**2.10 Molecular mass determination**

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 12.5 % gel in accordance with the Laemmli method (Laemmli, 1970). M. W. Marker "Daiichi" II (Daiichi Pure Chemicals, Tokyo, Japan) was used as a molecular mass marker. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250 (CBB).

#### **2.11 N-terminal sequence analysis**

SDS-PAGE of xylanases was performed according to the above-described method and then the xylanases on the gel were electroblotted to a commercial membrane (Immobilon-P;

Characterization of Enzymes Associated

cellulose and lignin.

not enhance the iturin A production.

diamonds, carboxymethyl cellulose.

**fermentation** 

with Degradation of Insoluble Fiber of Soybean Curd Residue by *Bacillus subtilis* 529

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,

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

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

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

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, 1949).

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