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Higashi, K., L. Kusakabe, T. Yasui, T. Ishiyama, and Y. Okimoto. 1983. Arabinan-degrading enzymes from Streptomyces diastato- chromogenes 065. Agric. Biol.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3425-3428

Vol. 60, No. 9

0099-2240/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Purification and Some Properties of oL-L-Arabinofuranosidase from Bacillus subtilis 3-6 SATOSHI KANEKO, MASAHARU SANO,t AND ISAO KUSAKABE* Institute ofApplied Biochemistry, University of Tsukuba, Tennoodai, Tsukuba, Ibaraki 305, Japan Received 1 February 1994/Accepted 8 July 1994

a-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6. The had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60°C. It released arabinose from O-a-L-arabinofuranosyl-(1-*3)-O-ji-D-xylopyranosyl-(1---*4)-D-xylopyranose (A1X2), O-II-D-Xy-

enzyme

lopyranosyl-(1-4)- [O--L-arabinofuranosYl- ( >3)] -0-3-D-xylopyranosyl-(l4)-D-Xylopyrnose (A1X3), and

arabinan, but not from O-13-D-xylopyranosyl-(1---2)-O-a-L-arabinofuranosyl-(1-*3)-O-.-D-xylopyranosyl(1--4)-O-I-D-xylopyranosyl-(1-4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan.

Hemicellulose is the second most common polysaccharide in are widely distributed in hemicelluloses such as arabinan, arabinoxylan, gum arabic, arabinogalactan, etc. In this connection, a-L-arabinofuranosidase (ct-L-AFase) becomes very important in the effective use and structural analysis of hemicellulose. Up to now, there have been many studies of a-L-AFase but only a few studies of the substrates which have well-defined structures. The elucidation of the substrate specificity of the enzyme will enable us to use it effectively for the degradation of hemicellulose. In our previous work, we defined the substrate specificity of the Ot-L-AFase from Aspergillus niger 5-16 (12). The enzyme hydrolyzed the terminal L-arabinosyl-linkage but did not cleave the L-arabinosyl side chains of arabinoxylooligosaccharides. Recently, we found that Bacillus subtilis 3-6 produced a-LAFase which hydrolyzed both terminal and stub L-arabinosyl linkages of arabinoxylooligosaccharides. In this article, we describe the purification and some properties including the substrate specificity of the a-L-AFase from B. subtilis 3-6. The strain 3-6 was isolated from soil and identified as B. subtilis at the Japan Food Research Laboratories. In a 5-liter jar fermenter, B. subtilis 3-6 was cultured in a medium described in a previous paper (12) at 30°C for 82 h with aeration of 1.5 liters/min and agitation at 600 rpm. Then, the culture broth was centrifuged (9,400 x g) for 20 min to remove cells. The culture supernatant (3,334 ml) was applied to a column (60 by 500 mm) of DEAE-Sephadex A-25 (Pharmacia LKB, Uppsala, Sweden) preequilibrated with 20 mM phosphate buffer, pH 6.5. The elution was done with a linear NaCl gradient from 0 to 0.5 M (2 liters each). a-L-AFase-containing fractions (fraction tubes 235 to 330, 10 ml each) were pooled and then dialyzed against 0.1 M NaCl in 20 mM phosphate buffer, pH 6.5. The dialyzed enzyme was applied to an anion exchanger (DEAE-Toyopearl 650M; Tosoh, Tokyo, Japan) packed in a column (55 by 225 mm) preequilibrated with 0.1 M NaCl in 20 mM phosphate buffer, pH 6.5. a-L-AFase was eluted by using an increasing NaCl gradient from 0.1 to 0.5 M

(1,000 ml each). a-L-AFase was separated into two fractions, fraction I (fraction tubes 51 to 75, 20 ml each) and fraction II (fraction tubes 77 to 90). Fraction II was concentrated by ultrafiltration (Amicon YM 10 membrane; Amicon Inc., Beverly, Mass.), because it liberated arabinose from both

nature. L-Arabinosyl residues

O-at-L-arabinofuranosyl-(1---3)-O-,B-D-xylopyranosyl-(l--14)-D-

xylopyranose (AlX2) (30) and 0-13-D-xylopyranosyl-(1-4)-[Oa-L-arabinofuranosyl - (1->3)] - 0-13 - D-xylopyranosyl - (1->4)-Dxylopyranose (A1X3) (30), but fraction I did so only from A1X2. The concentrate (5 ml) was put on a column (37 by 750 mm) packed with Ultrogel AcA 44 (IBF Biotechnics) preequilibrated with 0.4 M NaCl in phosphate buffer, pH 6.5. The enzyme was eluted with the same buffer. Ot-L-AFase-containing fractions (fraction tubes 33 to 45, 10 ml each) were combined and concentrated by ultrafiltration and then dialyzed against water. With the Model 491 Prep Cell (Bio-Rad, Richmond, Calif.) system, preparative polyacrylamide gel electrophoresis (PAGE) was performed by the method of Davis (3). A 7% acrylamide separation gel (2.5 cm high) and a 2.5% acrylamide stacking gel (1.0 cm high) were cast in the gel tube (28-mm inside diameter) of the Model 491 Prep Cell. A 9.4-ml portion of dialyzed enzyme solution was applied to the gel and then electrophoresed at a constant power of 10 W for 21.5 h. The elution chamber outlet was pumped at 1.5 ml/min to a fraction collector, and the eluate was fractionated (3.2 ml each). The Ot-L-AFase-containing fractions (fraction tubes 115 to 160) were pooled and dialyzed against water. The enzyme solution obtained was used as purified enzyme. The level of Ot-L-AFase activity was determined at pH 6.5 and 50°C according to the TABLE 1. Summary of purification of a-L-AFase from B. subtilis 3-6

Step

Culture supernatant

DEAE-Sephadex

Total

Total

activity

protein

(U) (mg) 12,014 20,838 8,325 1,558

Purifi-

(U/mg)

cation

(Um)(fold) 0.58 5.34

1 9

100 69

51.8

90

10

112.2 320

206 552

A-25

DEAE-Toyopearl

Corresponding author. Phone: 81-298-53-6623. Fax: 81-298-534605. t Present address: Snowbrand Milk Products Co., Ltd., Yokohama Cheese Plant, Midori-ku, Yokohama, Kanagawa 226, Japan. *

1,244

24

650M

Ultrogel AcA 44 Prep Cell 3425

1,010 96

9 0.3

8.4

0.8

3426

APPL. ENVIRON. MICROBIOL.

NOTES

TABLE 2. Amino acid composition of ot-L-AFase of B. subtilis 3-6

+

.4 .4

97,400 66,200

Composition

Amino acid

(mol%) 8.85

The .........................................

5.88

4

45,000

ASxa .........................................

4

31,000

Ser ......................................... Glxb-.............................

.4

21,500

.4

14,400

Ala ......................................... Val ......................................... Met .........................................

4.35 12.15 10.44 6.19 4.70 2.12

Ile .........................................

5.70

Leu .........................................

Phe ......................................... His .........................................

6.97 3.87 3.66 3.25

Lys ......................................... Arg .........................................

5.07 3.51

Gly .........................................

1

2

FIG. 1. SDS-PAGE of a-L-AFase from B. subtilis 3-6. Lane 1, standard proteins (each sample, 2 jig) including (molecular weights in parentheses correspond to those at the right of the figure) phosphorylase B (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme (14,400); lane 2, purified enzyme (2 jig).

method described in our previous paper (12). Protein concentration was determined by the method of Smith et al. (23). Table 1 summarizes the steps for the purification of Ot-L-AFase. The purified enzyme thus obtained produced a single band on Coomassie brilliant blue R-250 staining and exhibited a molecular weight of 61,000 on sodium dodecyl sulfate (SDS)PAGE (18) (Fig. 1). The first 8 amino acids of the N-terminal sequence, as determined by the automated Edman method, were Ser-Gln-X-Glu-Ala-Val-Ile-Glu-. The effects of pH on the activity and pH stability of oa-L-AFase were tested in a series of 0.2 M Na2HPO4-0.1 M citric acid buffers from pH 2 to 8 and a series of 0.2 M boric acid plus 0.2 M KCl-0.2 M Na2CO3 buffers from pH 8 to 11 according to the method described in our previous paper (12). The a-L-AFase showed maximum activity at pH 7.0. The enzyme became inactive rapidly at pHs above 10.0 and slowly at pHs below 7.0 (data not shown). The effects of temperature on the activity and thermal stability of Oa-L-AFase were tested at pH 6.5 from 30 to 80°C. The Ot-L-AFase showed maximum activity at 60°C. The enzyme rapidly became inactive at temperatures above 60°C (data not

shown). Amino acid analysis was done according to the method of Ploug et al. (20). Table 2 shows the amino acid composition of the ot-L-AFase. The substrate specificities of the enzyme were tested at pH 6.5, 30°C, by the method described in our previous paper (12). When the enzyme acted on p-nitrophenyl (PNP)-glycosides, namely PNP-a-L-arabinofuranoside (13), PNP-ao-L-arabinopyranoside, PNP-,B-D-galactopyranoside, and PNP-3-D-xylopyranoside, the Ot-L-AFase hydrolyzed only PNP-ao-L-arabinofuranoside. Figure 2 shows the courses of hydrolysis of arabinoxylan (Nihon Syokuhin Kakoh Co., Ltd., Fuji, Japan), arabinogalactan (Sigma), gum arabic (Wako Pure Chemical Industries, Tokyo, Japan), and arabinan (16) by t-L-AFase. The enzyme released L-arabinose as the sole hydrolysis product from arabinan (Fig. 2D), but not from arabinoxylan (Fig. 2A), arabinogalactan (Fig. 2B), or gum arabic (Fig. 2C) even after incubation for 24 h. Figure 3 shows the courses of hydrolysis of arabinoxylooligosaccharides. The Ot-L-AFase released arabinose from both A1X2 and A1X3 (Fig. 3A and B), but the enzyme did not act on 0- -D-xylopyranosyl-(1-- 2)-O-a-L-arabinofuranosyl-(1---3)-O-

Tyr .........................................

Asx includes Asp and Asn. bGlx includes Glu and Gln.

1-D -xylopyranosyl-(1--4) - O-I - D-xylopyranosyl-(1-4)-D-xylopyranose (A1X4) (15) (Fig. 3C). The limit of hydrolysis of arabinan by this enzyme was tested by the method described in our previous paper (12). The limit was 15% even when the enzyme was sufficiently in excess (data not shown). a-L-AFases were classified by Kaji (8) on the basis of their substrate specificities as follows. (i) The A. niger Kl type of cx-L-AFase (9) can hydrolyze stub L-arabinosyl linkages of arabinoxylan, arabinan, or arabinogalactan. Enzymes of this type have been purified from Streptomyces diastaticus (24), Ruminococcus albus 8 (4), Rhodotorula flava (26), Dichomitus squalens (2), Streptomyces massasporeus IFO 3841 (11), Streptomyces sp. strain 17-1 (10), Butyrivibriofibrisolvens GS113 (6), B. subtilis F-11 (27), Bacillus sp. strain 430 (28, 29), and Trichoderma reesei (21). (ii) The Streptomyces purpurascens IFO 3389 type of cx-L-AFase (14) does not hydrolyze the stub L-arabinosyl linkages of arabinoxylan, arabinan, or arabinogalactan. Enzymes of this type have been purified from Clostridium acetobutylicum ATCC 824 (19), A. niger (22), Aspergillus aculeatus (1), and cotyledons of soybean seedlings (5). CX-LAFase from B. subtilis 3-6 is also classed as an enzyme of this

A

B

C

D

A1A2 0 1 3 61224A2 0 1 3 61224A2 0 1 3 61224A2 0 1 3 61224

Reaction time (h) FIG. 2. Actions on arabinose-containing polysaccharides. (A) Arabinoxylan; (B) arabinogalactan; (C) gum arabic; (D) arabinan. Lane Al (from top to bottom), authentic xylose to xylohexaose; lanes A2, authentic arabinose. The members of the series authentic xylose to xylohexaose were prepared by the method described in a previous paper (17).

NOTES

VOL. 60, 1994

A

B

c 7.

8. 9.

10. A1A20 1 3 612240 1 3 61224A1A20 1 3 61224

11.

Reaction time (h) FIG. 3. Actions on arabinoxylooligosaccharides. (A) A1X2; (B) A1X3; (C) A1X4. Lanes Al (from top to bottom), authentic xylose to xylohexaose; lanes A2, authentic arabinose.

12.

type, but this classification is not enough to define the substrate specificities of a-L-AFases, because they act in quite unexpected manners on substrates. To define the substrate specificities of cO-L-AFases, it is necessary to prepare various kinds of oligosaccharides which have well-defined structures, but reports until now have described substrate specificities toward A1X2 (6, 7), arabinobiose (14, 22, 25), arabinotriose (14, 22), arabinotetraose (22), and arabinopentaose (22) only. We previously reported that intracellular Ot-L-AFase from A. niger 5-16 liberated L-arabinose from A1X2 only (12). However, a-L-AFase from B. subtilis 3-6 liberated L-arabinose from A1X2 and A1X3. That is to say, Ot-L-AFase from A. niger 5-16 hydrolyzed only the terminal L-arabinosyl linkage, but the enzyme from B. subtilis 3-6 hydrolyzed both the terminal and the stub L-arabinosyl linkages. We suggest that at-L-AFases have not been classified clearly, because there are still many unclear things about the substrate specificities of the enzymes. However, we claim that there are at least two kinds of Ox-L-AFases, which have different substrate specificities toward arabinosyl residue. One is the enzyme like that of A. niger 5-16, which hydrolyzes the terminal L-arabinosyl linkage, and the other is the enzyme like that of B. subtilis 3-6, which hydrolyzes both the terminal and the stub L-arabinosyl linkages.

14.

We thank M. Kakishima and Y. Yamaoka in the Institute of

13.

15.

16.

17.

18. 19.

20.

21. 22.

Agriculture and Forestry of the University of Tsukuba for their advice and for their operation of the electron microscope. REFERENCES

23.

1. Beldman, G., M. J. F. Searle-van Leeuwen, G. A. De Ruiter, H. A.

2. 3. 4.

5.

6.

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NOTES

28. Yasuda, M., and M. Tokuzato. 1988. Arabinan degrading enzymes produced by microorganisms. IV. Purification and properties of a-L-arabinofuranosidase II from Bacillus sp. no. 430. Sci. Bull. Coll. Agric. Univ. Ryukyus Okinawa 35:53-62. 29. Yasuda, M., M. Tokuzato, and M. Sesoko. 1983. Arabinanhydrolyzing enzymes in microorganisms. III. Purification and

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properties of a-L-arabinofuranosidase I from Bacillus sp. Sci. Bull. Coll. Agric. Univ. Ryukyus Okinawa 30:201-209. 30. Yoshida, S., I. Kusakabe, N. Matsuo, K. Shimizu, T. Yasui, and K. Murakami. 1990. Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan. Agric. Biol. Chem. 54:449-457.

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