**3. Results**

#### **3.1 Degradation of insoluble fibers in soybean curd residue by** *B. subtilis* **RB14-CS in SSF**

To evaluate the ability of *B. subtilis* RB14-CS to degrade insoluble fibers in soybean curd residue, residual fibers after SSF were analyzed by acid and neutral detergent fiber methods. The same analyses were repeated three times. The average values of three samples are shown in Figure 1. After SSF of RB14-CS, no change in content of cellulose and lignin was observed. On the other hand, the content of hemicellulose decreased to 15 % of initial one, indicating that RB14-CS degraded hemicellulose in soybean curd residue.

Millipore, Tokyo, Japan) with a horizontal blotting apparatus (ATTO, Tokyo, Japan). For the blotting of pure enzyme of Fraction II, 0.01 % of SDS was added to transfer buffer to improve protein transfer efficacy. Parts of the membrane blotted with xylanases were cut out and then amino acid sequencing analysis was performed with an amino acid sequencing apparatus (PPSQ-21; Shimadzu, Kyoto, Japan) according to the standard method (Edman,

Searches for homologous amino acid sequences were performed by a *B. subtilis* database BSORF (http://bacillus.genome.jp/) and the nonredundant database at The National Center

Xylanase activity was examined in pH range of 3.0 to 11.0. For pH from 3.0 to 4.0, 100 mM sodium citrate buffer was used. For pH from 4.0 to 6.0, 100 mM sodium acetate buffer was used. For pH from 6.0 to 8.0, 100 mM sodium phosphate buffer was used. For pH from 8.0 to 9.0, 100 mM Tris-HCl buffer was used. For pH from 9.0 to 11.0, 100 mM glycine-NaOH buffer was used. To investigate the effect of temperature, the xylanase activity was measured at 20-70°C at pH 6.5. Xylanase thermostability was measured at 50, 55 and 60°C.

The digestion products of xylan and xylooligosaccharides (Wako Pure Chemical Industries, Osaka, Japan) by xylanase were analyzed by thin layer chromatography (TLC) according to

As a substrate solution, 0.5 % xylan or 0.5 % xylooligosaccharides in 100 mM sodium phosphate buffer (pH 6.5) was used. In a test tube (15 mmΦ × 10.5 cm), 0.5 mL of substrate solution and 0.5 mL of enzyme solution containing 0.5 U of xylanase in 100 mM sodium phosphate buffer (pH 6.5) were mixed and the reaction mixture was incubated at 120 spm at 37°C. After 1, 3, and 16 h of incubation, 100 μL of reaction mixture was sampled to microtube, and mixed with 200 μL of ethanol. Then, the mixture was centrifuged at 18,000×*g* for 10 min and the supernatant obtained was evaporated with a centrifugal concentrator (VC-36N; Taitec, Saitama, Japan). The dried material was dissolved in distilled water and spotted on a Silica Gel 60 TLC plate (Merck, Tokyo, Japan), which was then developed with *n*-butanol/acetic acid/ water (10:5:1, by vol.). After development, the TLC plate was sprayed with aniline hydrogen phthalate reagent. The reagent consisted of 0.93 g of aniline, 1.48 g of phthalic anhydride, 84.5 mL of *n*-butanol and 15.5 mL of distilled water (Partridge,

**3.1 Degradation of insoluble fibers in soybean curd residue by** *B. subtilis* **RB14-CS in** 

To evaluate the ability of *B. subtilis* RB14-CS to degrade insoluble fibers in soybean curd residue, residual fibers after SSF were analyzed by acid and neutral detergent fiber methods. The same analyses were repeated three times. The average values of three samples are shown in Figure 1. After SSF of RB14-CS, no change in content of cellulose and lignin was observed. On the other hand, the content of hemicellulose decreased to 15 % of initial one,

for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) with the BLASTP.

**2.12 pH and temperature profiles and thermostability of xylanases** 

**2.13 Thin layer chromatography (TLC) analysis of the digestion products** 

the method previously reported (Kiyohara et al., 2005) with some modifications.

1949), and heated at 100°C to visualize the digestion products.

indicating that RB14-CS degraded hemicellulose in soybean curd residue.

1949).

**3. Results** 

**SSF** 

Fig. 1. Analysis of insoluble fiber contents in raw soybean curd residue and soybean curd residue cultured with *B. subtilis* RB14-CS (N=3). Gray bars, hemicellulose; Open bars, cellulose and lignin.

#### **3.2 Iturin A production by** *B. subtilis* **RB14-CS using insoluble fibers in submerged fermentation**

To investigate the effect of insoluble fibers on iturin A production of RB14-CS, each of insoluble fibers was added to a liquid medium as a carbon source and RB14-CS was cultivated in the medium. Results are shown in Figure 2. Xylan exhibited iturin A production at the same level with glucose which has been used as a carbon source for iturin A production in the previous reports (Asaka & Shoda, 1996; Tsuge et al., 2001). Other insoluble fibers, avicel and carboxymethyl cellulose, showed the similar level of iturin A production with control where no additional carbon was added. Pectin, a hardly-soluble or sometimes insoluble fiber which is contained in soybean curd residue (Kasai et al., 2004) did not enhance the iturin A production.

Fig. 2. Iturin A production during submerged fermentation in liquid medium containing fibers (N=3). Symbols: open circles, no additional carbon sources (control 1); open triangles, glucose (control 2); solid circles, pectin; solid triangles, xylan; solid squares, avicel; solid diamonds, carboxymethyl cellulose.

Characterization of Enzymes Associated

Fig. 4. SDS-PAGE of purified xylanases.(A) Xyl-I, (B) Xyl-II Lanes: M, molecular mass standards; I, Xyl-I; II, Xyl-II.

**3.5 Physicochemical properties of xylanases** 

approximately 40 min.

were not β-D-xylosidase.

**3.6 Analysis of hydrolytic products** 

with Degradation of Insoluble Fiber of Soybean Curd Residue by *Bacillus subtilis* 531

Effects of temperature and pH on xylanase activity and thermal stability of the two enzymes are shown in Figure 5. The optimal temperature and optimal pH of Xyl-I were 50-60°C and 6-7, respectively. At 50°C, approximately 30 % of the initial activity of Xyl-I remained after 3 h. At 55 and 60°C, Xyl-I was completely inactivated within 2 and 3 h and the half lives were approximately 18 and 8 min, respectively. The optimal temperature of Xyl-II was 70°C or higher and the optimum pH was 5.5-6. At 50°C, approximately 80 % of the initial activity of Xyl-II remained after 3 h. At 60°C, Xyl-II was inactivated within 3 h and the half life was

The hydrolysis products released from xylan or xylooligosaccharides by Xyl-I and Xyl-II were analyzed by TLC. From hydrolysis of xylan by both Xyl-I and Xyl-II xylotriose was liberated, but neither xylose nor xylobiose was released. This indicats that these xylanases

**3.7 Identification of xylanases by N-terminal sequencing and database matching**  The N-terminal sequences of Xyl-I and Xyl-II were determined by automated Edman degradation and compared with databases. Results are summarized in Table 1. Xyl-I displayed 90 % amino acid identity with endo-1,4-β-xylanase (XynA) of *B. subtilis* 168, a standard strain whose complete genome has been sequenced (Kunst et al., 1997). The molecular mass estimated by SDS-PAGE was similar to the database value. Moreover, pI

Xyl-II has exactly the same N-terminal sequence as α-amylase (AmyE) secreted by *B. subtilis* X-23 (Ohdan et al., 1999). Actually, Xyl-II exhibited α-amylase activity because reducing sugar was increased when soluble starch was treated with Xyl-II (data not shown). It is

value (9.64) of database was identical to that of purified Xyl-I.

### **3.3 Xylanase activity of** *B. subtilis* **RB14-CS during SSF**

As RB14-CS degraded xylan, a major hemicellulose in plant cell wall (Beg et al., 2001), in submerged fermentation, xylanase activity was measured during SSF, in which glucose was not added as medium component. Results are shown in Figure 3. The culture of RB14-CS exhibited xylanase activity in SSF. The activity increased after 12 h of incubation, reached the maximum value of approximately 50 U/g wet soybean curd residue at 3 d, and maintained the level during fermentation. When xylanase activity was detected, almost no reducing sugars were detected (data not shown), indicating that RB14-CS immediately utilized the saccharides released from hemicellulose as carbon sources. Changes in cell number and pH were similar to those in SSF of RB14-CS using soybean curd residue previously reported (Mizumoto et al., 2006).

Fig. 3. Xylanase activity of *B. subtilis* RB14-CS during SSF. Symbols: circles, xylanase activity; squares, pH; triangles, viable cell number.

#### **3.4 Purification of xylanases produced by** *B. subtilis* **RB14-CS in SSF**

Xylanases were purified as described in materials and methods. When the crude enzyme solution was applied to a cation exchange CM-Toyopearl column, xylanase activity was found in both the trapped fraction (Fraction I) and non-trapped fraction (Fraction II). From these fractions, two enzymes were purified and the two enzymes are homogeneous and have different sizes because each single protein band on SDS-PAGE was observed (Figure 4). This indicates that RB14-CS produces two different xylanases. Purified enzymes of Fraction I and II were designated as Xyl-I and Xyl-II, respectively. The molecular masses of the Xyl-I and Xyl-II estimated from SDS-PAGE were 24 and 58 kDa, respectively.

As RB14-CS degraded xylan, a major hemicellulose in plant cell wall (Beg et al., 2001), in submerged fermentation, xylanase activity was measured during SSF, in which glucose was not added as medium component. Results are shown in Figure 3. The culture of RB14-CS exhibited xylanase activity in SSF. The activity increased after 12 h of incubation, reached the maximum value of approximately 50 U/g wet soybean curd residue at 3 d, and maintained the level during fermentation. When xylanase activity was detected, almost no reducing sugars were detected (data not shown), indicating that RB14-CS immediately utilized the saccharides released from hemicellulose as carbon sources. Changes in cell number and pH were similar to those in SSF of RB14-CS using soybean curd residue

**3.3 Xylanase activity of** *B. subtilis* **RB14-CS during SSF** 

Fig. 3. Xylanase activity of *B. subtilis* RB14-CS during SSF.

respectively.

Symbols: circles, xylanase activity; squares, pH; triangles, viable cell number.

Xylanases were purified as described in materials and methods. When the crude enzyme solution was applied to a cation exchange CM-Toyopearl column, xylanase activity was found in both the trapped fraction (Fraction I) and non-trapped fraction (Fraction II). From these fractions, two enzymes were purified and the two enzymes are homogeneous and have different sizes because each single protein band on SDS-PAGE was observed (Figure 4). This indicates that RB14-CS produces two different xylanases. Purified enzymes of Fraction I and II were designated as Xyl-I and Xyl-II, respectively. The molecular masses of the Xyl-I and Xyl-II estimated from SDS-PAGE were 24 and 58 kDa,

**3.4 Purification of xylanases produced by** *B. subtilis* **RB14-CS in SSF** 

previously reported (Mizumoto et al., 2006).


Fig. 4. SDS-PAGE of purified xylanases.(A) Xyl-I, (B) Xyl-II Lanes: M, molecular mass standards; I, Xyl-I; II, Xyl-II.

#### **3.5 Physicochemical properties of xylanases**

Effects of temperature and pH on xylanase activity and thermal stability of the two enzymes are shown in Figure 5. The optimal temperature and optimal pH of Xyl-I were 50-60°C and 6-7, respectively. At 50°C, approximately 30 % of the initial activity of Xyl-I remained after 3 h. At 55 and 60°C, Xyl-I was completely inactivated within 2 and 3 h and the half lives were approximately 18 and 8 min, respectively. The optimal temperature of Xyl-II was 70°C or higher and the optimum pH was 5.5-6. At 50°C, approximately 80 % of the initial activity of Xyl-II remained after 3 h. At 60°C, Xyl-II was inactivated within 3 h and the half life was approximately 40 min.

#### **3.6 Analysis of hydrolytic products**

The hydrolysis products released from xylan or xylooligosaccharides by Xyl-I and Xyl-II were analyzed by TLC. From hydrolysis of xylan by both Xyl-I and Xyl-II xylotriose was liberated, but neither xylose nor xylobiose was released. This indicats that these xylanases were not β-D-xylosidase.

### **3.7 Identification of xylanases by N-terminal sequencing and database matching**

The N-terminal sequences of Xyl-I and Xyl-II were determined by automated Edman degradation and compared with databases. Results are summarized in Table 1. Xyl-I displayed 90 % amino acid identity with endo-1,4-β-xylanase (XynA) of *B. subtilis* 168, a standard strain whose complete genome has been sequenced (Kunst et al., 1997). The molecular mass estimated by SDS-PAGE was similar to the database value. Moreover, pI value (9.64) of database was identical to that of purified Xyl-I.

Xyl-II has exactly the same N-terminal sequence as α-amylase (AmyE) secreted by *B. subtilis* X-23 (Ohdan et al., 1999). Actually, Xyl-II exhibited α-amylase activity because reducing sugar was increased when soluble starch was treated with Xyl-II (data not shown). It is

Characterization of Enzymes Associated

Sequence Size

**4. Discussion** 

reaction.

This work Database

with Degradation of Insoluble Fiber of Soybean Curd Residue by *Bacillus subtilis* 533

(kDa) Sequence Gene Protein

Xyl-II SVKNGTILHA 58 SVKNGTILHA *amyE* -amylase 47, 67 - Ohdan et

SIKSGTILHA *amyE* -amylase 73 5.85 Kunst et al.

*B. subtilis* RB14-CS degraded xylan in soybean curd residue and utilized it as a carbon source during SSF by producing xylanases. Xylanases are produced from xylan by fungi, yeast and bacteria, including *Bacillus* sp. (Beg et al., 2001; Blanco et al.,1995; Gallardo et al., 2004;Heck et al., 2005; Sa-Pereira et al., 2003) and physicochemical properties, structures and

In this study, two xylanase-active enzymes were isolated. One of them (Xyl-I)was endo-1,4 β-xylanase (XynA), which has been found in many strains of *Bacillus* sp.( Blanco et al., 1995; Gallardo et al., 2004; Nishomoto et al., 2002). Characteristics of the Xyl-I obtained in this work are similar to those previously reported in that there is β-D-glucosidase activity and the values of optimum pH and temperature of Xyl-I are similar to those in other xylanases (Table 2). Another xylanase-active enzyme obtained (Xyl-II) was identified as α-amylase. As shown in Table 2, physicochemical properties of Xyl-II except for molecular mass were similar to those reported previously. Distribution of α-amylase is wide from common mesophilic bacteria to hyperthermophilic archaeon *Pyrococcus furiosus* (Jorgensen et al., 1997). Alpha-amylase of *B. subtilis* is used commercially in various categories such as starch hydrolysis in starch liquefaction process and additives to detergents for both washing machines and automated dish-washers because of its high thermo-stable activity (Nielsen & Borchert, 2000). As α-amylase, which catalyzes the hydrolysis and transglycosylation at α-1,4- and α-1,6-glycosidic linkages, it doesn't seem to be responsible for degradation of xylan. However, it has been shown that, due to the heterogeneity and structural complexity of xylan, the complete hydrolysis of xylan requires a large variety of cooperatively acting enzymes; such as endo-1,4-β-D-xylanases, β-D-xylosidase, α-L-arabinofuranosidases, α-Dglucuronidases, acetylxylan esterases, ferulic acid esterases and *p*-coumaric acid esterases (Collins et al., 2005). Thus, α-amylase of RB14-CS which hydrolyzed α-1,4- or 1,6-glucoside linkage in the reagent grade xylan used in this study may act as the cooperatively acting

Two enzymes isolated in this work liberated xylooligosaccharides but not xylose from xylan. However, almost no reducing sugars were detected when xylanase activity was detected in SSF. This indicates that RB14-CS degraded xylooligosaccharides into xylose and utilized it as a carbon source. RB14-CS may produce other enzymes such as β-D-xylosidase for this

In recent years, biomass containing hemicellulose, such as agricultural and forestry residues, waste paper, and industrial wastes, has been recognized as inexpensive and abundantly available sources of sugar (Katahira et al., 2004). Since the production of iturin A by RB14-

Xyl-I AGTDYWQNWT24 ASTDYWQNWT*xynA* endo-1,4-

Table 1. N-terminal amino acid sequences of purified xylanases.

specific activities of these xylanases were diverse.

enzymes to release reducing sugars from xylan.

identity

Size


(kDa) pI References

al.

Fig. 5. Effects of pH and temperature on xylanase activities of Xyl-I and Xyl-II. Effects of pH on Xyl-I (A) and -II (B), respectively; Effects of temperature on Xyl-I (C) and -II (D), respectively; Thermal stability of Xyl-I (E) and -II (F), respectively. Symbols in (E) and (F): circles, 50°C; triangles, 55°C; squares, 60°C.

assumed that 45 amino acid residues prior to these sequenced residues deduced from the nucleotide sequence of the *B. subtilis* X-23 are the signal peptide that is removed during the secretion process. Xyl-II also displayed 80 % amino acid identity with α-amylase of *B. subtilis* 168 (Kunst et al., 1997). Although the molecular mass of Xyl-II estimated from SDS-PAGE was different from those in the previous reports, the C-terminal structures of α-amylase of *B. subtilis* were reported to be variable (Ohdan, et al., 1999). The pI value of α-amylase of *B. subtilis* 168 (5.85) was identical with the value of purified Xyl-II. This also reflected in that Xyl-II was trapped in anion exchange chromatography when piperazine buffer of pH 9.5 was used for elution.


Table 1. N-terminal amino acid sequences of purified xylanases.
