**4. Discussion**

532 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Fig. 5. Effects of pH and temperature on xylanase activities of Xyl-I and Xyl-II. Effects of pH

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

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.

was used for elution.

*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 specific activities of these xylanases were diverse.

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 enzymes to release reducing sugars from xylan.

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 reaction.

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-

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#### (B) Xyl-II

Table 2. Comparison of characteristics of purified xylanases with previous reports.
