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Production of L-Asparaginase II by Escherichia Coli HOWARD CEDAR and JAMES H

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Production of L-Asparaginase II by Escherichia Coli HOWARD CEDAR and JAMES H JOURNAL OF BACTERIOLOGY, Dec. 1968, p. 2043-2048 Vol. 96, No. 6 Copyright @ 1968 American Society for Microbiology Printed in U.S.A. Production of L-Asparaginase II by Escherichia coli HOWARD CEDAR AND JAMES H. SCHWARTZ Department of Microbiology, New York University School ofMedicine, New York, New York 10016 Received for publication 30 July 1968 L-Asparaginase II was synthesized at constant rates by Escherichia coli under anaerobic conditions. The enzyme was produced optimally by bacteria grown between pH 7 and 8 at 37 C. Although some enzyme was formed aerobically, be- tween 100 and 1,000 times more asparaginase II was produced during anaerobic growth in media enriched with high concentrations of a variety of amino acids. Bacteria grown under these conditions should provide a rich starting material for the large-scale production of the enzyme. No single amino acid specifically induced the synthesis of the asparaginase, nor did L-asparagine, even when it was used as the only source of nitrogen. The enzyme was produced at lower rates in the presence of sugars; glucose was the most inhibitory. Deamidation of L-asparagine by extracts of the conditions which control the production of Escherichia coli was first reported in 1957 by asparaginase II. Tsuji (28). E. coli was later shown to produce two distinct asparaginases (L-asparagine amido- MATERIALS AND METHODS hydrolase, EC 3.5.1 .1) which differ in a number For most of the experiments, we used E. coli K-12 of properties, perhaps most significantly in wild type. We also used strain 22-64, which lacks their markedly different affinities for asparagine citrate synthase (10), and strain 309-1, which lacks (5, 24, 27). The enzyme with the greater affinity, a-ketoglutarate dehydrogenase (B. D. Davis et al., to be located in the Federation Proc., p. 211, 1959); both are mutants of asparaginase II, appears the W strain and were obtained from B. D. Davis. The periplasmic space between the bacterial plasma strain of E. coli B was obtained from J. Teller of membrane and the cell envelope (6). This as- Worthington Biochemical Corp., Freehold, N.J. paraginase has been purified to apparent ho- Mutants of K-12 which are unable to convert aspara- mogeneity (H. Whelan and J. C. Wriston, Jr., gine to aspartic acid were isolated after mutagenesis Federation Proc., p. 586, 1968). with ethylmethanesulfonic acid (18). After treatment Since the demonstration (2, 3) that an L- with the mutagen, cells were diluted and grown over- asparaginase is the component in guinea pig night on membrane filters (Millipore Co., New Bed- serum which inhibits the growth of certain ford, Mass.) overlying solid growth media. These has been filters were transferred to a detection medium, solidi- tumors (16, 17), there considerable fied with 2% agar (Difco), containing 10 mM aspara- interest in asparaginases from various sources. gine and 20% sucrose. After incubation for 6 hr at Asparaginase II of E. coli even more effectively 37 C, the membrane filters bearing the colonies were inhibits the growth of transplantable mouse and removed, and the underlying detection medium was rat tumors in vivo (5, 20, 26); it is also active carefully blotted for 1 min with Whatman no. 1 filter against lymphoma in the dog (14, 22) and against paper in order to absorb amino acids. After drying, human lymphoblastic leukemia (15, 21). This these papers were dipped in 0.5% ninhydrin in ace- asparaginase, which interferes with the incorpora- tone. The conversion of asparagine to aspartic acid tion of amino acids into tumor in cell was indicated by a blue spot given by aspartic acid protein corresponding to the position of a colony. Blue spots cultures (4), also inhibits the synthesis of protein were distinguishable from the brownish background under the direction of bacteriophage f2 RNA given by unconverted asparagine. Absence of a blue in cell-free extracts of E. coli (25). Asparaginase spot indicated the position of a colony with impaired I from E. coli is inactive against tumor growth conversion. Eleven mutants were isolated from 1,500 and does not interfere with protein synthesis colonies. in microbial extracts (26). Asparaginase I has Bacteria were grown aerobically on a rotary shaker the lower affinity for asparagine. in 250-ml Erlenmeyer flasks containing a volume of Whereas the production of asparaginase I is 20 ml of culture medium. Anaerobic growth was in jars (Torsion Balance Co., Clifton, N.J.) under a mix- unaffected by the conditions of growth, the ture of 90% nitrogen and 10% carbon dioxide gas at amount of asparaginase II in the bacterial cell a pressure of 3 psi. Bacteria were always adapted to varies greatly. In this paper, we report some of experimental conditions of growth for at least five 2043 2044 CEDAR AND SCHWARTZ J. BACTERIOL. generations. Cells were grown at 37 C, since growth at either higher or lower temperatures yielded lower - A amounts of asparaginase II. 5 4.0 For aerobic growth, the mineral-salts medium A of 0 Davis and Mingioli (7) was used without citrate. For o 3.0 anaerobic growth, the same medium was supple- mented with 2.4 mg of L-cystine per liter (11). Addi- z 2.0 tions to the minimal medium are indicated in the w legends to tables and figures. Tryptone, nutrient broth, 0 casein hydrolysate, and yeast extract were Difco U- 1.0 products. a-w Growth was measured turbidimetrically at a wave- 0 / length of 570 nm with a Coleman Junior spectro- *p pd * - photometer. One optical density unit corresponded to 0.93 mg of protein per ml, as determined, after lysis 0.24 B of the cells, by the method of Lowry et al. (19) with uo 0.12 . _. z egg white lysozyme as standard. For determination of w asparaginase II, 15-ml samples were pipetted into ice; CO 0.06 the cells were collected by centrifugation at o 0.03 14,000 X g, washed twice, and finally suspended in a-R 0.8 ml of 0.1 M NaCl buffered at pH 7.8 with 50 mM 0 tris(hydroxymethyl)aminomethane-chloride. Cells 20 40 60 80 100 120 140 160 180 were lysed by treatment with ethylenediaminetetra- TIME (MIN) acetic acid (EDTA) and lysozyme. The suspension was made 1 mm in EDTA and kept at 0 C for 8 min. FIG. 1. Time course of the appearance of aspara- After the addition of magnesium acetate to a concen- ginase II in standing cultures. Bacteria were grown tration of 1 mm, 1 mg of egg white lysozyme (Worth- aerobically with vigorous agitation in 0.05 M sodium ington Biochemical Corp.) was added to give a final phosphate buffer at pH 7.5, supplemented with 1% volume of 1 ml. The samples were incubated for 15 tryptone, 0.1% yeast extract, and 0.1% glucose. At the min at 37 C. Samples (0.01 ml) of these lysates were time indicated by the arrow, the shaker was stopped. assayed for asparaginase II either directly or after Samples were removedfor the assay ofasparaginase II dilution with buffered saline, in a final volume of 0.06 (A) andfor turbidity (B). ml, by following the conversion of '4C-asparagine to aspartic acid with rapid chromatography on ion-ex- in vitro. Incubation of asparaginase at pH 8 change paper (Ecteola, Whatman ET81) (6). The with 10 mm p-hydroxymercuribenzoate or with specific enzymatic activity is given as micromoles of any of the alkylating agents, iodoacetate, iodo- aspartic acid produced per hour per optical density acetamide, or N-ethylmaleimide, also at 10 mM, low unit of the culture. At the concentration of aspara- did not inhibit the enzyme. Asparaginase II in I does gine used this assay (0.1 mM), asparaginase was exposure to air. When not contribute significantly to the products formed not inactivated by (6, 26). the bacteria were aerated after a period of anaero- bic growth, the formation of the enzyme stopped. RESULTS Even so, enzyme previously formed in the ab- Formation of asparaginase II in standing cul- sence of air remained at a constant concentration tures. Schwartz et al. (26) observed that asparagi- in the aerated culture. nase II was formed in standing cultures during The production of asparaginase in standing the transition from aerobic to anaerobic growth. cultures depended upon protein synthesis. Cells The time course of the appearance of the enzyme were first grown aerobically. The addition of under these conditions is shown in Fig. 1. Little chloramphenicol at the time when the aeration enzyme was produced by cells during aerobic was discontinued prevented the appearance of growth. As soon as the aeration was discontinued, the enzyme. Dependence of enzyme formation the specific activity increased exponentially on protein synthesis indicated that its appearance until a plateau was reached by 40 min. The resulted from de novo synthesis rather than from form of these kinetics suggested that a factor, the conversion of a preexisting but inactive perhaps oxygen, inhibitory to the production protein precursor. of the asparaginase was disappearing during Roberts et al. (23) recently reported that, when the period following the cessation of aeration. E. coli is grown for 12 to 16 hr with shaking, These considerations led us to study the formation there is a greater yield of asparaginase in air of enzyme under strict anaerobiosis. than in an atmosphere of nitrogen. It was con- It was important to establish beforehand that cluded that anaerobiosis depressed the formation the enzyme is stable under aerobic conditions. of asparaginase II. The supply of oxygen is likely Sulfhydryl reagents did not affect the enzyme to be limiting to cultures at high cell densities VOL.
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