酵素材

The naringinase preparation contains pectinase activity as well as naringinase activity. When the enzyme preparation is employed to remove bitterness of some citrus fruits, pectinase brings about softening of the fruits. Therefore, prior to employing the preparation, pectinase activity contaminated must be removed or at least diminished without affecting upon the activity of naringinase. Before investigating this problem, it is necessary to examine concerning the properties of pectinase itself in the naringinase preparations. Pectinase was estimated by determining viscosimetrically the falling of viscosity of reaction mixture or iodometrically the formation of galacturonic acid, both pectin and pectic acid being as substrate. The naringinase preparations, A and B, were the same as those studied in the preceding paper. In the case of viscosimetry, the activity was expressed by the rate of decomposition of substrate which was derived from viscosity. Into 4 ml of 2 % pectin or pectic acid in a Ostwald's viscosimeter at 30℃, 1 ml of 1 % or 0.1 % enzyme solution of 30℃ was added and viscosity of the mixture was determined at definite intervals at 30℃ As solvent of both enzyme and substrate, 0.1 M Mcllvaine's buffer of pH 4.2 was employed. Degree of decomposition of substrate was shown in Fig. 1. Substrates were depolymerized very rapidly with 1 % enzyme solution, even though viscosity of pectin was reduced more quickly than that of pectic acid. At any rate, 1 % solution of the preparation seemed to be too strong for the enzymatic assay. However, it was found that reaction proceeds moderately if 0.1 % enzyme solution was used. When pectinase activity was determined iodometrically, the activity was shown with the amounts of galacturonic acid formed. Reaction mixture consisted of 40 ml of 1 % pectin or pectic acid and 10 ml of 1% or 0.1 % enzyme solution of pH 4.2. At definite intervals at 30℃, 5 ml of reaction mixture were taken off and the amounts of galacturonic acid were estimated. As indicated in Fig. 2, much galacturonic acid were formed from pectic acid than from pectin, on the contrary to viscosimetry. It was observed that 1% enzyme solution was suitable for the estimation of pectinase activity, since very less amounts of the acid were produced by 0.1 % enzyme solution in spite of using pectic acid as substrate. Furthermore, it was pointed out that the naringinase preparation A contained higher activity of pectinase than the preparation B. Regardless of the procedure of the assay, viscosimetry or iodometry, pectinase activity of both the preparations A and B was the highest at pH 4.0 to 4.2 as indicated in Figs. 3 and 4. Reaction was very slow, however, in acidic range below pH 3.0 and in alkaline media over pH 7.0. On the contrary, effect of temperature on the pectinase reaction of the preparation A was differ from that of the preparation B. As shown in Fig. 5, the pectinase activity of the preparation A to pectic acid had the wide reactive range from 35℃ to 50℃ with the peak at 40℃, while that to pectin showed the optimum temperature at 35°C. However, the optimum point of the preparation B was higher than that of the preparation A, 60℃ to pectic acid or 50℃ to pectin. Even at 70℃, fairly much amounts of galacturonic acid were formed from pectin as well as pectic acid. It was assumed from their optimum temperatures that pectinase of the preparation B had more resistance to heating than that of the preparation A. In fact, as indicated in Fig. 6, higher heat resistance of the preparation B at 60℃ was established, causing 70 % loss for 10 min. and 80 % for 30 min. On the other hand, about 85 % of pectinase activity of the preparation A were lost by heating at 60℃ for 5 min., but no more reduction of activity was provoked by treating of much longer time. Even at 50℃ for 30 min., half an activity was diminished. However similar falling of pectinase activity was brought about in both the preparations A and B by heating at 70℃, 90 % diminution for 5 min. and completely inactivation for 10 min. Pectinase was less stable than naringinase in alkaline range, and it is the most remarkable difference among the properties of pectinase and naringinase. The results in Fig. 7 indicated that pectinase in the preparation A is stable between pH 2.5 and 6.0, but inactivated below pH 1.5 and over pH 7.0. Similar reduction of the activity of the preparation B in alkaline media was also took place. However, alkaline inactivation was rather different between the activities to pectin and that to pectic acid as shown in Fig. 8. Stability of pectinase at various pHs was also influenced with temperature as in the case of naringinase. Similar to naringinase, stability was much lower at higher temperature. Fig. 9 is an example. At 5℃ for 20 hrs., decrease of the activity was not observed until pH 8.0 but 35 % loss at pH 8.5 and 70 % at pH 9.5. At 20℃, 60 % lowering of pectinase activity was brought about at pH 7.0 and 95 % at pH 8.0, while that was stable at pH 6.0. On the other hand, pectinase was instable at pH 6.0, retaining only 30 % of its activity and 10 % at pH 7.0, at 30℃. Of course, pectinase was the most instable at 37℃ and reduction of 60 % at pH 5.0, 80 % at pH 6.0 and almost complete inactivation at pH 7.0 were observed. Diminution of pectinase activity at alkaline pH was attained fairly quickly, at least within 2 to 5 hrs., as indicated in Fig. 10.