Asparaginase Production Research Articles

Sequential optimization and kinetic modeling of L‐asparaginase production by Pectobacteriumcarotovorum in submerged fermentation

AbstractL‐Asparaginase is a therapeutic enzyme widely used as an antitumor agent for the treatment of acute lymphoblastic leukemia and lymphosarcoma. Its potential applications as a therapeutic agent and food additive require production at high yields in a low‐cost medium with high purity and stability. Statistical experimental designs were applied to identify and optimize the significant parameters for the fermentative production of L‐asparaginase by Pectobacterium carotovorum Microbial Type Culture Collection (MTCC) 1428. Screening of different carbon sources identified galactose as the best carbon source for the production of L‐asparaginase. Twelve medium components were selected and sixteen experiments were carried out based on the Plackett–Burman (PB) design for identifying the most significant medium components. Yeast extract, galactose, monosodium glutamate, MgSO4.7H2O and CaCl2.2H2O were found to be the most significant components that affected the production of L‐asparaginase, and they were optimized by response surface methodology (RSM) using central composite design. This optimized medium showed maximum production rates of periplasmic and extracellular L‐asparaginase of 3.25 and 0.83 U ml−1, respectively. The periplasmic L‐asparaginase production in the optimized medium was twofold higher than in the basal medium. Unstructured kinetic models were applied and satisfactorily described the cell growth, product formation and substrate utilization profile, thus providing a better understanding and prediction of the fermentation profile. This paper details the application of PB experimental design and RSM for the optimization of L‐asparaginase production from Pe. carotovorum MTCC 1428 in submerged fermentation. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd.

Solid state fermentation for production of L – asparaginase in rice bran by Serratia marcescens SB08

L – asparaginase, produced by various genera under solid-state and submerged cultivation, is an important chemotherapeutic agent used for the treatment of a variety of lymphoproliferative disorders and lymphomas, acute lymphoblastic leukemia in particular. In this study, L – asparaginase production by Serratia marcescens SB08 in rice bran supplemented with different nutrient factors in broth medium was investigated. With 50 % initial moisture content in rice bran and supplemented with yeast extract (0.5%, w/v) and at 36 h incubation time, a maximum L asparagine production of 79.84U/g was achieved. When rice bran was combined with other starchy substances in the culture medium, quantum of L – asparaginase yield declined significantly. In rice bran, Serratia marcescens SB08 produced comparatively more L – asparaginase yield. Since rice bran is a very cheap, easily available source and more suited for L asparaginase production in solid state cultivation, a detailed study for the large-scale fermentation processes using rice bran are suggested.

Modification of recombinant asparaginase from Erwinia carotovora with polyethylene glycol 5000

A method for polyethylene conjugation with recombinant asparaginase has been developed to improve therapeutically important properties of this enzyme. Methoxy-nitrophenyl carbamate of polyethylene glycol with molecular weight 5000 was employed as the modification reagent. Optimization of pegylation procedure allowed achieving high level of the enzyme modification. Under 4.5 molar excess of modification reagent more than 10 molecules of methoxy-polyethylene bound per one asparaginase molecular. The modified asparaginase retained 57% of initial activity. A simple and efficient pegylation procedure described in this work can be used for asparaginase production with improved therapeutic properties.

Uso de matérias primas da agroindústria – garapa e extrato de levedura – na produção de asparaginase por Zymomonas mobilis CP4

Sugar cane juice and yeast extract have been used for asparaginase production by Z. mobilis CP4. A complete factorial design of two variables (yeast extract and asparagin) at three levels (1.0; 5.5 and 10.0 g/L) with one replication at the central point was used. Batch fermentation utilised sugar cane juice diluted at 8 % (W/V) of Total Sugars and an inoculum of 2 mg of cells/mL. After fermentation time of 18 hours, the highest production of asparaginase was 9.75 U/L using both yeast extract (5.5 g/L) and asparagin (1.0 g/l).

Production of L-Asparaginase, an Anticancer Agent, From Aspergillus niger Using Agricultural Waste in Solid State Fermentation

This article reports the production of high levels of L-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agro-wastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205-1405 micro range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9 +/- 3.35 U/g of dry substrate) at pH 6.5 and temperature 30 +/- 2 degrees C. The optimum temperature and pH for enzyme activity were 40 degrees C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.

Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor

Various host-vector combinations were tested to maximize the extracellular production of recombinant asparaginase in Escherichia coli. Expression of recombinant asparaginase fused to pelB leader sequence under the inducible T7lac promoter in BLR (DE3) host cells resulted in optimum extracellular production in shake-flasks. Fed-batch studies were carried out using this recombinant strain and an exponential feeding strategy was used to maintain a specific growth rate of 0.3 h(-1). To check the effect of the time of induction on expression, cultures were induced with 1 mM isopropyl-beta-D-thiogalactopyranoside at varying cell optical densities (OD(600): 33, 60, 90, 135). Although the specific product formation rates declined with increasing OD of induction, a maximum volumetric activity of 8.7 x 10(5) units l(-1), corresponding to approximately 5.24 g l(-1) of recombinant asparaginase, was obtained when induction was done at an OD(600) of 90. The recombinant protein was purified directly from the culture medium, using a rapid two-step purification strategy, which resulted in a recovery of approximately 70% and a specific activity of approximately 80% of that of the native enzyme.

Asparaginase II-GFP fusion as a tool for studying the secretion of the enzyme under nitrogen starvation

Production of asparaginase II of Saccharomyces cerevisiae is regulated by nitrogen and can be used as a model system for studying other secreted proteins in yeast. Green fluorescent protein (GFP) from Aequorea victoria was fused to the carboxy-terminus of the enzyme by genomic integration to the locus ASP3 of S. cerevisiae. We determined asparaginase II activity, mRNAASP3, mRNAASP3-GFP and GFP fluorescence. Nitrogen starvation in cells carrying the chimera ASP3-GFP caused an increase in fluorescence and in the expression of ASP3. We have shown that cells producing the chimera Asp3-GFPp displayed the same response to nitrogen starvation as control cells. We demonstrated that Asp3-GFPp can be used for studying asparaginase II secretion under nitrogen starvation in vivo.

Production and properties of asparaginase from a new Erwinia sp.

Asparaginase production by a mesophilic strain of Erwinia sp. was examined; the maximum of activity was found at 40 degrees C and pH 8.5. Among the various carbon sources, mannitol was shown to be the best for production of activity. Inorganic nitrogen sources were better than the organic ones. The enzyme activity was not inhibited by 10 mmol/L metal ions (Na+, K+, Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Zn2+); the activity was strongly inhibited by addition of EDTA. L-Arginine, DL-alanine, L-asparagine and L-glutamine stimulated the L-asparaginase production by 3.9, 1.7, 4.3 and 4.0 fold, respectively. The combination of L-arginine, L-asparagine and L-glutamine synergistically stimulated the asparaginase up to 5.8 fold.

Asparaginase production by chemostat cultures of Vibrio succinogenes

Asparaginase production by chemostat cultures of Vibrio succinogenes

Asparaginase production by chemostat cultures ofVibrio succinogenes

Asparaginase production by chemostat cultures of<i>Vibrio succinogenes</i>

An automated assay for the determination of asparaginase activity

Accurate and precise determinations of activity are required during the production of the drug asparaginase. Experience in a control laboratory situation has shown that manual enzymatic activity assays have a high degree of variability between chemists because of slight differences in technique. An automated assay for the determination of asparaginase activity was developed. It reproduces exactly the same assay conditions each time it is run. The procedure is fast, precise, accurate, and has eliminated technician to technician variability.

Asparaginase production by human clinical isolates of Vibrio succinogenes.

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.

Effect of medium composition on the growth and asparaginase production of Vibrio succinogenes.

Asparaginase synthesis by Vibrio succinogenes is induced by ammonium ions. Synthesis occurs throughout exponential phase, and in early stationary phase asparaginase accounts for about 5% of the total soluble protein. The organism grows best when fumarate is provided as the terminal electron acceptor of the formate-oxidizing cytochrome system. Yeast extract or enzyme-hydrolyzed proteins are effective nutrient sources. In an ammonium formate-sodium fumarate medium, where maximum growth and asparaginase synthesis occurs, the total enzyme yield (international units per liter of culture) is about one-tenth that obtainable with a good asparaginase-producing strain of Escherichia coli. The energetic inefficiency of V. succinogenes appears to cause a low yield of cells and therefore low total enzyme yield. However, the levels of asparaginase accumulated within cells raise questions about the organism's protein synthesizing system.

L-asparaginase production during static incubation of aerobically grown Escherichia coli B.

Glucose, galactose, or pyruvate independently supported biosynthesis of 1.5–2.6 IU/ml of antilymphoma L-asparaginase during static incubation of aerobically grown E. coli B cells. Fructose, lactate, and other compounds did not produce enzyme levels in excess of 0.12 IU/ml. Asparaginase synthesis by cells incubated in the presence of either glucose or pyruvate was inhibited by fluoride, iodoacetate, and sulfite. Amino acid analysis of the modified trypticase soy broth (MTSB) medium, used for aerobic cell proliferation and subsequent enzyme synthesis during static incubation, permitted development of a chemically defined (CD) medium. Washed cells, grown aerobically in the MTSB medium, produced equivalent quantities of L-asparaginase in both the MTSB and CD media. Low levels of chloramphenicol or puromycin reduced enzyme synthesis by as much as 95%.

Effect of oxygen-transfer rate on production of L-asparaginase by Erwinia aroideae.

A variety of aeration and agitation conditions were used to investigate the oxygen demand of Erwinia aroideae for growth and L-asparaginase production in a 14-liter fermenter. Maximum L-asparaginase activity of 4.25 IU/ml was obtained when the fermentation medium was aerated at 0.5 volumes air per volume medium per minute and agitated at 500 rpm. However, the specific L-asparaginase activity was not affected significantly by the rate of oxygen transfer. In most instances the level of dissolved oxygen decreased to zero before harvesting. KLa rather than dissolved oxygen as measured during the fermentation cycle is a more satisfactory index to determine the oxygen requirement for cell growth and asparaginase production. The optimum level of KLa for L-asparaginase production was 0.98 min−1. However, a higher value of KLa of 1.2–1.9 min−1 was needed to achieve the highest growth.

Production of tumor-inhibitory L-asparaginase by submerged growth of Serratia marcescens.

Production of a tumor-inhibitory asparaginase by submerged fermentation with Serratia marcescens ATCC 60 was studied to ascertain optimal nutritional conditions for large-scale production leading to enzyme purification studies. Five strains of S. marcescens were screened in shake-flask studies and were found to produce 0.8 to 3.7 IU/ml 48 hr after inoculation. The requirements for asparaginase production with S. marcescens ATCC 60, the high producing strain, included the following: 4% autolyzed yeast extract medium (initial pH 5.0), an incubation temperature of 26 C, and limited aeration for a zero level of dissolved oxygen during the fermentation. Addition of various carbohydrates to the fermentation medium did not enhance yields. The peak cell population in the fermentation medium and the maximal asparaginase yields occurred simultaneously. Highest enzyme yields were found when the pH of the fermentation cycle rose to approximately 8.5. Yields of 4 IU of asparaginase/ml of cell suspension have been obtained consistently in 40 to 42 hr from 10-liter volumes (500 ml/4-liter bottle) produced on a reciprocating shaker. Scale-up to a 60-liter fermentor yielded 3.1 IU/ml in 35 hr.

Production of Tumor-Inhibitory L-Asparaginase by Submerged Growth of Serratia marcescens1

Production of a tumor-inhibitory asparaginase by submerged fermentation with Serratia marcescens ATCC 60 was studied to ascertain optimal nutritional conditions for large-scale production leading to enzyme purification studies. Five strains of S. marcescens were screened in shake-flask studies and were found to produce 0.8 to 3.7 IU/ml 48 hr after inoculation. The requirements for asparaginase production with S. marcescens ATCC 60, the high producing strain, included the following: 4% autolyzed yeast extract medium (initial pH 5.0), an incubation temperature of 26 C, and limited aeration for a zero level of dissolved oxygen during the fermentation. Addition of various carbohydrates to the fermentation medium did not enhance yields. The peak cell population in the fermentation medium and the maximal asparaginase yields occurred simultaneously. Highest enzyme yields were found when the pH of the fermentation cycle rose to approximately 8.5. Yields of 4 IU of asparaginase/ml of cell suspension have been obtained consistently in 40 to 42 hr from 10-liter volumes (500 ml/4-liter bottle) produced on a reciprocating shaker. Scale-up to a 60-liter fermentor yielded 3.1 IU/ml in 35 hr.