**2.6 Xylanase activity assay**

Dinitrosalicylic acid (DNS) solution was prepared in the following manner. Solution A was prepared by mixing 300 mL of 4.5 % NaOH, 880 mL of 1 % 3,5- DNS and 225 g of potassium sodium (+)-tartrate tetrahydrate. For the preparation of solution B, 22 mL of 10 % NaOH and 10 g of phenol was mixed and filled up to 100 mL. To 69 mL of the mixture, 6.9 g of NaHCO3 was added. Solutions A and B were mixed thoroughly and placed at room temperature for 2 days. After filtration, the mixture was used as DNS solution.

Xylanase activity was determined by measuring the amount of reducing sugar released from xylan. One hundred μL of enzyme sample was added to 1 mL of 1 % xylan in 100 mM sodium phosphate buffer (pH 6.5) in a test tube (15 mmΦ × 10.5 cm) and incubated statically at 50°C for 5 min. Two mL of DNS solution was added and cooled immediately in an ice bath. Then the test tubes were boiled for 5 min and cooled in an ice bath. After centrifugation at 18,000×*g* at 4°C for 5 min, absorbance of the supernatant at 540 nm was measured by spectrophotometer (UV2400; Shimadzu, Kyoto, Japan). Xylose was used as the standard. One unit (U) of xylanase activity was defined as the amount of enzyme that liberates 1 μmol of reducing sugars (xylose equivalent) per min.

### **2.7 Measurement of xylanase activity during SSF**

SSF was carried out as described in Section 2.2 without addition of glucose. One gram of solid culture sample and 9 ml of sterile distilled water were mixed in a sterile 18-mmdiameter test tube, the test tube was vortexed thoroughly and shaken at 150 spm for 5 min at room temperature. The suspension was centrifuged at 18,000 ×*g* at 4 °C for 10 min and the supernatant obtained was used for xylanase assay.

#### **2.8 Measurement of concentration of protein**

Protein concentrations were determined by the Bradford method (Bradford, 1976) with the Protein Assay Kit II (Bio-Rad, Tokyo, Japan) with bovine serum albumin as the standard protein.

#### **2.9 Purification of xylanase**

Solid cultures (90 g) incubated for 5 days in SSF were mixed with 900 mL of distilled water and stirred for 10 min. The suspension was centrifuged at 6,500×*g* at 4°C for 20 min and the supernatant was frozen at -20°C and then thawed. The sample was centrifuged for removal of polysaccharides under the same condition. Ammonium sulfate was added to the

diameter× 100 mm height, Merck, Germany) to determine iturin A concentrations. The HPCL system was operated at a flow rate of 2.0 mL/min with acetonitrile-10 mM ammonium acetate (65:35 [vol/vol]) at a column temperature of 40°C. The elution was monitored at 205 nm by a UV detector (880-UV, Intelligent UV/VIS Detector, Jasco, Tokyo,

Although iturin A has 8 homologues with different side-chain structures (Asaka & Shoda, 1996), the concentration of iturin A was defined as the total amount of five major homologues. The correlation between the peak heights and the concentration of pure iturin A (Sigma-Aldorich, Tokyo, Japan) was used for quantification. Iturin A concentration was

Dinitrosalicylic acid (DNS) solution was prepared in the following manner. Solution A was prepared by mixing 300 mL of 4.5 % NaOH, 880 mL of 1 % 3,5- DNS and 225 g of potassium sodium (+)-tartrate tetrahydrate. For the preparation of solution B, 22 mL of 10 % NaOH and 10 g of phenol was mixed and filled up to 100 mL. To 69 mL of the mixture, 6.9 g of NaHCO3 was added. Solutions A and B were mixed thoroughly and placed at room

Xylanase activity was determined by measuring the amount of reducing sugar released from xylan. One hundred μL of enzyme sample was added to 1 mL of 1 % xylan in 100 mM sodium phosphate buffer (pH 6.5) in a test tube (15 mmΦ × 10.5 cm) and incubated statically at 50°C for 5 min. Two mL of DNS solution was added and cooled immediately in an ice bath. Then the test tubes were boiled for 5 min and cooled in an ice bath. After centrifugation at 18,000×*g* at 4°C for 5 min, absorbance of the supernatant at 540 nm was measured by spectrophotometer (UV2400; Shimadzu, Kyoto, Japan). Xylose was used as the standard. One unit (U) of xylanase activity was defined as the amount of enzyme that

SSF was carried out as described in Section 2.2 without addition of glucose. One gram of solid culture sample and 9 ml of sterile distilled water were mixed in a sterile 18-mmdiameter test tube, the test tube was vortexed thoroughly and shaken at 150 spm for 5 min at room temperature. The suspension was centrifuged at 18,000 ×*g* at 4 °C for 10 min and the

Protein concentrations were determined by the Bradford method (Bradford, 1976) with the Protein Assay Kit II (Bio-Rad, Tokyo, Japan) with bovine serum albumin as the standard

Solid cultures (90 g) incubated for 5 days in SSF were mixed with 900 mL of distilled water and stirred for 10 min. The suspension was centrifuged at 6,500×*g* at 4°C for 20 min and the supernatant was frozen at -20°C and then thawed. The sample was centrifuged for removal of polysaccharides under the same condition. Ammonium sulfate was added to the

temperature for 2 days. After filtration, the mixture was used as DNS solution.

liberates 1 μmol of reducing sugars (xylose equivalent) per min.

**2.7 Measurement of xylanase activity during SSF** 

supernatant obtained was used for xylanase assay.

**2.8 Measurement of concentration of protein** 

expressed as μg/ g initial wet soybean curd residue.

**2.6 Xylanase activity assay** 

Japan).

protein.

**2.9 Purification of xylanase** 

supernatant to 30 % saturation, and the precipitate was removed by centrifugation. Then, ammonium sulfate was added to 70 % saturation. The precipitate was recovered by centrifugation, suspended in 50 mM MES buffer (pH 6.0) and dialyzed overnight against the same buffer. Then the sample was concentrated by ultrafiltration with YM10 (molecular mass cut-off 10 kDa; Advantec, Tokyo, Japan).

The concentrate was applied to a CM-Toyopearl column (1.3 cm Φ×8.3 cm; Tosoh, Tokyo, Japan) pre-equilibrated with buffer A (50 mM MES buffer, pH 6.0), and fractions were eluted with a continuous linear gradient of 0-0.5 M NaCl in buffer A (total volume 120 mL). The flow speed and the volume of one fraction were 4 mL/min and 8 mL, respectively. In this process, xylanase activity was detected in two fractions, one of which was trapped in the column (Fraction I) and the other was not trapped in the column but passed through (Fraction II). These fractions were subjected to further purification processes.

Fraction I was concentrated using Centriprep YM-10 (molecular mass cut-off 10 kDa; Millipore, Tokyo, Japan), diluted with buffer A and applied to a RESOURCES column (0.6 cm Φ×3.0 cm; Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with buffer A. Fractions were eluted with a continuous linear gradient of 0-0.15 M NaCl in buffer A (total volume 30 mL). The flow speed and the volume of one fraction were 1 mL/min and 1 mL, respectively. The xylanase active fractions were concentrated with Centriprep YM-10 and applied to a Superdex 75 column (1.6Φ×60 cm; Amersham Bioscience, Tokyo, Japan) preequilibrated with buffer A containing 0.2 M NaCl. The elution was carried at a flow rate of 1 mL/min and a volume of one fraction was 2 mL.

The pH of the Fraction II was adjusted to 9.5 by adding NaOH and applied to a QAE-Toyopearl (1.6Φ×3.7 cm; Tosoh) pre-equilibrated with buffer B (25 mM piperazine buffer, pH 9.5), and fractions were eluted with a continuous linear gradient of 0-0.5 M NaCl in buffer B (total volume 120 mL). The flow speed and the volume of one fraction were 4 mL/min and 8 mL, respectively. The xylanase active fractions were concentrated with Centriprep YM-10, and fractions were diluted with buffer B and applied to a QAE column. Step elution was performed with 0.07 M NaCl (total elution volume 96 mL). The flow speed and the volume of one fraction were 4 mL/min and 8 mL, respectively.

The xylanase active fractions were supplied to the subsequent Butyl-Toyopearl chromatography. A column of Butyl-Toyopearl (1.6Φ×4.5 cm; Tosoh) pre-equilibrated with 25 mM piperazine buffer containing 1 M ammonium sulfate was used. Ammonium sulfate was added to the active fractions and its concentration was adjusted to 1 M. This solution was then applied to the column and the elution was carried out with a linear gradient of 1-0 M ammonium sulfate in 25 mM Piperazine buffer (total volume 180 mL). The flow speed and the volume of one fraction were 4.5 mL/min and 9 mL, respectively.
