Studies on the Bacterial Xylanase

Studies on the purification, crystallization, enzymic properties and enzyme formation conditions of Bacillus subtilis xylanase were investigated. 1. Aerobic mesophiles of 300 kinds, which Fukumoto (Bull. Osaka Municipal Technical Res. Inst, 9,1 (1943)) had isolated from various sources, were used in the screening test of xylanase-producing bacteria, and a strain (G 2) of Bac. subtilis was selected whose culture filtrate showed comparatively strong xylan-hydrolyzing activity. 2. In order to find the best culture medium, the effects of various materials on the xylanase formation were examined by using 5% soybean cake extract as the basal medium, and the addition of wheat bran extract was found to greatly stimulate the enzyme production. 3. The culture filtrate of Bac. subtilis (G 2) was used for the crystallization of xylanase. The enzyme in the culture filtrate was salted out with ammonium sulfate, dialysed, followed by submitting to anion resin (Duolite A 2) column treatment to decolor. Then, the decolored enzyme solution was passed through a cation resin column (Duolite C 10), whereby the enzyme was specifically adsorbed on the resin. The enzyme was subsequently eluted with a certain buffer, dialyzed, and then crystallized in a dilute acetone solution. 4. The main properties of the crystalline enzyme preparation obtained here are as follows : (1) The enzyme is stable only betweenpH 5.0 and 7.0 and at temperatures of less than 45°C. The optimum pH for enzyme action is atpH 6.0～6.2 and the optimum temperature for saccharifying and liquefying activity of the enzyme is in the vicinity of 37° and 45°C, respectively, in a thirty minutes' test. (2) The activity of the enzyme is markedly increased by various calcium salts or chlorides. In spite of these facts, neither of ions shows any protective action on the enzyme. (3) The enzyme is inhibited by heavy metals in the following order: Hg++ > Ag+ > Fe+++ > Cu++ > Fe++, but not so seriously inhibited by Co++ and Ni++. (4) A certain thiol group (s) of the enzyme protein was ascertained to be essential to enzymic activity. (5) The enzyme splits corn cob xylan-chiefly into L-arabinose and various xylooligosaccharides. D-xylose is also very slightly produced. (6) The maximum degree of hydrolysis of xylan by the crystalline enzyme is about 38 per cent. (7) The crystalline enzyme preparation, however, was certified not to be homogeneous as protein component by means of electrophoresis, but seemed to be enzymatically pure. 5. The chief conditions for formation of the enzyme are as follows: (1) The formation of the enzyme is inducible and xylose and xylan, especially the latter, are a good inducer and a very small quantity of xylan is sufficient to induce the enzyme. Xylan also serves as a carbon source for cellular respiration of the bacteria. (2) The conspicuous effect of xylan is reasoned from the fact that it is not only an excellent inducer for the enzyme formation, but also that it makes the respiration metabolism of the cells very moderate or rather sluggish, bringing forth an excellent physiological condition for the enzyme formation. (3) Also, it was found that the coexistence of galactose with xylan in the medium further stimulates the production of the enzyme, suggesting that the xylanase formation is coupled with the metabolism of certain kinds of carbon sources