FURTHER STUDIES UPON THE PURIFICATION AND PROPERTIES OF MALT AMYLASE

FURTHER STUDIES UPON THE PURIFICATION AND PROPERTIES OF MALT AMYLASE BY H. C. SHERMAN, M. L. CALDWELL, AND S. E. DOEBBELING (From the Department of Chemistry, Columbia University, New York) (Received for publication, January 9, 1934) Reports by Liiers and Sellner (1) upon the purification of malt amylase as well as our own experience (2) in the purification of pancreatic amylase led us to continued efforts to increase the activity and presumably the purity of preparations of malt amylase. These have resulted in the development of a new method by which preparations of much higher saccharogenic activity than any previously reported for this amylase may be consistently obtained. Additional insight into the properties of the enzyme has also resulted from this work. The method of obtaining these highly active preparations consists of fractional precipitation of extracts of barley malt by ammonium sulfate, solution of the most highly active fraction, dialysis to remove sulfate, repeated fractionation by ammonium sulfate with solution and dialysis of the best fraction in each case until no further increase in the activity per mg. of solid in the dialyzed solution occurs, concentration of the final dialyzed solution, fractional precipitation by alcohol, and finally precipitation by alcohol and ether. A typical product of such a procedure formed approximately 10,000 times its weight of maltose from 2 per cent starch in 30 minutes at 40 (3) at a dilution of 1:9,000,000. The activities of these preparations on the scale of Sherman, Kendall, and Clark (4) are 3800 to 4500. Thus, they show as high sugarforming activity as the most active preparations of pancreatic amylase so far obtained. 1 Barley malt of high diastatic activity was kindly supplied by Messrs. A. Schwill and Company through the courtesy of Mr. Robert Schwartz and Mr. Oscar Ruh. 591

502 Malt Amylase When freshly prepared, the material is readily soluble in water but becomes less soluble upon drying. It contains approximately 16 per cent nitrogen and gives the usual protein color reactions. Even in the relatively concentrated dialyzed solutions before the fractionation with alcohol, the Molisch test was negative. As purification is increased, the stability in solution is markedly decreased although to a much less extent than is observed with solutions of pancreatic amylase (5). The different conditions for optimal activity as well as other differences in properties show conclusively that malt and pancreatic amylases are different substances although of similarly high saccharogenic activity, and of protein nature. Some of the results obtained in developing the method, and observations made upon factors which influence the results may be of interest to other investigators. Injtuence of TemperatureIn order to obtain highly active preparations it is essential that the solutions be cold. This is emphasized by Liiers and Sellner (1) and has repeatedly been found to be true in our work. For this reason, solutions were kept at all times either in a refrigerator or in ice baths. We were fortunate in having available a mechanically cooled centrifuge2 which was regulated to approximately O so that even during the centrifuging the solutions remained cold. Dial~sisDialysis plays an important part in the purification. While this may be the source of large losses of enzyme (6) and of irregular results, it is a most useful step if suitably carried out. Many measurements of the activity of solutions of malt amylase before and after dialysis have shown that very little loss of this enzyme need occur during dialysis provided membranes free from mechanical defects are used, the temperature is maintained at approximately O, and the time of dialysis is kept as short as possible. We have found Thorns (7) dialyzers very helpful when sufficiently large volumes of solution are available for dialysis. The membranes were prepared as described by White (8) and soaked in 95 per cent alcohol for 24 hours to increase their permeability (9). For small volumes of solution, nitrocellulose membranes which * Manufactured by the International Equipment Company, Boston.

Sherman, Caldwell, and Doebbeling may be purchased3 in the form of tubing of different diameters and which may be cut and tied to any desired lengths have been found very satisfactory. By placing several such bags in a large volume of water in the refrigerator and changing the water frequently the dialysis can be made to proceed rapidly. Precipitation by Ammonium SulfateA systematic study was made of the use of ammonium sulfate for the separation of active from inert material in solutions of malt amylase. Fractional precipitation is more efficient than precipitation or reprecipitation with a single concentration of this salt and repeated fractionation with this reagent increases its efficiency. Typical data are given in Tables I and II. By treating aliquot portions of a malt extract4 each with a different weight of solid ammonium sulfate, including 10, 20, 30, 35,40, 45,50, 60, and 70 gm. per 100 cc., precipitates of different volumes and amylase activities were obtained. The greatest recovery of activity and the highest activity per volume of precipitate were found in the precipitates produced by the use of 20,30, and 35 gm. of ammonium sulfate per 100 cc. of extract. Fractional precipitation of malt extracts with use, consecutively, of the weights of ammonium sulfate given above, but with the removal of each precipitate as it formed, resulted in increased activity of the most active fractions which again followed the use of 20 to 35 gm. of the salt per 100 cc. of the extracts. In order to make possible quantitative comparisons of the activities of the different fractions, each precipitate was dissolved in water and dialyzed against cold distilled water until free from sulfate. Activity and total solids were then determined in the dialyzed solutions and activity per mg. of total solid calculated. The results were also calculated to activities on the scale of Sherman, Kendall, and Clark (4). Thus expressed, the original malt extract, in a typical case, had a power of 45 and the dialyzed solutions had powers of 80, 760, 954, and 103, respectively, after the fractional precipitation with 0 to 10, 10 to 20, 20 to 35, and 35 to 40 gm. of ammonium sulfate per 100 cc. of original extract. 3 Visking Corporation, Chicago. 4 The extracts were prepared in all cases by mixing finely ground barley malt with 2.5 times its weight of cold distilled water, letting it stand with occasional shaking in the refrigerator for 2 hours, and centrifuging.

504 Malt Amylase The solution remaining after the removal of the 35 to 40 gm. fraction had no measurable activity. Closer fractionation did not improve the results. The power of the most active dialyzed solutions thus obtained and of the products resulting from them by subsequent alcohol precipitation may be further increased by repeating the fractional precipitation with ammonium sulfate, solution, and dialysis, several times. In attempts to obtain as highly active final products as possible, the fractional precipitation with ammonium sulfate and dialysis are repeated until fractionation of inert material from the amylase no longer occurs and the activity of the resulting dialyzed solution fails to increase. The number of fractionations necessary to reach this point may differ with different solutions. Fractional Precipitation with AlcoholAfter being dialyzed free from sulfate and tested for activity, the solutions resulting from the treatment with ammonium sulfate were fractionally precipitated with alcohol. Even with such solutions, of an already relatively high degree of purification, a severalfold increase in activity is thus attained. In one case, a solution with a power of 1950 yielded a precipitate with a power of 4500 when fractionally precipitated with alcohol. This increase in saccharogenic activity per mg. of solid was accompanied by a correspondingly large increase in activity per mg. of nitrogen showing the removal of a relatively large amount of inactive nitrogenous material which had been carried along with the enzyme in the previous repeated fractionations with ammonium sulfate. The data assembled in Tables I and II confirm previous findings (10) that fractionation with alcohol is an efficient method for the separation of inert material from malt amylase either in crude extracts or in partially purified solutions, and show that, in general, the more highly purified the solution subjected to the alcohol treatment the higher will be the saccharogenic activity of the resulting products. They also show that repeated fractionation with ammonium sulfate as well as fractionation with alcohol is necessary to remove much of the inactive material which accompanies this enzyme in extracts of barley malt. In the fractionation with alcohol of solutions of malt amylase of widely different degrees of purity, the precipitates produced by

TABLE Influence of Ptedpitation and Fractional Precipitation with Ammonium Sulfate upon Purijication of Malt Amulase from Extracts of Barley Malt Experi. merit NO. Treatment ammonium with sulfate Solution after dialysis *In. par loo cc. power 1 None, malt extract 46* O45 744 2 None, malt extract 46* O10 80 1020 760 2035 954 3540 103 Remaining solution Negligible * Not dialyzed. TABLE I II T Fractional precipitation with alcohol O60 era?" Jcoho T 50w cr ten,icohol 6065 K?r call dcohol power PWe+ power pourer 80 562 142 Negligible 133 284 941 1328 70 500 432 32 725 715 106 121 175 951 477 2680 2307 326 Negligible Znjluence of Repeated Fractional Precipitation by Ammonium Sulfate upon Purification of Malt Amylase Treatment with ammonium sulfate *m. per loo 1. None, malt extract........... 2035 gm. fractiont...... Repeated, 2035 gm. fraction.. 2035 O35 ( i: 2. None, malt extract....... 2035 gm. fractiont..... Repeated, 2035 gm. fraction.. 2035.. Xl35.. 2035 o35 ;.. cc. I solution after dialysis &SO 6060 Br ten ler Cal t,lcohol rlcohol Power 54* 854 1157 1756 1513 45; 923 1320 1499 1644 2110 1951 Power 83 355 387 power 586 1363 1112 1363 p0ujtp pouw 270 82 2300 2958 2172 2814 2641 3172 3978 2ooo 1516 2750 4500 2441 l Not dialyzed. t In each case the precipitates obtained by the use of 0 to 10 and then 10 to 20 gm. of ammonium sulfate per 100 cc. were discarded. Those formed by the use of 20 to 35 gm. per 100 cc. were dissolved in as little water as possible and dialyzed free from sulfate. t No precipitate formed upon the addition of 20 gm. of the salt per 100 cc. of dialyzed solution. 505

506 Malt Amylase bringing the solutions to 50 and then to 60 per cent alcohol by volume were both invariably of low saccharogenic activity. More highly active final products resulted, however, if each of these fractions was precipitated and removed separately than if one 60 per cent alcohol fraction was obtained and discarded. After the removal of these precipitates, the solutions were brought to 65 per cent alcohol and after being centrifuged were treated with ether equal in volume to that of the original enzyme solution. The highest activity per mg. of solid may be found in the precipitate produced by the 65 per cent alcohol fraction, in the alcohol plus ether fraction, or in both. Precipitates produced by fractions of alcohol higher than 65 per cent were invariably low in saccharogenie activity. While fractional precipitation with alcohol is an efficient means for the purification of malt amylase, any prolonged contact of this enzyme with alcohol causes rapid loss of activity which becomes more noticeable as the purification increases. If the precipitates produced by the alcohol and ether are immediately redissolved in water and activity and total solids determined, higher powers will usually be obtained than if the precipitate is allowed to dry on a watch crystal over sulfuric acid in a dessicator kept in the refrigerator. It is, therefore, preferable, when feasible, to study this enzyme in freshly prepared solutions of its freshly precipitated undried preparations. The decrease in power upon drying, however, is variable and may be reduced by removing the supernat,ant alcohol as completely as possible from the precipitates before drying. Touching the precipitates on the watch crystal with the torn edges of filter paper to remove alcohol is helpful in this connection. The products are typically protein and it is probable that the alcohol causes a denaturation of the protein with resulting loss of activity. Influence of Concentration of Solutions of Malt Amylase upon Activity of Final ProductsThe activity of the product obtained by fractional precipitation by alcohol depends not only upon the degree of purification or activity per mg. of solid of the solution used for the fractionation as shown in Tables I and II but also upon its concentration or activity per volume of the solution. Solutions of malt amylase of approximately the same activity per mg. of solid will yield, upon alcohol fractionation, products of

Sherman, Caldwell, and Doebbeling 507 widely different activities depending upon their concentrations. This is shown by the data collected in Table III in which the data for the same solutions at different concentrations are compared. Concentration of Solutions of Malt Amy&xAs the activities of the final products are thus markedly influenced by the concentration of the solution used for the fractional precipitation by TABLE Injluence of Concentration of Solutions of Partially Purijled Malt Amylase upon Activities of Products Obtained by Fractional Precipitation by Alcohol Erperi O50 5060 6065 merit Treatment of solution* x?r cent mr cent mr cent No. alcohol slcohol rlcohol III Solutions Products of fractional precipitation 65 leer cant dcohol t ether. power Power polver power pout7 1 Dialyzed solution 867 343 863 1188 2310 Concentrated 50 per centt 936 381 990 2300 2710 Diluted 50 per cent 867 164 450 500 1897 2 Dialyzed solution 1368 364 820 1237 2460 Concentrated 50 per centt 930 403 2402 2648 1856 3 Dialyzed solution 1191 380 1105 1470 1880 Concentrated 75 per centt 1143 233 1553 2907 2100 4 Dialyzed solution 854 1363 2958 Concentrated 25 per cent? 925 381 708 2963 2140 50 t 893 544 1452 3173 1582 75 t 954 673 1790 3262 1772 * Each of these solutions was obtained by fractional precipitation of malt extract by ammonium sulfate, solution of most active precipitate, and dialysis until free from sulfate. t Concentrated at low temperature in nitrocellulose bags as explained in text. alcohol, the dialyses were arranged to take place with as little increase in volume of the enzyme solution as possible. The concentration of the dialyzed solution could be further increased by using, for the dialysis, saturated solutions or even suspensions instead of solutions of the precipitates produced by ammonium sulfate. The volume of water was just sufficient to transfer the suspended precipitates to the dialyzing membranes. As the sulfate was removed by dialysis the precipitates continued

Malt Amylase to dissolve. After such treatment the fractional precipitation by alcohol and ether gave products of higher activities than had previously been obtained. Lutz (11) has found it possible to concentrate solutions of invertase by evaporation brought about by fanning in air the nitrocellulose bags which contain the solutions. This is a suitable way in which to concentrate solutions of malt amylase also. The rapid evaporation keeps the solutions cool and usually little if any loss in activity occurred when the dialyzed solutions were thus concentrated 50 to 75 per cent. This is shown by the powers given in Table III for solutions before and after concentration. In a typical experiment a solution which was concentrated in this way to approximately onehalf its volume caused the formation of 344 mg. of maltose per 0.1 cc. before and of 685 mg. of maltose per 0.1 cc. after the concentration. Partial freezing has been suggested as a method for the concentration of enzyme solutions. It was found in this investigation that relatively dilute solutions of malt amylase may readily be concentrated in this way, the enzyme being concentrated in the unfrozen portion of the solutions. With dilute solutions very little loss of enzyme occurred either by removal with the ice or through inactivation. As the freezing of a solution was repeated and the solution became more concentrated, however, marked inactivation of the enzyme occurred. This is probably due to a denaturation of the protein such as occurs with other typical proteins under similar conditions. Adsorption by Alumina GelAdsorption of the enzyme from its solutions by alumina gel (1) with its subsequent elution (12) from the gel was introduced at different stages of the purification process. These steps were found to be relatively inefficient in the purification of this enzyme when compared with the precipitation by ammonium sulfate and alcohol described here, and their use in conjunction with the precipitation procedures did not increase the activity of the final products. SUMMARY A method for the purification of malt amylase which yields products of much higher saccharogenic activity than any previously reported for this enzyme has been developed. It is described briefly and discussed.

Sherman, Caldwell, and Doebbeling The material thus purified contains about 16 per cent nitrogen, shows all the ordinary protein color reactions, and also behaves like typical protein in respect to precipitation and denaturation. The denaturation of the protein and the loss of enzymic activity coincide. In respect to the relative prominence of its starchsplitting and sugarforming activities, the material purified by the method here described appears to correspond to the /3amylase discussed by Kuhn (13). BIBLIOGRAPHY 1. Ltiers, H., and Sellner, E., Woch. Brau., 49, 97 (1925). 2. Sherman, H. C., Caldwell, M. L., and Adams, M., J. Biol. Chem., 68, 295 (1930). 3. Sherman, H. C., Thomas, A. W., and Caldwell, M. L., J. Am. Chem. Sot., 46, 1712 (1924). 4. Sherman, H. C., Kendall, E. C., and Clark, E. D., J. Am. Chem. SOL, 32, 1073 (1910). 5. Sherman, H. C., and Schlesinger, M. D., J. Am. Chem. SOL, 33, 1195 (1911). 6. Sherman, H. C., Caldwell, M. L., and Adams, M., J. Am. Chem. Sot., 43, 2947 (1926). 7. Thorns, H., Ber. them. Ges., 69,1253 (1917). 8. White, W., Dissertation, Columbia University (1926). 9. Brown, W., Biochem. J., 9, 589 (1925). 10. Sherman, H. C., and Schlesinger, M. D., J. Am. Chem. Sot., 36, 1617 (1913). 11. Lutz, J. G., personal communication. 12. Caldwell, M. L., and Doebbeling, S. E., J. Biol. Chem., 98,553 (1932). 13. Kuhn, R., Ann. Chem., 443,1 (1925).

FURTHER STUDIES UPON THE PURIFICATION AND PROPERTIES OF MALT AMYLASE H. C. Sherman, M. L. Caldwell and S. E. Doebbeling J. Biol. Chem. 1934, 104:501509. Access the most updated version of this article at http://www.jbc.org/content/104/3/501.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's email alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/104/3/501.citation.full.h tml#reflist1