L-Asparaginase Production by Erwinia aroideae

Maximum yields of 1,250 IU (international unit)/g (dry weight of cells) of L-asparaginase were obtained in 8 hr from Erwinia aroideae NRRL B-138. Partial purification and concentration of the extracted L-asparaginase yielded a preparation with an activity of 275 IU/ml. Only one L-asparaginase was present as determined by electrophoresis, and the enzyme exhibited a pH optimum of 7.5 and a K(m) of 3 x 10(-3) M.     

Fermentation Kinetics and Continuous Process of L-Asparaginase Production

For the purpose of obtaining L-asparaginase in quantities from Erwinia aroideae, cell growth and enzyme formation were investigated in both batch and continuous fermentation. Using yeast extract as a growth-limiting substrate, the relationship between specific growth rate and substrate concentration was found to fit the Monod equation. The optimum temperature for enzyme production was 24 C, although cell growth was higher at 28 C. The enzyme yield reached its maximum of 4 IU/ml during the negative acceleration growth phase which occurs just prior to stationary growth. Compared to batch fermentations, the continuous fermentation process gave a lower enzyme yield except when the fermentation was conducted at a dilution rate of 0.1 hr(-1). The graphical method frequently used for prediction of continuous fermentation does not apply to L-asparaginase production by E. aroideae. The optimum temperature for enzyme production in continuous process was 24 C, which was the same as in batch process. Increasing the temperature from 24 to 28 C resulted in a 20% loss of enzyme yield.     

Purification of L-asparaginase from a bacteria Erwinia carotovora and effect of a dihydropyrimidine derivative on some of its kinetic parameters

L-Asparaginase shows antileukemic activity and is generally administered in the body in combination with other anticancer drugs like pyrimidine derivatives. In the present study, L-asparaginase was purified from a bacteria Erwinia carotovora and the effect of a dihydropyrimidine derivative (1-amino-6-methyl-4-phenyl-2-thioxo, 1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester) was studied on the kinetic parameters Km and Vmax of the enzyme using L-asparagine as substrate. The enzyme had optimum activity at pH 8.6 and temperature 35 degrees C, both in the absence and presence of pyrimidine derivative and substrate saturation concentration at 6 mg/ml. For the enzymatic reaction in the absence and presence (1 to 3 mg/ml) of dihydropyrimidine derivative, Km values were 7.14, 5.26, 4.0, and 5.22 M, and Vmax values were 0.05, 0.035, 0.027 and 0.021 mg/ml/min, respectively. The kinetic values suggested that activity of enzyme was enhanced in the presence of dihydropyrimidine derivative.     

A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy

The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). There is no simple method available for measuring the activity of this highly toxic drug. We incubated L-asparaginase from Erwinia chrysanthemi with L-aspartic acid beta-(7-amido-4-methylcoumarin) and measured the release of 7-amino-4-methylcoumarin fluorometrically for 30-300 min. The rate of the hydrolysis of the substrate was linear over a 50-fold range of the concentration of the enzyme. With increasing substrate concentration, the enzyme showed a saturable kinetic pattern with V(max) of 0.547 (SD 0.059) microM/min/mg of enzyme (n = 3) and Km of 0.302 (SD 0.095) mM (n = 3). This assay enables rapid analysis of L-asparaginase activity in biological samples and it can be used, for example, for monitoring of L-asparaginase activity in serum of ALL patients during their L-asparaginase therapy.     

PEGylated recombinant L-asparaginase from <Emphasis Type="Italic">Erwinia carotovora</Emphasis>: Production,properties, and potential applications

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Homogeneols L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 +/- 2 IU per mg of protein was obtained. The pure enzyme can be stored at -20 degrees for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit weight of approximately 37,000. The Km of the enzyme for L-asparagine is 4.78 X 10(-5) M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. L-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130- to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L-asparaginase (EC-2) of Escherichia coli.     

L-Asparaginase Production by Streptomyces griseus

Streptomyces griseus ATCC 10137 synthesizes about 1 IU of L-asparaginase/100 ml of a 4% peptone medium. The enzyme has a pH optimum of 8.5 which is comparable to that of the L-asparaginase derived from Escherichia coli which has antitumor properties.     

Removal of pleural liquid and protein by lymphatics in awake sheep

The contribution of the parietal pleural lymphatics to pleural liquid and protein removal is unclear. We asked two questions. What is the rate of removal of sterile, artificial hydrothoraxes in awake sheep? What percentage is removed through parietal pleural lymphatics? Three days after the placement of a rib capsule in 18 sheep, we instilled a 10 ml/kg 1.0 g/dl autologous protein solution with labeled albumin and erythrocytes through the capsule into the pleural space. Erythrocytes were used as a marker for lymphatic flow. We measured terminal pleural liquid volume and radioactivity at periods from 2 to 48 h. In three sheep, we obtained a third volume measurement at 6 h by the volume of dilution technique. We found that hydrothorax removal could be described by a linear function with a constant rate: 0.28 +/- 0.01 ml.kg-1.h-1 (mean +/- SE) for the grouped data, and 0.20, 0.28, and 0.31 ml.kg-1.h-1 for the individual sheep. At 24 h, erythrocyte clearance was 89 +/- 16% (mean +/- SD) that of liquid and albumin clearance. We conclude that in awake sheep with large hydrothoraxes, pleural liquid and protein are removed at a rate of 0.28 +/- 0.01 ml.kg-1.h-1 (mean +/- SE) and lymphatics are responsible for at least 89% of this removal.     

Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10

An L-asparaginase producing mesophilic fungus Cylindrocarpon obtusisporum MB-10 was isolated from soil. The constitutive intracellular L-asparaginase from the organism was purified. The enzyme after 65-fold purification with an overall yield of 11% and specific activity of 100 unit.mg-1 seemed to be homogeneous in native, SDS-PAGE and thin layer isoelectric focusing gel. The apparent Mr of the enzyme was 216,000, and it constituted four identical subunits. The pI of the enzyme was 5.5. It was a conjugate protein with 37.3% (w/w) carbohydrate. The enzyme was stable to storage at -20 degrees C and to repeated freezing and thawing. The L-asparaginase from the organism was very much specific for L-asparagine and did not hydrolyze D-asparagine and L-glutamine. The pH and temperature optima for the enzyme activity were 7.4 and 37 degrees C, respectively. The Km of the L-asparaginase was found to be 1 x 10(-3)M. Metal ions, such as Zn2+, Fe2+, Cu2+, Hg2+ and Ni2+ potentially inhibited the enzyme activity, while metal chelators like EDTA, CN-, cysteine, etc., enhanced the activity indicating that the enzyme was not a metalloprotein. Its activity was also enhanced in the presence of reduced glutathione but not with dithiothreitol and 2-mercaptoethanol. Differential inhibition of the enzyme activity was observed with iodoacetamide and p-chloromercuribenzoate, thus indicating possible involvement of free-SH group in the enzyme catalysis.     

新的分离纯化青霉素酰化酶方法的研究

按0．6％(w／v)的比例将皂土加到青霉索酰化酶发酵上清液中,可将酶100％吸附,而吸附的蛋白质仅占发酵上清液中的10％左右。吸附时的pH和无机盐对酶的吸附影响不大。使用不同pH和种类的缓冲液洗涤皂土－酶复合物,不能将酶洗脱,但可洗脱15％左右吸附的杂蛋白。使用含10％以上的PEG和NaCl的磷酸缓冲液可将酶全部洗脱．酶纯化25倍,浓缩6倍左右。此法特点是简便,酶活力收率高,可在常温下操作,也可直接从未除菌体的发酵液中提取酶,具有工业应用价值。     

Hydrolysis of oleuropein by recombinant beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus immobilised on chitosan matrix

The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1). For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.     

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Studies on pH and thermal stability of novel purified L-asparaginase from Pectobacterium carotojorum MTCC 1428

Glutaminase free L-asparaginase is known to be an excellent anticancer agent. In the present study, the combined effect of pH and temperature on the performance of purified novel L-asparaginase from Pectobacterium carotovorum MTCC 1428 was studied under assay conditions using response surface methodology (RSM). Deactivation studies and thermodynamic parameters of this therapeutically important enzyme were also investigated. The optimum pH and temperature of the purified L-asparaginase were found to be 8.49 and 39.3 degrees C, respectively. The minimum deactivation rate constant (k(d)) and maximum half life (t1/2) were found to be 0.041 min(-1) and 16.9 h, respectively at pH of 8.6 and 40 degreesC. Thermodynamic parameters (deltaG, deltaH, deltaS, and activation energies) were also evaluated for purified L-asparaginase. The probable mechanism of deactivation of purified L-asparaginase was explained to an extent on the basis of deactivation studies and thermodynamic parameters.     

Cloning, expression and characterisation of Erwinia carotovora L-asparaginase

Bacterial L-asparaginases (E.C. 3.5.1.1) have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. L-asparaginase from Erwinia carotovora NCYC 1526 (ErA) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilised L-asparagine. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of ErA, based on the known structure of the Erwinia chrysanthemi enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The kinetic parameters of selected substrates were determined at various pH values, and the pH-dependence profiles of V(max) and V(max)/K(m) were analyzed. The pH-dependence of V(max) shows one transition in the acidic pH range with pK(a)=5.4, and the pH-dependence of V(max)/K(m) exhibits two transitions with pK(a)=5.4 and 8.5. Based on analysis of alternative substrates and molecular modelling studies, it was concluded that the pK(a) at the acidic pH range corresponds to the active site residues Asp115 or Glu82, whereas the pK(a) observed at the alkaline pH range is not due to substrate amino group ionisation, but rather is the result of enzyme ionisation. The effect of temperature and viscosity on the catalytic activity of the enzyme was also investigated and it was concluded that the rate-limiting step of the catalytic reaction is relevant to structural transitions of the protein. Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity.     

Withania somnifera (Ashwagandha): a Novel Source of L-asparaginase

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. Was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. Somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72±0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37℃. The Km value for the enzyme is 6.1×10-2 mmol/L. This is the first report for L-asparaginase from W. Somnifera, a traditionally used Indian medicinal plant.     

L-Asparaginase in Treatment of Acute Leukaemia and Lymphosarcoma

L-Asparaginase was used to treat 40 patients with acute leukaemia or lymphosarcoma. Fifteen with acute lymphoblastic leukaemia either untreated or in relapse after previous therapy were given “Squibb,” “Bayer,” or “Porton” L-asparaginase. Five of these patients had complete remission of their disease, and four had good partial remission. Eleven patients with acute myeloid leukaemia were treated for a short period with L-asparaginase alone. None of them went into remission though a pronounced fall in the numbers of circulating white cells was seen. Six patients with lymphosarcoma received L-asparaginase, two of them having good partial remissions.The toxic side-effects of the L-asparaginase from the three sources seemed to vary, and L-asparaginase from Erwinia carotovora appeared to be antigenically different from the enzyme produced by Escherichia coli.The way in which leukaemic cells become resistant to the action of the enzyme requires further investigation. To overcome this resistance asparaginase should be used in combination with other drugs in the treatment of acute leukaemia.     

Physical properties of L-asparaginase from Serratia marcescens.

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.     

Activity and operational stability of immobilized mandelonitrile lyase in methanol/water mixtures

Summary The enzyme mandelonitrile lyase was covalently immobilized on solid support materials using different methods. Immobilization on porous silica using coupling with glutaraldehyde afforded preparations with high enzyme loading (up to 9% (w/w)). The immobilized enzyme was used in a packed bed reactor for the continuous production of d-mandelonitrile from benzaldehyde and cyanide. The influence of the flow rate, pH, substrate concentrations and enzyme loading on the reaction yield and the enantiomeric purity of the product was investigated. In order to suppress the competing spontaneous reaction, the enzymatic reaction must be rapid. A flow rate of 9.5 ml/min (0.1 M benzaldehyde and 0.3 M HCN) through a 3 ml reactor afforded a 86% yield of mandelonitrile with 92% enantiomeric excess. No leakage of enzyme occurred under continuous operation. One column was used continuously for 200 h without any decrease in yield or enantiomeric purity of the product. High concentrations of benzoic acid were shown to decrease the operational stability of the system.     

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.