concentrations was alleviated in the presence of glycerol, and sorbitol. The rate of conversion could be manipulated still further, even with high trnas-cinnamic acid concentrations, by elevating the reaction pH to, 12 in the presence of polyhydric alcohol. When cells were also treated first with glutaradehyde (0.1% v/v) and then polyethylene glycol (15% v/v), although neither compound stimulated the actual rate of bioconversion, the reaction was markedly stabilised and gave a 73% yield after 28 days of continuous operation.     

Effects of medium glucose concentration and pH on docosahexaenoic acid content of heterotrophic Crypthecodinium cohnii

The effects of medium glucose concentration and pH on growth and docosahexaenoic acid (DHA, C22:6 ω-3) content of Crypthecodinium cohnii were investigated. Over a range of glucose concentrations (5–40 g l⁻¹) investigated, the highest specific growth rate (0.12 h⁻¹), highest cell dry weight concentration (3.13 g l⁻¹) and highest growth yield on glucose (0.6 g g⁻¹) were obtained at 20 g l⁻¹ glucose. However, the highest degree of fatty acid unsaturation (3.2) and highest DHA proportion (53.4% of total fatty acids) were achieved at 5 g l⁻¹ glucose. Low glucose concentrations enhanced the degree of fatty acid unsaturation and DHA formation. Medium pH also affected cell growth, fatty acid unsaturation and DHA proportion. When medium pH was 7.2, the highest specific growth rate (0.089 h⁻¹), highest cell dry weight concentration (2.73 g l⁻¹), highest growth yield on glucose (0.564 g g⁻¹), highest degree of fatty acid unsaturation (3.4) and highest DHA proportion (56.8% of total fatty acids) were obtained. Results suggest that glucose concentration and pH value could be effectively manipulated to achieve maximum DHA production by C. cohnii.     

血卟啉衍生物光敏产生的单线态氧量子产额的测定及其影响因素的研究

用色氨酸降解法测定了给定条件下含血卟啉衍生物(Hpd)的溶液系统中产生的单线态氧相对量子产额,并由单线态氧相对量子产额与色氨酸浓度之间的关系推算出单线态氧的实际产额及单线态氧与色氨酸反应的速率常数(1.0×10~8M~(-1)S~(-1)。研究了溶液pH、温度、Hpd浓度、光照波长等因素对单线态氧产额的影响。实验结果表明,单线态氧量子产额随温度升高而增大;随pH值升高而增大;随Hpd浓度增加而减小;并与光照波长有关。讨论了各个因素影响单线态氧量子产额的可能原因。     

Demethylation of theophylline (1,3-dimethylxanthine) to 1-methylxanthine: the first step of an antioxidising cascade

Abstract

The reaction of theophylline with HO^? radical, produced by photolytic methods at pH 7, was studied in aqueous solution and the products characterised by HPLC and GC-MS. In addition to the expected 1,3-dimethyluric acid, the formation of 1-methylxanthine and, to a lesser extent, of 3-methylxanthine was observed. Theoretical calculations confirmed the preferred formation of the former compound. Both demethylated products were also observed upon reaction of theophylline with O^?– radical anion at pH～13, and 1-methylxanthine was consumed faster than 3-methylxanthine after its formation. Molecular oxygen had no significant effect on the formation of the mono-methylxanthine derivatives. A reaction mechanism for the demethylation of theophylline by oxidising radicals is proposed. This demethylation reaction can play an important role in the protection of biological targets against oxidative stress as the first step of an antioxidising cascade.     

Myoglobin-catalyzed tyrosine nitration: no need for peroxynitrite.

The nitration of tyrosine residues in protein to yield 3-nitrotyrosine derivatives has been suggested to represent a specific footprint for peroxynitrite formation in vivo. However, recent studies suggest that certain hemoproteins such as peroxidases catalyze the H(2)O(2)-dependent nitration of tyrosine to yield 3-nitrotyrosine in a peroxynitrite-independent reaction. Because 3-nitrotyrosine has been shown to be present in the postischemic myocardium, we wished to assess the ability of myoglobin to catalyze the nitration of tyrosine in vitro. We found that myoglobin catalyzed the oxidation of nitrite and promoted the nitration of tyrosine. Both nitrite oxidation and tyrosine nitration were H(2)O(2)-dependent and required the formation of ferryl (Fe(+4)) myoglobin. In addition, nitrite oxidation and tyrosine nitration were pH-dependent with a pH optimum of approximately 6.0. Taken together, these data suggest that the acidic pH and low oxygen tension produced during myocardial ischemia will facilitate myoglobin-catalyzed, peroxyntrite-independent formation of 3-nitrotyrosine.     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

Reduction of ferricytochrome c by tyrosyltyrosylphenylalanine

Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0–8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4×10^–4 and 8.1×10^–4 s^–1 at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15°C. The reciprocal initial rate constant (1/k_int) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logk_int decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)^–, presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt c_ox/(TyrTyrPhe)^–cyt c_red/(TyrTyrPhe) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides.     

Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: use of citric acid as the carbon source

The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C_c = 9.0 kg m⁻³ citric acid as sole carbon source were investigated in 3.5 dm³ batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. At Q_o/V = 1 vvm air flow rate, the effect of agitation rate on DO concentration, pH, product, and by-product concentrations and SAP activity were investigated at N = 150, 500, and 750 min⁻¹; these are named as low-(LOT), medium-(MOT), and high oxygen transfer (HOT) conditions. LOT conditions favor biomass concentration; however, substrate consumption was highest at HOT conditions. MOT was optimum for maximum SAP activity which was 441 U cm⁻³ at t = 37 h. The total amino acid concentration was maximum in LOT and minimum in MOT conditions; lysine had the highest concentration under all oxygen transfer conditions. Among organic acids, acetic acid had the highest concentration and its concentration increased with oxygen transfer rate. The oxygen transfer coefficient increases with the agitation rate and the oxygen consumption rate increased almost linearly with the biomass concentration.     

Asymmetric synthesis of unnaturall-amino acids using thermophilic aromaticl-amino acid transaminase

Aromaticl-amino acid transaminase is an enzyme that is able to transfer the amino group froml-glutamate to unnatural aromatic α-keto acids to generate α-ketoglutarate and unnatural aromaticl-amino acids, respectively. Enrichment culture was used to isolate thermophilicBacillus sp. T30 expressing this enzyme for use in the synthesis of unnaturall-amino acids. The asymmetric syntheses ofl-homophenylalanine andl-phenylglycine resulted in conversion yields of >95% and >93% from 150 mM 2-oxo-4-phenylbutyrate and phenylglyoxylate, respectively, usingl-glutamate as an amino donor at 60°C. Synthesizedl-homophenylalanine andl-phenylglycine were optically pure (>99% enantiomeric excess) and continuously pre-cipitated in the reaction solution due to their low solubility at the given reaction pH. While the solubility of the α-keto acid substrates is dependent on temperature, the solubility of the unnaturall-amino acid products is dependent on the reaction pH. As the solubility difference between substrate and product at the given reaction pH is therefore larger at higher temperature, the thermophilic transaminase was successfully used to shift the reaction equilibrium toward rapid product formation.     

Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species

After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1). At the beginning of the process the Damk?hler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).     

Photodynamic potentialities of some phenothiazine derivatives

Summary The quantum yields of fluorescence and phosphorescence and the triplet lifetimes were determined for 29 phenothiazine derivatives; the _p values are varying from 0.2 to 1. The irradiation of phenothiazine derivatives in aerated solutions yields from the triplet state to the formation of singlet oxygen and more especially of superoxide ions and cation-radicals characteristic of the dye. Relative values of the formation rate were determined for these two mechanisms. Production of cation-radicals was correlated with phototoxicity in vivo. Chlorination enhances the two phenomena and acetylation reduces to nothing. A maximum value was estimated for the quantum yield of photoionization.     

Studies on steroid monooxygenase from Cylindrocarpon radicicola ATCC 11011. Oxygenative lactonization of androstenedione to testololactone

A steroid monooxygenase of Cylindrocarpon radicicola was found to catalyze oxygenative lactonization of 17-ketosteroid, androstenedione, to yield D-homo-17 alpha-oxasteroid, testololactone, i.e., the androstenedione monooxygenase reaction, in addition to catalyzing the progesterone monooxygenase reaction. The reaction product was identified by TLC, GLC, and mass spectrometry. The oxygenation proceeded with unitary stoichiometry for 17-ketosteroid, NADPH, and molecular oxygen, indicating that it is a typical monooxygenase reaction of the external electron donor type. The enzyme catalyzed successively the side chain cleavage reaction of 17 alpha-hydroxy-20-ketosteroid to produce its 17-keto derivative and the lactonization of the product. The effects of pH and of the concentration of substrate steroids on the androstenedione monooxygenase reaction were different from those on the progesterone monooxygenase reaction. Progesterone is a strong and competitive inhibitor of the lactonization of 17-ketosteroids. The steroid monooxygenase is concluded to have the activities of both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids.     

Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp.

Redox conversions of exogenic C₁₉-steroids (androstenedione (AD), androstadienedione (ADD), testosterone and 1(2)-dehydrotestosterone) were studied using a mutant strain of Mycobacterium sp. Et1 with high level of 17β-hydroxysteroid dehydrogenase (17-HSD) activity. Factors affecting target 17β-reduction and side reactions by resting and growing cells were estimated. The effects of glucose supplementation, pH, mode of substrate addition were identified. The results confirmed that double reduction of androstadienedione, both of 17-keto group and 1(2)-double bond, is more effective for testosterone formation than a single reduction of 17-keto group of AD. These findings argued for the application of the strain capable of sterol side chain degradation and expressed 17-HSD, 3-ketosteroid-1(2)-dehydrogenase and 1-ene-reductase activities, for testosterone obtaining from sitosterol. Under optimal conditions, the conversion of sitosterol (5 g/l) by Mycobacterium sp. VKM Ac-1816D in laboratory fermenter resulted in 50–55% molar yield of testosterone.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Production of l-serine from methanol and glycine by resting cells of a methylotroph under automatically controlled conditions

Serine production from methanol and glycine was tried using frozen-thawed resting cells of a methylotroph, Protomonas extorquens NR-1 under multi-variable controlled conditions. The conditions for l-serine formation were optimized at 30°C. The production of l-serine in 0.4% CaCl₂ solution (initial pH 8.2) was the same as in 0.1 M Tris-HCl buffer (initial pH 8.3). Increasing the initial glycine concentration promoted l-serine formation. A high aeration rate decreased l-serine production. The optimum concentrations of dissolved oxygen and methanol were 0.5 ppm and 10 g/l, respectively. The highest l-serine, 24.9 g/l, was obtained at 24 h from 30.94 gl (as dry weight) resting cells using 100 g/l initial glycine with controlled pH. The relationship between the initial rate of l-serine formation and cell concentration indicated an unusual curve due to the effects of the added NaOH which was used for controlling the pH. In similar experiments without control of pH, a normal profile was observed with respect to the relationship between the initial rate of l-serine formation and cell concentration. The highest l-serine, 54.5 g/l, was obtained at 48 h by 36.4 g/l (as dry weight) resting cells. The yield (mol of l-serine/mol of added substrate) of l-serine from methanol and glycine were 8.3% and 39.3%, respectively. The selectivity of l-serine (mol of l-serine/mol of glycine consumed) was 67.9%. The stoichiometry of the maximum l-serine formation showed that the resting cells carried the highly active methanol dehydrogenase while serine transhydroxymethylase was rather low. Serine glyoxalate aminotransferase was not completely inhibited by the high concentration of glycine (about 68% of synthesized l-serine was detected in the supernatant.