α-Chymotrypsin (EC 3.4.21.1 from bovine pancreas) (Type II, 52 units/mg solid) was purchased from Sig- ma-Aldrich Co. (St. Louis, USA). p-Nitrophenyl acetate was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Under nitrogen atmosphere, dumped bamboos were dried at 180˚C for 2 hr, were pyrolyzed at 450˚C for 2 hr, were carbonized at 350˚C for 3 hr, and then were cooled at 100˚C for 1 hr by pyrolyzer (EE21 Pyrolyzer, EEN Co. Ltd., Japan), as shown in Figure 1. Biomass charcoal powder (BCP) was obtained by grinding the resultant biomass charcoal (BC) with jet mill (100AS, Fuji Sangyo Co. Ltd., Japan). BCP of waste adzuki beans or woods was prepared by the same method.

The SEM micrograph was obtained using a scanning electron microscope (JSM-7500FA, JEOL, Japan) oper- ating at 15 kV. The sample for SEM was prepared on a carbon tape without vapor deposition.

As a typical procedure, 5 mL of 0.01 M phosphate buffer solution at pH 7 containing 300 μM α-chymotrypsin and 3 g/L BCP was placed in a 10-mL test tube with a screw cup, and was incubated at 25˚C and 120 rpm for 24 h. After adsorption, the mixture was filtrated with a membrane filter (pore size: 0.1 μm, Millipore Co. Ltd.) to col-

lect α-chymotrypsin adsorbed onto BCP. The amount of α-chymotrypsin adsorbed onto BCP was calculated by subtracting the amount of α-chymotrypsin included in the supernatant liquid after adsorption from the amount of α-chymotrypsin in the aqueous solution before adsorption. The amount of α-chymotrypsin was measured at 280 nm by UV/vis spectrophotometer (UV-1800, Shimadzu Co. Ltd.).

α-Chymotrypsin activity was determined using p-nitrophenyl acetate as a substrate [15] . Four mL of 0.01 M phosphate buffer solution at pH 7.5 containing free or adsorbed α-chymotrypsin (29.5 μM) was added to 16 mL of 0.01 M phosphate buffer solution at pH 7.5 containing 750 μM p-nitrophenyl acetate, and the mixture was incubated at 25˚C and 120 rpm. The absorbance of the mixture was periodically measured at 400 nm by UV/vis spectrophotometer (UV-1800, Shimadzu Co. Ltd.).

In order to assess the thermal stability of free and adsorbed α-chymotrypsin, the activity of free or adsorbed α-chymotrypsin was measured after free or adsorbed α-chymotrypsin was stored in 0.01 M phosphate buffer so- lution at pH 7.5 and 45˚C for 10 min, and then was cooled at 25˚C for 60 min. The residual activity was obtained by Equation (1).

The present method for preparing BCP is an environmentally benign process, since plant biomass wastes are not burned in the process of pyrolysis at low temperatures. As seen in Figure 2, the morphology in BC of bamboos was kept, similar to that in native bamboos. Additionally, functional groups containing oxygen atoms are formed by thermal decomposition of cellulose and hemicelluloses at low temperatures, and are useful for the adsorption of proteins [16] .

Figure 3 shows the scanning electron micrograph of BCP of bamboos. As seen in the figure, any distin- guishing roughness was not observed on the surface of BCP of bamboos. In our previous work [10] , the mean diameter of BCP of bamboos was 7 μm. Specific surface area of BCP of bamboos was 294 m²/g [13] . The pore

diameter peak was less than 2.6 nm [13] . The ζ potential of BCP of bamboos was −53.5 mV at solution pH 7 [10] . Moreover, from the results of ¹³C-NMR and X-ray photoelectron spectroscopy (XPS), acidic functional groups such as phenols, carbonyl groups, and carboxyl groups were detected in BCP [10] .

The surface chemical and pore properties of BCP are strongly dependent upon a kind of plant biomass wastes [10] [13] . In order to assess the influence of the kind of BCP on the activity of α-chymotrypsin adsorbed onto BCP, the activities of α-chymotrypsin adsorbed onto BCP prepared from different kinds of plant biomass wastes were investigated. As seen in Figure 4, the activity of adsorbed α-chymotrypsin was smaller than that of free one. Moreover, the activity of adsorbed α-chymotrypsin markedly depended upon the kind of BCP, and the ac- tivity of α-chymotrypsin adsorbed onto BCP of bamboos was the highest value. The activity of α-chymotrypsin adsorbed onto BCP of bamboos was 1.48 μmol/min·g of enzyme, while that of free α-chymotrypsin was 8.67 μmol/min·g of enzyme. Therefore, the effectiveness factor, which was defined as the ratio of the activity of α-chymotrypsin adsorbed onto BCP to that of free one, exhibited 0.17. On the other hand, from the results of ¹³C-NMR and XPS, acidic functional groups such as phenols, carbonyl groups, and carboxyl groups are detected on the surface of BCP [10] . Moreover, since the amount of α-chymotrypsin adsorbed onto BCP is strongly in- fluenced by the solution pH and the salt concentration, electrostatic interactions and hydrogen bonds via func- tional groups largely contribute to the adsorption of enzymes onto BCP [13] . Thus, it is suggested that the flexi- bility of enzymes might be limited by the adsorption. Additionally, it is possible that enzymes are partially de- natured, and the active site of enzymes is shielded due to the undesired orientation of enzyme molecules through the adsorption.

Figure 5 shows the relationship between the incubation temperature and the remaining activity of free α-chy- motrypsin (α-CT) or α-chymotrypsin adsorbed onto BCP of bamboos after the incubation time for 10 min. As seen in the figure, the dependence of the remaining activity on the temperature exhibited the sigmoid curve. The remaining activity of α-chymotrypsin adsorbed onto BCP of bamboos gradually dropped in the range from 35˚C to 55˚C, and still exhibited 12% at 55˚C, which was four times larger than that of free enzyme, although the re- maining activity of free α-chymotrypsin dramatically decreased with an increase in temperature in the range from 35˚C to 55˚C. Additionally, the transition temperature of α-chymotrypsin adsorbed onto BCP of bamboos was around 46˚C, whereas that of free α-chymotrypsin was around 41˚C, similar to that measured by fluores- cence analysis [17] . These results indicated that adsorbing α-chymotrypsin onto BCP of bamboos effectively enhanced the thermal stability of α-chymotrypsin. Specifically, the difference of the remaining activity between adsorbed enzyme and free enzyme was observed around 50˚C, where is the optimum temperature of α-chymo- trypsin [14] . In addition to the maintenance of enzyme structures due to the adsorption of enzyme molecules, it

was suggested that the autolysis of enzymes was inhibited by adsorbing enzyme molecules onto BCP of bam- boos, while free enzyme was gradually denatured in water by autolysis.

In general, the activity of enzymes gradually decreases with an increase in incubation time. Figure 6 shows the time course of remaining activities of free α-chymotrypsin and α-chymotrypsin adsorbed onto BCP of bamboos at pH 7.5 and 45˚C. Free α-chymotrypsin solution was transparent during the incubation. On the other hand, any cohesion of adsorbed enzymes was not observed during the incubation. Thus, the intermolecular aggregation among unfolded proteins was not observed at the present enzyme concentration. The figure revealed that the adsorbed enzyme retained relatively high catalytic activity. In the figure, the relationship of the remaining activ- ities of free and adsorbed enzymes with incubation time can be correlated by first-order kinetics. As shown in Table 1, the half-life of adsorbed enzymes was 2.6 times longer than that of free enzymes. Thus, the robust sta- bility of enzymes was obtained by adsorbing enzyme molecules onto BCP.

In order to extend our study, the remaining activities of α-chymotrypsin adsorbed onto BCP prepared from dif- ferent kinds of plant biomass wastes after the incubation at 45˚C for 10 min were investigated. The stability of α-chymotrypsin was sufficiently enhanced by adsorbing α-chymotrypsin onto BCP, as shown in Figure 7, com- pared to that of free α-chymotrypsin. The sequence of the remaining activity of BCP-adsorbed α-chymotrypsin went as follows: BCP of bamboos > BCP of woods > BCP of adzuki beans. On the other hand, the sequence of the remaining activity of BCP-adsorbed HEWL went as follows: BCP of adzuki beans > BCP of woods > BCP of bamboos in our previous work [12] . These results indicate that a suitable kind of BCP to one protein depends upon a kind of protein.