Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Yeast glutathione reductase. Studies of the kinetics and stability of the enzyme as a function of pH and salt concentration.

1. The pH dependencies of the apparent Michaelis constant for oxidized glutathione and the apparent turnover number of yeast glutathione reductase (EC 1.6.4.2) have been determined at a fixed concentration of 0.1 mM NADPH in the range pH 4.5--8.0. Between pH 5.5 and 7.6, both of these parameters are relatively constant. The principal effect of low pH on the kinetics of the enzyme-catalyzed reaction is the observation of a pH-dependent substrate inhibition by oxidized glutathione at pH less than or equal 7, which is shown to correlate with the binding of oxidized glutathione to the oxidized form of the enzyme. 2. The catalytic activity of yeast glutathione reductase at pH 5.5 is affected by the sodium acetate buffer concentration. The stability of the oxidized and reduced forms of the enzyme at pH 5.5 and 25 degrees C in the absence of bovine serum albumin was studied as a function of sodium acetate concentration. The results show that activation of the catalytic activity of the enzyme at low sodium acetate concentration correlates with an effect of sodium acetate on a reduced form of the enzyme. In contrast, inhibition of the catalytic activity of the enzyme at high sodium acetate concentration correlates with an effect of sodium acetate on the oxidized form of the enzyme.     

On Extraction and Quantitation of Plant Peroxidase Isoenzymes

Peroxidase in tobacco callus tissue differed in extract-ability depending on the subcellular distribution of the enzyme. Based on extractability it consisted of four fractions: freely soluble and less freely soluble in phosphate buffer, KCl-soluble, and insoluble. The latter two fractions were un-extractable by a phosphate buffer alone. The different fractions contained varied proportions of peroxidase isoenzymes. The extractability of indoleacetic acid oxidase was similar. A medium of high ionic strength is essential for quantitative extraction of peroxidase and indoleacetic acid oxidase isoenzymes. For quantitation of isoperoxidase activity on polyacryl-amide gel following electrophoretic separation, benzidine and o-dianisidine were better hydrogen donors than guaiacol and pyrogallol. The optimum pH was 4.5, but a citrate buffer was inhibitory. The optimum conditions included an acetate buffer at pH 4.5, a substrate concentration of 0.03 %, benzidine as the hydrogen donor, and a 3-minute treatment with 7 % acetic acid after staining. The color intensity of the bands remained unchanged for at least three days. With appropriate sample size and reaction time there was a linear relationship between enzyme concentration and activity.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Fluorimetric determination of d-amino acid oxidase

1. A sensitive fluorimetric procedure for the assay of d-amino acid oxidase has been developed. 2. The method depends on the formation of a fluorescent derivative, 2-hydroxy-3-methylquinoxaline, on condensation of pyruvate with o-phenylenediamine in acid medium. 3. 2-Hydroxy-3-methylquinoxaline fluoresces strongly in 50% (v/v) sulphuric acid after excitation at 375mmu. A single emission peak is observed at 480mmu. 4. Formation of the quinoxaline is dependent on time, temperature, acidity and relative concentration of reactants. 5. A particulate preparation from mouse kidney required FAD for optimum activity at pH8.5; K(m) was 3.8x10(-3)m; K(FAD) was 1.4x10(-7)m and the reaction was strongly inhibited by p-chloromercuribenzoate and phenylmercuric acetate. 6. Subcellular fractionation on a sucrose density gradient confirmed that the d-amino acid oxidase was localized on small granules.     

The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Modulation of radiation-induced changes in the xanthine oxidoreductase system in the livers of mice by its inhibitors

The xanthine oxidoreductase (XOD) system, which consists of xanthine dehydrogenase (XDH) and xanthine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOD system is present predominantly in the normal tissues as XDH. In damaged tissues, XDH is converted into XO, the form that generates free radicals. Therefore, the XO form of the XOD system is expected to be found mainly in radiolytically damaged tissue. In this case, XO may catalyze the generation of free radicals and potentiate the effect of radiation. Inhibition of the XOD system is likely to attenuate the detrimental effects of ionizing radiation. We have examined this possibility using allopurinol and folic acid, which are known inhibitors of the XOD system. Swiss albino mice (7-8 weeks old) were given single doses of allopurinol and folic acid (12.5-50 mg/kg) intraperitoneally and irradiated with different doses of gamma radiation at a dose rate of 0.023 Gy/s. The XO and XDH activities as well as peroxidative damage and lactate dehydrogenase (LDH) were determined in the liver. An enhancement of the activity of XO and a simultaneous decrease in the activity of XDH were observed at doses above 3 Gy. The decrease in the ratio XDH/XO and the unchanged total activity (XDH + XO) suggested the conversion of XDH into XO. The enhanced activity of XO may potentiate radiation damage. The increased levels of peroxidative damage and the specific activity of LDH in the livers of irradiated mice supported this possibility. Allopurinol and folic acid inhibited the activities of XDH and XO, decreased their ratio (XDH/XO), and lowered the levels of peroxidative damage and the specific activity of LDH. These results suggested that allopurinol and folic acid have the ability to inhibit the radiation-induced changes in the activities of XDH and XO and to attenuate the detrimental effect of this conversion, as is evident from the diminished levels of peroxidative damage and the decreased activity of LDH.     

Immobilization of alpha-chymotrypsin on poly(urethane-graft-acrylic acid)

A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

High rates of [1-14C]acetate incorporation into the lipid of isolated spinach chloroplasts.

Spinach chloroplasts, isolated by techniques yielding preparations with high O2- evolving activity, showed rates of light-dependent acetate incorporation into lipids 3-4 fold higher than any previously reported. Incorporation rates as high as 500 nmol of acetate/h per mg of chlorophyll were measured in buffered sorbitol solutions containing only NaHCO3 and [1-14C]acetate, and as high as 800 nmol/h per mg of chlorophyll when 0.13 mM-Triton X-100 was also included in the reaction media. The fatty acids synthesized were predominantly oleic (70-80% of the total fatty acid radioactivity) and palmitic (20-25%) with only minor amounts (1-5%) of linoleic acid. Linolenic acid synthesis was not detected in the system in vitro. Free fatty acids accounted for 70-90% of the radioactivity incorporated and the remainder was shared fairly evenly between 1,2-diacylglycerols and polar lipids. Oleic acid constituted 80-90% of the free fatty acids synthesized, but the diacylglycerols and polar lipids contained slightly more palmitic acid than oleic acid. Triton X-100 stimulated the synthesis of diacylglycerols 3-6 fold, but stimulated free fatty acid synthesis only 1-1.5-fold. Added glycerol 1-phosphate stimulated both the synthesis of diacylglycerols and palmitic acid relative to oleic acid, but did not increase acetate incorporation into total chloroplast lipids. CoA and ATP, when added separately, stimulated acetate incorporation into chloroplast lipids to variable extents and had no effect on the types of lipid synthesized, but when added together resulted in 34% of the incorporated acetate appearing in long-chain acyl-CoA. Pyruvate was a much less effective precursor of chloroplast fatty acids than was acetate.     

Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.     

Partially deacetylated chitin as an acid-stable support for enzyme immobilization

Partially deacetylated chitin (PDAC) obtained by boiling chitin in 28.6% (w/w) sodium hydroxide was not dissolved when it was suspended in 2% acetic acid (pH 2.6) at 60°C for 12 h or autoclaved in acetate buffer (pH 5.0) for 20 min. The enzyme binding ability of the PDAC with glutaraldehyde was similar to that of chitosan. Immobilized pullulanase had low enzyme activity for high-molecular-weight material such as pullulan, but its activity for maltosyl β-cyclodextrin was almost the same as that of the free enzyme. The immobilized enzyme produced branched cyclodextrin through a reverse reaction in acetate buffer of pH 3.75 at 53°C.     

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.     

Mechanisms of Fatty Acid Toxicity for Yeast.

Neal, A. L. (Rutgers, The State University, New Brunswick, N.J.), Joan O. Weinstock, and J. Oliver Lampen. Mechanisms of fatty acid toxicity for yeast. J. Bacteriol. 90:126-131. 1965.-The internal pH of stationary- and log-phase yeast cells dropped quite rapidly when the cells were exposed to acetate buffers at pH 4 and 3, whereas no, or much less, acidification occurred with pyruvate or phosphate. Although inhibition of respiration and glycolysis was almost instantaneous when the cells were exposed to 0.2 m acetate at pH 4, the effect was not permanent and could be reversed by washing them with water or phosphate buffer. Irreversible inhibition did occur, however, at 0.5 m acetate under the same conditions; there was a marked decrease in several glycolytic enzyme systems, which undoubtedly contributed to the irreversible nature of the inhibition. In cell-free homogenates, various low-molecular-weight monocarboxylic acids exhibited about the same inhibitory effect on glycolysis; structural differences such as branching or unsaturation did not cause a marked change in their inhibitory effect. Also, glycolysis was much more sensitive to dicarboxylic acids such as succinate and phthalate than to acetate; phthalate was more inhibitory than succinate. This is in contrast with the noninhibitory nature of succinate and phthalate to whole cells, even at pH 4. Pyruvic acid decarboxylation was inhibited by phthalate but not by succinate. The greater toxic effect of phthalic acid may be due to the fixed steric configuration of its carboxyl groups, as compared with those of succinic acid.     

A sensitive method for quantitative analysis of hydrogen peroxide and oxidase substrates in biological samples]

The sensitive method for peroxidative assays of hydrogen peroxide (up to 5 nmole in 1 ml) and oxidases' substrates (glucose, ethanol) has been developed by use of a non-cancerogenic chromogen--3,3', 5,5'-tetramethylbenzidine (TMB), horseradish peroxidase and corresponding oxidases. The proposed modification of the known method consists in using decreased concentration of TMB (0.05-0.1 mM) and stopping the reaction by adding of acid to pH 1.4-2.1. This approach allowed to enhance the sensitivity of assays of corresponding analytes (7-10 times) and decrease the costs of analysis.     

Determination of L-dopa using electropolymerized 3,3',3',3'-tetraaminophthalocyanatonickel(II) film on glassy carbon electrode

Electropolymerized film of 3,3',3',3'-tetraaminophthalocyanatonickel(II) (p-Ni(II)TAPc) on glassy carbon (GC) electrode was used for the selective and stable determination of 3,4-dihydroxy-L-phenylalanine (L-dopa) in acetate buffer (pH 4.0) solution. Bare GC electrode fails to determine the concentration of L-dopa accurately in acetate buffer solution due to the cyclization reaction of dopaquinone to cyclodopa in solution. On the other hand, p-Ni(II)TAPc electrode successfully determines the concentration of L-dopa accurately because the cyclization reaction was prevented at this electrode. It was found that the electrochemical reaction of L-dopa at the modified electrode is faster than that at the bare GC electrode. This was confirmed from the higher heterogeneous electron transfer rate constant (k(0)) of L-dopa at p-Ni(II)TAPc electrode (3.35 x 10(-2) cms(-1)) when compared to that at the bare GC electrode (5.18 x 10(-3) cms(-1)). Further, it was found that p-Ni(II)TAPc electrode separates the signals of ascorbic acid (AA) and L-dopa in a mixture with a peak separation of 220 mV. Lowest detection limit of 100 nM was achieved at the modified electrode using amperometric method. Common physiological interferents like uric acid, glucose and urea does not show any interference within the potential window of L-dopa oxidation. The present electrode system was also successfully applied to estimate the concentration of L-dopa in the commercially available tablets.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Intracellular aminopeptidase from Xanthomonas rubrilineans, hydrolyzing alpha-amino acid esters and cefalexin

The aminopeptidase was isolated from cell-free extracts of Xanthomonas rubrilineans by protein precipitation by isopropyl ester with subsequent purification by affinity chromatography on CABS-Sepharose, bacitracin-Sepharose, gel filtration through Sephadex G-200 and ultrafiltration, the total yield being 32% with 2200-fold purification. The enzyme was homogeneous during SDS-PAAG electrophoresis. Apart from the broad spectrum of the peptidase activity, aminopeptidase possesses a hydrolase activity towards beta-lactam antibiotics and an esterase activity towards L- and D-amino acids. Besides, this enzyme catalyzes the acetyl transfer reaction during cephalexin synthesis from the D-phenylglycine ester and 7-aminodesacetoxycephalosporanic acid. The maximal enzyme activity during L-Ala-pNA and cephalexin hydrolysis is manifested at pH 6.5. The enzyme is stable at pH 4.0-8.0 and is inhibited by o-phenanthroline, p-chloromercuribenzoate, hydrogen acetate and N-bromosuccinimide. The molecular mass of the enzyme is 270-280 kDa. The enzyme is a tetramer; the molecular mass of each of its four subunits is 70 +/- 2 kDa. The isoelectric point for the enzyme is 6.8. The amino acid composition of the enzyme appears as follows: Asp63, Thr33, Ser32, Glu72, Gly55, 1/2Cys3-4, Val45, Ile24, Leu53, Tyr23, Phe24, Lys23, His16, Arg36, Pro60, Met25, Ala55.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.