Indian Journal of BiotechnologyVol 2, April 2003, p184-194

Microbial L-Asparaginase: A Potent Antitumour Enzyme

Savitri, Neeta Asthana and Wamik Azrni"Department of Biotechnology, Himachal Pradesh University, Summer-Hill, Shimla 171 005, India

Received 3 May 2002; accepted 29 August 2002

L-asparaginase (LA) can be effectively used for the treatment of acute lymphoblastic leukemia and tumour cells.Interest in this enzyme arose few decades ago when it was discovered that the antilymphoma activity of the guineapig serum was due to LA. For pharmacological and clinical tests, microbial sources are best for the bulk productionof LA. Initially this drug failed to fall in antineoplastic category due to the immunogenic reactions caused by theforeign protein. With the development of protein engineering, modifications carried out in purified LA eitherreduced or completely eliminated the immunogenicity of LA in test model. The improved therapeutic activity anddecreased immunogenicity of LA can be of immense use as an antineoplastic agent.

Keywords: L-asparaginase, intracellular enzyme, anti tumour, antilymphoma, acute lymphoblastic leukemia

IntroductionL-Asparaginase (L-asparagine amino hydrolase,

E.C. 3.5.1.1, LA) catalyzes the hydrolysis ofL-asparagine into L-aspartic acid and ammonia.L-asparagine is an essential amino acid for the growthof tumour celIs whereas the growth of normal celIs is •independent of its requirement. Most normal tissuesynthesizes L-asparagine in amounts sufficient fortheir metabolic needs with their own enzyme,L-asparagine synthetase but the malignant celIsrequire an external source of L-asparagine for growthand multiplication. In the presence of LA, the tumourcelIs get deprived of an important growth factor andcannot survi ve. This fact suggested the developmentof this enzyme as a potent anti tumour or antileukemicdrug.

LA is broadly distributed among the plants, animalsand microorganisms. The microbes are a better sourceof LA, because they can be cultured easily and theextraction and purification of LA from them is alsoconvenient, facilitating the large-scale production.The most economical and the most commonly usedmicroorganisms to produce LA are Erwiniacaratovora, Bacillus sp., Corynebacteriumglutamicum, Pseudomonas stutzeri and Escherichiacoli. LA from E. coli has a tumour inhibitory effectand that from E. chrysanthemi is alsopharmacologicalIy active. Since these two LAspossess different immunological specifications, the

*Author for correspondence:Tel: 0177-231948; Fax: 0177-230775E-mail: wamikazmi@rediffmail.com

availability of both provides an important alternativetherapy if a patient happens to develophypersensitivity for one enzyme, a commonphenomenon associated with the administration ofany foreign protein (Lee et al, 1988).

LAs also playa very critical role in the biosynthesisof the aspartic family of amino acids. Corynebacteria-producing amino acids are of great industrial interestas they excrete large amounts of various amino acids(Martin, 1989). Lysine, threonine and methionine,commercially important amino acids produced by C.glutamicum, are derived from aspartic acid, which,under normal physiological conditions, might belimiting for lysine and/or threonine biosynthesis.Apart from Kreb's cycle (using glutamic acid asamino acid donor), aspartic acid is formed fromasparagine by the action of asparaginase. LA wasproduced constitutively and its role may be that of anoverflow enzyme, converting excess asparagine intoaspartic acid, the direct precursor of lysine andthreonine. A very active LA was found in C.glutamicum under lysine producing fermentationconditions (Mesas et al, 1990). In addition to theirrole in amino acid metabolism, several asparaginaseare of interest because of their antitumour properties.For clinical use, LA has to be produced in largerquantities.

LA as an Antitumour DrugLA is the first enzyme with antitumour activity to

be intensively studied in human beings. It is anenzyme drug of choice for acute lymphoblastic

SAVITRI et al: MICROBIAL L-ASPARAGINASE: AN ANTITUMOUR ENZYME

leukemia in children used in combination therapy(Schemer & Holcenberg, 1981). Since 1922, LA hasbeen considered as a therapeutic agent againstmalignant tumours (Clementi, 1922). High LAactivity was observed in guinea pig serum, whereasother mammals were found devoid of this enzyme. Acomponent exclusively present in guinea pig serumwas proposed to be responsible for tumouricidaleffects (Kidd, 1953). The growth of a cell line derivedfrom Walker carcinosarcoma was shown to bedependent on L-asparagine (Neuman & McCoy,1956). Haley et al (1961) showed the same resultsusing a mouse leukemia cell line. It was furthershown that growth of normal cells was not dependenton L-asparagine, making LA a potential tumourspecific drug. LA purified from guinea pig serum wasa tumouricidal agent (Broome, 1961) againstimplanted 6C3HED cells (serum from newbornguinea pigs lacking LA was devoid of anti tumouractivity).

Consequently the therapeutic potential of LA wasconfirmed. Using salt precipitation, starch blockelectrophoresis and DEAE-cellulose columnchromatography, two isoforms of LA were partiallypurified (Yellin & Wriston, 1966) and only oneisoform had antilymphoma activity in vivo. Sinceextraction of LA from mammalian cells is difficult,microorganisms have proved to be a better alternativefor LA, thus facilitating its large-scale production.Mashburn and Wriston (1964) successfully purified E.coli LA and demonstrated its tumouricidal activity.Oettgen et al (1967) were the first to show theefficacy of LA in human beings with leukemias. Itturned out that LA is not an anticancer drug in generalbut is effective against certain types of tumours. Forthese reasons, it has become an essential componentof chemotherapy strategies.

LA treatment for acute lymphoblastic leukemia is amajor breakthrough in modern oncology as it inducescomplete remissions in over 90% children within fourweeks (Gallagher et al 1989).

Microbial Sources of LAWide range of bacteria, fungi, yeast, actinomycetes

and algae are very efficient producers of LA(Table 1). Asparaginase from Gram +ve bacteriareceived little attention. This prompted researchers toscreen large number of bacteria for activity.

LA by BacteriaLA has been studied in E. coli and other Gram -ve

bacteria such as Achromobacteriaceae (Roberts et al,

IX5

Table I-List of major L-asparaginase producing microorganisms

Microorganisms ReferencesBacteriaAcinetobacter calcoaceticusBacillus sp.B. mesenteric usB. polymyxaCitrobacter sp.Corynebacterium glutamicumEscherichia coliEnterobacter aerogenesE. cloaceaeErwinia aroideaeE. carotovoraE. chrysanthemiHelicobacter pyloriKlebsiella pneumoniaeMycobacterium phleiPseudomonas ovalisP. stutzeriSerratia marcescensStaphylococcus sp.S. au reusStreptococcus albusTetrahymena pyriformisThermus thermophilusT. aquaticusVibrio succinogenesYeastCandida uti/isC. guilliermondiiPichia polymorphaSaccharomyces cerevisiaeActinomycetesStreptomyces karnatakensisS. venezuelaeS. collinusThermoactinomyces vulgarisFungiAspergillus nidulansA. terreusCylidrocapron obtusisporumMucor sp.AlgaeChlamydomonas sp.

Joner (1976)Mohapatra et al (J 995)TiuJ'panova et al (1972)Nefelova et al (J 978)Bascomb et al (1975)Mesas et al (1990)Netrval (1977)Mukherjee et al (2000)Nawaz et al (1998)Tiwari & Dua (1996)Maladkar et al (1993)Moola et al (1994)Stark et al (1997)Reddy & Reddy (1990)Pasterzak & Szymona (1976)Badr & Foda (1976)Manna et al (J 995)Rowly & Wriston (1967)Mikucki et al (1977)Rozalska & Mikucki (1992)Reddy & Reddy (1990)Tsirka (1990)Pritsa et al (200 I)Curran et al (1986)Disteasio et al (1976)

Kil et al (1995)Stepanyan & Davtyan (1988)Foda et al (1980)Bon et al (1997)

Mostafa (1979a)Mostafa (1979b)Mostafa & Salama (1979)Mostafa & Ali (1983)

Drainas & Drainas (1985)Ali et al (1994)Raha et al (J 990)Mohapatra et al (1997)

Paul (1982)

1972) and Vibrio succinogenes (Kafkewitz &Goodman, 1974), but little work was carried out onLA from Gram +ve bacteria. There are a number ofreports on LA from terrestrial microorganisms, littleinformation is available on LA from marine bacteria(Benny & Kurup, 1991), which are considered to be

186 INDIAN J BIOTECHNOL, APRIL 2003

an important source of bioactive enzymes (Williams& Vickers, 1986). Due to halophilic nature, marinebacteria can be exploited industrially. LA of aBacillus strain was isolated from the intertidal marinealga, Sargassum sp (Mohapatra et al, 1995). A newGram +ve bacterial isolate produced intracellular LAunder aerobic conditions using glucose as a carbonsource (Azmi et al, 2001).

The LAs currently in use are obtained from variousmembers of Enterobacteria. Kafkewitz and Goodman(1974) reported the constitutive LA production by therumen anaerobe V. succinogenes. Unlike theEnterobacteria, in which the enzyme apparently has ascavenger function, V. succinogenes is grown underconditions such that the LA reaction is potentiallygrowth limiting. Therefore, this organism has thepotential to produce large quantities of enzyme with alow Km value and high substrate specificity.L-Glutaminase and LA, produced simultaneously byPseudomonas ovalis upon induction by L-glutamineor L-asparagine in the growth medium (Badr et al,1976), are confined to the cells during active growthand are not released into the medium. The process ofenzyme formation showed a firm link to the activecell growth, as evidenced by the use of growthinhibitors.

LAfrom YeastAsparaginase II is a periplasmic enzyme of

Saccharomyces cerevisiae encoded by the ASP3 gene(Bon et al, 1997). The enzyme activity, which is notfound in the cells grown in ammonia, glutamine andglutamate, is found in the cells subjected to nitrogenstarvation or grown on a poor source of nitrogen suchas proline. LA was also extracted from the cell culturebroth of Candida utilis by treatment with reducingagent (Kil et al, 1995). This LA may be used for thetreatment of acute lymphoblastic leukemia. Aascogenous yeast, Pichia polymorpha with highpotential for LA formation, was isolated fromEgyptian soils by the application of cultureenrichment method (Foda et al, 1980).

LA from ActinomycetesLike bacteria, actinomycetes are also good source

of LA. Five actinomycetes isolates (all belonging tothe genus Streptomyces), capable of producingdetectable amounts of LA, were isolated afterenrichment from the soil of Kuwait (Mostafa &Salama, 1979). The three most potent enzymeproducers were identified as different strains ofStreptomyces collinus. LA was also produced by two

soil isolates, S. karnatakensis and S. venezuelae,under different environmental and nutritionalconditions (Mostafa, 1979b). The LA, extracted fromthe whole cells of S. karnatakensis by differentprocedures (Mostafa, 1982), showed stereospecificityfor L-asparagine, however, some activity was alsonoticed with the D-isomer. Mostafa (l979a) foundthat S. karnatakensis produces LA intracellularly.Presence of L-asparagine in the culture mediuminduces the enzyme production but it is not essentialfor the enzyme biosynthesis. Cells grown on L-asparagine showed amidase activity with other amidesbut at a reduced rate.

LA from FungiMucor sp, isolated from the marine sponge

Spirastrella sp, produces extracellular LA (Mohapatraet al, 1997). Aspergillus nidulans is also reported toproduce LA (Drainas & Drainas, 1985). A strain of A.terreus, isolated from decomposing vegetablesubstrate (Ali et al, 1994), produced LA, which wasnot toxic and appeared to have myelosuppressive andimmunosuppressive activity. A mesophilic fungus,Cylindrocarpon obtusisporum MB-I0 (Raha et al,1990), also produces LA intracellularly. This LA wasvery specific for L-asparagine and did not hydrolyseD-asparagine or L-glutamine.

LA from AlgaeLA specific for L-asparagine has been purified

from a marine Chlamydomonas sp (Paul, 1982), thefirst such enzyme to be purified from a microalgae.This LA showed a limited antitumour activity in anantilymphoma assay in vivo. Properties of the enzymecontrasted with those of asparaginases fromprokaryotic and eukaryotic sources.

Production of Microbial LALA producing microorganisms either produce this

enzyme constitutively or after induction. The physico-chemical conditions for LA production vary with themicroorganism. Effect of media composition on thegrowth and LA production of V. succinogenes wasobserved (Albanase & Kafkewitz, 1978). LA isinducible and ammonium ions plays as inducer. Theorganism grows best when fumarate is provided as theterminal electron acceptor of the fumarate oxidizingcytochrome system. Yeast extract or enzymehydrolyzed proteins were effective nutrient sources.Synthesis of LA occurs throughout the exponentialphase, and in early stationary phase. LA accounts forabout 5% of the total soluble protein.

SAVITRI et al: MICROBIAL L-ASPARAGINASE: AN ANTITUMOUR ENZYME

Various carbon and nitrogen sources for theproduction of LA by E. carotovora were optimized(Maladkar et al, 1993). In comparison to control withno carbon source at 37°C, lactose showed themaximum activity (16-fold) followed by monosodiumglutamate (12-fold). Out of different nitrogenoussubstances added to the fermentation medium, com-steep liquor showed 14-fold higher activity followedby tryptone 03-fold) and yeast extract (I I-fold). Dueto practical reasons, the removal of impurities fromcorn-steep liquor during isolation was very difficult,hence, tryptone and yeast extract were selected aseffective nitrogen sources. The Bacillus sp, isolatedfrom an intertidal marine alga, Sargassum sp,produces constitutive LA in the cultivation media (pH8.0) at 28°C using peptone as the main nutrient(Mohapatra et al, 1995). C. glutamicum produced LAaerobically (pH 7.3) using casein and soya peptonesas main nutrients (Mesas et al, 1990). Helicobacterpylori, which can utilize amino acids as the solecarbon and energy source (Stark et al, 1997), wasgrown in continuous culture using defined mediacontaining glucose and amino acids. All the 13 strainsof H. pylori tested produced both glutaminase and LAactivity. The effect of various amino acids andorganic acids on the production of LA by a strain ofE. coli in a chemically defined medium wasinvestigated (Netrval, 1977) under moderate aeration.Bascomb et al (975), who optimized the conditionsfor the production of intracellular LA with anantitumour activity by Citrobacter, produced theenzyme up to 2700 L scale yielding highest LA usingcorn steep liquor at 37°C. They observed that oxygenlimitation was not necessary for the high enzymeyield.

For the production of LA by Enterobacteraerogenes using 12 C and 21 N sources (Mukherjee etal, 2000), most suitable C and N sources were sodiumcitrate and di-ammonium hydrogen phosphate,respectively. Nitrogen catabolite repression onenzyme formation was absent in this bacteria butglucose was a repressor of this biosynthesis. Duringthe cultivation in a fermenter, the dissolved oxygenlevel was the limiting factor for LA production.Asparagine was absent intracellularly at high level ofLA.

Maximum yield of LA by Staphylococci wasobtained in the stationary phase of growth in a batchculture when casein hydrolysate and yeast extract.were supplied as C and N sources (Mikucki et al,1997). The highest LA yield was obtained when the

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culture were aerated during the exponential phase ofgrowth and further incubated in the stationary phase.Repression by L-asparagine and L-aspartic acid wasabsent but glucose inhibited the enzyme formation.Rozalska & Mikucki (1992) confirmed the role ofcAMP in the regulation of Staphylococcal LAsynthesis. The enzyme production was inhibited by allthe carbon sources added to the growth medium. Thestrongest inhibition was caused by saccharose andmaltose. The best C sources for Klebsiellapneumoniae growth were melibiose, maltose andsorbitol, and for LA production mannitol and sucrose(Reddy & Reddy, 1990). No correlation was foundbetween bacterial growth and enzyme production.

Synthetic media with asparagine as an N sourcestimulated more enzyme production than naturalmedia by Streptomyces (Mostafa & Salama, 1979).Strach 0.0%) as C and asparagine (0.8%) as N sourcewere optimum for enzyme production at pH 8.5.Incubation was carried out at 28-30°C in a staticculture for six days to get the maximum LA. Same Cand N sources were found suitable for LA productionby S. karanatakensis and S. venezuelae (Mostafa,1979b). In both the cases, presence of C sources otherthan starch or N sources other than L-asparagine inthe growth medium inhibited the enzymebiosynthesis. Aeration stimulated the growth but notenzyme production and both organisms producedmore enzyme in static culture than in shaken culture.

S. cerevisiae produced LA under N starvingcondition (Bon et al, 1997). LA formation isdependent upon the functional GLN3 gene and thatthe response to N availability is under the control ofURE2 gene product. Kil et al (995) producedintracellular LA by cultivating the cells of Candidautilis in medium containing glucose, yeast nitrogenbase and peptone at 30°C. So, after 18 hrs, cells werecollected by centrifugation and LA activity wasmeasured. Pichia polymorpha, an ascogenous yeast,was obtained through the enrichment of soil samplewith a simple medium containing 0.5% L-glutamineas a major C and N source at low pH values (Foda etal, 1980). The amidase was produced constitutivelyon a variety of media irrespective of the presence ofsubstrates in the growth medium.

Purification of LAThe major proposed application of LA is as an

injectible drug for the treatment of tumour orlymphoblastic leukemia in human beings. Thesensitivity of application demands high degree of

188 INDIAN J BIOTECHNOL, APRIL 2003

purity of this enzyme. Most of the microbial LA isintracellular in nature except few, which are secretedoutside the cells (Kil et al, 1995; Mohapatra et al,1995). In case of extracellular LA from C. utilis, theenzyme was extracted from the cell precipitate or cellculture broth by the treatment of 2-mercaptoethanol,dithiothreitol or cysteine as a reducing agent (Kilet al, 1995). LA was typically extracted by incubatingthe cells at 50°C for 4 hrs in extraction solutioncontaining 2-mercaptoehanol in potassium phosphatebuffer. E. coli cell permeabilization was also carriedout with the help of K2HP04 and triton X-lOO and therelease of LA was over 70% (Zhao & Yu, 2001).Maladkar et al (1993) extracted an intracellular LAfrom the cells of E. carotovora and purified about30-fold to apparent homogeneity employingpolyacrylamide gel electrophoresis. The methods usedin the sequence were DEAE cellulosechromatography, sephadex G-200 gel filtration,hydroxylapatite ion-exchange and affinitychromatography on sepharose CL-6B (Table 2).Extraction of LA (yield, 60%) was done by treatingenzyme with acetone. This acetone treated powder ofcells was extracted in potassium phosphate buffer (pH8.0) containing NaCl for obtaining cell freepreparation. Crude extracts of Thermoactinomycesvulgaris 13 MES (Mostafa & Ali, 1983) wereprepared after filtration of cultures and grinding thecelis with sand, alumina or glass beads, by rapidfreezing and thawing, by rapid mixing and also byexposure to ultrasonic waves.

Tiwari & Dua (1996) purified LA from Erwiniaaroideae NRRL B-138 to apparent homogeneity byammonium sulphate precipitation, chromatography onsulphopropyl-sephadex C-50 and sephadex G-200with 22% recovery and 567-fold purification. LAfrom Psedomonas stutzeri, after initial ammoniumsulphate fractionation, was purified by consecutivecolumn chromatography on sephadex G-200, calciumhydroxyapatite and DEAE sephadex A-50 (Manna etal, 1995). Mycobaterium phlei LA was purified(Pastuszak & Szymona, 1976) about 170-fold with an11% yield by fractionation with ammonium sulphate,adsorption of contaminating proteins on calciumphosphate gel, and chromatography on sephadex G-150 and DEAE-cellulose. Joner (1976) purified LAfrom Acinetobacter calcoaceticus using varioustechniques, which include precipitation withstreptomycin, chromatography on DEAE-celluloseand CM-cellulose, gel filtration on agarose andchromatography on phosphocellulose.

Lee et al (1985) purified LA from crude acetonetreated extracts of Erwinia carotovora by CM-sepharose fast flow column chromatography with apre-column of CDR (cell debris remover) modifiedcellulose. An affinity column of L-asparagine coupledepoxy-activated sepharose CL-4B further purified thepartially purified enzyme. In a patent report (Goward,1994), a detailed process of E. chrysanthemi LA(yield, 59%) purification comprises the alkalineextraction of enzymes from the cells, acidprecipitation followed by Sepharose fast flow

Table 2-Purification of L-asparaginase from Erwinia carotovora and Citrobacter C6

Purification steps Total protein Total units Specific activity Purification Yield(mg) (IU) (IU/mg protein) fold (0/0)

Citrobacter C6Cell suspension 6,104 1,735 0.24Ruptured cells 6,150 1,410 0.24MnCI2 supernatant 5,270 790 0.25 100Acetone precipitation 534 529 0.99 4.0 67Ammonium sulphate precipitation & dialysis 160 273 1.88 7.5 35DEAE II column fractionation 54 197 3.65 15.0 25HAP column fractionation 6.0 132 21.90 88.0 17Sephadex G-200 0.75 34 45.28 181.0 4.3Erwinia carotovoraWhole broth 18,000 100Cell free extract 3,154 17,568 5.57 97.6DEAE-Cellulose 1.500 16,690 11.13 2.0 95.0Sephadex G-I 00 740 15,690 21.20 3.8 87.1Hydroxylapatite 288 14,400 50.00 9.0 80.0Sepharose CL-6B 65 10,854 167.00 30.0 60.3

Maladkar et al, 1993; Bascomb et al, 1975

SAVITRI et al: MICROBIAL L-ASPARAGINASE: AN ANTITUMOUR ENZYME

chromatography. Corynebacterium glutamicum LAwas purified 98-fold by protamine sulphateprecipitation, DEAE-sephacel anion exchange,ammonium sulphate precipitation and sephacryl S-200 gel filtration. Partially purified preparations kepttheir activity when frozen at -80°C but lost their 50%activity at 5 d at 4°C (Mesas et al, 1990). Theintracellular LA of Cylindrocarpon obtusisporumMB-lO was purified 65-fold from cell free extractwith an overall yield of 11.4% by acetonefractionation, ammonium sulphate precipitation andtwo chromatographic runs on DEAE sephadexA-40 columns (Raha et al, 1990). Guy et al (1984)reported a method for separation of streospecificD-asparaginase from a mixture of D- and LA.

Properties of Microbial LAMostly LAs are intracellular and the pH and

temperature optima for LA production are the same asthat for the growth of the enzyme-producing organism(Mesas et al, 1990). Presence of metal ions does notaffect LA production indicating that it is not ametalloprotein or does not require cofactors. Presenceof chelating agents such as EDT A and compoundshaving thiol protecting groups such as glutathione,dithiothreitol, 2-mercaptoethanol, etc. markedlyenhance the enzyme activity (Raha et al, 1990). LAsfrom different sources vary in their physico-chemicalproperties. Generally LA is found in more than oneisoforms in different organisms but all the isoformsdo not have antitumour activity. Guinea pig serum LA(PH, 7.5-8.5) has a molecular weight of 1,38,000 Da.It is stable for at least 6 months at -20°C to repeatedfreezing and thawing; and to heating to 55°C for 10min but is labile under the conditions, which promotesurface denaturation. Guinea pig serum LA alsocatalyzes the hydrolysis of L-f3-aspartyl

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hydroxylamine and synthesis of hydroxamate fromasparagine and hydroxylamine, but all these reactionsproceed much more slowly than the hydrolysis of L-asparagine.

LAs in E. coli B cells are designated as EC-1 andEC-2 (Campbell et al, 1967). Only EC-2 (mol wt,1,25,000 Da) possesses antilymphoma activity. ForEC-2, the pH-activity profile is a broad range with amaximum at 8.0, while EC-1 shows a rapid decreasein activity below pH 8.4. EC-1 is precipitated fromcrude extracts at 2.0 M ammonium sulphate, whileEC-2 remains largely in solution. The content of LAvaries widely in different strains of E. coli (Campbellet al, 1967). EC-2 with greater substrate affinity,possessing anti lymphoma activity, appears to belocated in the periplasmic space between the bacterialplasma membrane and the cell envelope.

The mol wt of Corynebacterium glutamicum LAdetermined by gel filtration was 81,000 Da (Mesas etal, 1990) and the apparent Km of LA for L-asparaginewas 2.5 mM (Table 3). LA from Erwinia carotovorashowed maximum activity at pH 8.0 and at 50°Ctemp. The Km value of purified enzyme was1.8xlO-5M. Purified enzyme showed significantantitumour activity on experimental animal models(Maladkar et al, 1993). The LA activity from Bacillussp. was found to be optimum at pH 8.0 (Mohapatra etal, 1995) and temp. at 3rc. The enzyme activitydecreased sharply above 40°C and the enzyme wasinactivated at 50°C and exhibited a half-life period ofabout 1 hr. Km value of LA from Bacillus sp wasfound to be 2.4xlO-4 M.

Mechanism of Antineoplastic ActionLA converts L-asparagine to L-aspartic acid. As

several types of tumour cells require L-asparagine asan essential amino acid for protein synthesis, they are

Table 3-Properties of some of the L-asparaginases

S Microorganism Production Activity Km(M)No pH Temp pH Temp

1. Erwinia carotovora 7.3 37 8.0 50 1.8xlO-5

2. Pseudomonas 7A 7.0 28 7.2 37 4.4xlO-6-

3. Corynebacterium glutamicum 7.3 30 7.0 40 2.5xlO-3

4. Vibrio succinogenes 7.0 37 8.5 37 I.7x 10-5

5. Bacillus sp. 8_0 28 8.0 37 2.4xlO-4

6. Aspergillus terreus 4.5 28 5-7 40-45 5.88xlO-4

7. Pseudomonas stutzeri 7.2 30 9.0 37 1.45xlO-4

Specific activityof purified

enzyme (IU/mgof protein)

167

162

2020

5.6

References

Maladkar et al, 1993

Roberts, 1976

Mesas et al, 1990

Goodman, 1974

Mohapatra et al,1995

Ali et al, 1994

732 Manna et al, 1995

190 INDIAN J BIOTECHNOL, APRIL 2003

deprived of an essential growth factor in presence ofLA. Effective depletion of L-asparagine results incytotoxicity for leukemic cells. Asselin et al (1989)have quantified cell kill both in vitro and in vivo inpatients with acute lymphoblastic leukemiaundergoing treatment with LA as a single agent.Besides these specific effects, LA can exertimmumosuppressive effects in general. Chakrabarty& Friedman (1970), using an animal model,demonstrated the depression of both humoral andcellular immune reactions.

In human, acute lymphoblastic leukemia cell linesis markedly inhibited by Asparaginases, the effectbeing lO-fold higher for Erwinia carotovora LA (Han& Ohnuma, 1972). While these in vitro resultsindicate significant differences among preparations invivo, the immunosuppressive effects in animals arecomparable. LA, as a single agent, is not used incurrent treatment protocols. It is always a part ofmultiple agent regimens and thus combined withdrugs with definitive immunosuppressive effects.

Resistance to LATumour cells can develop the potential to

synthesize L-asparagine intracellularly, which makesthe cells resistant to the action of LA (Broome, 1963).The degree of methylation of cytosine residues inDNA regulates the expression of asparaginesynthetase, the enzyme responsible for L-asparaginesynthesis (Andrulis & Barrett, 1989; Greco et al,1989). Accelerated cleavage of LA followinginduction of specific antibodies has also been foundas a potential reason for resistance (Capizzi, 1993).However, the other groups reported that despiteimmunization of patients, the drug's efficacy remainsunimpaired (Killander et al, 1976). The pool of LAsensitive cells may produce cytokines that control theexpansion of the resistant cells (Gallagher et al,1989). As soon as sensitive cells are killed by LA,resistant cells escape from regulatory control.

Immunogenicity and Stability of Native LA andNeed for the Modified Enzyme

The native enzyme is having high immunogenicityand very less half-life value. Attempts have beenmade to reduce the immunogenicity of the drug bycovalently linking the enzyme with polyethyleneglycol (PEG) and the modified drug seems to have aunique therapeutic potential. The purified LA fromErwinia aroideae was unstable and lost activitywithin few hours but addition of glycerol helped inrestoring the activity of the enzyme (Tiwari & Dua,

1996). Structural and functional stabilization of LAfrom E. coli was also done by multisubunit covalentimmobilization onto agarose-glutaraldehyde (Balcaoet al, 2001). The higher the density of reactive groups,the higher the stabilization attained. Boiling ofmodified enzyme in the presence of SDS and ~-mercaptoethanol did not lead to release of anyenzyme into the medium. This type of immobilizedenzyme may be useful for extracorporeal devices inthe clinical treatment of acute leukemia, with longerhalf-life and virtually nil risk of subunit release intothe circulating blood stream. For similar purpose, LAcan also be immobilized by use of a water-solublephotocrosslinking resin of high sensitivity (Ichimura,1981). This method is capable of immobilizing LAwithout impairing the molecular structure of theenzyme.

With LA, some glutaminase (up to 10%) is alsoproduced (Campbell & Mashburn, 1969). All thepurification procedures attempting to get rid of thisactivity have been unsuccessful so far and evenrecombinant LA has glutaminase activity; it seemslikely that the hydrolysis of L-glutamine is notmediated by a contaminating enzyme but by LA itself.Parenteral therapy with foreign proteins in humanshas limitations due to the fundamental problem of thedrug's immunogenicity. As real immunologicaltolerance that would require antigen specific T-cellmediated immunosuppression is difficult to achieve,other approaches have been made towardimmunological non-responsiveness rather thantolerance (Wahn, 1997). One-way to overcome thisproblem for a limited time is to switch to anotherpreparation i.e. not cross-reacting. The chemicalmodification of the enzyme is other way, whichreduces its immunogenicity but preserves the enzymeactivity.

Enzymes Modification by Polyethylene Glycol (PEG)Abuchowski et al (1979) successfully coupled PEG

to LA, which almost abolished the drug'simmunogenicity. The antileukemic activity wasshown in the L5178Y tumour bearing BDF mousemodel (Yashimoto et al, 1986). The mol wt of PEGused and the degree of modification of this enzymeturned out to be crucial for obtaining optimum results.Biochemical properties of PEGylated asparaginasemarkedly differ from the native enzyme. Its mol wt ishigher and SDS is unable to separate the subunits ofthe enzyme due to cross-linking by PEG. The enzymeis having antitumour activity clinically both in

SAVITRI et al: MICROBIAL L-ASPARAGINASE: AN ANTITUMOUR ENZYME

animals and humans (Abuchowski et al, 1979;Yashimoto et al, 1986; Jurgens et al, 1988). Its lowimmunogenicity has been confirmed in vivo even inhighly sensitized children with multiple acutelymphoblastic leukemia relapses (McEwen et al,1987).

Kodera et al (1992) used a comb shaped co-polymer of PEG-derivative and malic anhydride toabolish the enzyme's immunogenicity. Following themodification process, there was no longerdemonstrable immunoreactivity towards anti-LAantiserum.

Enzymes Modified with DextranTo improve thermal stability, provide resistance to

proteolytic digestion, prolong half-life and reduceimmunogenicity, LA has been coupled to dextran.The mol wt of dextran determines the degree ofenzyme inactivation (up to 65%) but the reduction ofimmunogenicity by dextran seems to be less effectivethan by PEG (Davis et al, 1991).

Enzymes Modified with Poly-DL-Alanyl Peptides orHuman Albumin

Uren & Ragin (197.9) used poly-DL-alanyl peptidesto block the immunogenic epitopes within the LAmolecule derived from both E. coli and Erwinia.Modified enzyme has shown reasonable retention ofcatalytic activity, protease stability, therapeuticactivity, decreased immunogenicity and cross-reactingantigenicity compared to their native counterparts.The half-life of enzyme in mice was markedlyprolonged compared to the native enzyme even if theanimals had previously been immunized with the laterpreparation. Nerkar & Gangadharan (1989) coupledErwinia LA with human serum albumin. The complexwas enzymatically active but had a lO-fold lower Kmvalue. Acylation has also been applied as a methodfor LA modification (Martins et al, 1990). Theenzyme turned hydrophobic after modification.Immunological and pharmacokinetic data regardingthis procedure is not yet available.

Delivery of LA by Erythrocytes or with LiposomesLA entrapped in red blood cells was quite stable

and the half-life in vivo markedly prolonged (Nagi etal, 1988, Deloach et al, 1990). Intravenous infusion inmice resulted in an effective elimination of L-asparagine for a period of two weeks (Kravtzoff et al,1990). However, the immunogenicity of the enzymeseems to be only slightly diminished (Kravtzoff et al,1992). The doses of 150-200 IU per kg body wt

191

results in effective elimination of L-asparagine for 50days (Kravtzoff et al, 1996).

LA entrapped in liposomes can be expected todisplay reduced immunogenicity and toxicity.Encapsulation of LA in small liposomes with amedian diam of 158-180 nm resulted in markedprolongation of in vivo half-life in animals, avoidanceof induction of LA antibodies and enhancedantitumour activity (Gaspar et al, 1994). PalmitoylLA, a chemically modified derivative of the enzymeencapsulated in Iiposomes, has also been studied inanimals (Jorge et al, 1994) and shown to exhibit about10-fold prolonged half-life, no acute toxicity butpreservation of antitumour activity in vivo.

Recombinant LAMoola et al (1994) studied epitopes on Erwinia

chrysanthemi using synthetic hexapeptides andpolyclonal antisera from rabbits and mice.Elimination of immunodominant epitopes in theenzyme by site directed mutagenesis resulted inmarkedly decreased binding of the antibodiesindicating reduced immunogenicity while theenzymatic activity remained unchanged. Harry et al(1986) cloned and expressed the E. crysanthemiasparaginase gene in E. coli and Erwinia carotovora.The enzyme was produced at high level in E. coli (5%of soluble protein) and was shown to be exported toperiplasmic space. Expression of cloned gene wassubject to glucose repression in E. coli but was notsignificantly repressed by glycerol. The isolatedErwinia asparaginase gene was successfullyintroduced in E. carotovora and enzyme expressionwas approximately three-fold higher than theproduction strain of E. crysanthemi.

Prospects of LALA carries out the cleavage of L-asparagine into L-

aspartic acid and ammonia. L-asparagine acts as anessential amino acid for the growth of tumour cells.They require an external input of this amino acidwhereas the normal cells are independent of itsrequirement because they can synthesize L-asparagineby the enzyme L-asparagine synthetase. When LA isprovided to the tumour cells it causes the deprivationof the cells, as an important growth factor becomesunavailable to them and the cells cannot survive anymore. This fact suggests that the enzyme can be usedas an anti tumour drug.

Extensive studies have been done on the therapy byeliminating L-asparagine, which is non-essential for

192 INDIAN] BIOTECHNOL, APRIL 2003

normal cells from blood. This therapy is expected tocause no damage to normal cells. It is demonstratedthat a variety of LAs are useful for the therapy ofcertain leukemias and solid tumours including acutelymphoblastic leukemia (Masao, 1986). This therapygot attention as a specific and favourable therapeuticidea, which represents inhibition (prevention) ofgrowth of leukemic cells without damaging normalcells. If means provided for avoiding theimmunoreaction caused by foreign protein, it isexpected that the therapy will recover great hope. Asa solution, a scheme has been proposed, in whichblood is temporarily drawn out of the body, contactedwith immobilized LA to decompose the asparaginedissolved in blood and then returned to the body(Maladkar et al, 1993). The whole potential of LA hasprobably not been fully elucidated due to itstreatment-limiting immunogenecity. Two major waysmight be able to reduce this problem. One is to reducethe drug immunogenecity by chemical modificationof relevant epitopes. The other would be to activateimmune mechanisms involved in immune tolerance.If the details for such an approach will work out in thefuture, that will lead to an even more effective LA.

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