Production of two types of phytase from Aspergillus oryzae during industrial koji making

J~UKNALOF BKXXIENCE AND BIOENGINEEKING Vol. 95, No. 5, 460465.

2003

Production of Two Types of Phytase from AspergiEZusoryzae during Industrial Koj i Making JIN FUJITA,‘*S SEIKO SHIGETA,’ YU-ICHI YAMANE,’ HISASHI FUKUDA,’ YASUZO KIZAKI,3”s SABURO WAKABAYASHI,3ss AND KAZUHISA ONO’ Department of Molecular Biotechnology Graduate School of Advanced Sciences qfMattev, Hiroshima UniversifL l-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan,‘Suishin Yamane Honten Co.. Ltd.. l-5-58 Higashi-machi, Mihara, Hiroshima 723-0011, Japaq2and National Research Institute of Brewing, 3-7-l Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan’ Received 7 November 2002/Accepted

15 January 2003

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an important role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH,-terminal sequence of Phy-I, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy-I, 120 kDa; ACP-II, 58 kDa). From the NH,-terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p-nitro phenyl phosphate, but also glucose 6-phosphate and glycerol l-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.

[Key words: Aspergillus otyzae, solid state culture, koji, phytase, purification] Aspergillus oryzae belongs to a group of well-known and important filamentous fimgi producing various traditional Japanese beverages and foods, such as sake, miso, and soy sauce (1). In our previous study, it was shown that when A. oryzae was cultivated in a submerged culture, to which phytic acid was added as the sole phosphate source, A. oryzae produced phytase (myo-inositol hexakisphosphate phosphohydrolase) and two types of acid phosphatase. Phytase activity in the submerged culture was detected during the early phase of logarithm growth (around 24 h), ahead of the activity of the other acid phosphatases (2). Phytase production during the early growth phase was expected to release inorganic phosphate from phytic acid and lead to the growth of mycelia. Sake, which is a traditional Japanese alcoholic beverage, is produced from steamed rice (1). The process of sake making is comprised of koji making and alcohol fermentation. During the stage of koji making, A. oryzae is inoculated as spores onto steamed rice and cultured for 2 d. At the alcohol fermentation stage, Saccharomyces cerevisiae is inoculated

into the moromi mush, which contains koji and steamed rice, and the culture period lasts for approximately 1 month. The steamed rice in the moromi mush is digested by various extracellular enzymes supplied by the koji during fermentation. The various nutrients released are fed to yeast to promote growth and alcohol fermentation. Phosphorous is an important nutrient for yeast. In rice grains, around 80% of the phosphorous exists as phytic acid (3), and accordingly, the main source of phosphate for sake yeast in the moromi mush is phytic acid released by phytase in koji (4). In this study, phytase production by A. oryzae in koji culture and the characterization of purified phytases were investigated; the role of phytases in kqji cultures is also discussed. MATERIALS

AND METHODS

Microorganism A. oryzae RIB-128 was employed for this study. This strain was maintained at 4°C on an agar slant containing 39 g/l of Bacto Potato Dextrose Agar (Difco Laboratories, Detroit, MI, USA). Chemicals Unless otherwise stated, all chemicals were obtained from Wako Pure Chemical Industries (Osaka). Koji culture The koji rice was produced by the method of Yamane et al. (5). Cultivation was performed according to industrial procedures as follows. Brown rice (Nipponhare) was polished until it reached 70% of its original weight, and the polished rice was washed and soaked for 30min at 9°C. The rice was then

* Corresponding author. e-mail: [email protected],ip phone: +81-(0)22-7 19-5979 fax: +81-(0)22-7 19-5980 Present address: 5Innovation Plaza Miyagi, Japan Science and Technology Corporation, 6-6-5 Minami-yoshinari, Aoba-ku, Sendai, Miyagi 989-3204, Japan and Q National Research Institute of Brewing, 2-6-30 Takinogawa, Kita-ku, Tokyo 114-0023, Japan. 460

VOI>.95.2003 allowed to stand overnight, and was subsequently steamed for 50 min. After cooling the rice to 35”C, the steamed rice was inoculated with 0.1 g of spores of A. oryzue per gram of steamed rice. The initial temperature of the cultivated rice was maintained at 32°C for 24 h, then the temperature was increased at rate of 1“C per hour to 42”C, and was then maintained at 42°C. Phytase assay Phytase activity was assayed according to a procedure described in our previous report (6). An aliquot of enzyme solution was incubated with 2 mM phytic acid (Sigma, St. Louis, MO, USA) in 200 mM acetate buffer (pH 5.0) at 40°C for 20 min. The amount of liberated inorganic phosphate was measured using a Phosphor C test kit (Wako). One unit of phytase activity was defined as the amount of enzyme liberating 1 Fmol of inorganic phosphate per min under the present assay conditions. The mycelial growth on a Measurement of mycelial weight koji culture was evaluated by an assay for glucosamine contained in the mycelium (7). Intracellular glucosamine in the mycelium was extracted and measured using a glucosamine estimating kit (Kikkoman, Nagoya). The dry mycelial weight was calculated by assuming a mycelia glucosamine content of 139 mg of glucosamine per 1 g of dry mycelium (8). Purification of phytase from koji culture The protein elution profile was monitored spectrophotometrically by the absorbance at 280 nm. Step 1. Enzyme extraction Koji rice (50 g) was steeped in 200 ml of IO mM acetate buffer (pH 5.0) containing 0.9% NaCl and shaken on a rotating shaker (100 rpm) at 4°C for 3 h and then centrifuged (6000 xg, 20 min). The supernatant was filtered through a cellulose nitrate membrane (pore size, 0.45 pm; Advantec Toyo, Osaka), and the filtrate was concentrated to approximately 50 ml by a centrifugal concentrator (I 0 K; Pall filtron, Northborough, Ml, USA), followed by dialysis against 10 mM acetate buffer (pH 5.0). The dialyzed solution was used for subsequent fractionation. Step 2. Anion exchange chromatography The dialysate in step 1 was applied to a Poros-PI anion exchange column (4.6 x 50 mm; Applied Biosystems, Tokyo) equilibrated with IO mM acetate buffer (pH 5.0) at a flow rate of 5 ml per min. After the column was washed with 10 ml of equilibration buffer, the absorbed enzyme was eluted with 50 ml of a linear gradient of 0 to 1.5 M NaCl in the same buffer. Each fraction (I ml) was collected and the phytase activity was measured. The active fractions were separately pooled, concentrated to approximately 15 ml by a centrifugal concentrator (10 K; Pall filtron), and dialyzed against 10 mM acetate buffer at pH 4.5. Step 3. Cation exchange chromatography The respective desalted fractions from step 2 were loaded on a Poros-HS cation exchange column (4.6 x 50 mm; Applied Biosystems) using a similar method to that used in step 2, except that the buffer pH was 4.5 in this case. The active fractions were pooled and concentrated to approximately IO ml by a centrifugal concentrator (10 K; Pall filtron). Step 4. GelfiltrationThe concentrated fractions obtained from step 3 were subjected to gel filtration on a TSK-gel C-2000SW column (7.5 mm x 30 cm; Tosoh, Tokyo) using 40 mM acetate buffer containing 0.2 M NaCl (pH 5.0) at a flow rate of 3 ml per min. Each fraction (3 ml) was collected. The phytase-active fractions were pooled separately. Step 5. Gel filtration-2 The phytase-active fractions from step 4 were subjected to gel filtration on a TSK-gel G-4000-SW column (7.5 mm x 30 cm; Tosoh) according to the same procedure as that used in step 4. Protein assay The protein concentration was measured by the Bradford method (9) using bovine serum albumin as the standard. Estimation of molecular weight by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE

PHYTASE FROM A. ORYZAE IN KOJI CULTURE

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was performed according to the procedure described by Laemmli (10). The purified enzyme was loaded on a precast SDS l&20% gradient gel (Daiichi Pure Chemicals, Tokyo) and was visualized by the silver stain method (2D-Silver stain I1 Daiichi; Daiichi). The molecular weight of the purified enzyme was determined using a molecular weight marker kit (Daiichi II-III; Daiichi) as the standard. Analysis of the NE&-terminal amino acid sequence The NH,-terminal amino acid sequence of the purified enzyme was analyzed by automated Edman degradation (model 491; Applied Biosystems). Measurement of sugar content The sugar content was measured by the phenol-sulfuric acid method as glucose equivalents (11). General properties of purified phytases Effect ofpH on enzyme activity and stability The pH of the reaction solution was adjusted with the following buffers: for pH 2.0 to 3.0, 40 mM glycine-HCI; for pH 3.0 to 6.0; 40 mM acetic acid-NaOH; for pH 6.0 to 8.0, citric acid-NaOH. The pH stability of each enzyme was determined by an assay for residual activity after incubation at the respective pH values at 40°C for I h. Effect of temperature on enzyme activity and stability The dependence of temperature of the activity of each enzyme was determined at 10°C to 80°C at pH 5.0. The thermal stability of each enzyme was determined by an assay for residual activity after incubation under the above mentioned thermal conditions at pH 5.0 for 30 min. Substrate specificity Each enzyme was incubated with 10 mM of each substrate for 40 min at 40°C in 0. I M acetate buffer (pH 5.0). The amount of released inorganic phosphate was measured using the Phosphor C test kit (Wako). RESULTS Phytase production As phytase produced in a submerged culture was found to play an important role in mycelial growth in our previous study (2), the phytase production of A. oryzae during koji culture was determined in the present study. As shown in Fig. 1, although phytase activity was not detected during the lag phase from 0 to 12 h, it increased subsequent to the logarithmic growth of the mycelia. Phytase activity reached a plateau at 30 h, and the maximum phytase activity was obtained at 6.6 units/g koji rice at 45 h of cultivation.

Culture time (h) FIG. I. The time course of phytase production by A. otyzae in kqji culture. Symbols: closed circles, phytase activity: open circles, mycelial weight.

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1 HiOScl. HlOl

20

2.0

(kDa .)

Ml

2

NC,..

3

200 -^ E 2 .2 .z

10

116

1.0

66 0

0.0 cE z

42.4 0.3 0.2

0

10

20 30 Elution volume (ml)

40

50

FIG 2. Ion exchange chromatograms of the phytases of A. oryzae in koji culture. (a) Anion exchange chromatogram of the crude solution from the koji culture, and (b) cation exchange chromatogram of the active fraction from the anion exchange chromatography (Fig. 2a., fraction nos. 4 to 8). After elution of non-absorbed proteins with IO ml of equilibrating buffer, a linear salt gradient of 0 to I .5 M NaCI in the equilibration buffer (50ml) was used to release the bound proteins. Symbols: closed circles, phytase activity; open circles, the absorbance at 280 nm.

Since high amounts of phytase Purification of phytase were produced during the logarithmic growth of the myCelia, it was concluded that phytase played an important role in mycelial growth. To determine the type of phytase that was active in the koji culture, we purified the phytase. Typical elution patterns for anion and cation exchange chromatography are shown in Fig. 2. Initially, the koji extract was applied to the anion exchange column (Poros PI) (Fig. 2a). The phytase activity was detected as a single activity peak in an elution volume from 23 to 30 ml. Following cation exchange column (Poros HS) chromatography, two peaks of phytase activity were detected in elution volumes from 2 to 10 ml (FAP-I) and 24 to 3 1 ml (FAP-II) (Fig. 2b). The FAP-II fraction contained more than 80% of the total phytase activity. FAP-I and FAP-II were further purified on TSK-gel G-2000 SW, followed by TSK-gel G-4000 SW columns. After two steps of gel chromatography, purified FAP-I and FAP-II were referred to as Phy-I and Phy-II, respectively. Phy-I gave a single band, and Phy-II presented a single but slightly broad band (Fig. 3, lanes 1 and 2). Lane 3 in Fig. 3 shows the results of the SDS-PAGE of ACP-II that was previously purified from a submerged culture. The estimated ‘1‘ABI.T:I Step Crude enzyme FAP-I (Phy I)

FAP-II (Phy II)

Pros-PI/M Pros-HUM TSK gel G-2000 TSK gel G-4000 Pros-HSiM TSK gel G-2000 TSK gel G-4000

Summary

FIG. 3. SDS-PAGE of purified acid phosphatases produced b> A otyzue. Lane M, Molecular weight standard proteins (myosin, 200 kDa: P-galactosidase, I 16kDa; albumin, 66 kDa; aldolase, 42.4 kDa; carbonic anhydrase, 30 kDa; myoglobin, 17.2 kDa; Daiichi II); lane I, Phy-I; lane 2, Phy-11; lane 3, ACP-II. SDS-PAGE was performed with a I O-20% gradient gel containing 0.1% SDS.

molecular weights of Phy-I, Phy-11, and ACP-II were approximately 120, 116, and 58 kDa, respectively. Each of the purification steps is summarized in Table 1. Phy-1 and Phy-II were purified to approximately 614- and 2950-fold, with a recovery of 2.6% and 11.7%, respectively. Their specific activities were approximately 1106 and 5309 units/mg-protein, respectively. NH,-terminal amino acid sequences and sugar content The NH,-terminal amino acid sequences of Phy-I and Phy-II were ASRNQSS and AALPKAN, respectively. The sugar contents (%) of Phy-I and Phy-II were 53% and 47%, respectively. In comparison with ACP-II (8% sugar content). the koji produced phytases with relatively high sugar contents. Effects of pH and temperature on enzyme activity and stability The activity of Phy-I and Phy-11 was determined at different pHs and different temperatures, as described in Materials and Methods. The optimum pH of both phytases (Phy-I and Phy-II) was 5.0, and both phytases were virtually inactive above pH 7.5 (Fig. 4a). Moreover, both enzymes were completely stable between pH 5 and 6. Phy-II retained more than 80% of its activity within a pH range of 3 to 7.4. More than 80% of the maximum activity was retained at pH 3.8 to 6.9 in the case of Phy-1, but in the case of ACP-II, the pH range for the activity was relatively

of purification of phytase from A. u,:)w~~

Total activity (W 6875 3573 390 230 177 1668 1456 1221

‘l’otal protein (mg) 3750.1 348.0 75.5 2.22 0.16 i x 2.3 0.23

Specific activity (U/mg-protein) I .x IO.3 15.3 104 I106 339 633 5309

Yield (%) 100.0 52.0 5.7 i. i 3.6 2-l. i 71.1 17.7

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T

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.$ 2 50 2 P lz!

0 2

3

4

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6

7

8

2

3

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5

6

8

7

PH

PH

FIG. 4. Effect of pH on phytase activity and stability. (a) Optimum temperatures for purified Phy-I, Phy-II, and ACP-II. The reactions were carried out at 30°C at various pHs, as described in Materials and Methods. Symbols: circles, Phy-I; triangles, Phy-II; dotted line, ACP-II.

b

20

30

40

50

Temperature

60

70

80

10

20

30

40

50

Temperature

(“C)

60

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(“C)

FIG. 5. Effects of temperature on phytase activity and stability. (a) Optimum temperatures for purified Phy-I, Phy-II, and ACP-II. The reactions were carried out in 40 mM acetate buffer (pH 5.0) at various temperatures. Symbols: circles, Phy-I; triangles, Phy-II; dotted line, ACP-II.

narrow (pH 4.5 to 6.5) (Fig. 4b). Figure 5 shows the effect of temperature on phytase activity and stability. The optimum temperatures for Phy-I and Phy-II activity were 65°C and 55”C, respectively. On the other hand, the optimum temperature for ACP-II activity was 40°C (Fig. 5a). Regarding stability, the optimum temperature of both phytases ranged from 10°C to 5O”C, and they were more stable than ACP-II (10°C to 30°C). Both Phy-I and Phy-II retained 100% activity at temperatures between 10°C to 50°C; this activity decreased gradually with increases in temperature, and was ultimately lost at 75°C TABLE 2. Substrate specificity of each Phy-I, Phy-II, and ACP-II Substrate (10 mM) p-Nitro phenyl phosphate Phytic acid myo-lnositol- I -phosphate D-Glucose-6-phosphate Glycerol- I -phosphate ATP

Relative activity” (%) Phv-I

Phv-II

ACP-Ilb

100 290 30 0 0 0

100 I51 IS 90 36 0

100 315 I5 0 0 0

aHydrolysis rate ofp-nitro phenyl phosphate was taken as 100%. b The results of ACP-II were taken from our previous report (2).

(Fig. Sb). More than 90% of the Phy-I and Phy-II activity was observed after the enzymes were incubated at 10°C for 192 h, whereas ACP-II activity decreased rapidly and all activity was lost at 120 h of incubation (Fig. 6). Substrate specificity Substrate specificities for 6 different substrates are listed in Table 2. Phytic acid was the best substrate for Phy-I and Phy-II. Phy-II released inorganic phosphate from D-glUCOS-6 phosphate and glyceroll-phosphate; on the other hand, Phy-I did not release inorganic phosphate from either of these substrates. Production profile of Phy-I and Phy-II during koji culture In the context of koji production, the production profiles of both types of phytase are shown in Fig. 7. Initially, Phy-I was detected at an early stage of cultivation (18 h), and reached maximum activity at 27 h, i.e., during the middle phase of logarithmic growth; on the other hand, Phy-II was detected at 27 h, following Phy-I production, and Phy-II activity was responsible for the majority of phytase activity at 45 h.

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Incuvation period (h) FIG. 6. Stability of phytase activity at different incubation times. Phy-I, Phy-II, and ACP-II in 40 mM acetate buffer (pH 5.0) at 10°C for various periods of time; the residual activity was then measured. Symbols: circles, Phy-I; triangles, Phy-II; dotted line, ACP-II

r

U

18

27 36 Culture time (h)

II 45

FIG. 7. Phytase production during koji culture. Changes in activities of Phy-I and Phy-II during koji culture. The activities of the two enzymes at different times during the cultivation period were divided by the total phytase activity in the crude extract to give the ratio of Phy-I and Phy-II activities separated on Poros HS/M. Symbols: open rectangle, Phy-I; closed rectangle, Phy-II.

DISCUSSION

In the present study, we investigated the phytase production and properties of A. oryzae in koji culture (solid-state cultivation). Two types of phytase (Phy-I and Phy-II) were produced during koji culture. The seven amino acids of the NH,-terminal sequence of Phy-I were identical to those of the NH,-terminal sequence of ACP-II produced from A. oryzae in submerged culture (2). Moreover, the sequence is almost identical to those of A. niger- (12) and A. niger var. awamori-3-phytase A (13). On the other hand, the sequence of Phy-II exhibited homology neither to the internal region of the two above-mentioned 3-phytases A for which the entire sequence has previously been determined, nor the sequences listed in a BLAST database. These results suggested that Phy-II was not a fragment of Phy-I but a novel phytase. The entire sequences of the two present phytases have to be determined.

Phy-I was detected initially at 18 h of cultivation (Fig. 7), during the lag phase of mycelia growth. The optimum pH and the optimum temperature of Phy-I agreed with those of ACP-II, but the molecular weight of Phy-1 (120 kDa), estimated by SDS-PAGE, was twofold higher than that of ACP-II (58 kDa) (Fig. 3). The difference in the molecular weights of these enzymes results from their respective sugar contents (Phy-I, 53%; ACP-II, 8%). Phy-I, with a higher sugar content, was superior in terms of pH and thermal stability to ACP-11 (Figs. 4 and 5). Nakamura e/ ~1. reported previously that several properties of lysozyme were affected by their sugar chains (14-17). The carbohydrate moiety of Phy-I determines the pH and thermal stabilities. The major phytase, Phy-II, was newly discovered in koji, which was cultivated in the solid-state condition. Phy-II, a glycoprotein, was also stable over broad pH and temperature, as was Phy-I. The inorganic phosphate liberation spectrum of Phy-11 was somewhat wider than that of Phy-I at the point of phosphate liberation from glucose 6-phosphate and glycerol l-phosphate. The substrate specificities of Phy-11 resemble those of the appA (18) and appA2 ( 19) phytases from Escherichia coli and the phytase from Bacillus suhtilis (20); however, the molecular weight, as well as the pH and heat resistance properties of Phy-II were quite different from those of the other enzymes. Previously, glucoamylase (gla B) (21) from A. oryzae was reported as a solid statespecific enzyme. Akao et al. noted that A. oryzue incubated in the solid state showed different regulation from that in submerged culture (22). Yamane et ul. indicated that different mycelial features were observed when various moisture contents were used during cultivation (5). Two mycelial features noted by Hata namely, the basal and air-type mycelia, were observed in A. oryzue cultured in the solid state (23). The basal type mycelia could be further categorized into two types; in one type, the mycelia elongated into the solid matter used in the cultivation and in the other type. the mycelia extended towards the solid. Ito cv 01. and Matsunaga et ul. reported that the mycelial penetration in koji affected enzyme activity, such as a-amylase, glucoamylase, acid protease, and acid carboxy peptidase (24, 25). Solid state-specific enzymes, such as Phy-11. and glucoamylase B, would be produced from such solid state-specific mycelia. In sake brewing, phytic acid in rice is the main source of phosphorous for the yeast in sake moromi mush. As Phy-I and Phy-II could release inorganic phosphate from phytic acid in sake brewing at a pH close to 4 (Fig. 4) these enzymes are considered to function as the main enzymes supplying inorganic phosphate to yeast in moromi mush. In particular, Phy-I1 which accounted for approximately 90% of the total phytase activity at 45 h of cultivation (Fig. 7) is presumed to function as the key enzyme.

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