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3,5i42,584 ire States Patented July 3, 1962 1 2 converted liquors, these consist essentially of reversion 3,ti42,5?4 TREATMENT AND USE 9F ENZYMES FOR THE HYDRULYSIS 0F STARCH Earl R. Kooi, La Grange, Clarence F. Harjes, Hinsdale, and John S. Gilkison, Chicago, Ill., assignors to Corn Products Company, New York, N.Y., a corporation of Deiaware No Drawing. Filed Dec. 22, 1960, Ser. No. 77,531 2 Claims. (Cl. 195—-31) products and ash. In liquors converted by means of amylase preparations, the impurities include the synthetic dextrose polymers resulting from the action of trans glucosidase' and the incompletely hydrolzed starchy ma terials. . An object of this invention is to remove trans~ glucosidase activity from amylase preparations in order to increase the ef?ciency and usefulness of such prepara 10 tions. A further object is to provide an improved proc ess for the hydrolysis of starch or conversion products This invention relates to a method for treating starch thereof to dextrose-containing liquors having an excep hydrolyzing enzyme preparations and to an improved tionally high concentration of dextrose by means of such process for the production of hydrolyzates of starch con improved amylase preparations. Other objects will ap taining an exceptionally high concentration of dextrose by means of such enzyme preparations. 15 pear hereinafter. Fungal amylase preparations, particularly those derived All of the fungal amylase preparations examined by us contained appreciable. amounts of transglucosidase activ ity, as measured by the extent of synthesis of unferment genus, are known to convert starchy materials to sub able sugars from maltose. We have found further that stantial amounts of dextrose. However, experience has shown that except in very dilute solutions, the conversion 20 the ratio of transglucosidase:glucamylase in the enzyme preparation, measured as described below, greatly a?ects stops far short of complete conversion of the starch to the amount of dextrose that can be obtained from dextrose. amylaceous materials upon hydrolysis with enzyme prepa Amylase preparations of microbiological origin, par ration. ticularly those derived from the members of the Aspergil Determination of glucamylase aczivity.—The substrate lus genus and the Rhizopus genus, contain three major 25 is a l5~l8 D.E. spray-dried acid hydrolyzate of corn types of enzyme activity concerned with the hydrolysis of from members of the Aspergillus genus and the Rhizopus alpha-1,4-linked glucose polymers. These three types of activity may be classi?ed as alpha~amylase activity, glu camylase activity, and transglucosidase activity. Alphaaamylase action on starch pastes causes consider able reduction in viscosity. In the absence of appreciable amounts of glucamylase (or maltase) activity, consider able amounts of maltose are produced by alpha-amylase action. Glucamylase activity on starch, partially hydrolyzed starch, or on maltose results in the formation of dextrose. This type of action has also been referred to as maltase activity, amyloglucosidase activity, glucogenic activity, or starch-glucogenase activity. Transglucosidase activity results in the formation, par ticularly from maltose, of unfermentable dextrose poly starch. This material is dissolved in water and diluted to 4.0 grams of dry substance per 100 ml. of solution. Exactly 50 ml. of the solution is pipetted into a 100-ml. volumetric ?ask. To the ?ask is added 5.0 ml. of pH ‘4.3, 1.0 molar sodium acetate-acetic acid buffer. The ?ask is placed in a Water bath .at 60° C., and after 10 minutes, the proper amount of enzyme preparation is added. At exactly 120 minutes after addition of the enzyme preparation, the solution is adjusted to a phenol phthalein end point with one normal sodium hydroxide. The solution is then cooled to room temperature, and diluted to volume. A reducing sugar value, calculated as dextrose, is determined on the diluted sample and on a control with no enzyme preparation added. Glucamylase activity is calculated as follows: mers containing alpha-1,6-glucosidic linkages. Pan et al. (Arch. Biochem. Biophys, 42, 421-434 (1953)) tested 2H the transglucosidase activity of various laboratory and industrial enzyme preparations and found that fungal 45 » where: amylase preparations exhibited considerable transglucosi dase action. Pazur and French (J. Biol. Chem, 196, A=glucamylase activity, units per ml. or per gram of 265-272 (1952)) showed that the most probable action enzyme preparation. of the transglucosidase was to transfer a glucosyl radical from maltose to the 6-position of a glucose molecule, or 50 S=reducing sugars in enzyme converted sample, grams per 100 ml. to the 6-position of the non-reducing end of a maltose B=reducing sugars in control, grams per 100 ml. molecule, resulting in the formation of isomaltose, (6—(a E: amount of enzyme preparation used, ml. or grams. D-glucopyranosyl)-D-glucose) and panose, (4~(6-(a-D glucopyranosyl) - (alpha-D-glucopyranosyl)-D~glucose), respectively. We have now discovered that the transglucosidase con tent of fungal enzyme preparations is a considerable fac tor in limiting the extent to which amylaceous materials can be converted to dextrose by the enzyme preparation. When amylaceous materials in a reasonably concentrated solution are hydrolyzed by fungal enzyme preparations, the enzymatic resynthesis of carbohydrates which are not hydrolyzed to dextrose at an appreciable rate by the enzymes present in the enzyme preparation reduces the extent to which the amylaceous material can be con verted to dextrose. The reducing sugar concentration in the enzyme-con verted sample should be not more than 1.0 gram per 100 ml. Determination of Transglucosidase Activity-A \solu- ' tion of maltose is prepared by dissolving 200 grams of Pfanstiehl C.P. maltose in water and diluting to 500 rnl. Exactly 50 ml. is pipetted into a 100-ml. volumetric ?ask. To the ?ask is added 5.0 ml. of pH 4.3, 1.0‘ molar sodium acetate-acetic acid buffer. After mixing, an amount of enzyme preparation containing 2.8 units of glucamylase activity is added. The ?ask is placed in a 60° C. water 65 bath. After 72 hours, the ?ask is placed in a boiling water bath for 15 minutes, then cooled, and the contents are transferred quantitatively to a ISO-ml. beaker. The solu tion is adjusted to pH 4.8 with two normal sodium hy obtainable by successive evaporations and crystallizations of dextrose-containing liquors, resulting from. either the 70 droxide solution, transferred to a 500-ml. Erlenmeyer ?ask, and diluted to about 200 ml. Ten grams of Fleisch enzyme or acid hydrolysis of starch is limited by the mann’s active dry yeast is added, and the ?ask is shaken buildup of impurities in the mother liquors. In acid In prior art processes of obtaining crystalline dextrose from amylaccous materials, the yield of pure dextrose 3,042,584 3 4 continuously for 5 hours at 30° C. The contents are then transferred to a 250-ml. volumetric ?ask and diluted to volume. Two-hundred ml. are then centrifuged at 2000 r.p.m. for 15 minutes, and the supernatant liquor is decanted into a dry ?ask. Fifty ml. of this liquor is pipet ted into a 70-ml. test tube, 5 .ml. of 5.0 normal hydro crystallization from hydrolyzates of various dextrose con tents has been found to be as‘follows: Dextrose content of hydrolyzate, per cent dry basis _____________________ ._ Recoverable dextrose, anhydrous basis, percent of hydrolyzate dry substance 86 88 90 92 94 72 76 80 84 88 chloric acid is added, the test tube is stoppered loosely, and is heated in a boiling water bath for 3 hours, then cooled For each percent increase in the dextrose content of the in an ice bath. The contents of the tube are transferred hydrolyzate, a 2 percent increase in dextrose yield is ‘ob to a 100-mi. volumetric ?ask, and adjusted with two nor 10 tained. The signi?cance of the increased dextrose con mal sodium hydroxide to a phenolphthalein end point. tent of the enzyme hydrolyzate is apparent. A reducing sugar value, calculated as dextrose, is deter The practice of treating enzyme preparations or at mined on an aliquot of the ?nal solution. To obtain a tempting to separate enzymes by the use of clay minerals correction for reducing sugar contributed 'by the yeast, a is not new. However, none of the prior art practices has control sample is included in which 20 grams of pure 15 been directed toward solving our problem nor to our dextrose are used in the place of maltose, no enzyme method of solving it. The prior art practice relating to being added. The reducing sugar value of the enzyme the use of clay minerals in the treatment of fungal enzyme converted sample, corrected for reducing sugars contrib preparations has been directed toward the removal of ex uted by the yeast, represents the unfermentable material traneous protein, toward the separation of protease from synthesized by the enzyme preparation. Results are calcu alpha-amylase, or consists of strictly experimental iat lated as grams of .unfermentable sugars synthesized per tempts to isolate pure, single enzymes. None of the prior 100 grams of maltose hydrate added. art has been directed toward the removal of transgluco Since the two enzymes, glucamylase and transglucosi dase, compete for the same substrate (maltose), the above results may be expressed as the ratio of transglucosidase activity to glucamylase activity. This ratio is obtained by dividing the grams of unfermentable sugars synthesized per 100 grams of maltose hydrate by 100 minus this value. ' ‘We have found that in the enzymatic conversion of amylaceous materials under practical conditions, the 'sidase from amylase preparations by the use of clay min erals, nor does the prior art contain evidence that trans glucosidase can be removed from amylase preparations by. treatment with a clay mineral. None of the prior art has been directed toward removal of transglucosidase from enzyme preparations as a means of increasing the yield of dextrose obtained upon use of the enzyme preparation in the hydrolysis of amylaceous materials. In any event, we are aware of no prior instance where transglucosidase amount of dextrose ?nally formed is governed by this activity has ‘been removed substantially quantitatively from ratio of transglucosidase activity to glucamylase activity. enzyme preparations without substantial loss of the de We have found further that all fungal enzyme prepara sirable enzyme activity, such as glucamylase and alpha tions examined by us exhibit transglucosidase: glucamyl 35 amylase activity. ase ratios in the range of approximately 0.2-0.1. These In carrying out the invention, the enzyme preparation fungal enzyme preparations, used under practical condi is ?rst treated by adding a clay mineral to a solution or tions of substrate concentration, amount of enzyme, and time of conversion, convert partial hydrolyzates of starch to hydrolyzates containing in the range of 84 to 90 per cent dextrose, based on the dry substance content of the hydrolyzate. The disadvantages of processes proposed in ‘the prior art for the production of dextrose involving enzymatic suspension of the enzyme preparation. ‘The enzyme prep aration may consist of the whole culture liquor obtained from submerged growth of an amylase-producing micro organism; the clari?ed liquor obtained from the sub merged culture, a suspension of a dried or partially dried preparation which may contain bran, starch, or various other adulterants used in standardizing the amylase prep hydrolysis of starchy materials are many fold. In order 45 aration, or a solution of a completely soluble enzyme to obtain a high extent of conversion of the starchy mate— preparation. Following addition of the clay mineral rials to dextrose, such dilute concentrations of the starchy to the solution or suspension of the enzyme preparation, materials were used that the cost of evaporation ren the mixture is agitated and then the solid and liquid phases dered such processes uneconomicals. Other processes are separated. The transglucosidase activity is retained made use of highly puri?ed enzyme preparations, still 50 in the solid phase while the desirable carbohydrases, in without attaining the improvements described herein. We have made the startling discovery that we can re move transglucosidase activity substantially completely from fungal amylase preparations, thus remarkably lower ing the transglucosidase:glucamylase ratio, by treating cluding, for example glucamylase and alpha-amylase, re main in the liquid phase. Other methods may be usedfor bringing about contact of the clay mineral with the liquid enzyme preparation, provided that intimate contact between the clay mineral and. liquid enzyme preparation occurs, and that the clay the amylase preparation with a clay mineral. We have also discovered that amylase preparations treated in this mineral is then separated from the liquid phase containing manner produce unexpectedly high yields of dextrose the desirable enzymes. when used to hydrolyze starchy materials under practical The resulting liquid enzyme preparation in which the conditions. Enzyme preparations which will convert 60 activity of the desired glucamylase and alpha'amylase is partial hydrolyzates of starch to hydrolyzates containing retained may then be used directly for the hydrolysis of starchy materials, or may be processed in known manner only 85 to 86 percent dextrose, dry basis, will after treat to :obtain a solid enzyme preparation. ment with a clay mineral, thereupon convert partial hy In ‘general, any clay mineral may be used for the re drolyzates of starch to hydrolyzates containing as much 65 moval of transglucosidase activity. The clay minerals, as 93 to 94 percent dextrose, dry basis. as a group, are described as “silicates of alumina.” The The signi?cance of this improvement is apparent when principal clay minerals are classi?ed as montmorillonite, it is recognized that the yield of pure crystalline dextrose attapulgite, kaolinite, and illite. The clay minerals are obtainable from hydrolyzates of starchy materials is, in “essentially hydrous aluminum silicates, with magnesium general, governed by the amount of non-dextrose mate 70 or iron proxying Wholly or in part for the aluminum in rial present in the hydrolyzates. Each part of non-dextrose some minerals, with alkailies or alkaline earths present as impurity in the hydrolyzate will prevent the recovery or" essential ‘constituents in some of them.” .Some are fur approximately an equal weight of dextrose. For example, ther described as fuller’s earth, ?oridin, sub-bentonite, the yield of pure crystalline dextrose, expressed on an china clay, and ball clay. They may range in composi anhydrous basis, which can be economically obtained by 75 tion from nearly pure magnesium silicate to aluminum 3,042,584 5 amples which follow. They are for illustrative purposes silicate, and may also be synthesized from the component oxides or salts, or by reaction of crystalline minerals with chemical reagents. For details see data sheet No. only and are not to be construed as limiting our invention. 204, American Colloid Company (1945); Industrial Min erals and Rocks, Amer. Inst‘. Mining and Met. ‘Eng. (1949 ed.), and Clay Minerolog (R. E. Grim, McGraw Hill A series of enzyme preparations containing various ratios of transglucosidase: glucamylase activity was pre EXAMPLE I pared by ammonium sulfate fractionation, by solvent (1953) pages 18, 19). fractionation, and by clay mineral treatment. The bene?ts of transglucosidase removal are obtained when the clay-treated enzyme preparation is used to con These zyme preparation will convert the amylaceous materials enzyme preparations, derived from cultures of the Aspar gillus niger group, were used to convert a partial hydrol yzate of starch as follows: A 35 percent by weight sus to a higher dextrose yield than will the untreated enzyme pension of corn starch was acid hydrolyzed to a dex vert amylaceous materials to dextrose. The treated en preparation. The amylaceous material may range from trose equivalent (D.E. value) of 16 (dextrose equivalent gelatinized starch, partial acid hydrolyzates of starch, refers to the reducing sugar content of the hydrolyzate, CI: calculated as dextrose, and expressed as percent by weight of the dry substance present), The thinned starch was adjusted to pH 4.5, brought to 60° C., and an amount partial enzyme hydrolyzates of starch, to pure maltose. While the ?nal dextrose content attainable with a given enzyme preparation will depend somewhat on the speci?c substrate material, the ‘bene?ts of transglucosidase re— moval will be apparent on all of the above mentioned of enzyme preparation was added calculated to contain 14 units of glucarnylase activity per 100 grams of thinned starch dry substance. After 72 hours of incubation at 60° C., the liquors were analyzed for DE. and dex‘ trose content, with the results shown in Table I. The rela tion between the transglucosidase:iglucamylase ratio and the extent of conversion is well established. substrate materials. _ In the case of fungal amylase preparations, conversion of the amylaceous material should be carried out under conditions of pH, temperature, enzyme dosage, and time of conversion conducive to the formation of the max imum amount of dextrose by the enzyme preparation used. ‘In the pH range of 3.5 to 5.5, at temperatures of 50°-70° C., it will be found that maximal dextrose yields will be obtained in about 72 hours if the glucamylase dosage is about 14 units per 100 grams of substrate dry substance. It will be apparent tothose skilled in the art that lesser enzyme dosages may be ‘used for longer periods Table I Composition of Enzyme Converted Liquor Ratio, Transglucosidase: Glucamylase ' Dextrose, D .E . of time, and greater dosages for shorter periods of time to obtain essentially equivalent results. The bene?ts of transglucosidase removal will be most readily apparent if the substrate concentration is ‘greater percent , 90. 1 91. 1 91. 2 90. 7 90. 9 than about 5 grams per 100 ml. The more concentrated the substrate, the greater the bene?cial e?ect of transglu 91. 6 ‘ccsidase removal. The amount of clay mineral used in the treatment of the enzyme preparation will depend to a certain extent upon the species of clay mineral used, the amount of en zyme activity present, the amount of extraneous material present, and the pH value during treatment. In general, a pH value of about 4 will be most suitable for treatment. The time of treatment is not critical, as long as the clay is well dispersed in the enzyme preparation. D. 83. 85. 85. 85. 85. 5 6 6 6 8 86. 6 92. 1 , 88.1 92. 2 92. 2 92. '7 92. 6 92. 8 93. 6 94. l 93. 7 95. l. 94. 7 95. 0 95. 4 87. 7 87. (i S8. 7 S8. 5 88. S 89. 8 90. 9 90. 3 92. 8 Q2. 5 92. 8 93. 2 The elTects of the clay mineral treatment are the result of the greater adsorptive capacity for transglucosidase EXAMPLE II activity than for glucamylase activity. While it may be Clay minerals as a group are effective in the selective desirable to use somewhat more than the minimum removal of transglucosidase activity Without removal of appreciable quantities of glucamylase activity. A culture of Aspergillus niger Corn Products Company amount necessary to effect substantially complete removal of transglucosidase, in order to insure maximum conver sion of the substrate to dextrose with the treated enzyme preparation; in no case has it been found newssary or de sirable to use more than the amount which will adsorb more than 20 percent of the glucamylase activity. Culture No. M-370, isolated from a soil sample from Louisiana, was grown in a fermentor under submerged, aerobic conditions on a medium composed of 14 per cent ground corn and 1.0 percent corn steep liquor ’ The adsorptive preference of clay minerals is sur'li ciently selective for transglucosidase so that transgluccsi dase removal will be essentially complete at a ‘clay con’ centration which will adsorb less than 20 percent of the glucamylase activity. In most cases, transglucosidase ac tivity can be completely adsorbed with a loss of glucamyl ase activity not greater than 10 to 15 percent, even a loss of 20 percent of the glucamylase activity is not a substan tial loss in view of the considerable improvement obtained, particularly since it is true that eleven units of a clay treated preparation per 100 grams of starch will give hydrolyzates of higher dextrose contents than will four~ teen units of the untreated preparation. It will be noted from the foregoing description and the examples which follow that we have made a decided im~ dry substance. After completion of the fermentation, the liquor was ?ltered to remove the myceliurn and other suspended matter. The ?ltrate was divided into several I parts. To 100-ml. portions of the culture ?ltrate at pH 4.0 was added, with agitation, 5 grams of ?nely divided clay mineral. After 30 minutes agitation, the clay mineral was separated by ?ltration. The resulting ?ltrates were examined for glucamylase activity, for alpha-amylase activity, and for transglucosidase activity. There was no appreciable decrease in alpha-amylase activity. The treated ?ltrates, along with the untreated control, were used to convert a 16 DE. hydrolyzate of corn starch as described in Example I. In this example, the amount of clay used was 2.4 grams per 100 units or‘ glucamylase provement in the art by -a simple, inexpensive, yet highly activity. The results shown in Table II demonstate the effective expedient. applicability of clay minerals as a group to the removal The invention may be further illustrated by the ex of transglucosidase, and the effectiveness of transgluco she 2,58/l 8 sidase removal by clay minerals in increasing the dextrose yield attainable by the enzyme preparation. the ?ltrates from clay mineral treatment as described in Example I. The amount of clay required for eifective removal of transglucosidase, as shown in Table ill, will EXAMPLE III depend to a certain extent upon the species of clay min While it may be desirable to ?rst remove any in— 5 61.211 Used‘ soluble material present in the enzyme preparation prior Table. H] to treatment with a clay mineral, it is not essential to do so. A culture of Aspergillus niger M~370 was grown in a fermentor as described in Example II, After completion of the fermentation, the whole culture liquor 10 was removed from the fermentor and or nded into .our Amount 0; Clay Mmmn Clay Mineral Trademark Used G v 11 Ht 1 F1 P m rams 0.0 ay 1 3.1)“ OI‘0X 8.21 101‘ Grams pm. 100 Damon Bentomte gas may XXF Creek 100-ml. portions. The ?rst part was not treated in any ' ; way. The second part was agitated at pH 4.0 with 2.0 ml. ofenzyme re 3.1'3. 1011 P p - ‘ "‘ ' ‘I 41 glul units CBJHY 215G Demos? ContentofEmymeaqnvcrtgd grams of clay mineral, sold under the trademark ‘Volclay Bentonite.” The mixture containing the mold my- Liquor, Percent Dry Basis ' celium and clay mineral was ?ltered and the ?lter cake was discarded. The third part was ?ltered without clay treatment. 0 18 01;; The fourth part was ?ltered; the ?ltrate 8‘ was agitated with 2.0 grams of “Volclay Bentonite” added. 2% Table II . Dextrose Major Clay Mineral Trademark Glucatn- Transglu- Content of Content of G-lucam— Converted Units per ml. Ratio ylase Mineral Type Component Treatment of Clay Filtrate, Untreated Control __________________________________________________ ,_ Volclay Bentonite ________ __ MontmorilSwelling None _____ __ lonite. Bentonite. ' 2. 08 2.04 Panther Creek _________________ “do ____ .. None _____ __ Calcined____ Impreg- Nonswelllng cosidase ylase Enzyme Hydroly zato, percent dry basis 0. 16 0.05 86. 4 93.0 2.01 0.05 93.3 2. 04 1.89 0.05 0.03 93.0 94.2 92.1 Bontonite. Adsorbol A~565 ________________ __do __________________ __ AdsorbolA-46 _________________ __do __________________ __ hated 2. 04 0. 00 Pikes Peak 9877 _______________ __do_ _ -_.. Filtrol 105 _____________________ __do ____ __ None _____ __ Acid Acti- 1. 91 0. 03 93.1 Pikes Peak 9T77 _______________ __d0_ _ ___. Galetnod__._ 1. 93 0.07 91. 8 Florex XXF ______________ __ Floridin. _. Caleined Florex XXIL __. _____do ____ __ None_______ Caleined____ 1.96 1.80 0.05 0.05 93.0 93.3 _ vated Attapulgus Clay __________ __ Attspulgitm F1§lert’1.s__.-. None _____ __ Bandy Tan _______________ __ Ball Clay 1.91 0.05 93.8 1. 97 0.05 93. 1 1. 98 0.13 88. 7 1. 92 0.07 92.0 ar 1. Illinois Fire Clay _ Grundlte. _-_ i _ _ _ . __ . _ _ _ _ _ _ __ The suspension was then ?ltered. Conversion of :1 16 DB. thinned starch was, as described under Example I, carried out with the four preparations. This example shows that whole culture liquor may be e?ectively treated with a clay mineral. Dextrose Clay Mineral Glueamylase Filtration of Culture Liquor Treatment Activity, Content of Enzyme Converted Units per ml. Liquor, Per cent dry basis EXAMPLE V While some transglucosidase can be removed over a considerable range of pH values, it will be found that the treatment is most effective in the pH range of 3 to 5, and a pH value of about 4.0 is recommended. With some enzyme preparations, it may be necessary to make periodic adjustments of the pH value during the treat ment period, since the addition of the clay may result in a change of the pH value. At pH values above 6, either transglucosidase removal will be incomplete, or such an 55 excessive amount of clay will be required to obtain sub Un?ltered ________________ __ None 2% 2. 24 92. 3 Filtered __________________ __ None 2. 30 2. 3O 86. 1 84. 7 2% 2. 33 92. 8 EXAMPLE IV stantially complete removal of transglucosidase that a considerable loss of glucamylase and alpha-amylase ac tivity will occur. At pH values below 3, substantial in activation or adsorption of glucamylase may occur. Separate portions of a culture ?ltrate from a sub merged culture of Aspergillus niger were adjusted with While clay minerals as a group are effective in the hydrochloric acid or with sodium hydroxide to the pH removal of transglucosidase from fungal enzyme prepara values shown below. To each 100 ml. portion was added tions, they are not all equally effective at a given amount. 2.0 grams of Volclay Bentonite. The mixtures were In an Aspergz‘llus niger culture liquor ?ltrate containing 65 agitated for 30‘ minutes and ?ltered. Results of conver about 2 units of glucamylase activity per mL, for ex sions conducted with the treated enzyme preparations, ample, it will be found that where 0.5 gram of Volclay carried out as described in Example I, are shown below. Bentonite will accomplish effective removal of trans glucosidase, as much as 5 grams or more of other clay pH value prior to addition of ________________________ __ minerals will be required to accomplish the same extent 70 pHclay value of ?ltrate after treat of removal. ment _______________________ _. Glueamylase Activity of ?l Portions of a culture ?ltrate of Aspergillus niger M—370 trate, percent of original ____ _. were treated at pH 4.0 with various amounts of several Dextrose content of Enzyme Converted Liquor, percent clay minerals as described in Example II. Conversion D.B ________________________ ._ of a 16 DE. hydrolyzate of starch was carried out with 75 2.0 2.4 3.0 4.0 4.3 2.2 2.5 3.1 4.0 4.6 5.0 5.2 26.6 42.8 75.5 100 96.0 93.0 91.9 92.7 93.2 93.0 92.1 87.9 3,042,584 The initial glucamylase content of the culture ?ltrate was 2.33 units per ml. The dextrose content of hydroly zate obtained with the untreated preparation was 85.9%, eral dried preparations derived from Aspergillus ?avus dry basis. ?ltered. The ?ltrates, as well as the untreated prepara tions, were used to convert a 16 DE. hydrolyzate of oryzae group were dissolved or suspended in water, treat ed with Volclay Bentonite at pH 4.0 for 30 minutes, and EXAMPLE VI starch as described in Example I, except that the con versions were carried out at pH 4.6-4.8, 50° C., for 72 hours, using an amount of enzyme preparation equivalent Was vtreated essentially as described in Example V, but to 14 glucamylase units per 100 grams of starch dry sub with various amounts of Volclay Bentonite. 10 stance. Activity determinations were also carried out Another sample of Aspergillus niger M-—370 culture ?ltrate, with a glucamylase activity of 2.20 units per ml., at pH 4.6-4.8, and 50° C. Amount of Clay Mineral Used: g./100 ml ______________ __ 0 0.5 1 2 5 g./100 units ____________ _. 0 0.23 0. 45 0.91 2. 3 Rhozymes Enzyme Preparation Mylase Dextri 112158 GLUCAMYLASE ACTIVITY OF FILTRATE, PERCENT OF ORIGINAL 15 Glucamylase Activity of solution, Units/ml: PP max Before Clay Treatment _____ __ After Clay Treatment ______ __ pH value during treatment: 3 ______________________ _. 100 98. 2 98. 2 98. 2 97. 9 100 100 100 100 100 98. 2 100 100 97. 8 100 100 98. 2 100 100 99. 0 Amount of Clay used: g 100 ml. . _ g/100 units cC0am1 10. ____________ .. 20 Dextrose Content of enzyme converted liquor: 86. 6 92. l Untreated preparation ______ __ DEXTROSE CONTENT OF EN Clay-treated preparation _ _ . . _ ZYME-CONVERTED LIQUOR, PERCENT D.B. EXAh/IPLE IX A dried enzyme preparation derived from a culture of Rhizopus delemar was, dissolved in water. One por tion of the solution was not treated, the other was treated at pH 4.0 with 2 grams of Volclay Bentonite per 100 ml. EXAMPLE VII of solution. Conversions of 16 DE. thinned starch were The cultures described as Aspergillus niger NRRL 330 30 conducted as described in Example VII. Dextrose con and NRRL 337 have been studied very exhaustively for ‘tents of hydrolyzates resulting from conversion with the production of amylases (Tsuchiya et al., Cereal Chem. untreated and clay treated preparations were 85.1 and 27, 322 (1950)). These and other members of the Asper 93.3, respectively. gillus niger group (see Thom and Raper, “A Manual of EXAMPLE X the Aspergilli,” Williams and Wilkins Co. (1945)) Were 35 In the case of substantially puri?ed enzyme prepara grown on a corn and corn steep liquor medium as de tions, the pH range over which transglucosidase can be scribed in Example II. The culture ?ltrates therefrom e?ectively removed is extended up to at least pH 6. were divided into two portions. One portion was un However, it Will also be found that at a given glucarnylase treated, the other portion was treated with Volclay concentration, less clay will be required to e?ect sub Bentonite at pH 4.0 for 30 minutes, then ?ltered. The 85.0 85.0 85. 0 85. 0 85. 8 88. 7 85. 5 84. 8 91. 3 91. 8 88. 2 84. 6 92. 5 92. 5 90.8 85. 5 92. 5 92. 5 92. 1 88. 7 25 stantially complete removal of transglucosidase. untreated culture ?ltrates, and the‘ clay-treated ?ltrates Also, clay concentrations at which substantially complete re moval of transglucosidase from an unpuri?ed prepara drolyzate of starch, adjusted to pH 4.3, and held at 60° tion is attained without substantial loss of glucamylase C. for 48 hours. The enzyme dosage used was in each case 28 glucamylase units per 100 grams of starch dry 45 activity may cause inactivation or adsorption of glucamyl ase in the puri?ed preparation. substance. The enzyme-converted hydrolyzates were an An enzyme preparation derived from Aspcrgillus niger alyzed for dextrose content with the results shown below: M—370 was puri?ed by solvent precipitation. The puri In all cases, the hydrolyzate from the clay ‘treated enzyme ?ed preparation contained essentially all of the glucarnyl preparation had a substantially higher dextrose content than that obtained with the untreated preparation, show» 50 arse, alpha-amylase, and transglucosidase activity of the original preparation, ‘out only 5 percent of the total solids. ing the general applicability of transglucosidase removal The puri?ed preparation was dissolved in water to give through clay-mineral treatment to members of the As were then added to a 20% solution of a 16 DE. hy a glucarnylase concentration of 1.76 units per ml. Por tions of the solution were treated at pH 3, 4, 5, or 6 for 30 minutes with various amounts of Calcined'Florex pergillus niger group. Dextrose Content of Enzyme-Converted Culture XXF, with the results shown below. Hydrolyzate, Percent D13. Untreated Preparation Amount of Clay Clay treated Mineral used; Preparation 60 Aspergillus awamm'i NRRL 2042 ________ __ Aspcrgillus fonsecaerts NRRL 67 _ 85.0 90. 4 Aspergillus Aspergillus Aspergillus Aspergz'llus _ _ r __ S6. 0 86. 0 86. 7 87.0 92. 8 92. 8 92.1 92. 5 Aspergillus niger N REL 363 _____________ __ 89. 7 95. 1 86.0 90. 8 84. 3 90. 4 88. 8 91.5 niger niger niger nz'ger GLPC M—370-_ NRRL 330.. NRRL 3.-.NRRL 337 > 89. 7 92. 8 merit: ____________________________________ __ Aspergillus phoem'cis CPC M-381. __ 84. 0 93.1 Aspergillus p/Loenicis ATCC 13156 _______ __ 86.0 90. 4 0. 10 0. 057 0. 5 0.28 l. 0 0. 57 2. O l. 14 5.0 2. 8 GLUCAMYLASE ACTIVITY OF FIL TRATE, PERCENT OF ORIGINAL 100 98. 3 95. 5 89. 3 ____ ._ 76. 5 100 100 100 100 99. 0 100 93. 2 100 92. 5 96. 0 .... _ _ ____ _ _ 78. 3 84. 5 100 97. l ____ _. 94. 4 85. 4 100 Aspergz’llus niger mut Schismanii NRRL Aspergz'llus phoenicis NRRL 1956. 0 0. O5 0 0. 028 ph value during treat~ Aspergz'llus m‘ger mut cinnamomeus NRRL 34 ____________________________________ .. g/l00 11:11: _________ .t g/100 uruts _______ __ ____ _ _ ____ __ DEX’I‘ROSE CONTENT OF ENZYME 70 CONVERTED Lroguon PERCENT D EXAMPLE VIII Enzyme preparations derived from the Aspergillus ?avus-oryzae group are also known to convert amyl acc‘ous materials to substantial amounts of dextrose. Sev 75 88.0 92.3 ____ _. 93. 3 86. 3 85. 5 91.8 88.0 93.0 90. 6 ____ __ ____ _- .... __ 93. 3 93. l’ 85. 5 85. 5 ____ _ _ 89. 9 92. (l 3,042,584: '11 ing from gelatinized starch to starch which has previously EXAMPLE X A culture ?ltrate of Aspergillus niger M~370, and a puri?ed preparation like the one described in Example been subjected to appreciable acid or enzyme conversion.‘ An 18% d.s. suspension of corn starch was ?rst gelati nized and then cooked at 121° C. for one hour. One 1.66 units per ml. with, at pH 4.0, various amounts of UK portion of the cooked starch was converted for 90 hours at pH 4.5 with an Aspergillus niger M-37O enzyme prep Volclay Bentonite. aration which had not been treated with a clay mineral. X were each treated at a glucarnylase concentration of Amount of Volclay Bentonite Used Results with Unpuri?ed Enzyme I gap/100 g./l00 ml. units Loss of Dextrose Content of Loss of Dextrose Content of Glu- Enzyme- Glu- Enzyme camylase Activity, Percent Ooncamylase verted Activity, Liquor, Percent Con verted Liquor, Percent Percent .B. O 0 85.0 0 and 98.0% dextrose, respectively. 15 86. 0 __________________ __ 0 0. 03 __________________ _ - - l. 8 88. 4 4. 2 G. 0 10.8 27. 7 93.0 93. 3 93. 3 93. 3 0 0 0 O 0 1 2 hydrolyzates in conversions with the untreated and treat ed enzyme preparations contained, on a dry basis, 93.3 .B 0. 015 0. G6 0. 15 0. 3 0. 6 1.2 2. 4 4. 8 Another portion was converted in the same manner with a preparation which had been treated with Volclay Ben tonite. In each case, the enzyme ‘dosage was 20 glu camylase units per 100 grams of starch. The ?ltered Results with Puri?ed Enzyme 86. G 87.0 88. 7 92.1 92. 92. 92. 87. 0 EXAMPLE XII In the case of dilute enzyme preparations, it may be 25 This application is a continuation-in-part of applica tion Serial No. 666,469, ?led June 18, 1957, and now abandoned. We claim: 1. A process for selective removal of transglucosidase activity from a fungal amylase preparation obtained from an organism selected from the group consisting of mem bers of the Aspergillus genus and members of the Rhizo pus genus, said preparation containing transglucosidase and glucamylase activities, which comprises treating said amylase preparation in aqueous medium with a clay min eral in an amount up to 50 grams of clay mineral per 100 units of glucamylase activity and not greater than that a necessary to use a lesser amount of clay to remove trans glucosidase selectively. For example, Aspergillus niger which will remove 46 percent of the glucamylase ac M-370 culture ?ltrate was diluted to glucamylase con 30 tivity to adsorb the transglucosidase on the clay mineral, centrations of 1.7, and 0.1 units of glucamylase per ml. separating the medium from the clay mineral, and re covering said medium substantially free from transglu cosidase activity, said recovered medium containing at least 54 percent of the glucamylase activity of the original Each portion was then treated with various amounts of Volclay Bentonite at pH 4.0. For simplicity of expres sion, the following values were read from plots of the data. Values in parenthesis were obtained by extrapola 35 tion because of the di?iculty of dispersing these amounts of Volclay Bentonite in the volume shown. preparation. ’ 2. A process for hydrolyzing starch to dextrose which comprises partially hydrolyzing the starch to reduce the The amount of alpha-amylase adsorption was nearly viscosity thereof and thereafter subjecting it to the action the same value as shown for glucamylase adsorption. Similar results were obtained with the clay minerals 40 of a fungal amylase preparation treated by the process of claim 1. Florex XXF and Adsorbol A-420. Initial Gluoamylase Concentration, Units/ml. Amount of 0.1 1.7 ‘ Volclay Used, gJlOO units glucamylase Transgln- Glue Transglu- Gluc centration, Clay con- cosidase amylase centration, cosidase amylase g./100 ml. removed, loss, . g./10O ml. removed, 1 2 3 0.17 0.42 0. 85 11 39 100 5 percent Clay 0on4 percent percent loss, percent 0 0 O 1. 7 __________ __ 9 4. 2 _ l 13 8.5 0 19 31 46 67 (17) (42) (85) (170) _ _ _ . (4) (10) (19) (37) 2 EXAMPLE XIII The bene?ts of transglucosidase removal from fungal amylase preparations may be attained on substrates rang References Cited in the ?le of this patent 60 Cereal Chemistry, vol. 26, No. 2, March 1949, pp. 98-; 1‘09.