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Purification Andsomeproperties of Cytosine Deaminase from Bakers

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Purification Andsomeproperties of Cytosine Deaminase from Bakers Agric. Biol. Chem., 53 (5), 1313-1319, 1989 1313 Purification and SomeProperties of Cytosine Deaminase from Bakers' Yeast Tohoru Katsuragi, Toshihiro Sonoda, Kin'ya Matsumoto, Takuo Sakai and Kenzo Tonomura Laboratory of Fermentation Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka 591, Japan Received November 24, 1988 Cytosine deaminase (EC 3.5.4.1) was extracted from commercial compressed bakers' yeast and purified to an almost homogeneous state. The enzyme activity was more than 200U/mg of protein, which was several times higher than reported before. The molecular weight was 41,000 by gel permeation. The pi was at pH4.7. 5-Fluorocytosine, 5-methylcytosine, and creatinine were other substrates for the enzyme.An experiment with inhibitors suggested that the enzyme was an SH- enzyme. The enzyme was unstable to heat, with a half-life of about 0.5hr at 37°C. Characteristics of the enzyme, especially its substrate specificity, were compared with those reported earlier for other cytosine deaminases from bacteria and a mold. Local chemotherapy of cancer with the com- (5MC), a 5-substituted cytosine.4) 5FC, an- bined use of 5-fluorocytosine (5FC) given oral- other 5-substituted cytosine, is deaminated to ly and a cytosine deaminase capsule implant- 5FU in Saccharomyces cerevisiae.5) So, cy- ed locally may be possible.1} However, al- tosine deaminase of bakers' yeast should con- though this approach is successful in animal vert 5FCto 5FU, and could be used in place of experiments,1'2) there are problems when we E. coli cytosine deaminase. Although the yeast use the enzyme from Escherichia coli,3) which enzyme is unstable to heat (at 37.5°C),4) which is thermostable,1'3) and which can deaminate would prevent its use in long-term therapy in 5FC to 5-fluorouracil (5FU).1>3) First, it is the body, it might be stabilized by immobili- difficult to culture the bacteria on a large scale zation or other techniques. to obtain enough activity.3) As often occurs Anextract from compressed yeast deami- during purification of enzymes, E. coli cytosine nated 5FC to form 5FUin a preliminary ex- deaminase was obtained only in low yield periment, so we prepared the enzyme from when highly purified.3) Second, because of the yeast. possible pyrogenic reactions, it is preferable to The enzyme from a commercial bakers' use a highly purified preparation of the enzyme yeast has been reported to be purified, 340-fold or else to use an enzymefromother, safer at first,6) and "completely" in a second re- sources than bacteria. port,7) although only a few times more pure Cytosine deaminase can, however, be ob- than the first preparation. We purified this tained in large quantities from commercial enzyme much more, and describe the pro- compressed bakers' yeast.4) Yeast has been cedure and some characteristics of the enzyme used as an agent of alcoholic fermentation preparation here. from ancient times, and also as an agent for bread leavening in the Western world; so, it Materials and Methods seems to be a safe source. Yeast cytosine deaminase deaminates 5-methylcytosine General. Unless otherwise noted, all experiments were 1314 T. Katsuragi et al. done as described before,1} and at below 10°C, with a scribed before.3) Densitograms for stained disc gels were 50mMpotassium phosphate buffer, pH 7.0. recorded at a wavelength of 550nmwith a densitometer (model 39433; Gelman Sciences Japan, Ltd., Tokyo). Materials. Compressed bakers' yeast was obtained from Oriental Yeast Co. (Tokyo). DEAE-Sephacel, Octyl- Results Sepharose CL-6B, LKBAmpholine carrier ampholytes, and a pi caliberation kit of marker proteins (range pH 3~10) for isoelectrofocusing were obtained from Purification of enzyme Pharmacia KK (Tokyo). Toyopearl HW-50S was from Cytosine deaminase was extracted from Tosoh Corp. (Tokyo). Other chemicals used were pur- compressed yeast by plasmolysis. The enzyme chased from WakoPure Chemical Industries (Osaka), was purified from the extract by fractional and were of guaranteed reagent grade. precipitation with ammoniumsulfate, anion- exchange chromatography (with DEAE- Enzymeassay. The enzyme activity was assayed in a cellulose or DEAE-Sephacel), hydrophobic mixture of an adequate amount of the enzyme and 3mM chromatography (with Octyl-Sepharose CL- cytosine in 0.20 m potassium phosphate buffer (pH 8.0) as described previously.1} After 30 min of incubation at 37°C, 4B), and gel-permeation chromatography the mixture was diluted with 0. 1 n HC1to stop the reaction (with Toyopearl HW-50S). A typical pro- andto measurethe conversionof the substrate cytosine to cedure was as follows, and the results are the product uracil spectrophotometrically (at 280 nm). For the study of the substrate specificity, the substrates and the summarized in Table I. products were assayed by dual-wavelength spectropho- Cell extract. Compressed yeast (2.0 kg) was tometry also described there1}: cytosine and uracil at 250 broken into flakes with the fingers, mixed with and 280nm; 5FC and 5FU, or 5MC and thymine, at 255 ethyl acetate (200 ml) with a spatula, stirred for and 290nm. The creatinine specificity was established 30min with a magnetic stirrer, and further through the enzymatic assay of the ammonia that had stirred for a few hours with the addition of formed as described before,3) and compared with the 2.00 1 of buffer that had been 15% saturated cytosine value by the same method for the reference. with ammoniumsulfate and adjusted to pH Ultrafiltration and diafiltration. Concentration and 7.0, so that the cells were plasmolyzed. To the diafiltration of a small volume of enzyme solution were autolysate obtained, 8.00 1 of buffer was add- done with an ultra filter with a molecular-weight cutoff of ed; the mixture was left in a cold room and about 10,000 (UK-10 filter, fitted in a UHP-25 holder stirred for a few minutes every day. Cell debris equipped with a spinner; Toyo Roshi Co., Tokyo) under was removed by centrifugation after 3 days. pressure with nitrogen gas. The clarified lysate (1 1 1) was referred to as the Electrophoreses. Discontinuous (disc) and isofocusing cell extract. electrophoreses on polyacrylamide gels were run as de- Ammoniumsulfatefractionation. The extract Table I. Purification of Cytosine Deaminase Specific Activity activity Purification Yield Step (U) (U/mg (fold) (%) (mg)(A280 x ml) protein) Autolysis (2.0 kg 98,000 3,600 0.037 1 100 of compressed yeast) 1st ammoniumsulfate 39,000 3,000 0.077 2.1 82 (50 - 73% saturation) 2ndammoniumsulfate 1 1,000 1,500 0.14 3.8 42 (56 - 70% saturation) DEAE-Sephacel 78 420 5.5 150 12 Octyl-Sepharose CL-4B 4.9 210 42 1,100 5.7 Toyopearl HW-50S 0.41" 1 00 250a 6,800" 2.9 The amount of protein was calculated with the assumption that the concentration was proportional to A280. Cytosine Deaminase from Bakers' Yeast 1315 was brought to 70% saturation. The pre- Instruments, Inc., Osaka). The active fractions cipitate that formed overnight was collected by were combined (12ml). They contained 100U centrifugation, dissolved in 3.50 1 of buffer, ofenzyme activity, corresponding to a yield of and dialyzed thoroughly against the buffer. 2.9%, and had an absorbance of 0.043 at The dialyzed solution (3.99 1) was fractionally 280nm, from which we calculated that there precipitated with ammoniumsulfate from 50 was about 0.41mg of protein, with the as- to 73% saturation. The precipitate was col- sumption that absorbance and protein con- lected with 1.00 1 of 20mMbuffer, dialyzed as above, and diluted to 4.001 with the same centration were proportional. buffer. Fractionation was again done between Purity 56 and 70%saturation, and the precipitate was The fraction above had a specific activity of dissolved in 350ml of 10mMbuffer, followed over 200 U/mg (Table I). This corresponds to a by dialysis. 6800-fold purification over the cell extract, Anion-exchange chromatography. The above calculated from the purification factors from fraction (350ml) was put onto a column of step to step. Whenthe gel was stained, discon- DEAE-Sephacel (4.4x 14cm) previously tinuous gel electrophoresis gave one dense equilibrated with buffer. The column was main band, and two very faint bands on both washed thoroughly with buffer, and the en- sides of the main band. Figure la shows a zymewas chromatographed with a linear gra- densitogram. To identify which of the three dient of potassium chloride from 0 to 100 bands was the enzymeprotein, another gel rod mMin 1000ml of buffer at a flow rate of was cut lengthwise into two almost equal about 50ml/hr. Active fractions were collect- parts. One piece was studied by dye-staining, ed and combined (40ml). and the other was studied by activity-staining, Hydrophobic chromatography. The above as follows. This piece was further cut crosswise 40ml was dialyzed against 3.1 m ammonium into 1.7-mmslices. Every slice was transferred sulfate solution buffered at pH 7 with 100mM potassium phosphate, centrifuged to remove the precipitates formed, and put onto a col- umn of Octyl-Sepharose CL-4B (2.2x 12.5 cm). Elution was forced by application of a linear gradient of ammoniumsulfate from 3.1 to 0m in 200ml of 100him.buffer at a flow rate of about 25ml/hr. The active fractions (18 ml) were combined, concentrated by ultra- filtration to about 1.5ml, and diafiltrated with 50mMbuffer containing 100mMpotas- sium chloride. Gel-permeation chromatography. The dia- lyzed solution was chromatographed on a column ofToyopearl HW-50S (1.3 x 6.3 cm) at a flow rate of about 14ml/hr. The active fractions were collected (8ml; 130U; 0.130 absorbance at 280 nm), concentrated by ultra- filtration to about 1.4ml, and passed again Fig.
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