INTRODUCTION

Asparaginase (EC 3.5.1.1) is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid. Asparaginases are naturally occurring enzymes expressed and produced

by microorganisms. Different types of asparaginases can be used for different industrial and pharmaceutical. Intense interest in asparaginase has resulted from the discovery of its ability to inhibit growth of tumors in the mouse, rat, and dog and to suppress human leukemias in clinical trials. The most common therapeutic indications of L-asparaginase are: treatment of Hodgkin’s disease, treatment of acute lymphocytic leukemia mainly in children, acute myelocytic leukemia, acute myelomonocytic leukemia and chronic lymphocytic leukemia, lymphosarcoma treatment and melanosarcoma Studies on the mechanism of antineoplastic action of this enzyme suggest that a number of tumor cells responding to it lack adequate Lasparagine synthetase activity and require an exogenous supply of L-asparagine. Upon depletion of these amino acid tumour cell die, are recently summarized by Bloome. However, the use of a large protein as a drug poses several challenges. Our immune system is designed to destroy large, multisubunit proteins, so L- asparaginase is rapidly cleared from the blood in a day or so. Also, the immune system occasionally responds too strongly to a large influx of foreign protein, leading to allergic reactions.(David S.goodshell,march 2005). L-Asparaginase catalyzes the deamination of asparagine to aspartic acid and ammonia. LAsparaginase is used in combination therapy to treat childhood acute lymphocytic leukemia.

In the 1950's Kidd reported a biochemical difference in metabolism related to the amino acid asparagine was found. Normal cells apparently can synthesize asparagine while leukemia cells cannot. If leukemia cells are deprived of asparagine, they will eventually die. In an

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Some blood cells. Most cells use the enzyme asparagine synthetase (Fig. rely instead on the blood for their supply of asparagine. If the enzyme L-asparaginase is given to humans. The enzyme L-asparaginase was eventually identified as the anticancer agent. coli. malignant growth. 2 . This means they use both asparagine from the diet as well as what they can make themselves (which is limited) to satisfy their large asparagines demand. top) to make their own asparagine. forming the characteristic amide group of asparagine. trusting that there will always be enough and not bothering with making their own. The enzyme takes aspartate and adds an amine. most cells can make their own supplies of asparagine and do not need to obtain it in their diet. more specifically lymphatic tumor cells. L-asparaginase was isolated and tested successfully on human leukemias. however. 1⇓. require huge amounts of asparagines to keep up with their rapid. Eventually the enzyme asparaginase was also found and isolated from the bacteria. Tumor cells. Mechanism of action of drug Our cells require a steady supply of the amino acid asparagine to build proteins. E. various types of leukemias can be controlled. it was found that blood serum from guinea pigs and other South American rodents had antileukemia properties.almost unrecognized and parallel discovery. Thus.

cleaving its amino group off at trusting that there will always be enough and not bothering with making their own.Asparagine synthetase (top) is a large enzyme composed of two identical subunits.The enzyme converts asparagine in the blood into aspartic acid by a deamination reaction. The enzyme is also active with glutamine. Normal cells are able to make all the asparagine they need internally whereas tumor cells become depleted rapidly and die. The one shown here is from bacteria. L-Asparaginase (bottom) purified from bacterial cells is used for chemotherapy. The leukemia cells are thus deprived of their supply of asparagine and will die. The active sites grip asparagine (red) and use a well-placed threonine amino acid (green) to perform the cleavage reaction. This enzyme is composed of four identical subunits. 3 . Our own enzyme uses glutamine to provide the amine instead of ammonia. It connects an ammonia molecule directly to aspartate to form asparagine. L-asparaginase is an enzyme that destroys asparagine external to the cell.

These sideeffects are partially attributed to the glutaminase activity of asparaginase obtained from these sources [9].Apart from its therapeutic application Lasparaginase is also being used in Food industry.leucopoenia pancreatitis. neurologicalseizures and coagulation abnormalities . Erwinia aroideae. The enzyme is produced by a large number of micro organisms that include fungi. Serratia marcescens and Enterobacter aerogenes .now the production will be affected by the different media composition to increase the production. 4 . Enterobacter cloacae.enzyme produced through out the world by submerged culture . but thus far tumourinhibitory activity has been demonstrated only with the asparaginases obtained from Escherichia coli. It has been observed that from previous work the therapeutic effect of L-asparaginase from these two species is accompanied by sideeffects such as anaphylaxis. Recently it has been found that L-asparaginase reduces the formation of carcinogenic acryl amides in deep fried potato recipes.yeast. and Serratia marcescens. diabetes.

OBJECTIVE The objective of the dissertation work is : Production of L-asparaginase from Serratia mercescens     Using different carbon sources Using different nitrogen sources At different temperatures. At different pH 5 .

Plan of workTo meet the said objective are organized carefully under the following options.       Literature survey Selection of microorganism Revival and subculturing Growth in broath media containing different carbon & nitrogen sources Growth in broath media at different pH and temprature Cell harvesting and production 6 .

Enzymes are highly specific 7 . are converted into different molecules.. with an important example being some parts of the ribosome.1 Characteristics of enzymes 1.but enzymes have several characteristics that make them unique. called substrates. products are formed faster and reactions reach their equilibrium state more rapidly. enzymes are not consumed by the reactions they catalyze. 2.1 Biological enzymes. However. As a result. Chemist find to synthesize compounds that even approach this catalytic efficiency. enzymes work by lowering the activation energy (Ea‡) for a reaction. As with all catalysts. 2. increase the rates of) chemical reactions. thus dramatically increasing the rate of the reaction. nor do they alter the equilibrium of these reactions. called products. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities. Enzyme exhibit enormous catalytic power extremely small quantity of enzymes as little as a millionth of mole can accelerate reaction rates by factors of 103 to 106. In enzymatic reactions.which function as biological catalyst. Almost all chemical reactions in a biological cellneed enzymes in order to occur at rates sufficient for life. enzymes do differ from most other catalysts in that they are highly specific for their substrates.000 biochemical reactions. the molecules at the beginning of the process. Enzymes are known to catalyze about 4.Literature Review Enzymes are biological molecules that catalyze (i. the set of enzymes made in a cell determines which metabolic pathways occur in that cell. The million of chemical reactions that take place within our bodies occur rapidly and in a highly specific and organized fashion.1. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. Like all catalysts. Synthetic molecules called artificial enzymesalso display enzyme-like catalysis. They are controlled by class of proteins called enzymes.e. 2. A few RNA molecules called ribozymes also catalyze reactions.

A different asparaginase is marketed as a drug under the brand name Elspar for the treatment of acute lymphoblastic leukemia (ALL)[2] and is also used in some mast cell tumor protocols. particularly in starchy variants which provide .production rate is different in different microbes. The most common use of asparaginases is as a processing aid in the manufacture of food[1]. erwinia. It is usually derived from Escherichia coli. serratia mersescens.coli. and is available in the United Kingdom under the trade name Erwinase.5.1) is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid. marcescens that had been assigned by Bizio in 1823. or intravenous injection without fear of tissue irritation. 3. and was often.amminoacid. While this organism was known formerly by a variety of names. has been recognised as a cause of hospital-acquired infection for the last two decades. [3] Unlike other chemotherapy agents. it can be given as an intramuscular. 2.1. Marketed under the brand names Acrylaway and PreventASe.[2] Production of l-asparaginase from different microbes like-e. The activity of most enzyme are closely realated This regulation cause each reaction to occur at its appropriate time and place and to the appropriate extent.1 Serratia mercescensSerratia marcescens. a suspected carcinogen. so that cellular activity proceeds in a coordinated way. actinomycetes etc. 2. and has been found in food. including Chromobacterium prodigiosum [ 11. in starchy food products such as snacks and biscuits. [2] used the name S. a gram-negative bacillus classified as a member of the Enterobacteriaceae. Asparaginases are naturally occurring enzymes expressed and produced by microorganisms.2 L-Asparaginase Asparaginase (EC 3. subcutaneous.Some enzymes can differentiate the subtle stereo-chemical differences between a Dand L. used as a biological marker because of its easily recognised red 8 . asparaginases are used to reduce the formation of acrylamide. an excellent growth environm’ent. Gaughran et al. Different types of asparaginases can be used for different industrial and pharmaceutical purposes. Almost every biochemical reaction is controlled by specific enzyme.2. It is a widely distributed saprophytic bacterium. Asparaginase produced by Erwinia chrysanthemi instead is known ascrisantaspase (BAN).

marcescens is unable to ferment arabinose in peptone water. liquefaciens strains are arabinose-positive.2. marcescens characteristically produce a red pigment. S. marcescens was defined by Grimont and Grimont [ 121 as an oxidase-negative gramnegative bacillus producing DNAase. and in early times such growth was often mistaken for fresh blood [2]. McCormack and Kunin [ 5 ] reported a nursery epidemic involving 27 babies. marcescens has now been implicated as an aetiological agent in every conceivable kind of infection. In contrast to other gram-negative bacteria. The ability to form prodigiosin is characteristic of S. The first description of nosocomial infection caused by S. marcescens has attained the status of'a hlly fledged pathogen that causes infections particularly in two disparate groups: heroin addicts and hospitalized patients. marcescens was Wheat’s report of 11 cases over a 6month period in 195 1 at Stanford University Hospital [3].colonies.2 Culture and identification S. marcescens has been reported to cause infective endocarditis acquired in the community [lo] and in hospitals. meningitis and wound infections [7-91. but this system was found to correctly identi@ only 85% of S. Its ability to cause infection was once thought to be limited to patients with chronic debilitating disorders. prodigiosin. s 9 . S. including respiratory tract infection. S. water. although only 15 cases of Serratia bacteraemia had been recorded by 1968 [ 6 ] . Therefore. marcescens [12]. Environmental isolates of S. air. giving a false-positive reaction leading to misidentification as S. including arabinose and raffinose. plants and animals [12]. S. it usually affects the left side of the heart [ll]. The pigmented bacterium is found in various ecological niches. S. Professor Scheurlen of the University of Strasbourg concluded that this organism contributed to more deaths than many pathogenic bacteria. to confirm the identity of S. urinary tract infection (UTI). marcescens.but S. marcescens isolates to the species level (personal unpublished results). Today. In 1966. but the hnction of this red pigment remains unclear because clinical isolates are rarely pigmented. Ziquefuciens [ 1 31. Over the last two decades. 2.marcescens endocarditis acquired in the hospital is usually an exogenous infection associated with cardiac surgery [ 1 13. whereas all S. the API 20E system has been used widely for the identification of gram-negative bacilli. including soil. a short series of sugars. After a review in 1896 of a small number of incidents. However. marcescens strains can oxidise arabinose in the API system. Infections caused by this organism have been reported with increasing frequency since 1960 [4]. 'septicaemia.

with light influencing these pigments. Rjazantseva et al. However.2. there was no growth in a culture medium containing NaCl 8% w/v. at different oxygen concentrations in de-ionised water containing materials derived from the blood bags. including in disinfectant [ 14. thus pigment would not appear to be a virulence factor of advantage to clinical isolates. In contrast. The rate of survival and growth was highest under anaerobic conditions. 10 .3 Growth in the environment The potential of S. [17] studied the survival and growth of S. marcescens strain K202. respectively. S. The pigment has a role in respiration [33] and has some antibiotic properties [34]. as with other physiological processes. A culture medium containing glucose as the carbon and energy source did not allow prodigiosin synthesis. marcescens differ in their sensitivity to the effect of NaCl on prodigiosin biosynthesis. [32] found that pigment synthesis started later in the presence of low concentrations of NaCl. isolated from blood bags. marcescens did 2. However. It is presumed that pigment biosynthesis acts as a protective mechanism in unfavourable conditions when the growth of cells is delayed [35]. 151. The highest increase in biomass and maximal pigmentation was found in cultures grown on glycerol medium.2. marcexens has been suggested [32]. Although proline was present ' in the medium. Growing cells contain mono and dimer forms of prodigiosin in glycerol. marcescens to utilise a wide range of nutrients is expressed clearly by its ability to survive and grow under extreme conditions. ions [38]. S. marcescens grown on mineral media did not produce pigment when the carbon source was glucose or the nitrogen source was ammonium chloride. but that prodigiosin accumulatiodbiomass unit was increased at NaCl concentrations of up to 5% wlv. in which growth occurred with all materials and even in de-ionised water alone. Szewzyk et al. Visible light (2000 lux) influenced pigmentation without changing the growth characteristics of the culture.2. The investigation supported the conclusion that strains of S. anionic detergents [39] and amino acids [40]. most strains that cause infection fail to produce pigment and form colourless colonies which are difficult to distinguish from other coliform organisms [41]. The maximum prodigiosin content in dark and light cultures was observed on days 3-4 and 2-3. Light affects the pigment synthesised by the culture directly. marcescens depends on growth conditions [32]. antiseptics [ 161 and doubledistilled water [17]. Pigment biosynthesis. is affected significantly by factors such as temperature [37]. marcescens was also demonstrated. attempts to exploit the pigment as a commercial dye have failed because of its sensitivity to light [36].4 Pigmentation Prodigiosin biosynthesis in S. While the utilisation of light energy by pigmented S. The influence of illumination on pigmentation of S.

the length of time the bacteria are exposed to antibiotic concentrations above the MIC is an important parameter when assessing likely clinical outcome. Despite the unique mechanism of action of this cytotoxic substance which shows relative selectivity with regard to the metabolism of malignant cells. Recently initiated clinical trials have still confirmed the eminent value of asparaginase in the combination chemotherapy of acute lymphatic leukaemia and of some subtypes of non-Hodgkin lymphoma.2. As the killing effect of lactam antibiotics is time dependent. marcescens than for the other gram-negative bacilli studied. The extended activity of cefepime results from its low affinity for type I /3-lactamase and the ease with which the molecule passes through porin channels [55]. Aminoglycosides have good activity against S. Immunological reactions toward the foreign protein include enzyme inactivation without any clinical manifestations as well as anaphylactic shock. is active against organisms such as E. marcescens may be difficult to treat because of resistance to a variety of antibiotics. some patients experience toxic effects during asparaginase therapy.5 Antibiotic susceptibility Infections caused by S.2. Data obtained from a rabbit model suggest that if a P-lactam and aminoglycoside are combined. an extended-spectrum cephalosporin. Use of L-asparaginase in childhood ALL. The changes affecting the proteins of the coagulation system have considerable 11 . cloacae and S. it has therefore been described as a fourth-generation cephalosporin [54]. but resistant strains have-also been reported recently [43]. Cefepime . Since asparaginase monotherapy was associated with a high response rate but short remission duration. Severe functional disorders of organ systems result from the impaired homeostasis of the amino acids asparagine and glutamine. rnarcescens. [56] found that the MIC90s of cefepime and amikacin (both 8 mg/L) were lower for S. marcescens which are frequently resistant to broadspectrum cephalosporins. Chong et al. and its important role as an essential component of multimodal treatment protocols. the enzyme is currently employed within the framework of combination chemotherapy schedules which achieve treatment response in about 90% and long-term remissions in the majority of patients.including ampicillin and first and second generation cephalosporins [42]. the aminoglycoside induces rapid killing and reduction of the inoculum. Owing to the high efficacy of L-asparaginase in the treatment of acute lymphatic leukaemia the enzyme was introduced into the chemotherapy schedules for remission induction of this disease shortly after results of large-scale clinical trials had become available.

Studies on the mechanisms of action and the occurrence of resistance phenomena have shown that a treatment response may only be expected if the malignant cells are unable to increase their asparagine synthetase activity to an extent providing enough asparagine to the cell. Besides this.coli and Erwinia carotovora are now being used in the treatment of acute lymphoblastic leukaemia. L-asparaginase. Other organ systems potentially affected by relevant functional disorders are the central nervous system. Clinical studies using enzymes from E.. the findings of recently published clinical trials indicate that the therapeutic efficacy is affected when different asparaginase preparations are given by identical therapy schedules. and the pancreas. the liver. there are other mechanisms of clinical relevance like induction of apoptosis. The enzyme is produced by a large number of micro organisms that include Enterobacter cloacae. Only few publications have dealt with the question of minimum trough activities to be ensured before each subsequent asparaginase dose in order to maintain uninterrupted asparagine depletion under treatment. 12 .clinical impact as they may induce bleeding as well as thromboembolic events and may be associated with life-threatening complications when the central nervous system is involved. the enzyme which converts L-asparagine to L-aspartic acid and ammonia has been used as a chemotherapeutic agent. While pharmacokinetic studies showed clinically relevant differences in biological activity and activity half-lives for enzymes from different biological sources. The enzymes isolated from E. The dosage and administration schedule of the various enzyme preparations required for complete asparagine depletion over a period of time have been insufficiently defined. and answers to this problem are not definitive. Serratia marcescens and Enterobacter aerogenes. coli asparaginases. coli strains as well as those isolated from Erwinia chrysanthemi and to the PEG-conjugated E. These findings have been transferred to enzymes from other E. one may thus conclude that the enzyme-induced asparagine depletion of the serum constitutes the decisive cytotoxic mechanism. further influences on signal transduction cannot be excluded. Risk factors predisposing to thromboembolic complications are hereditary resistance against activated protein C and any other hereditary thrombophilia. with patients who have a history of pancreatic disorders carrying an especially high risk of developing pancreatitis. coli strains indicate that a trough activity of 100 U/l will suffice for complete asparagine depletion of the fluid body compartments with the preparations studied. Independent of the asparagine depletion related cytotoxicity however. It might be desirable to countercheck the results for confirmation or correction.

lactic acid or both in WF and 933.5-9.In addition to glycerol. it enhanced the enzyme production in mutant 933..0) of the medium.The effect of changing the composition of the medium used for growing two mutants WF and 933 of S. Nutrient broth and CSL medium were equally efficient.Production from Serratia marcescens The production of L-asparginase enzyme by S. glutamic acid and glutamine resulted in increased synthesis of L-asparaginase. Maximal stimulation was observed when L-asparagine or yeast extract were added to glycerol-peptone medium. Production of L-asparaginase was carried out in groundnut cake extract. glycerol-peptone medium was chosen for further studies on enzyme production. while succinic and malic acids had a marginal effect. Maximum enzyme production occurred in both the mutants in glycerol peptone medium. The enzyme is produced by both submerged and solid-state cultures. incubation 13 . Semiaerobic conditions created by the addition of agar and sodium thioglycollate had no effect on enzyme production. 1970. fumaric. While lactose inhibited in WF. It was observed that the enzyme synthesis was stimulated by the addition of glucose in glycerol-peptone medium while soluble starch and dextrin had no effect. aspartic acid. Marcescens is done by Khan et al. α-Ketoglutaric acid slightly inhibited the enzyme production in WF but was marginally stimulatory in the mutant 933. L-asparaginase activity was increased by the addition of L-asparagine to synthetic medium. Extra-cellular asparaginases are more advantageous than intracellular since they could be produced abundantly in the culture broth under normal conditions and could be purified economically. mannitol and sorbitol were inhibitory for enzyme production by the two mutants. Maltose and galactose were marginally favourable while fructose. Yeast extract further activated L-asparaginase production. The parameters studied for submerged culture were initial pH (6. The enzyme activity could not be detected when the mutants were grown in the synthetic medium. marcescens on the levels of the enzyme in the medium is observed. Lactic.The effect on 933 was more pronounced than on WF. In view of the maximal activity observed. pyruvic and citric acids increased enzyme production in both the mutants. glucose and sucrose were the best carbon sources for enzyme production by the two mutants. theree occurred only marginal increase in enzyme production by the addition of L-asparagine. Production from Streptomyces gulbargensis The production of extra-cellular L-asparaginase from Streptomyces gulbargensis using groundnut cake extract. oxalic. In CSL medium. Supplementing the medium with amino acids like methionine.

Maximum Lasparaginase production (12. Maximum yield of L-asparaginase (10.1 M borate buffer pH 7.temperature (25-55oC). solid-state fermentation (SSF) is a more effective 14 . while the lowest yield of enzyme was observed with an inoculum size of 1x105 spores/ml.4 IU) at pH 6. However. The gel was stained with coomassie brilliant blue R-250 and destained with a solution of methanol. acetic acid and water in the ratio of 4:1:5. Optimization of inoculum size is necessary because too few spores lead to insufficient biomass. SDS-PAGE was performed with a separating acrylamide gel of 10% and stacking gel 5% containing 0.9 IU) at temperature 55oC. elution is done with NaCl gradient (0.1 IU) at 140 rev/min.5% maltose. Narayana et al. it was kept constant at that level while varying the other parameters individually. Sephacryl S-200 column in which the protein elution was done with the tris HCl buffer at a flow rate of 5ml/30 min.1% SDS.Purification steps include ammonium sulphate purification. and the lowest (4. Production from marine actinomycetes Production of L-asparginase from marine actinomycetes is done by the technique of submerged fermentation. The effect of addition of various carbon and nitrogen sources on L-asparaginase production was determined in groundnut cake extract. Once a given parameter was optimized.5 and minimum (6.1-0. Maximum production of L-asparaginase by S. CM Sephadex C-50 column. The purification was carried out using crude enzyme extract. albidoflavus was found to be at 35oC (14).5 M) and 0.8 IU) was obtained at pH 8.5.The L-asparaginase activity was associated with the fraction precipitated at 40-60% saturation. inoculum size (1x105-1x109 spores/ml) and agitation speed (140-200 rev/min).1IU) was obtained at an agitation speed of 200rev/min and lowest yield (7. whereas too many spores lead to overproduction of biomass resulting in quick depletion of nutrients.0.5 IU) was observed at a temperature of 40oC. (14) have reported the optimum pH for L-asparaginase production by Streptomyces albidoflavus to be 7.0 IU) of Lasparaginase. The yield of L-asparaginase increased with increase in initial pH of the medium up to 8. The activity of L-asparaginase was evaluated at different pH values and temperature.5 and thereafter it decreased.5. The yield of enzyme increased with increase in agitation speed from 140-200 rev/min and decreased later. The maximum yield (9. The carbon sources were studied at a concentration of 0.9 IU of L-asparaginase.5%. The effect of different concentrations of nitrogen sources was studied by adding a nitrogen source to the extract supplemented with 0. producing 6. An inoculum size of 1x108spores/ml showed the highest yield (11.

Mg2+ ion slightly stimulated activity while Cu2+. (1997). After 30 min of incubation. All three marine soil isolates synthesized asparaginase with yield ranging from 24. 0. the absorbance was measured at 660 nm using a double beam UV – visible and the protein content determined.2 IU/ml. The apparent Km value for the substrate was 25 μM. It also offers many other advantages including resistance to contamination. Among 10 marine isolates subjected to preliminary screening. S4 and K8 showed potential for L-asparaginase activity. solid-state media.5 and 50 ºC. The enzyme was purified to near homogeneity by ammonium sulphate precipitation. gel filtration on Sephadex G-100 column and SDS-PAGE. Zn2+ and EDTA were inhibitory. at a concentration of 1000 μg/ml was made. 1 ml of folins cocatteau reagent was added to each test tube. bovine serum albumin (BSA). Production of Lasparaginase was carried out in three different media.technique as the yield of the product is several times higher than that of submerged fermentation (SF). Marine actinomycetes were isolated from sediment samples obtained from Tamilnadu and Kerala in India. Tryptone Glucose Yeast extract (TGY) broth and Tryptone Fructose Yeast extract (TFY) broth.6 to 49. The determination of protease production was performed to explain the decrease of L-asparaginase activity. After 60 h. Isolation of Lasparaginase from marine actinomycetes by solid-state fermentation using soybean meal and submerged fermentations well as partial purification. Production from filamentous fungi The production of L-asparginase from filamentous fungi. Soil isolate S3 showed the highest productivity of 49. A stock solution of standard protein. only isolates S3. it was demonstrated that the protease activity could be responsible for decrease in L-asparaginase activity since protease levels increase 15 . Twenty-six strains belonging to Aspergillus. ease of product extraction and simpler methods for treating the fermented residue. The isolates were identified as actinomycetes by microscopical and biochemical tests.2 to 1 ml of working standard solution at concentration of 100 μg/ml was taken in test tubes.2 IU/ml with a protein content of 65 μg/ml and optimum activity at pH 7. From this solution.and Fusarium genera were submitted to this systematic investigation. In the screening the optimal chromogenic substrate concentration was 0. and characterization of the crude enzyme extract is done.. namely.04%(V/V). Protein content was determined according to the method of Lowry et al. The volume was made up to 1 ml wi th distilled water to give concentrations ranging from 20 to 100 μg/ml. Protein activity is determined by release of ammonia by nessler’s reagent. dialysis. Penicillium. The screening of filamentous fungi is based on the semi-qualitative method described by Gulati et al.

Different amino acids were used since microorganisms can utilize a wide variety of nitrogen sources. By using different standard proteins with known molecular weights. The effect of the incubation time on L-asparaginase activity was studied in the ranges of zero to 50 min (Figs. The reaction rate of L-asparaginase was measured at various temperatures.900 IU/mg after the Sephadex G-100 and Sephadex C50 steps. respectively. The purified 16 . A Lineweaver-Burk analysis gave Km of 0. different nitrogen sources can be used to promote high enzyme production.7 IU The quaity of P. L-asparaginase production is nitrogen regulated. it was discovered that the apparent molecular weight of Pseudomonas aeruginosa 50071 Lasparaginase was 160 kDa. The medium that was used for the cultivation of Pseudomonas aeruginosa 50071 under (SSF) had the following composition: 10 g Soya bean meal of 0. SDSPAGE showed that the enzyme is one band with electrophoretic mobility of 0.150 to 674 mg in the ammonium sulfate precipitation step. Production from Pseudomonas aeruginosa Cultivation of Bacterial strain of Pseudomonas aeruginosa 50071 was achieved by solidstate fermentation (SSF) as previously reported by Ramesh and Lonsane (1987).4. the enzyme activity decreased (Fig.01 M phosphate buffer of different pH values. regulate gene transcription according to nitrogen availability.8 cm particle size were moistened with 10 ml of 0. similar by to enzyme production in Saccharomyces cerevisae. At higher pH.when L-asparaginase levels decrease. Maximum activity was obtained at 37oC. The specific activity increased to 497 and 1. The proline 2% medium was observed to be the best condition for L. At higher temperatures.0 to 11.asparaginase production. LAsparaginase activity increased as the incubation time increased. 6).01M phosphate buffer pH 7.The enzyme activity gradually increased until pH 9. at which time the maximum activity was observed. The pH influence on the L-asparaginase activity was studied using a 0. It has been observed that the structural gene expression is under GATA factor control as the activators ARE-A and NIT-2.4-0.147 mM and a Vmax value of 35. Indeed. these activity levels could be improved with the utilization of protease inhibitors. The activity ran at maximum for 30 min and decreased as the time increased. aeruginosa 50071 L-asparaginase was assessed for its amino acid contents. The total protein decreased from 4. terreus at 48 h. The partial purification of the L-asparaginase crude extract that was affected by the ammonium sulfate (80%) precipitation showed that most of the enzyme activity was preserved in the precipitate.48. However. ranging from 2. the reaction rate declined sharply. 7 and 8). the highest Lasparaginase activity level was present by A. The nitrogen regulation mechanism in filamentous fungi has been carefully studied. As a rule.

lysine. and serine were present. methionine. this work gives promising results on the possible production of l. such as the activity at the alkaline pH range at 37oC. leucine. Relatively higher amounts of aspartic acid. (1996) reported that aspartic acid protects the active site of E. 17 . Economically. make it extremely valuable in the chemotherapeutic treatment of leukemia. Qian et al. coli L-asparaginase. The excellent properties of this enzyme.asparaginase from Pseudomonas aeruginosa 50071 under solid-state fermentation.enzyme was rich in glycine and glutamic acid. this enzyme could be produced from cheap. Finally. untreated biomass residues.

mercescens MTCC #97 obtained from Institute of Microbial Technology(IMETCH) was maintained by cultivation on YBP agar slant at 30 C and stored at 4 C.marcescens.Materials and Methods Bacterial strain S. the microorganism was grown in an incubator at 30C for 24 h. Media preparation Yeast extract . Cell disruption for release of intracellular L asparaginase Cell harvesting Cell were disrupt using ultracentrifugation at 2000 rpm for 20 mintes  discard supernanat and mixed with tris buffer to dilute pellet  again centrifuge at 10000 rpm for 10 mints. separate the pellet  pellet is dissolved in tris buffer sonication sonicate at 50 Hz  exposure time is 1 second and relaxation time is 10 second.0. The inoculated Erlenmeyer flasks were kept on an orbital shaker at 150 rmp for 24 h and were used as inoculum. Sample is collected after 30 second of total exposure. Inoculum preparation Inoculum was prepared in 250ml Erlenmeyer flasks containing 50 ml of YBP liquid media of pH 7.2 g Beef extract – 1 g Peptone – 5 g NaCl – 5 g Agar – 15 g Distilled water -1 L Culture maintenanceThe culture was maintained on YBP medium at Ph -7 . The media was autoclaved at 121 ºC (15lbs) for 20 min and inoculated with S.  After soinication detect the protein and chek activity of enzyme 18 .

Enzyme assay- 19 .