Communication MULTIPLE FORMS OF ACID PHOSPHATASE PRODUCED BY ASPERGJLL US OR YZAE YONEKICHI SAKURAI AND HIDEO SHIOTA

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1 Short Communication J. Gen. Appl. Microbiol., 16, (1970) MULTIPLE FORMS OF ACID PHOSPHATASE PRODUCED BY ASPERGJLL US OR YZAE YONEKICHI SAKURAI AND HIDEO SHIOTA Faculty of Agriculture, Iwate University, Morioka, Japan (Received December 16, 1969) Phosphatase is present widely in higher organisms and occurs in multiple forms called isozymes (1, 2). In microorganisms, phosphatase is produced in many species and its production is varied by culture conditions. On multiple forms of phosphatase in Aspergilli, it was reported that phosphatase activity of Aspergillus flavus (3) showed two ph optima in acid and alkaline ranges and acid phosphatase of A, nidulans (4) and A, niger (5) was separated into two components by gel electrophoresis. Further, four fractions of acid phosphatase were obtained from the culture of A. awamori var. kawachii (6) on very low phosphate medium. The present studies were carried out on changes in the pattern of acid phosphatase activity of A, oryzae during incubation which seemed to be due to the multiple forms of acid phosphatase produced and on separation of these components by gel filtration. A. oryzae A 1-5 (7) maintained in our laboratory was cultured in 50 ml of a medium composed of 50 g glucose, 20 g peptone, 1 g KH2PO4, 0.5 g MgSO4 7H2O and CaCl2 2H2O per liter on reciprocating shaker (7 cm/stroke, 120 rpm) at 30. Enzyme activity was measured by the modified method of TORRIANI (8) using p-nitrophenyl phosphate (p-npp) as substrate. The reaction mixture was composed of 1 ml of the enzyme solution, 3 ml of buffer solution (0.1 M citric acid-sodium citrate) and 1 ml of substrate solution (0.02 M p-npp). The reaction was carried out at 40 for an appropriate period and terminated by the addition of 5 ml of Na2CO3 (1.0 M). p-nitrophenol liberated was determined by HITACHI Model 101 spectrophotometer at 400 mp. Enzyme activity of the culture filtrate and that of the extract of tolueneautolysed cell for 24 hr at 30 were named the exocellular and endocellular phosphatase activity, respectively. The enzyme activity was expressed as pmole/min/flask of p-nitrophenol. As shown in Fig. 1, the exocellular acid phosphatase activity was observed over a wide ph range between ph 3 and ph 6, and increased with incubation. The shape of ph activity curve obtained varied and ph of the 335

2 336 SAKURAI AND SHIOTA VOL H 0 C E a, 0 10 E 0 Z a >.-. } 5 U Q ph Fig. 1. Exocellular acid ncubation periods. phosphatase production on various Incubation period for -0-1 day, days. -A- 3 days. maximum activity shifted toward alkaline side as incubation period was prolonged ; the peak was present at ph in 1 day incubation, about ph 4.5 in 2 days, and about ph 5.5 in 3 days. As shown in Fig. 2, the endocellular acid phosphatase activity was much higher than the exocellular and its activity was in similar ph ranges as the exocellular activity, ph of the maximum activity of endocellular acid phosphatase was at ph and did not shift with incubation, being different from the exocellular one. The ratio of the endocellular activity to the total activity (sum of the exocellular and endocellular activity) was calculated throughout the incubation, and the values showed about 85-90% in ph 3-5 and about 60-70% in ph Although the cause of these differences between the exocellular and endocellular is not obvious, but it seems that a greater part of acid phosphatase produced is present in the cell, and activities at ph 3-5 and at ph do not depend on the same enzyme and are probably due to the different component of acid phosphatase produced by A. oryzae A 1-5. The exocellular and endocellular acid phosphatase solutions prepared from the culture of 3 days' incubation were fractionated by gel filtration with Sephadex G-200. Each enzyme solution was concentrated to about 1/10 of

3 F 1970 Acid Phosphatase of Aspergillus oryzae o c 100 0~ ~-o 0 Z a } 50 U Q Fig. 2. Endocellular acid phosphatase production on various incubation periods. Endocellular acid phosphatase activities were estimated on the same culture shown in Fig. 1 Symbols are the same as in Fig. 1. Ten-fold dimension of Fig. 1 is used 1 r expression of phosphatase activity. the original volume by membrane filtration Diaflo Ultra Membrane type UM-10, Amicon Corp., U.S.A.). Twenty ml of a concentrated enzyme solution was applied to the Sephadex G-200 column (50 x 850 mm) equilibrated to ph 7.0 with 0.01 M citric acid-sodium citrate buffer solution and divided into 20 g fractions with the same buffer, the flow rate being 20 g/45 min. Ultraviolet absorption at 280 mp and the enzyme activity at ph 4.0 and ph 5.5 were estimated for each fraction. The exocellular acid phosphatase was separated into two peaks by gel filtration in fraction No and No as shown in Fig. 3. In the former fraction, the activity at ph 4.0 was greater than that at ph 5.5 and, in the latter fraction, the activity at ph 5.5 was better than that at ph 4.0. The elution pattern of endocellular enzyme solution showed one peak in fraction No of whose activity at ph 4.0 was greater than that at ph 5.5, and the chromatogram of endocellular enzyme solution was distinguished from that of the exocellular. Results are shown in Fig. 4. In fraction No , the activity at ph 5.5 was somewhat better than that at ph 4.0. It seemed that, although the endocellular acid phosphatase was not separated into two peaks, there might be a small amount of the enzyme in which the activity at ph 5.5 better than that at ph 4.0. In this experiment, the activity of exocellular or endocellular acid

4 338 SAKURAI AND SHIOTA VOL 16 E X C- k Fig. 3 Dhatase Gel filtration chromatogram Extinction of each fraction at Phosphatase activity at ph 4.0, activity was expressed as the Fraction number of exocellular acid phosphatase preparation. 280 m i (E280). -s- phosphatase activity at ph 5.5. Phosextinction at 400 m i (E400) per 1 ml of eluate. E280 E x10 C s k Fraction number Fig. 4. symbols The Gel filtration chromatogram of endocellular acid phosphat ase preparation sample was made from the same culture used in Fig. 3, and the same and expression of phosphatase activity are used as in Fig. 3.

5 1970 Acid Phosphatase of Aspergillus oryzae 339 phosphatase was separated into two fractions by gel filtration, one fraction showing its higher activity at ph 4.0 than at ph 5.5, and, in contrast, the other fraction was better at ph 5.5 than at ph 4.0. From these results, it is pointed out that at least two components of acid phosphatase were produced by i i. oryzae A f--5 and greater part of the enzyme was present in the cell. REFERENCES 1) NO. KAPLAN, Bacteriol. Rev., 27, 155 (1963). 2) J.H. WILKINSON, Isoenzyme, E. and F. Spon Ltd., London (1965). 3) T.N.R. VARMA and KS. SRINIVASAN, Enzymologia Acta Biocatalytica, 17, 116 (1954) 4) G. DoRN and W. RIvERA, J. Bacteriol., 92, 1618 (1966). 5) S. NAGASAKI, J. Gen. Appl. Microbiol., 14, 263 (1968). 6) Y. OHTA, K. IKEDA and S. VEDA, Appl. Microbiol., 16, 973 (1968). 7) K. SAKAGUCHI and K. YAMADA, Nippon Nogeikagaku Kaishi, 20, 65, 141 (1944), in Japanese. 8) A. ToRRtANI, Biochim. Biophys. Acto, (1960).