A radiometric method for the measurement of l-asparaginase at concentrations below 1 × 10−5 I.U./ml: Technique and application

A radiometric method for the measurement of low levels of l-asparaginase activity (EC 3.5.1.1) has been devised. This technique uses a protracted incubation at 37°C to magnify amidohydrolytic activity. During this time, in most of the cases examined, l-[U-¹⁴C]asparagine is hydrolyzed to l-[U-¹⁴C]aspartic acid in a linear way; the l-[U-¹⁴C]aspartic acid so generated is transaminated with α-ketoglutaric acid by l-glutamic acid oxaloacetate transaminase (EC 2.6.1.1) and the [U-¹⁴C]oxaloacetic acid so formed is β-decarboxylated by Zn²⁺ at pH 5.0. Using this procedure, low levels of l-asparaginase have been detected in the serum of the chicken, horse, and ox. Use also has been made of maleimide, which inhibits mammalian l-asparaginase without affecting the enzymes from bacterial sources, to discount the possibility that bacterial contamination of mammalian samples was responsible for the activity seen. When a survey was conducted of the distribution of l-asparaginase in the organs of Mus musculus, testis was found to contain surprisingly high levels of the analogous hydrolase from liver. The applicability of this technique to the measurement of feeble activities of l-asparaginase leeched from Dacron tubing to which l-asparaginase has been covalently bound, also has been demonstrated.     

Localization and production of novel l-asparaginase from Pectobacterium carotovorum MTCC 1428

Bacterial l-asparaginase has been widely used as therapeutic agent in the treatment of various lymphoblastic leukemia diseases. Studies on localization and production of novel glutaminase-free l-asparaginase were performed using Pectobacterium carotovorum MTCC 1428. The localization of l-asparaginase was carried out using cell fractionation techniques. The activity of l-asparaginase was found to be 85 and 77% in the cytoplasm of P. carotovorum MTCC 1428 grown on medium containing l-asparagine and combination of l-asparagine and glucose respectively. Among the tested carbon sources, l-asparagine or the combination of l-asparagine and glucose was found to be the most suitable carbon sources to maximize the production of l-asparaginase. The maximum production of l-asparaginase was observed to be 14.56 U/ml (26.92 U/mg of protein) at 4 and 2 g/l of l-asparagine and glucose respectively. Yeast extract, l-asparagine and peptone have shown significant effect on the production of l-asparaginase. P. carotovorum MTCC 1428 has assimilated l-asparagine as an essential carbon source for maximizing the production of l-asparaginase.     

New procedures for purification of L-asparaginase with high yield from Escherichia coli

l-Asparaginase is now known to be a potent antineoplastic agent in animals and has given complete remission in some human leukemias. Extensive clinical trials of this enzyme, however, were not possible in the past because of inadequate production of this substance. We have developed practical procedures for producing l-asparaginase in yields of sufficient quantity and purity for more extensive clinical evaluation. The nutritional requirements for optimal production of biologically active l-asparaginase by a strain of Escherichia coli have been ascertained. The highest yields of enzyme were obtained when cells were grown aerobically in a corn steep medium. Good enzyme production was associated with media containing l-glutamic acid, l-methionine, and lactic acid. The addition of glucose to the medium, however, resulted in depressed production of l-asparaginase. Sodium ion appeared to suppress l-asparaginase production. With the procedure described for isolation of biologically active l-asparaginase from E. coli, stable l-asparaginase preparations with a specific activity of 620 IU per mg of protein (1,240-fold purification with 40% total recovery) were obtained.     

Guinea pig serum l-asparaginase: Purification, and immunological relationship to liver l-asparaginase and serum l-asparaginases in other mammals

l-asparaginase, an enzyme used in the treatment of acute lymphocytic leukemia, is found in the serum of only a few mammalian groups, including the guinea pig and its close relatives in the superfamily Cavioidea. This report describes the purification and characterization of l-asparaginase from guinea pig serum. Antiserum against the purified enzyme cross-reacted with sera from other Cavioidean species but not with mouse serum. Relatively weak cross-reaction with unpurified l-asparaginase in guinea pig liver indicates a significant degree of evolutionary divergence.     

Continuous assays of L-asparaginase by coupling with glutamic dehydrogenase and by cationic glass electrode

Continuous assays of l-asparaginase by coupling with the glutamic dehydrogenase reaction and by cationic glass electrode are described. The procedures are about equally sensitive although the latter is simpler and a more direct assay. The two methods gave identical specific activity values for a purified preparation of Escherichia colil-asparaginase.     

l-Asparaginase from Proteus vulgaris

To produce an immunologically and enzymologically new type of l-asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium, and Proteus were found to produce l-asparaginases in high levels. Among these l-asparaginases, partially purified l-asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified l-asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia colil-asparaginase on the Ouchterlony agar plate. Culture conditions for the production of l-asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of l-asparaginase. Under the optimum conditions, 3,700 international units of l-asparaginase was obtained from 1 liter of culture medium.     

粪便微生物宏基因组来源L-天冬酰胺酶的性质表征及应用研究

l-天冬酰胺酶是氨基酸代谢的关键酶,广泛应用于食品和医药领域,肠道菌群及其产生的l-天冬酰胺酶与宿主健康和疾病关系密切。【目的】获取肠道微生物来源的新型l-天冬酰胺酶,并对其进行性质表征和应用研究。【方法】以西黑冠长臂猿粪便微生物宏基因组为模板,克隆l-天冬酰胺酶基因,并在大肠杆菌中表达;对表达出的酶进行酶学性质研究,并用于处理薯条和癌细胞。【结果】克隆获得l-天冬酰胺酶基因NCasn5,全长996 bp,重组酶NCasn5分子量大小为37.296 kDa,最适pH为8.0,最适温度为60 ℃,K_m和V_max值分别为(3.33±0.21) mmol/L和(836.30±13.91)µmol/(min·mg),37 ℃体外血清半衰期约69 h。NCasn5能降低薯条中69.35%的丙烯酰胺含量,抑制人肝癌细胞QGY-7703和人恶性黑色素瘤细胞A-375细胞的生长。【结论】本研究获得的新型l-天冬酰胺酶,具有良好的热稳定性和较长的血清半衰期,不仅无谷氨酰胺酶活性,还能减少油炸薯条中丙烯酰胺的含量,也能诱导癌细胞QGY-7703和A-375凋亡,在食品加工及医药领域具有潜在的应用价值。     

Effect of Glucose and Low Oxygen Tension on l-Asparaginase Production by a Strain of Escherichia coli B

Cultural and nutritional requirements for maximum l-asparaginase synthesis were determined. Conventional aerobic and anaerobic fermentations were not satisfactory. The former yielded larger quantities of cells containing minimal amounts of l-asparaginase, whereas the latter supplied only minute amounts of bacteria that contained an abundance of enzyme. However, the combination of these classical methods, i.e., allowing growth to proceed aerobically until the mid to late exponential phase and then forcing the facultative microbial cells toward anaerobic metabolism by static incubation, produced 2.6 international units of enzyme per ml of fermentation broth when glucose was present. Enzyme synthesis was not induced by terminating aeration-agitation in the absence of glucose, nor was it induced in the presence of glucose when aeration was continued. Use of 0.2 m phosphate buffer resulted in a constant pH near the optimum value of 7.5 during l-asparaginase formation. Addition of 0.05% l-asparagine prior to induction was also beneficial, but other amino acids or their catabolites failed to increase biosynthesis of l-asparaginase.     

Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi l-asparaginase

During the manufacture of the chemotherapeutic enzyme Erwinia chrysanthemi l-asparaginase, a small proportion (approximately 5–15%) of acidic variants, including deamidated species, are observed. Although the deamidated forms appear to have similar specific activity and quaternary structure to the unmodified enzyme, monitoring and control of these forms is important from a regulatory perspective. The extent of Asn to Asp deamidation directly correlates with the time taken to thaw the Erwinia cells. Erwinia l-asparaginase is a tetrameric enzyme containing one site, Asn₂₈₁, theoretically very labile to deamidation due to the sequence Asn-Gly. Structurally, this part of the protein sequence is completely buried inside the tetramer, but solvent-exposed upon tetramer dissociation. During the cell thawing and alkaline lysis sequence of the process, lengthening the cell thaw times by up to 24 h allowed tetramer to reassociate, protected Asn₂₈₁ from deamidation and reduced the acidic species content of the l-asparaginase from approximately 17% to 9% as measured by weak cation-exchange (WCX) HPLC. The correlation of cell thaw time with acidic species content was also confirmed using capillary zone electrophoresis (CZE) and peptide mapping. These studies demonstrate that cell thaw time is an important, if unexpected, control variable for l-asparaginase deamidation.     

Immunological aspects in the L-asparaginase treatment of children with lymphoproliferative diseases]

A group of 20 children, including 14 with acute lymphoblastic leukemia and 6 with lymphosarcoma, was studied. 24 cures of l-asparaginase therapy were carried out. The increase of serum immunoglobulin (IgG, IgA, IgM) levels was found in children treated with smaller (from 300 to 500 I.U./kg b. w.) doses of asparaginase. In the group treated with higher doses (from 501 to 760 I.U./kg b. w.) the maximal increase of immunoglobulins was observed in the second half of the cure with l-asparaginase, followed by a decrease of the immunoglobulins levels at the end of treatment. The presence of anti-asparaginase antibodies in two children with anaphylactic shocks after l-asparaginase has been shown. In these two children and 6 others the lymphocyte count significantly dropped down on the day of shock before l-asparaginase injection.     

A novel multiple reactor system for the long-term production of L-asparaginase by Penicillium sp. LAMAI 505

In this paper a novel system of multiple reactors with immobilized cells (MRICs) to produce l-asparaginase by Penicillium sp. LAMAI 505 is presented. The effects of the number of associated reactors, nutrient composition, residence time for nutrient cycling, and extraction cycles were investigated using an artificial neural network (ANN). The ANN model was satisfactory (R² > 0.85 with an error lower than 2.4%) and the MRICs performed well when compared to the literature. The cyclic operation of the MRICs can regenerate cells within the inert support, allowing for the long-term production of l-asparaginase. l-asparaginase activity of 13.7 U/gds was achieved under optimised conditions that included a residence time of 33.5 min, pH of 5.1, and concentrations of l-asparagine and glucose of 1.2 and 3.0 g/L, respectively. Furthermore, we demonstrate that MRICs are effective with other fungi and can be used for other products.     

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Thermophilic l-asparaginases display high stability and activity at elevated temperatures. However, they are of limited use in leukemia therapy because of their low substrate affinity and reduced activity under physiological conditions. In an attempt to combine stability with activity at physiological conditions, 3 active-site mutants of Pyrococcus furiosus l-asparaginase (PfA) were developed. The mutants, specifically K274E, showed improved enzymatic properties at physiological conditions as compared to the wild type. All variants were thermodynamically stable and resistant to proteolytic digestion. None of the enzymes displayed glutaminase activity, a highly desirable therapeutic property. All variants showed higher and significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli l-asparaginase. Our study revealed that increased substrate accessibility through the active site loop plays a major role in determining activity. A new mechanistic insight has been proposed based on molecular dynamics simulated structures, where dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic l-asparaginases. Our study not only resulted in development of PfA mutants with combination of desirable properties but also gave a mechanistic insight about their activity.     

An Amplex Red-based fluorometric and spectrophotometric assay for l-asparaginase using its natural substrate

We report on the development of a sensitive real-time assay for monitoring the activity of l-asparaginase that hydrolyzes l-asparagine to l-aspartate and ammonia. In this method, l-aspartate is oxidized by l-aspartate oxidase to iminoaspartate and hydrogen peroxide (H₂O₂), and in the detection step horseradish peroxidase uses H₂O₂ to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing l-asparaginase.     

Cytoplasmic L-asparaginase: isolation of a defective strain and mapping of ansA

An Escherichia coli mutant defective in the production of cytoplasmic l-asparaginase I has been isolated. The mutation (ansA) has been mapped between the gap and pncA loci at 39 min on the E. coli map.     

L-asparaginase production by isolated Staphylococcus sp. - 6A: design of experiment considering interaction effect for process parameter optimization

AIMS: Evaluation of fermentation process parameter interactions for the production of l-asparaginase by isolated Staphylococcus sp. - 6A. METHODS AND RESULTS: Fractional factorial design of experimentation (L18 orthogonal array of Taguchi methodology) was adopted to optimize nutritional (carbon and nitrogen sources), physiological (incubation temperature, medium pH, aeration and agitation) and microbial (inoculum level) fermentation factors. The experimental results and software predicted enzyme production values were comparable. CONCLUSION: Incubation temperature, inoculum level and medium pH, among all fermentation factors, were major influential parameters at their individual level, and contributed to more than 60% of total l-asparaginase production. Interaction data of selected fermentation parameters could be classified as least and most significant at individual and interactive levels. Aeration and agitation were most significant at interactive level, but least significant at individual level, and showed maximum severity index and vice versa at enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: All selected factors showed impact on l-asparaginase enzyme production by this isolated microbial strain either at the individual or interactive level. Incubation temperature, inoculum concentration, pH of the medium and nutritional source (glucose and ammonium chloride) had impact at individual level, while aeration, agitation and incubation time showed influence at interactive level. Significant improvement (ninefold increase) in enzyme production by this microbial isolate was noted under optimized environment.     

Preparation and characterization of antigen and antibody absorbents covalently coupled to an inorganic carrier

Immunoadsorbents were prepared by the use of an inorganic carrier. The proteins were covalently coupled to the carrier through a silane coupling agent. An immunoadsorbent prepared with human gamma-globulin was found to complex with specific antibodies. These antibodies could be recovered in high yield and purity from the adsorbent. Similarly, insolubilized antibodies to l-asparaginase were used to isolate and purify the enzyme from crude extracts. These insolubilized antibodies are extremely stable with time and may be reused many times.     

L-glutamine as a substrate for L-asparaginase from Serratia marcescens

l-Asparaginase from Serratia marcescens was found to hydrolyze l-glutamine at 5% of the rate of l-asparagine hydrolysis. The ratio of the two activities did not change through several stages of purification, anionic and cationic polyacrylamide disk gel electrophoresis, and partial thermal inactivation. The two activities had parallel blood clearance rates in mice. l-glutamine was found to be a competitive inhibitor of l-asparagine hydrolysis. A separate l-glutaminase enzyme free of l-asparaginase activity was separated by diethylaminoethyl-cellulose chromatography.     

Catabolite repression during single and multiple induction inEscherichia coli

Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of beta-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction in Escherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of l-tryptophanase was most sensitive, whereas l-asparaginase was not influenced at all. Exogenous cAMP was found to overcome partially the catabolite repression of beta-galactosidase and D-serine deaminase, both during single induction. The synthesis of l-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of l-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP.     

Purification and properties of L-asparaginase from Serratia marcescens

The purification and properties of a tumor inhibitory l-asparaginase from Serratia marcescens are described. The following properties of the enzyme were examined: kinetics of the enzyme reaction, catalytic activity as a function of pH, boundary sedimentation velocity, electrophoresis on polyacrylamide gel, immuno-electrophoresis against homologous and heterologous antisera, immunodiffusion, blood clearance rate in mice, and inhibition of the 6C3HED lymphoma in C3H mice. Complete regression of this tumor was obtained with a smaller dose of the enzyme from S. marcescens than with enzyme from Escherichia coli. The reason for this difference was not evident from a comparison of several properties of the two enzymes.     

Regulation of Escherichia colil-asparaginase II and l-aspartase by the fnr gene-product

l-asparaginase II and l-aspartase, which are known to be regulated by oxygen in Escherichia coli, are shown, by an examination of known fnr mutants, to be under the control of the fnr gene product. Mutants obtained from a procedure devised to select for asparaginase II-deficiency are fnr⁻.