Production of Amylolytic Enzymes in Natural and Synthetic Media"2C. G. DUNN, G. J. FULD, K. YAMADA,3 J. MAS URIOSTE,4 AND P. R. CASEY5

Department of Food Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts

Received for publication January 15, 1959

The production of microbial amylases has been re-viewed by Underkofler (1954), Hoogerheide (1954),Forbath (1957), Underkofler et al. (1958), Prescott andDunn (1959), and others. Fungal amylase production bysubmerged culture methods has been described by theforegoing and by scientists such as Le Mense and associ-ates (1947), Adams and co-workers (1947), Erb andcollaborators (1948), Tsuchiya et al. (1950), members ofthe U. S. Department of Agriculture (1950), Shu andBlackwood (1951), and Shu (1952). Bacterial amylaseproduction by submerged culture methods has also beendescribed by a number of researchers, including Im-henekii and Solnteva (1944), Terui and Fugiwara(1948), Smythe and associates (1950), Hoogerheide andLaughery (1951), and Nakanishi and Murata (1953).

In the studies described herein, submerged amylaseproduction was carried out with three different micro-organisms: Aspergillus niger strain NRRL 337, Bacillussubtilis strain NRRL B-941, and Bacillus polymyxastrain NRRL B-372. The mold and bacterial studiesare reported separately in view of the different methodsused.

FUNGAL AMYLASE PRODUCTION

Methods and EquipmentAll of the fungal studies, with the exception of one,

were carried out with A. niger strain NRRL 337, using500-ml Erlenmeyer flasks, each containing 100 ml ofmedium and incubated at 30 C on a rotary shaker (NewBrunswick Scientific model VS)6 set to rotate at 240rpm.The mold was cultivated in a medium containing

glucose, 2.5 g; glycerol, 2.5 g; yeast extract (Difco),70.5 g; and agar (Difco), 1.5 g; in 100 ml of distilledwater (Tsuchiya et al., 1950). The inoculum for each

I Presented at Division of Agricultural and Food Chemistry,Fermentation Subdivision, Symposium on Industrial Enzymes,134th Meeting, American Chemical Society, Chicago, Illinois,September, 1958.

2 Contribution no. 323 from the Department of Food Tech-nology.

3Present address: University of Tokyo, Japan. (See Ya-mada, 1958.)

4Present address: Carace 521, Montevideo, Uruguay. (SeeMas Urioste, 1957.)

6 Present address: 200 Cabrini Blvd., New York, New York.6 New Brunswick Scientific Company, New Brunswick,

New Jersey.7Difco Laboratories, Inc., Detroit, Michigan.

212

500-ml flask consisted of 2 loopfuls of spores from themold grown for 3 days on a slant of this medium at30 C.

Dextrinogenic amylase activity was measured in thefollowing manner: 20 ml of a 2 per cent soluble starchsolution and 2 ml of an acetate buffer at pH 4.8 wereplaced into a 100-ml Erlenmeyer flask. Two ml of thefiltered production medium broth at 30 C were addedand all of the ingredients were mixed. Samples weretaken at specified intervals and permitted to react with1 ml of 0.1 N iodine and 0.1 N potassium iodide solutionat 30 C. The time in min required for the color of thehydrolyzed starch solution mixture to match a red-brown dextrin-iodine color was measured and recorded.This procedure differs from the standard methoddescribed by Sandstedt et al. (1939).

Saccharogenic activity was measured by determiningthe amount of reducing sugars produced during 30 minin 2 ml of the test solution used to determine dextrino-genic amylase activity. The reducing sugars were deter-mined by the modified Somogyi method (Somogyi,1945). The activity was reported as follows:

Activity = mg of reducing sugar X 100mg starch (calculated as glucose)

The basal synthetic medium used contained 2 percent corn starch (Swansdown),8 0.2 per cent ammoniumnitrate, 0.1 per cent sodium phosphate (dibasic), and0.05 per cent magnesium sulfate heptahydrate. Gener-ally 1 to 2 g of calcium carbonate per 100 ml mediumwas added after 24 hr of fermentation.

Results and DiscussionA series of experiments was conducted with A. niger

strain NRRL 337 using the basal medium with theaddition of calcium phytate and various nitrogensources. The results of these experiments are sum-marized in table 1 and indicate that 0.3 per cent wheatgerm and calcium phytate gave the highest yields ofdextrinogenic and saccharogenic amylase activities. Theuse of wheat derivatives has been previously recom-mended (Fukimbara, 1950; Murota et al., 1954).Phytic acid derivatives occur quite commonly in wheatproducts.An effort was then made to determine if inositol plus

phosphate could be used to replace the phytic acid. The8 Available at food stores.

PRODUCTION OF AMYLOLYTIC ENZYMES

results of a typical series of experiments are shown intable 2. It may be seen that inositol did not replacephytic acid.A number of vitamins, such as thiamin, riboflavin,

TABLE 1Comparison of the effects of various nitrogen sources and

phytic acid on the amylase activity of Aspergillusniger

Source of Nitrogen*Incu-

Phytic Acid bation pHPeriod

Ammonium nitrate Not added

Ammonium nitrate Addedt

Yeast extract

Peptone (Difco)

Distiller's dried sol-ubles (Nadrisol)

Wheat germ 0.3%(Kretschmer)

Not added

Not added

Not added

Not added

days

23452345234523452345

123

5.55.55.55.05.25.5

5.57.57.57.57.56.55.55.55.55.55.05.55.53.05.5

Dextrino-genic

AmylaseActivity

min

>30>30>30>30

423

>3014159

>30>30

1914168129

>32816

Saccharo-genic

AmylaseActivityt

1.210.650

25.329.919.5

1.12.33.34.71.31.42.35.25.66.67.89.31.16.93.6

* Each ingredient used in a 0.5 per cent concentration,except for wheat germ.

t Activity equals mg of reducing sugar (multiplied by 100)divided by mg of starch (calculated as glucose).

$ Added in 0.05 per cent concentration.

TABLE 3Comparison of amylase activity by different molds,

biotin, niacin, pyridoxal, cobalamin, pantothenic acid,and folic acid, were added to the medium to determinewhether they affected amylolytic activity. None of thesevitamins, with the possible exception of thiamin andpantothenic acid, produced dextrinogenic and sac-charogenic amylase activities higher than those ob-tained with controls containing no added vitamins orphytic acid. Each vitamin was also added to a basalmedium containing added phytic acid, but none pro-duced amylase activity greater than that observed in thephytic acid control. In the cases of thiamin and panto-thenic acid, the saccharogenic amylase activity wasslightly higher than that of the control containing noadded vitamins, but it did not approach the value ob-tained in the presence of phytic acid.

In an effort to ascertain whether or not the phytic

TABLE 2Comparison of the effects of inositol plus phosphateand phytic acid on amylase activity by Aspergillus

niger

Mdbincu- pH Dextrino- Saccharo- Mg ofIncu- eni~~Sccro MyceliumMediumbation pH genic geni per 10 MI

Medium bationd Amylase Amlase of BrothActivity Actiity* (Dry Wt)

hr min

Basal medium only 40 6.0 29 1.3 8064 6.0 28 1.7 8588 5.5 27 0.8 50

Basal medium plus 40 6.0 29 1.3 70inositol plus 64 6.0 18 0 85Na2HPO4 88 5.5 27 0 75

Basal medium plus 40 4.0 10 5.3 75phytic acid 64 5.0 8 8.3 60

88 5.0 5 9.2 77

* See footnote table 1.

with and without added phytic acid

Dextrino- Mg of Myce-Molds Phytic Acid* ~~~~~Incubation genie Saccharogenic lium per SugarMolds Phytic Acid* Period pH Amylase Amylase 10 Ml of Consumed

Activity Activityt Broth(Dry Wt)

days min %Aspergillus oryzae Not added 2 6.0o 18 1.7 45 31.2

3 5.5 28 3.4 45 43.7Added 2 6.0 2 21.1 110 98.9

3 7.0 3 26.8 115 98.1Aspergillus awamori Not added 2 5.0 18 1.9 50 72.5

3 5.5 33 1.3 35 92.9Added 2 6.0 10 8.5 125 98.9

3 7.0 5 18.7 99 97.4Rhizopus javanicus Not added 2 5.0 32 0.4 50 70.3

3 5.0 33 0.6 62.2Added 2 5.0 32 0 32 74.8

3 5.0 33 2.6 - 99.0

* Added in 0.01 per cent amounts.t See footnote table 1.

1959] 213

DUNN, FULD, YAMADA, MAS URIOSTE, AND CASEY

acid was capable of increasing the amylase activity ofother fungi, research was carried out with three otheramylase-producing molds. The results obtained withthese microbes, Aspergillus oryzae strain NRRL 458(Tsuchiya et al., 1950), Aspergillus awamori var.fumeus,9 and Rhizopus jaianicus var. kawasakiensis,9are summarized in table 3. It may be observed thatphytic acid increased the amylase activity in each case.An investigation of the effects of various sugars (as

carbon sources) on the amylase activity of A. nigerstraini NRRL 337 with and without the addition of 0.01per cent calcium phytate was also made. A standard

9 This mold is used industrially in Japan. It was obtainedfrom the stock collection of the University of Tokyo.

TABLE 4

Conmparison of the effects of various sugars, with andwithout added calciumtt phytate, on the amtiylase

activity of Aspergillus niger

MIonosaccharideUsed*

Glucose

Fructose

Mannose

Galactose

CalciumPhytatet

Not added

Added

Not added

Addecl

Not added

Addecl

Not added

Added

Incu-bationPeriod

days

23452345234523452345234523452345

pH

5.85.55.55.56.06.57.07.05.55.55.55.56.07.07.27.05.55.55.55.56.07.07.07.06.05.55.55.55.55.55.55.5

Dextrino-genic

AmvlaseActivity

inin

>30>30>30>30

15544

>30>30>30>30>30

966

>30>30>30>30>30

1365

>30>30>30>30201919

>30

Saccharo-genic

AmylaseActivity$

0.401.52.19.616.818.919.10.40.60.60.32.17.18.89.700.20.40.82.13.78.910.30.200

2.73.32.51.2

* Two per cent of sugar was added to a basal medium con-taining 0.5 per cent NH4NO3, 0.1 per cent Na2HPO4, 0.05 percent KCI, and 0.05 per cent MgSO4-7H20. Initial pH (beforesterilization), 5.0.

t Added in 0.01 per cent amounts.I See footnote table 1.

basal medium was utilized to which was added a 2 percent concentration of a mono- and disaccharide. Theresults obtained with glucose, fructose, mannose, andgalactose are shown in table 4, and indicate that in theabsence of phytic acid there was very little or no dex-trinogeniic or saccharogenic activity produced. The pro-duction of amylase activity in media containinghexoses as the carbon source and calcium phytate maybe ranked in the following descending order of effective-ness:

glucose > (fructose = mannose) > galactose

Several disaccharides were also metabolized by A-niger and the results are summarized in table 5. Therewas no significant production of dextrinogenic or sac-charogenic amylase activity when the sugars were usedin the absence of added phytic acid, and there was nodiscernible production of amylase activity even in thepresence of phytic acid when lactose was the source ofcarbon. NIaltose appeared to be a slightly better carbonsource than sucrose in respect to amylase activity.

Studies were also conducted on the utilizaton of un-cooked starch as a carbon source. A summary of theresults of a typical experiment is given in table 6. Thebasal medium contained 0.2 per cent NH4N'O3, 0.:3 percent Casamino Acids (Difco), 0.1 per cent Na2HPO4,

TABLE 5Comparison of the effects of some disaccharides, withand without added catcium phytate, on the amylase

activity of Aspergittus niger

DisaccharidesUsed*

Maltose

Stuerose

Lactose

Incu-Calcium Phytatet bation

Period

Not added

Added

Not adde(

Added

Not added

Added

days

235235235235235235

pH

5.55.55.05.67.07.35.65.65.66.07.07.36.06.86.55.55.85.5

Dextrino-genic

AmylaseActivity

miin>30>30>30

934

>30>30>322156

>30>30>30>30>30>30

Saccharo-genic

AmylaseActivitYv

1.080.433.6816.7021.7020.830.860.993.474.7714.5315.970.860.210

00

* Added in 2 per cent amount to basal nmedium which con-tained 0.5 per cent NH4NO3, 0.1 per cent Na2HPO4, 0.05 percent KCI, and 0.05 per cent MIgSO,7H20. Initial pH (beforesterilizing), 5.0.

t Added in 0.01 per cent amounts.t See footnote table 1.

214 [VOL. 7

PRODUCTION OF AMYLOLYTIC ENZYMES

0.05 per cent KCl, and 0.05 per cent MIgSO4- 7H20 indistilled water. The medium was sterilized by steam,and, where indicated, 1 g of uncooked starch, irradiatedat 150,000 rep10 by cobalt60 gamma rays, was added per100 ml of medium. One g of CaCO3 was added to eachflask after 24 hr, and calcium phytate and glucose wereadded at levels of 0.5 and 1.0 per cent, respectively,when used. The mold used was A. niger strain NRRL337. In the presence of phytic acid, uncooked starchmay be utilized as a carbon source for amylase pro-duction, but amylase activity was somewhat higher inthe presence of added glucose.

In summary, it may be stated that the addition ofphytic acid or calcium phytate to synthetic media coIn-siderably enhanced the production of both dextrinogenicand saccharogenic amylase activities by A. niger insubmerged culture. The phytate molecule stimulatedproduction of amylolytic enzymes from various carbonsources, including both cooked and uncooked starchesand several mono- and disaccharides.

0 One rep = 93 ergs of energy absorbed per g.

TABLE 6

Comparison of the effects of uncooked starch, wvith andwithoutt added glucose and/or calciuim phytate, on the

amlylase activity of A spergill us niger

Incuba- Dxrn-SaccharogenicMedium* tion pH genic Amylase

Period Amylase AcityActivity Aciiy

days mnin

Basal plus vuncooked 1 6.0 >33 0starch 2 7.0 > 30 0

3 7.5 >30 04 7.2 >30 0.425 7.0 >30 06 7.3 >30 1.27

Basal plus uncooked 1 6.5 >33 0.42starch plus Ca phy- 2 7.0 >30 0.85tate 3 7.5 29 1.27

4 7.5 29 1.705 7.5 12 1.706 7.5 12 2.34

Basal plus uncooked 1 4.5 >33 0starch plus glucose 2 5.8 >30 0

3 5.5 >30 04 6.5 >30 1.(065 7.0 6 9.786 7.5 >30 0.85

Basal plus uncooke(d 1 5.0 12 3.82starch pltus glucose 2 6.7 30 1.27plus Ca phytate 3 7.0 3 16.16

4 7.2 3 19.145 7.5 2 18.726 7.3 4 19.14

* The basal medium contained 0.2 per cent N1H4N03, 0.3per cent Casamino Acids (Difco), 0.1 per cent Na2HPO4, 0.05per cent KCl, and 0.05 per cent MgSO4*7H20 in distilled water.

t See footnote table 1.

BACTERIAL AMYLASE PRODUCTION

Mlethods and Equipment

Two organisms were used in the bacterial amylasestudies: B. subtilis strain NRRL B-941 for dextrinogenicamylase; and B. polymyxa strain NRRL B-372, the sameas N. R. Smiths' no. 354 (Kneen and Beckord, 1946),for saccharogenic activity. Surface culture studies wereconducted with (a) 350-ml portions of media in 1000-mlErlenmeyer flasks, each of which was equipped with asampler (Beckord et al., 1946); or, (b) 100-ml portionsof media in 250-ml Erlenmeyer flasks. All of the shake-flask studies were carried out with 100-ml portions ofmedia in 500-ml Erlenmeyer flasks which were incu-bated on a rotary shaker at 290 rpm (model VS). Pilot-plant studies were carried out in a 50-gal fermentor,which was equipped with automatic pH control (Fuldand Dunn, 1958).

Cultures were carried on nutrient agar slants (Difco).The B. subtilis inoculum was prepared in inoculatingflasks containing diluted production medium with aspore suspension of the test organism and incubatingthem for 24 hr on the shake table. The B. polymyxainoculum was prepared by inoculating two loopfuls ofspore-containing bacteria from a slant culture into 100ml of nutrient broth in a shake flask and incubating for24 hr. Most of the studies with these organisms werecarried out at 30 C.The basal medium for the B. subtilis study was,

generally, dried distiller's solubles. The basal mediumfor B. polymyxa was essentially that described by Lulla(1951) and contained the following ingredients in theamounts indicated:

KH2P04.........................................K2HP(4.........................................NaCl ............................................

CaC12...........................................MNgSO4-7H20....................................MInSO4-4H20....................................FeS04-71120.....................................D)istilled water to 1 L

gIL0.10.10.050.010.010.0010.001

Various carbon and/or nitrogen sources were addedto this basal medium.

Dextrinogenic amylase activity was determined bythe following procedure: 20 ml of a buffered 1 per centsoluble starch solution (pH 6.0) was added to a 50-mlErlenmeyer flask, together with sufficient water tomake a final volume of .30 ml after the sample wasadded. The flask was held at 30 i 0.01 C. Whenequilibrium was reached, the sample containing theenzymes was added. One-ml aliquots were taken atspecified time intervals and mixed with 5 ml of diluteiodine solution and the time to approach a red-browncolor was ascertained. An arbitrary unit for dex-trinogenic activity was established by one of the authors

1959] 215

DUNN, FULD, YAMADA, MAS URIOSTE, AND CASEY[

(J. M.) in which the dextrinizinig units per ml =

100/TV, wherein T is the time in min and V is thevolume of the sample in ml.

Saccharogenic enzyme activity was determined byadding 2 ml of broth to 20 ml of a soluble starch solutionof known concentration, allowing reaction to take placefor 30 min at 30 C, and determining the reducing sugars

by the Somogyi (1945) method.

Results and Discussion

When B. subtilis strain NRRL B-941 was grown insurface culture in a medium containing 5 per cent ofdried distiller's solubles, the maximal dextrinogenicamylase activity was obtained after a fermentationperiod of 100 to 120 hr and averaged 14.5 dextrinizationunits per ml. Similarly, it was found, under identicalsurface culture conditions, that maximal dextrinogenicamylase activity was about 40 per cent higher at 30 Cthan at 37 C, although growth was faster at the lattertemperature. There was poor dextrinogenic amylase ac-

tivity at 20 C and no growth at 59 C. Under submergedculture conditions, the dextrinogenic amylase activityat 30 C was about 140 per cent greater than that at37 C.Using identical media, the yield by the surface culture

method with a medium depth of 1.5 cm was 38 unitsper ml, whereas the yield by the submerged culturemethod was 40 units per ml after 97 hr. Thus, at an

optimal area: volume ratio (Beckord et al., 1946), thereseemed to be no essential differences in yields by thesurface and submerged culture methods.

Various nonsynthetic media were used for productionin submerged culture. The best media, in order of effi-ciency, were distiller's solubles plus yeast extract; corn

TABLE 7Effect of the composition of the medium on detrinogenic

amylase produiction by Bacillus subtilis strainNIRRL B-941

DextrinizingMedium

Units per Nil afterIncubation for

72 Hr*

Linseed meal, 4% 4.9

Cottonseed meal, 8%/- .......................... 17.2Soybean meal, 8% ............................. 14.7

Distiller's solubles, 8% 37

Distiller's solubles, 4%/c, pltis yeast extract, 4%c... 52

Linseed meal, 4%/0, plus distiller's solubles, 4%... 24Linseed meal, 4%/c, plus yeast extract, 4% 11.9

Yeast extract,8% 25

Peanut meal, 8% 1.83

Peanut meal, 4%, plus linseed meal, 4 7.7

Peanut meal, 4%, plus yeast extract, 4%c 27

Corn steep liquor, 25% 4.9

Corn steep liquor, 12%, plus distiller's solubles,6%cl ........................................ 42

* Inoculum size, 5 per cent by volume.

steep liquor plus distiller's solubles; distiller's solubles;peanut meal plus yeast extract; yeast extract; linseedmeal plus distiller's solubles; cottonseed meal; and soy-bean meal. Data obtained during the study are sum-marized in table 7.

Variation of the initial pH of the medium in the rangeof pH 5.0 to 8.5 had no appreciable effect on the finalyield of dextrinogenic amylase. This finding is in agree-ment with the claims of a recent patent (Hoogerheideand Laughery, 1951).

Variation of the size of the inoculum within the rangeof 0.1 to 3 per cent (25 to 750 million bacteria per 100 gof medium) likewise showed no significant effect ondextrinogenic amylase yield. At inoculum levels of over5 per cent, there was a slight, but not significant, de-crease in the yield. This finding is in agreement withpublished reports concerning the surface culture use ofB. subtilis (Beckord et al., 1945).

It was found that the addition of various minerals,such as dibasic potassium phosphate, and/or a mixtureof calcium chloride, sodium chloride, magnesium sulfate,manganese sulfate, and ferrous sulfate, did not affectyields of the dextrinogenic amylase produced in adistiller's solubles medium under submerged cultureconditions. This fact is in agreement with the findingsof others in respect to surface culture investigations(Beckord et al., 1946; Lulla, 1951).A study of the effect of phytic acid on the production

of dextrinogenic amylase by B. subtilis in natural andsynthetic media was undertaken. The synthetic mediumused was essentially that employed for the studies withB. polymyxa. The results are summarized in table 8.The addition of calcium phytate in amounts of 0.075

per cent or less to a basal medium containing 12 per

TABLE 8Effect of calcimni phytate on the suibmerged cultureproduction of dextrino9enic amylase by Bacillus

subtilis

Dextrinizing Activitv,Units per MI, with

Medium* Fermentation at:

50 Hr 72 Hr

12%G l)S 21 4612%c/ DS + 0.015% ph 20 4712% DS + 0.075% ph 22 4812%l DS + 0.225% ph 19.2 5912%, I)S + 0.375%,/O ph 18.8 73

SB 0.1 1.5SB + 0.015%c ph 1.9 2.9SB + 0. 075%/c ph 2.1 2.9SB + 0.15% ph 2.2 2.9SB + 0.225% ph 2.5 3.3SB + 0.375%c ph 2.1 2.7

* Abbreviations: DS = distiller's soluhies; pil = calCiumphytate; and SB = synthetic basal mediuim.

[VOL. 7216

PRODUCTION OF ANIYLOLYTIC ENZYMIES

cent of distiller's solubles did not enhance the dex-trinizing activity of B. subtilis in 50 hr, but the additionof 0.225 and 0.375 per cent amounts of calcium phytate,respectively, caused a significant increase in activityduring a period of 72 hr.

In the case of the synthetic basal medium, the addi-tion of calcium phytate increased the dextrinizing ac-tivity, but the activity was low in comparison with thatproduced in media containing distiller's solubles.A number of experiments were carried out in a 50-

gal fermentor, using a medium containing 12 per centof distiller's solubles. The inoculum for 150 L of mediumin the fermentor was 2 L of a culture of B. subtiliswhich was propagated in shake flasks in a medium con-sisting of 4 per cent of distiller's solubles.

In the first experiment, with an aeration rate of 34 ftper hr (eu ft of air per sq ft of surface area per hr), themaximal production of dextrinizing amylase (55 unitsper ml) occurred at 39.5 hr and when the pH had risento 8.05. In the second experiment, in which the pH wasnot controlled and in which the aeration rate was 45 ftper hr for the first 47 hr and 90 ft per hr thereafter, themaximal production of amylase (50 units per ml) oc-curred after 48 hr at a pH of 7.8. In a third experiment,the pH was controlled at 7.7 between the 44th and 63rdhr. The initial pH of the medium was 6.4. At the end of44 hr, the dextrinizing activity was 28 units per ml andpH control was initiated. After 63 hr, the control of pHwas discontinued and the dextrinizing activity haddropped to 20 units per ml. Thus, this preliminary ex-periment indicated that holding the pH at 7.7 was notconducive to good yields of dextrinizing activity by B.subtilis strain NRRL B-941 under the conditions used.A limited number of experiments were carried out with

B. polymyxa strain NRRL B-372. Three nitrogensources, ammonium nitrate, urea, and asparagine, werecompared at different concentrations for their abilitiesto increase the saccharogenic activity of B. polymyxa,using 5 per cent starch as the carbon source and thesalts listed earlier. The best nitrogen source was foundto be asparagine and the most effective concentrationwas 0.33 per cent.

TABLE 9

Effect of calcium phytate on the subnmerged cultureproduction of saccharoqenic activity in synthetic

media by Bacillius polytnyxa*

Amount of Calcium Phytate Added Saccharogenic Activity (Approx)

0.0 40.1 160.2 170.5 151.0 16

* Results obtained after 125 hr fermentations.

The effects of the volume of the inoculum on sac-charogenic activity was studied. A 5 per cent inoculumwas found to be superior to both a 2 and 10 per centinoculum.The effects of various concentrations of calcium

phytate on the production of saccharogenic activity inmedia containing the basal salts, 0.33 per cent as-paragine, and 5 per cent starch were investigated and theresults are summarized in table 9. It may be observedthat calcium phytate markedly increased the sac-charogenic activity of the basal production medium andthat a concentration of calcium phytate greater than0.2 per cent did not appear to enhance the results.Details of these studies have been published (Casey,1958).

SUMMARYCalcium phytate and phytic acid, when added to

synthetic and nonsynthetic media, increased the pro-duction of dextrinogenic and saccharogenic amylase ac-tivity by Aspergillus niger strain NRRL 337 under sub-merged culture conditions. When added to syntheticmedia, calcium phytate brought about similar resultswhen glucose, fructose, mannose, sucrose, or maltosewas the sole source of carbon.Wheat germ, in the absence of added calcium phytate

or phytic acid, produced higher amylase activity than anumber of other nutrient sources.

Yields of dextrinogenic amylase by Bacillus subtilisstrain NRRL B-941 appeared to be as high by sub-merged culture as by surface culture methods and wereindependent of inoculum size in the range of 0.1 to 3.0per cent and of initial pH in the range of 5.5 to 8.4.Calcium phytate in low concentrations increased theyields of dextrinogenic amylase in natural and syntheticmedia.

Bacillus polymyxa strain NRRL B-372 was studiedin synthetic media. The best nitrogen source for optimalsaccharogenic activity was found to be asparagine at aconcentration of 0.33 per cent; the best level of inoculumwas found to be 5 per cent. Phytic acid significantlyimproved the saccharogenic activity in a syntheticmedium.

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