Purification and characterization of an endo-1,4-β-d -galactanase from Aspergillus sojae

JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 85. No. 1. 48-52. 1998

Purification and Characterization of an Endo-l&3-D-Galactanase from Aspergillus sojae ISA0 KIMURA,‘”

NAOMI

YOSHIOKA,’

AND

SHIGEYUKI

TAJIMA*

Kagawa Prefectural Fermentation and Food Experimental Station, 1351-I Nouma, Uchinomi-cho, Shouzu-gun 761-44’ and Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Mki-cho, Kita-gun, Kagawa 761-07,? Japan Received

An endo-I,4-,8-o-galactanase

28 August

1997/Accepted

15 October

1997

(EC 3.2.1.89) was purified to homogeneity from a solid-state culture of

Aspergillus sojae. The molecular weight of the galactanase was estimated to be 39,700 by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration chromatography indicated the native enzyme to be a monomer. The isoelectric point of the galactanase was 3.60. The optimum pH and temperature of the enzyme activity were 4.5 and 5O”C, respectively. The galactanase was stable from pH 6.0 to 10.0, and up to 35°C. The K, value for arabinogalactan from soybean was 0.82 mg/ml. The activity of the enzyme was significantly inhibited by Mn2+, Hg2+, Ag+, and Fe3+, and no stimulation by metal ions was apparent. After the hydrolysis of arabinogalactan from soybean, the major products were galactobiose and galactose, and no liberation of arabinose was observed in the reaction mixture. [Key words: galactanase, arabinogalactan, Aspergillus sojae] Pentoses in soy sauce mash are believed to contribute to the browning of the sauce (1). These pentoses, e.g., xylose and arabinose, are released from the soy sauce starting materials-soybeans (Glycine max) and a cereal such as wheat-by hemicellulases contained in the koji mold. With the aim of being able to control the color of soy sauce, we have been investigating the role of hemicellulases from Aspergillus sojae in its production (2, 3). which is mainly composed of Arabinogalactan, arabinose and galactose, is a major cell wall component of the soybean cotyledon (4-7). Although the arabinogalactan is hydrolyzed into monosaccharides during the soy sauce manufacturing process (S), little is known of the properties and action pattern of the arabinogalactan-degrading enzymes of yellow aspergilli, i.e., A. oryzae, A. sojae, and A. tamarii, which are important in the food industry, e.g., in the production of soy sauce and miso (fermented soybean paste). Arabinogalactan-degrading enzymes have been purified from commercial enzyme preparations of black aspergilli, e.g., A. aculeatus (9, 10) and A. niger (9, 11, 12). However, the enzymatic characteristics and roles of arabinogalactandegrading enzymes in the production of soy sauce have not been compared with those reported in the literature. Here, we describe the purification and properties of an endo-1,4-P-D-galactanase from a solid-state culture of A. sojae, and compare its characteristics with those of other galactanases of aspergilli. MATERIALS

and 1,5-arabinan from beet pulp, which were prepared according to the method of Tagawa and Kaji (14), were kindly provided by Professor M. Sato, Kagawa University. Other polysaccharides and synthetic substrates were obtained from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). Affinity gel was prepared according to the as a method of Nakano et al. (15), using arabinose ligand for the gel. Biochemical assays Enzyme activity was measured on the basis of the release of reducing sugar from arabinogalactan. The assay mixture contained 0.4 ml of 0.625% arabinogalactan (from soybean cotyledon) solution in 0.05 M sodium acetate buffer (pH 4.5) and 0.1 ml of an appropriately diluted enzyme solution. It was incubated at 30°C for 10min. The reducing sugar released was measured as galactose by the Somogyi-Nelson method (16). One unit of enzyme activity was defined as the amount of enzyme which liberated 1 pmol equivalent of galactose per minute from arabinogalactan. Native-polyacrylamide gel electrophoresis (nativePAGE) was performed according to the method of Davis (17), and sodium dodecyl sulfate (SDS)-PAGE according to the method of Laemmli (18). The protein in the gel was stained by the method of Oakley et al. (19), and an electrophoresis LMW calibration kit (Pharmacia Fine Chemicals, Uppsala, Sweden) was used to provide marker proteins. Gel isoelectric focusing was done on a thin-layer gel (Bio-Rad Labs., Richmond, CA, USA). The method used for p1 determination was based on the procedure reported by Lz& et al. (20). The molecular weight of the purified enzyme was determined by gel filtration chromatography (GFC) and SDS-PAGE. GFC was performed on a G3000SWxL column (Toyo Soda Co. Ltd., Tokyo) equilibrated with 0.05 M sodium phosphate buffer (pH 7.0) containing 0.3 M NaCl at 10°C. The column was calibrated with an LMW gel filtration calibration kit (Pharmacia). Protein was hydrolyzed in vacua at 110°C for 24, 48, and 72 h with double-distilled HCl. Half-cysteine was determined by the method of Ellman (21), and the tryptophan content by the method

AND METHODS

A. sojae no. 3 (3) was cultured Biological materials on potato dextrose agar at 30°C and the stock culture was kept in a refrigerator at 4°C. The strain was cultured in a 500-ml Erlenmeyer flask containing 20g beet pulp, 0.4 g urea, and 12 ml tap water at 27°C for 48 h. Arabinogalactan from soybean was preChemicals pared according to the method of Morita (13). Arabinan

*

Corresponding

author. 48

GALACTANASE

VOL. 85, 1998

of Simpson et al. (22). The hydrolyzates were analyzed by a Hitachi reaction LC-OPA amino acid system. Protein sequencing was done with a Milligen/Biosearch ProSequencer system. The protein content of the enzyme was measured according to the method of Bradford (23) with bovine serum albumin as a standard. The total carbohydrate content of the protein was determined by the phenol-sulfuric acid method (24) with mannose as a standard. For the hydrolysis of arabinogalactan from soybean, 5 units of the purified enzyme were mixed with 15 mg of arabinogalactan in 15 ml of 0.02 M sodium acetate buffer (pH4.5). The mixture was then incubated at 30°C for 72 h with one drop of toluene. Aliquots (2ml) of the reaction mixtures were collected and placed in a boiling water bath for 3 min, and the samples were analyzed by high-performance liquid chromatography (HPLC). The neutral sugar composition of various polysaccharides was analyzed according to the method of Pazur (25) using a Shodex sugar KS-801 column (Showa Denko Co. Ltd., Tokyo); uranic acids were determined accordet al. (26) with galacing to the method of Blumenkrantz turonic acid as a standard. The products of the enzymatic hydrolysis of arabinogalactan were analyzed by HPLC using a TSK gel G-Oligo-PW column (Tosoh Co. Ltd., Tokyo), which was calibrated with standard glucose oligomers (Seikagaku Co. Ltd., Tokyo). The sugars were detected by a refractive index monitor (RI-8010; Tosoh). RESULTS AND DISCUSSION Purification of arabinogalactan-degrading enzyme All the purification steps were done at 10°C unless otherwise specified. The solid-state culture (500g) was soaked in distilled water (2.5 r) at 4°C for 3 h, and then filtered through four layers of cheesecloth. The filtrate was fractionated by acetone precipitation (70x, v/v) at 0°C. After 30mi1-1, the precipitate was collected by centrifugation (10,OOOxg) at 0°C for 30min, and dialyzed against 0.05 M sodium acetate buffer, pH 5.5. The enzyme solution was loaded onto a DEAE-Sepharose FF (Pharmacia) column (2.6 x 40 cm), which was equilibrated with 0.05 M sodium acetate buffer (pH 5.5). The column was washed with an equilibrating buffer (4OOml), and then the enzyme in the column was eluted with a linear gradient of NaCl from 0 to 1 .O M in the sodium acetate buffer at a rate of 60ml/h. Four peaks exhibiting enzyme activity (G-l, G-2, G-3 and G-4) were obtained, but the major fraction of the enzyme activity (55%) was localized in the G-3 fraction. The G-3 fraction was concentrated and loaded onto a Sepharose CL-6B (PharmaTABLE Step Crude extract Acetone precipitate DEAE-Sepharose FF G-l G-2 G-3 G-4 G-3 purification Sepharose CL-6B Bio gel HPT Affinity chromatography

1.

Summary

of purification

OF

A. SOJAE

49

cia) column (2.6 x 100 cm) equilibrated with 0.05 M sodium acetate buffer (pH 5.5) containing 0.1 M NaCl, and then eluted with the same buffer. The active fraction was then applied onto a Bio gel HPT (Bio-Rad) column (1.6x 30 cm) equilibrated with 0.001 M potassium phosphate buffer (pH 6.8). The enzyme in the column was eluted with a linear gradient of potassium phosphate buffer (pH 6.8) from 0.001 M to 0.2 M. For affinity chromatography, the sample was loaded onto an Epoxy-activated Sepharose 6B (Pharmacia) column using arabinose as a ligand (15) (1.6X 15 cm), equilibrated with 0.01 M sodium acetate buffer (pH 5.5). The enzyme in the column was eluted with a linear gradient of NaCl from 0 to 0.5 M in sodium acetate buffer at a rate of 10 ml/h. The purified enzyme was stored on an ice bath. The galactanase purification steps are summarized in Table 1. The final purified enzyme had a specific activity of 660.0 units per mg protein. Molecular characteristics of the purified enzyme The purified enzyme showed a single protein band after native PAGE (Fig. IA). The molecular weight of native enzyme was estimated to be 39,000 by GFC. On SDSPAGE gel, the purified enzyme gave a single protein band (Fig. 1B) with a molecular weight of 39,700. The isoelectric point (PI) of the purified enzyme was estimated to be 3.60 by thin-layer polyacrylamide gel isoelectrophoretic analysis. Amino acid and carbohydrate analysis Table 2 shows the amino acid composition of the purified enzyme. The average values for 24, 48, and 72 h hydrolysis were used for all amino acids except serine, threonine, and tyrosine, for which “zero time” values were calculated by first-order extrapolation, and histidine, proline, and isoleucine, for which 72 h values were used. No half-cysteine content could be detected in the purified enzyme by the method of Elleman (21), or by that of Simpson et al. (22). The amino acid sequence at the amino-terminal end of the purified enzyme was V-Y-; further amino acid residues could not be identified. Since the purified enzyme contained 26.6.9: carbohydrate by weight, the result suggested that the third amino acid residue might be glycosylated. Enzymatic properties A Lineweaver-Burk plot showed that the K,,, value was 0.82 mg/ml for arabinogalactan from soybean cotyledon (data not shown). The optimum pH and temperature of the purified enzyme for the enzyme reaction were 4.5 and 5O”C, respectively. The purified enzyme was stable at 30°C for 6 h in the pH range 6.0-10.0. The activity was stable up to 35°C on incubation at pH4.5 for 15 min, and was completely lost at temperatures above 60°C. of galactanase

from

A. sojae

Volume (ml)

Total activity (units)

Total protein (mg)

2,150 116

7,151 3,220

700.9 660.9

10.2 4.9

100 45

Specific activity (units/mg)

Yield (%)

70 110 70 70

40 170 422 140

42.1 109.9 47.5 29.6

1.0 1.5 8.9 4.7

2 6 2

55 25 14

318 236 132

10.9 3.7 0.2

29.2 63.7 660.0

4 3 2

1

so

KIMURA

ET AL.

.I. FERMEW. HIOEY(;..

Origin

-

Origin

-

94,000

-

67,000

-

43,000

@w!sWV -

Front

.-

30,000

-

20,100 14,400 Front

(+I

FIG. 1. Native and SDS-polyacrylamide gel electrophoresis of galactanase. (A) Native-PAGE. The purified enzyme (5 ,fg) was electrophoresed according to the method of Davis (13) using IO!6 polyacryiamide gel. (B) SDS-PAGE. Lane 1, purified enzyme; lane 2, standard proteins: phosphorylase (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30.000), trypsin inhibitor (20,100), tr-lactoalbumin (14,400). The purified enzyme (5 pg) and standard proteins were treated with SDS at 100°C for 2 min, and then electrophoresis was carried out at 10 mA for 3 h with O.l,‘% SDS.

Purified enzyme solutions (0.04 units) were preincubated in a mixture containing various chemicals at 30°C for 15 min and the residual activity was then measured under the standard assay conditions. Addition of Pb2+, MtQ+, Hg*‘, Ag+, or Fe3+ at a concentration of 1 mM caused 69.2, 71.5, 98.8, 99.1 and 82.6% inhibition of the enzyme activity, respectively, whereas Znzi , Mg**, NiZ+, Ba*+, Ca2’, and ethylenediaminetetraacetic acid disodium salt at the same concentration resulted in 2560% inhibition. Group-specific reagents-p-chloromercuribenzoate (0.1 mM), sodium azide (0.1 mM), sodium cyanide (0.1 mM), and iodoacetate (1 mM)-exhibited no inhibitory effect on the enzyme activity. Table 3 shows the substrate specificity of the purified TABLE Amino

2. acid

Asxh Thr Ser GlXb Pro GlY Ala l/ZCys Val Met Be Leu Tyr Phe His Lys Trp Arg

Amino

acid composition

of galactanase Residue

from A. sojae per moleculea 36.2 24.5 23.3 26.4 5.9 23.4 23.5 0 13.8 1.1 9.0 20.7 6.9 8.6 3.4 11.3 0.2 3.6

(36) (25) (23) (26) (6) (23) (24) (0) (14) (1) (9) (21) (7) (9) (3) (11) (0) (4)

B Calculated on the basis of an Mr of 39,700. b The values include aspargine and glutamine, respectively. Numbers in parentheses are the rounded-off values.

enzyme. The enzyme showed strong activity toward arabinogalactan from soybean, and weak activity toward arabinan, polygalacturonic acid, and pectin. Analysis of the hydrolyzates of these substrates revealed that the purified enzyme released galactobiose and galactose from arabinogalactan from soybean, arabinan, polygalacturonic acid, and pectin (data not shown). However, the enzyme showed no activity toward arabinogalactan from larch wood, 1,5-arabinan, gum arabic, and the synthetic substrates. The weak activity toward arabinan, polygalacturonic acid, and pectin may have been due to galactan contaminants, since these polysaccharides contained small amounts of galactose in their sugar components (Table 3). The findings therefore suggested that the purified enzyme acts specifically on the ,i-1,4-galactopyranose linkage. Hydrolysis of arabinogaiactan In hydrolysis of arabinogalactan from soybean cotyledon, the major products in the early stage of the reaction (1 h) were galactose and oligosaccharides, corresponding to standard glucose oligomers (G6G2). Oligomers higher than G2 fractions were subsequently degraded by further incubation. In the later stage of the reaction (72 h), galactobiose and galactose were mainly accumulated, but arabinose was not liberated from arabinogalactan, and the maximum degree of hydrolysis by the purified enzyme was 6 1,?G . The results of the substrate specificity experiments and the action pattern of the arabinogalactan-degrading enzyme strongly suggested it to be endo-l,4-,i-n-galactanase (EC 3.2.1.89). Although endo-1,4-,i-u-galactanases have already been purified from several organisms (9-12, 15, 27-29), the details of the properties and molecular characteristics of the endo-1,4-,i-n-galactanase from A. so&e are reported here for the first time. In TABLE

3.

Substrate

Substrate (molar ratio of major

specificity

sugar)”

Arabinogalactan: Soybean (Gal/Ara: 3.0) Larch wood (Gal/Ara: 8.1)

of galactanase

Major

linkage”

,3- 1,4-Galp, ,S-1,3-Galp, CL-1,3-Araf

from .-1. sojae --_-_ Relative degree of hydrolysis

tr-1,5-Araj +1,6-Galp,

Arabinan: Beet pulp (Gal/Ara: 0.06) (t-1,5-Araf, (t-1,3-Araf 1,5-Arabinan: Beet pulp (Gal/Ara: 0.02) o-1,5-Araf Gum arabic: Acacia tree (Gal/Ara: 2.2) ,5- 1,3-Galp, ,i- 1-6.Gaip Polygalacturonic acid: Orange (Gal/GalUA: 0.5) IY-1,4-Galp UA Pectin: Citrus fruits (Gal/GalUA: 0.3) (r-1,4-Galp UA ?-Nitrophenyl tr-L-arabinopyranoside p-Nitrophenyl cr-L-arabinofuranoside o-Nitrophenyl tr-D-galactopyranoside p-Nitrophenyl ,i-D-galactopyranoside

( ‘&‘)L

100 0

16.4 0 0 17.7 10.8 0 0 0 0

,’ Ara, arabinose; Gal, galactose; GalUA, galacturonic acid. h Galp, galactopyranose; Araf, arabinofuranose; Galp UA, galactopyranosyluroic acid. L (Reducing sugar/total sugar) x 100. The enzyme was reacted with the various polysaccharides in a mixture containing 0.04 units of purified enzyme, and 2.5 mg of substrate in 0.5 ml of 0.05 M sodium acetate buffer (PH 4.5) at 30°C for 30 min. The enzyme was reacted with the synthetic substrates in a mixture containing 0.02 units of purified enzyme and 1.25 /~mol of substrate in 0.5 ml of 0.05 M sodium acetate buffer (pH 4.5) at 30°C for 30 min.

VOL. 85, 1998

GALACTANASE

TABLE Property

A. sojae No. 3”

Molecular weight: SDS-PAGE 39,700 Gel filtration 39,000 Isoelectric point (pl) 3.60 26.6b Sugar content Optimum pH 4.5 50 Optimum temperature (“C) pH stability 6.0-10.0 Thermal stability (“C) up to 35 Inhibitor’ Mn’~ ,Hg”,Fej+,Ag Reaction products G2 and Gld on arabinogalactan K, value (mg/ml) 0.82

4.

Properties

of galactanases

from various

OF A. SOJAE

AspergiNus spp.

A. niger var. aculeatus (11)

A. aculeatus

A. niger

A. aculeatus

A. niger

(9)

(9)

(10)

(12)

38,000

42,000

43,000 -

43.000

32,000

2.8 -

4-6

3.5-4.0 50-55 4.0-7.0 up to 30 -

4-6 n.d. 4.25 50 5.0-7.0 up to 35 Pb’-

4.0 50-55 5.0-7.0 up to 60 Pb’

’ 3.0 bind GNA lectin’ 4.0-4.5 40-65 2.0-8.0 up 10 50

G2 and Gl 0.60

G2 and Cl 0.31

G2 and Gl 0.77

a In this study. h Expressed as mannose (‘6). c Indicates less than 30x of residual activitv when 1 mM inhibitor L’G2 and Gl indicate gaiactobiose and galactose, respectively. c Lectin from Giganthus nivalis. ‘< x - , not determined; n.d., noI detected.

regard to the analysis of the hydrolysis products of arabinogalactan, the endo-1,4-f-D-galactanase purified from A. sojae behaved in a strictly endo-fashion, and the final products after the enzyme reaction were in agreement with those of A. aculeutus (9, lo), A. niger (9, 11, 12), and Penicillium citrunum (15). The reaction pattern of the endo-1,4-,!?-D-galactanase from A. soj, differed from those of the bacterial galactanases of Bacillus subtilis K-SO (27) and B. subtilis WT-168 (28), which exhibit both exo- and endo-type activity, with gaiactobiose and galactotetraose, respectively, as the major reaction products. In Table 4, some properties of endo-1,4-/3-D-galactanases from Aspergillus spp. are compared. Although A. sojae is distinguished from A. niger and A. aculeatus taxonomically, the properties of their endo- 1,4-p-D-galactanases are similar. However, the enzyme purified from A. sojae differs from the galactanases of the other aspergilli in terms of its carbohydrate content and its pH and thermal stabilities. Degradation of arabinogalactan by the combined action of galactanase and arabinofuranosidase We previously and reported that the galactanase arabinofuranosidase of shoyu koji remained active in the mash at high salt concentrations during the early stage of brewing (the first 50 d of aging) in soy sauce production (2). It might be that the endo-1,4-$-D-galactanase from A. soj, acts on the galactan region of arabinogalactan together with other enzymes e.g. arabinanases. Arabinofuranosidase has been proposed as a key enzyme for the degradation of arabinoxylan, which has a single arabinofuranosyl unit linked to the main xylan chain, since the arabinofuranosyl residues of xylan hamper the action of endo-1,4-B-D-xylanase (3). When the N-L-arabinofuranosidase (0.02 units) from A. sojae (3) was reacted in a mixture containing 0.01 unit endo-1,4-,3-D-galactanase under the standard assay conditions, the ratio of each enzyme in the reaction mixture adjusted to those of the endo-1,4-,%D-galactanase and rr-L-arabinofuranosidase in the soy sauce masha (2, 3). The K, and V,,,, values calculated by LineweaverBurk plor were little altered with (0.78 mg/ml, 0.37 /fM/s) or without (0.82 mg/ml, 0.36 /‘M/s) tr+arabinofuranosidase. These results indicated the tr-L-arabino-

51

G2andGl data not shown

3.5 55 4.5.-7.0 up to 70 G2 and Gl -

was used.

furanosidase did not enhance arabinogalactan degradation with endo- 1,4-,3-D-galactanase under the conditions employed. Labavich et al. (28) and Misaki et al. (Misaki, A. et al., Abstr. Annu. Meet. Nippon Denpun Gakkai p. 259, 1987) reported that the structure of arabinogalactan from soybean is different from that proposed earlier (32): mono- or disaccharide arabinofuranosyl units are linked to the C3 of the main galactan chain, but arabinose in arabinogalactan is organized primarily in rather large oligo- or polyarabinosides. Moreover, Ronbouts et al. (33) reported that endo-1,4-,3-D-galactanase in combination with endo-arabinanase accelerates the degradation of potato arabinogalactan. Therefore, in soy sauce mash, endo-arabinanase might play a key role in the degradation of soybean arabinogalactan. We are presently engaged in the further purification of other arabinogalactan-degrading enzymes from A. sojae in order to clarify the mode of liberation of arabinose from arabinogalactan. ACKNOWLEDGMENTS This work was supported in part by a grant from the Technical Development Association Project of Kagawa Prefectural Government, Japan. We wish to thank Professor M. Sato for supplying arabinans. REFERENCES

Motai,

Browning of shoyu. Nippon Shokuhin Kogyo 23, 372-384 (1976). (in Japanese) Kimura, I. and Sasahara, H.: Effect of enzyme systems of “koji” mold on the browing of soy sauce. Nippon Jozokyokai Shi, 87, 566-572 (1992). (in Japanese) Kimura, I., Sasahara, H., and Tajima, S.: Purification and characterization of two xylanases and an arabinofuranosidase from Aspergillus sojae. J. Ferment. Bioeng., 80, 334-339 (1995). Kawamura, S.: A review on the chemistry of soybean polysaccharides (I). Nippon Shokuhin Kogyo Gakkaishi. 14, 514-523 (1967). (in Japanese) Kawamura, S.: A review on the chemistry of soybean polysaccharides (II). Nippon Shokuhin Kogyo Gakkaishi. 14, 553-562 (1967). (in Japanese) Kikuchi, T., Ishii, S., Fukushima, D.. and Yokotsuka, T.: Gakkaishi,

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.[.FEKMENl.

21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

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