Isolation And Characterization Of Enterobacter Cloacae

Appl Microbiol Biotechnol (1998) 50: 568±572

Ó Springer-Verlag 1998

ORIGINAL PAPER

M. S. Nawaz á D. Zhang á A. A. Khan á C. E. Cerniglia

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine

Received: 19 February 1998 / Received last revision: 4 June 1998 / Accepted: 10 July 1998

Abstract A gram-negative, rod-shaped bacterium capable of utilizing L-asparagine as its sole source of carbon and nitrogen was isolated from soil and identi®ed as Enterobacter cloacae. An intracellularly expressed Lasparaginase was detected and it deaminated L-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM L-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on L-fructose, D-galactose, saccharose, or maltose, and in cells cultured on L-asparagine as the sole nitrogen source. The pH and temperature optimum of L-asparaginase was 8.5 and 37±42 °C, respectively. The halflife of the enzyme at 30 °C and 37 °C was 10 and 8 h, respectively.

Introduction L-Asparaginase is widely used as an antioncolytic agent for the treatment of acute lymphoblastic leukemia and lymphosarcoma (Goldberg 1992) and treatment of these two types of cancers with L-asparaginase results in complete remission (Keating et al. 1993). The enzyme deprives these tumors of L-asparagine because it transforms L-asparagine to aspartic acid and ammonia (Capizzi et al. 1981) and thus shrinks these tumors. Indeed, several microorganisms, such as Pseudomonas sp. (Manna et al. 1995), Escherichia coli (Barnes et al. 1977, 1978), Erwinia sp. (Maladkar et al. 1993; Moola et al. 1994), Citrobacter freundii (Davidson et al. 1977), Bacillus sp. (El-Shora and Ashour 1993; Mohapatra et al.

M. S. Nawaz (&) á D. Zhang á A. A. Khan á C. E. Cerniglia Division of Microbiology, National Center for Toxicological Research, Je€erson, AR 72079, USA Tel.: +1-870-543-7341 Fax: +1-870-543-7307 e-mail: [email protected]

1995), Staphylococcus sp. (Rozalska and Mikucki 1992) and Proteus vulgaris (Tosa et al. 1972), have served as sources of the enzyme for clinical trials. Successful treatment of tumors requires the administration of large quantities (3000±9000 IU á kg)1 á day)1) of the enzyme (Capizzi 1993). However, the crude extract of the majority of the strains used to obtain puri®ed asparaginase have poor enzyme activity (less than 1.0 IU of L-asparaginase); therefore large quantities of puri®ed protein must be administered and this causes deleterious side e€ects (Wada et al. 1990). In addition, hypersensitivity to the enzyme and the accelerated clearance time of the enzyme from the plasma have been reported for asparaginases from some strains (Moola et al. 1994). Furthermore, a complex and cumbersome set of culture conditions is recommended for the optimal synthesis of the enzyme in some microbes (Barnes et al. 1977, 1978). Thus there is a need to isolate and identify bacteria which produce high levels of asparaginase under simple culture conditions. We report herein the isolation and characterization of a bacterium, identi®ed as Enterobacter cloacae, that can utilize L-asparagine as the sole growth substrate. The in¯uence of several growth substrates on asparaginase biosynthesis is also reported.

Materials and methods Isolation of bacteria An enrichment culture technique with phosphate bu€er medium (Nawaz et al. 1991) amended with asparagine as the sole growth substrate was used to isolate asparagine-utilizing microorganisms. Isolation and identi®cation of pure cultures from mixed cultures that produced ammonia were performed as described previously (Nawaz et al. 1991). Utilization of L-asparagine by bacteria The isolates were cultured in 125-ml ¯asks containing 50 ml of the growth medium supplemented with 0.1% glucose. The growth medium was inoculated with 1 ml of a 36-h bacterial suspension

569 (A600 ˆ 1.0). One millilitre of the bacterial suspension contained 630 lg of protein. Samples (2 ml) were removed periodically. After determining bacterial growth at 600 nm and ammonia production, the cells were centrifuged at 10,000 g for 15 min. The supernatant was used for identi®cation and quanti®cation of L-asparagine and L-aspartic acid by high-pressure liquid chromatography (HPLC; Sieciechowicz and Ireland 1989). Enzyme assay Cell extracts (10,000 g supernatant), prepared as described previously (Nawaz et al. 1991, 1993), served as the crude enzyme source. Asparaginase activity was assayed by measuring the ammonia released from asparagine (Manna et al. 1995; Mesas et al. 1990; Raha et al. 1990) at 37 °C for 10 min. One unit of asparaginase activity (IU) was de®ned as the amount of enzyme that catalyzed the formation of 1 lmol of ammonia per minute. Speci®c activity is expressed as units per milligram of protein. Analytical methods Bacterial growth was determined by measuring the turbidity at 600 nm with a DU-7 spectrometer (Beckman Instruments, Fullerton, Calif., USA). The protein content of the cell extracts was determined with Folin phenol reagent (Lowry et al. 1951), using bovine serum albumin as a standard. The production of ammonia was estimated colorimetrically by the Bertholett procedure (Kaplan 1969). Samples were assayed in triplicate and diluted when necessary. Asparagine and aspartic acid were identi®ed and quanti®ed as detailed by Sieciechowicz and Ireland (1989). Peaks were identi®ed and quanti®ed based on the retention times and areas of known quantities of authentic standards.

Results Enrichment and isolation of axenic cultures Asparagine-utilizing microorganisms were isolated by enrichment from farm soil samples. Six isolates consistently utilized asparagine as the sole growth substrate. One isolate was a gram-negative, rod-shaped, motile bacterium that was positive for o-nitrophenyl-b-D-galactopyranosidase, arginine dihydrolase, ornithine decarboxylase, and Voges-Proskauer reactions. The organism tested negative for lysine decarboxylase, urea utilization, and hydrogen sul®de and indole production. Based on these characteristics, the bacterium was identi®ed as E. cloacae and was designated as strain NCTR 5. The e€ect of carbon and nitrogen substrates on the asparaginase activity The in¯uence of various sugars on the asparaginase activity was investigated (Table 1). These cultures were supplemented with L-asparagine (1.0 g/l) along with the sugar at a concentration of 1.0 g/l. Although substantial growth (A600 P 2.0) was observed in the presence of these sugars, asparaginase activity was poor in cultures grow with lactose and sorbose. Bacteria grown on saccharose, galactose, maltose and fructose had the highest enzyme activities (Table 1). Asparaginase activity was

Table 1 The e€ect of various carbon substrates on asparaginase production in Enterobacter cloacae. Values are given as the average of three replicates, ‹SD Carbon source (1 g/l)

Speci®c activity (IU/mg)

Fructose Galactose Glucose Lactose Maltose Saccharose Sorbose Sucrose Asparagine

23.9 25.1 18.6 13.9 25.5 24.6 8.8 17.8 14.9

‹ ‹ ‹ ‹ ‹ ‹ ‹ ‹ ‹

2.44 2.32 0.58 1.64 1.16 0.53 1.79 0.84 0.14

directly proportional to the concentration of the sugar, and cultures grown on 4 g/l of fructose had the highest asparaginase activity (asp. activity of 23.9). Thus all subsequent investigations were conducted with fructose at 4 g/l. Among the di€erent nitrogen substrates tested (sodium nitrate, ammonium chloride, ammonium citrate, ammonium nitrate, and ammonium sulphate) asparaginase activity was highest in cultures grown with asparagine as the sole nitrogen source (Table 2). Metabolism of asparagine HPLC analysis of culture ®ltrates recovered after 10 min of incubation indicated the transformation of asparagine to a compound with a retention time of 6.1 min (Fig. 1). This compound was identi®ed as aspartic acid. Filtrates of the reaction mixture taken after 25 min of incubation indicated the complete disappearance of asparagine (originally 2.8 mM) and the presence of 2.6 mM aspartic acid. Another metabolite found in the reaction mixture was determined to be ammonia. Ammonia accumulation increased with increased incubation time and reached a maximum of 2.65 mM after 25 min. Characterization of asparaginase Asparaginase activity was a function of cell age and the maximum activity occurred in cells harvested after 24 h Table 2 E€ect of di€erent nitrogenous substrates on the asparaginase activity of E. cloacae. Values are given as the average of three replicates, ‹SD Nitrogen source (1 g/l)

Speci®c activity (IU/mg)

Asparagine Sodium nitrate Ammonium chloride Ammonium citrate Ammonium nitrate ammonium sulfate

34.8 25.5 24.0 24.6 28.4 24.4

‹ ‹ ‹ ‹ ‹ ‹

0.78 1.59 2.82 2.17 1.59 3.14

570

of incubation. However, cell growth peaked after 48 h of incubation (A600 P 2.0). Thus enzyme assays were always performed with cells that were harvested after 24 h of incubation with 2.8 mM asparagine. Asparaginase activity increased with increasing incubation temperatures, up to a maximum at between 37 and 42 °C (Fig. 2A). Asparaginase activity was detected at neutral and alkaline pH values (6.5±9.5). No activity was detected at acidic pH values (<6.0). Asparaginase activity peaked at pH 8.5 and then declined (Fig. 2B). The temperature stability of the enzyme at 4 °C, 30 °C and 37 °C was determined for longer periods of time (Fig. 3). At 4 °C the enzyme retained 100% activity for 12 h. At 30 °C and 37 °C, 50% of the activity was lost after 10 h and 8 h of incubation, respectively. The e€ects of various concentrations of divalent metals on the enzyme were investigated (Table 3). Manganese and zinc completely inhibited the enzyme activity at all concentrations tested. Similarly, various

Fig. 1A±C High-pressure liquid chromatography (HPLC) pro®les of asparagine hydrolysis. The peaks shown in A are standards for asparagine and its metabolite, aspartic acid. Asparagine had a retention time of 8.4 min, whereas aspartic acid had a retention time of 6.1 min. HPLC of the culture ®ltrate was undertaken after 0 (B) and 10 (C) min of incubation

Fig. 2A, B E€ects of temperatures (A) and pH (B) on the asparaginase activity of Enterobacter cloacae

571

Fig. 3 The e€ect of temperature on the stability of asparaginase activity. Enzyme samples (containing 1 mg/ml of protein) were incubated at 4 °C (s), 30 °C (d), and 37 °C (h), respectively

concentrations of copper also inhibited the enzyme activity. Lower concentrations of iron (0.5 and 1.0 mM) increased the enzyme activity whereas a higher concentration (2.0 mM) inhibited it. Although a lower concentration of nickel (0.5 mM) enhanced the enzyme activity, higher concentrations (1.0 and 2.0 mM) inhibited it. Regardless of the concentration, both cobalt and magnesium increased the enzyme activity.

Discussion The speci®c activity of most asparaginases is between 0.1 and 5.0 IU á mg)1 protein (Curran et al. 1985; Davidson et al. 1977; Kitto et al. 1979; Maladkar et al. 1993; Manna et al. 1995; Raha et al. 1990; Tosa et al. 1972). In contrast, E. cloacae produces high levels of asparaginase (35.0 IU á mg)1 protein) under simple culture conditions, in media with 0.1% maltose, saccharose, fructose or galactose supplemented with asparagine. Table 3 The e€ect of various metals on the activity of asparaginase from E cloacae. A reaction mixture containing no metal had an enzyme activity of »35.0 IU; this was considered as 100% activity metal

Copper Manganese Iron Nickel Cobalt Magnesium Zinc

% Relative activity with di€erent metal concentrations (mM) 0.5

1.0

2.0

95.2 0.0 144.1 127.0 119.8 142.0 0.0

91.2 0.0 159.6 83.8 131.6 152.2 0.0

63.9 0.0 0.0 0.0 149.3 165.4 0.0

Although earlier studies had indicated the inhibition of asparaginase in Escherichia coli B cells cultured with fructose (Boeck and Ho 1973), our studies indicate that fructose promotes the synthesis of the enzyme in E. cloacae. The physicochemical properties of L-asparaginase from E. cloacae are di€erent from the L-asparaginases from other microorganisms. The enzyme from E. cloacae had a pH and temperature optimum of 8.5 and 37± 42 °C, respectively. The asparaginase isoenzymes from Pseudomonas geniculata (Kitto et al. 1979) have pH optima of 9.0 and 9.5, respectively. The asparaginase from Erwinia carotovora had an optimal pH at 8.0 and an optimal temperature of 50 °C (Maladkar et al. 1993). The asparaginases from Pseudomonas acidovorans (Davidson et al. 1977) and Thermus aquaticus (Curran et al. 1985) had a pH optimum of 9.5, whereas the maximum L-asparaginase activity from Proteus vulgaris (Tosa et al. 1972) was observed between pH 7.0±8.0. The puri®ed enzyme from Pseudomonas stutzeri displayed optimum activity at pH 9.0 and 37 °C (Manna et al. 1995). The thermostable asparaginase from T. aquaticus had an optimum temperature of 75 °C (Curran et al. 1985). Some assumptions can be made based on the e€ects of di€erent metals on the asparaginase activity. The enhancement of asparaginase activity by iron, cobalt and magnesium indicates a crucial role for these metals at the active site of the enzyme. The inhibition of the enzyme by other heavy metals (copper, manganese and zinc) may indicate the involvement of cysteine, histidine and carboxyl groups at the enzyme active site, and their modi®cation may lead to enzyme inhibition (Dixon and Webb 1964; Nawaz et al. 1994). Several factors in¯uence the selection of asparaginase as a therapeutant (Keating et al. 1993). Critical among these are: (1) the relative ease of isolation of the enzyme in large quantities, (2) maximum activity at physiological pH and temperature, (3) persistence of the enzyme in the bloodstream, and (4) appreciable activity at the Lasparagine levels found in blood (approximately 0.03± 0.05 mM asparagine). This study indicates that large quantities of the enzyme with the highest activity reported can be obtained overnight in a simple growth medium. In addition, the enzyme from E. cloacae also displays high catalytic activity under a wide range of pH and temperatures, making it attractive for further screening as a potential anticancer agent.

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