Proteolytic enzymes of microorganisms. Evaluation of proteinases produced by molds of the Aspergillus flavus-oryzae group in submerged culture

Proteolytic Enzymes of Microorganisms. Evaluation of Proteinases Produced by Molds of the Aspergillus jZuvus-oryzae Group in Submerged Culture R G. Dworschack, H. J. Koepsell and A. A. Lagoda From the Fermentation

Division,

Northern Regional Illinois

Received

March

Research Laboratory,’

Peoria,

27, 1952

INTRODUCTION

The production and properties of microbial proteinases have been reported by Ayres and Tobie (l), Berger et al. (2), Dion, (3), Dox (4), Haines (5), Oshima and Church (6), Waksman (7, S), Wallerstein (9), and Wehmer (10) among others. These studies have concerned only a comparatively small number of molds, cultured on the surface of unagitated liquid or on solid substrates. Such methods for growing molds to produce enzymes on a commercial scale present operational difficulties in the maintenance of aseptic conditions and optimum temperatures, and involve the extravagant use of equipment and personnel. Submerged, or deep culture, fermentation methods tend to alleviate these problems and are more applicable to present-day industrial fermentation equipment. Successful production of penicillin and fungal amylases by the submerged culture technique suggests that the manufacture of proteinases in like manner should be feasible. A program to develop such a process has been initiated. LeMense and co-workers (11) found that the elaboration of amylases by molds in submerged culture is markedly dependent on the strain used. Only a few strains among many investigated were found to be suitable. It seemed probable that the production of proteinases in submerged cultures might also be a strain-specific characteristic of certain molds; hence a survey of mold strains for this property was undertaken. The Aspergillus Jlavusoryzae group and related species were 1 One of the laboratories try, Agricultural Research

of the Bureau Administration, 48

of Agricultural and Industrial ChemisU. S. Department of Agriculture.

PROTEOLYTIC

ENZYMES

49

OF MICROORGANISMS

first studied, since some members of this group were known to produce high yields of proteinases under surface culture conditions. Four hundred and ninety-one strains were tested. The results of this survey as well as a discussion of analytical procedures are given in this paper. MATERIALS

AND METHODS

Production of Mold Culture Filtrates The mold cultures were obtained from the culture collection maintained at this laboratory.2 Spores were used to inoculate 100 ml. of survey medium contained in a 500-ml. Erlenmeyer flask. The survey medium consisted of 2oJ0whole corn meal, 1% whole soybean meal, and 0.5% calcium carbonate, sterilized at 120°C. for 30 min. The flask was shaken on a reciprocating shaker at the rate of ninety 3-in. strokes/min. at 28°C. for 2 days. Ten milliliters of this culture, now containing submerged mycelium, was used to inoculate 260 ml. of survey medium in a l-l. Erlenmeyer flask. This flask was incubated on the shaker for 4 days. The fermented medium was filtered, and the filtrate was assayed for proteolytic enzymes. In general, filtration was rapid and led to essentially clear, amber culture filtrates. The mold mycelium was also examined for proteolytic activity in several instances. The mycelium was ground in a mortar, autolyzed under toluene, or frozen and thawed repeatedly, in attempts to release intracellular enzymes. Only traces of activity were found.

Methods for Proteinase Assay It was anticipated that proteinases varying in pH optima, substrate specificity, and mode of attack might be encountered in the survey. Detection of all types of proteinases was desired. However, if a large number of mold strains were to be tested, the assay methods had to be rapid, simple, and direct. These opposing requirements necessitated considerable compromise in selecting the analytical methods to be used. Suitable basic proteinase methods were selected from the literature. In most cases, the methods required modification to adapt them to survey use. Gelatin and casein hydrolysis tests were conducted at pH 2.5,5.0, and 7.5. Both viscosity reduction and amino nitrogen formation in gelatin hydrolysis were measured. Casein digestion was estimated by sulfosalicylic acid precipitation of unreacted substrate. Milk-clotting activity at pH 4.6 was also determined. The procedures used are described in detail below. To orient the reader as to the extent of proteolytic activity that can be expected from well-known enzymes under our experimental conditions, the results obtained when purified preparations of pepsin, papain activated with hydrogen sulfide, and trypsin were used as standard test enzymes are shown in Table I. The proteinases of mold culture filtrates undoubtedly differ in important characteristics from- the test enzymes, and the 2 We are indebted to Dr. Kenneth B. Raper, Miss Dorothy May Flickinger for selecting and preparing the cultures.

Fennell,

and Mrs.

50

DWORSCHSCK,

KOEPSELL

.4ND

LAGODA

filtrates probably contain varying ratios of proteinases and peptidases. For these reasons the results obtained with mold filtrates should not and are not intended to be compared directly with those obtained with the test enzymes.

Gelatin Liquejactim The procedure used in this work is modified from that of Lennox and Ellis (12). The substrate was purified calfskin gelatin.3, 4 No buffer is required, since the gelatin contains sufficient buffer capacity to maintain the reaction pH at the desired level. Thymol (0.5 g/l.) was added as a preservative. For the enzyme assay, 10 ml. of a 10% gelatin solution adjusted to pH 2.5, 5.0, or 7.5 with HCl or NaOH and 4 ml. of distilled water, contained in a digestion TABLE Proteolytic

Activities

I

of Pepsin, Papain, and l’rypsin” Conditions Employed

Under

Experimental

-

Amounts of enzyme ~___ m.

0.1 0.3 0.6 1.0 5.0 10.0 20.0

%mino nitrogen from gelatin

Gelatin liquefaction

-

Pepsin unifs

13.6 27.5 43.7 / 56.7

-

Papain - ______ unifs

Trypsin

-

14.6 37.5 57.3 71.3 --

5.8 47.0 66.6 75.4

Pepsin ’ Papain ---_I--~ m. w.

units

-

-

-

-

-

0.1 0.2 0.3 0.4

0.3 0.8 1.9 3.2

Casein digestion

Trypsin

Pepsin

Papain

-~ Trypsin

w.

WT.

w.

w.

1.0 1.4 1.8 2.0 -

25 57 76 85 -

-

-

2 33 53 74

7 48 63 76

a Pepsin (crystalline, porcine origin) and trypsin (crystalline, bovine origin) were obtained from Armour Laboratories, Chicago, 111. The papain concentrate (Optimo brand) was obtained from the S. B. Penick and Co., Inc., New York, N. Y. The percentages of nitrogen in these three preparations before and after dialysis were as follows: pepsin, 12.84, 13.09; trypsin, 9.23, 13.68; papain, 10.11, 10.85. beaker, are equilibrated to temperature in a 40°C. water bath. The viscosity of this solution is measured exactly 3 min. after adding 1 ml. of culture filtrate. The measurement is made with a viscometer shown in Fig. 1, essentially similar to that described by Landis and Redfern (13). The viscometer consists of a bulb of 2-ml. capacity, a capillary tube 3 cm. long having an inner diameter of 0.5 mm., a digestion beaker made by cutting off the top of a M-ml. beaker, and a vent, s Differences in substrate were avoided by obtaining large lots of Eastman’s purified calfskin gelatin. It contained 0.05y0 ash (dry basis) and had an isoelectric point at pH 4.85. 4 The use of names of commercial products in this paper is solely for product identification.

PROTEOLYTIC

ENZYMES

OF

51

MICROORGANISMS

tube through the stopper to maintain atmospheric pressure inside the digestion beaker. The rubber stopper is weighted with lead to anchor the apparatus while it is submerged in a water bath. The time required for 2 ml. of reaction mixture to drain through the capillary tubing is noted. If it is apparent that the culture filtrate contains little or no gelatin-liquefying enzyme, the reaction mixture is blown from the bulb and the flow time is recorded as being greater than 3 min. A viscometric determination can be made every 4 min. A blank measurement, using distilled wat,er instead of enzl’me, is made in a similar manner.

Bulb, 2 ml Capacity Between Graduations

Vent Tube Leod Weight --i

L.1

I

Rubber Stopper Digestion

Beoker

Tik

FIG. 1. Viscometer for measuring gelatin liquefaction Gelatin liquefaction activity is expressed as a percentage reduction of the initial viscosity, as discussed by Lennox (14). One unit of activity comprises a viscosity reduction of 1%. Mold filtrates varying 1 unit/ml. in activity can be distinguished.

Form&m

of Amino Nitrogen in Gelatin Hydrolysis

Amino nitrogen liberation during gelatin hydrolysis is measured by a modification of Brensen’s (15) form01 titration. The reaction mixture employed in the gelatin liquefaction determination described above is incubated further in the 40°C. bath for a total of 4 hr. The stoppered beaker is then removed from the bath

52

DWORSCHACK,

KOEPSELL

AND

LAGODA

and the pH is adjusted to 8.5 (glass electrode) with 2 N NaOH. Five milliliters of neutralized formalin (407, formaldehyde) is added, resuhing in a drop in pH. The solution is titrated back to pH 8.5, with 0.07 A’ NaOH. One milliliter titration represents 1 mg. amino nitrogen. As a blank, the amino nitrogen content of the gelatin solution without enzymatic hydrolysis is determined. The increase in milligrams of amino nitrogen in the reaction mixture brought about by 1 ml. culture filtrate is recorded. Enzyme activities differing by 5% are distinguishable.

Casein Digesiion Casein has been employed as substrate for various types of proteolytic enzyme assay methods. Kriggsman (16) and Wyss (private communication) have described turbidimetric methods for the determination of casein digestion. A simple, rapid modification of the latter, suitable for routine analysis with equipment at hand, was developed and used. The substrate is sodium caseinate.6 The limited solubility of casein, especially at its isoelectric point (pH 4.8), necessitates the use of a peptizing agent (6.6 M urea) to form stable solutions at pH values as low as 2.5. It was also used in reaction mixtures at pH 7.5 to keep this condition uniform and thus allow direct comparison of results. The pH of the substrate solution is adjusted to 2.5,5.0, or 7.5 with HCI or NaOH. No buffer is necessary to maintain the desired pH during the enzyme reaction. Substrate solutions are stable for weeks when stored under refrigeration with a few drops of toluene as preservative. For the. assay, 5 ml. substrate solution and 2 ml. water are equilibrated in a 40°C. water bath. One-half milliliter culture filtrate is added. After 30 min., 2 ml. of the reaction mixture is pipetted into an 18 X 150 mm. test tube containing 5 ml. of approximately 2.5% gum ghatti solution, and the solutions are mixed well. Five milliliters of a 5% sulfosalicylic acid solution is added and mixed by gently inverting the tube two or three times. Undigested casein precipitates as a colloidal suspension. After 5 min., precipitation is complete, and the turbidity of the solution is determined with a Lumetron calorimeter fitted with a red (656 rnp) filter. The amount of casein digested is obtained from a standard curve previously constructed for the specimen of substrate in use. At pH 5.0, small casein digestion values cannot be determined accurately because of the low slope of the standard curve in this region. However, activity above 30 units can be ascertained satisfactorily. Results of the enzyme assay are reported as milligrams of casein digested by 0.5 ml. of culture filtrate. With the exception of low activity at pH 5.0, enzyme potencies differing by 3% may be distinguished. Gum ghatti solution used in this procedure is difficult to prepare in exact concentration. Variations up to about 20% in concentration can be tolerated, however, without materially affecting assay results. Other vegetable gums have proved less satisfactory. The gum solution is prepared most easily by grinding the resinlike pellets in a burr mill. Twenty-five grams of the resulting material and 756 ml. distilled water are heated in a water bath at 70-80°C. for 2-3 hr. with a “Nutrose,” used.

manufactured

by the Difco

Laboratories,

Detroit,

Mich.,

was

PROTEOLTTIC

ENZYMES

OF MICROORGANISMS

53

stirring. Twenty grams of filter-aid is :~ddcd, rind the solution is filtered with vacuum. Completely colorless solutions (*:uI be obt:rincd by t rcntmc>nt with adsorbent ch:lrco:ll or selection of only clear pellets of gum for grinding. Thct filtrrcd solution is storrd undcxr tolucbnc at room tc‘mpcr:iturc.

M:lny mct.hods for proteinnscb ass:~y hascd on milk clotting, using :I v:Gt.y of milk substrates ant1 clotting end points, nre described in the litcraturc. The tncthotl tl~~viaetl for this survey has t.hc advantages of being simple, rapid, and c:lsily reproducible. The sul)str:ite is dried whole milk6 reconstituted in 0.1 M acet:itc buffer :lt. pI1 4.6 ncrording to the proccdurc of Balls nnd Hoover (17). Five milliliters milk in n 25 X 200 mm. test tube is cquilibrntcd at 40°C. in 3 wntcr bnth. One milliliter of culture filtrate, previously diluted 1:l with acrter, is added, and the t.ube is rotated slowly by a small motor nt about a 45” rrnglc in the water bath. As the tube revolves, a uniform layer of milk clings to the surface. At the end point. the milk forms small clots which aggregate quickly. The end points are sharp and easily reproducible. Duplicate samples do not vary by more than 39& Enzyme activity is expressed as 10 times the reciprocal of the clotting time in minutes.

a-Amyhse Activity Since the work of LeMense et al. (ll), considerable interest has developed in submerged production of fungal amylascs, especially for distillery use. Advantage was taken of the present opportunity to extend the amylase survey to include molds in our culture collection. It haa additional members of the A.jZauus-oryzae also been desirable to ascertain the relationship of amylase to proteinase production by the vrrrious mold strains under our growth conditions. The a-amylase content of culture filtrates was determined by the procedure of Sandstedt, Kneen, and Blish (18) ttt 20°C. PRELIMINARY

EXPERIMENTS

The selection of the survey medium previously described was based on a series of experiments to determine the effects of nutritional factors on proteinase production, and to ascertain the course of enzyme production. These experiments will be summarized briefly. Nutritional

Factors Aflecting Proteinuse Production

Twelve members of the A. jlavusoryzae group, known to produce proteolytic enzymes, were chosen for a series of nutritional studies. Only materials which are readily available and easily stored were used in the medium. 6 “Klim” milk powder, manufactured N. Y., was used.

by the Borden Company, New York,

.)‘4

DWOFUXHACK,

KOEPSELL

AND

LAGOD,4

The effects of the corn meal and calcium carbonate in the survey medium are shown in Table 11. In general, maximal enzyme formation occurred at the 2% corn meal level, and this level was chosen for our standard survey medium. The removal of corn meal resulted in sharply decreased enzyme yields except as measured by form01 titration, although there was usually no apparent decreasein mold growth or significant variation in terminal pH. The presence of calcium carbonate in the survey medium was found to be necessaryfor enzyme production. This may be due to the buffer action of calcium carbonate, since, in the absence of the carbonate, the pH of the medium dropped to low TABLE The Effects

--

IX

C&0P

-

i.-

of Corn Meal

II

and CaCOa in the Survey Analytical

Gelatin liq~ue;tjon

units

--

Medium

data*

Amino nitrogen pH 7.5

Terminal PH

w.

w.

units

0.5 0.5 0.5 0.5

63.6 78.3 76.2 73.0

2.4 2.4 2.3 2.0

42 77 79 75

4.4 10.9 12.8 10.2

8.0 7.5 7.2 7.0

0 1 2

65.3 77.0 75.6

0.8 1.9 1.8

39

5.1 11.0 10.0

7.4 7.5 7.4

%

-

-

79 75

0 Whole corn meal and CaC03 as indicated plus 1% whole soybean meal. b hnalyses of A. jlauus-oryrae NRRL strain 491 rafter 5 days’ incubation.

values (about pH 5.0) on the third day and returned to the usual value of about pH 7.0-7.5 on the fifth day. A level of 0.5% calcium carbonate prevented the pH drop and resulted in the greatest enzyme yields. Various specimens of distillers’ solubles and corn steep liquor could be substituted for soybean meal in this medium without appreciably affecting proteinase production, as shown in Table III. Enzyme yields, in general, were maximum when levels between 0.5 and 2.0% were used. Soybean meal (loJo) was chosen for the survey medium because it is generally constant in composition, is stored easily, and contributes little to the color of culture filtrates.

PROTEOLYTIC

ENZYMES

55

OF MICROORGANISMS

The above series of experiments indicated that a medium consisting of 2% whole corn meal, 1% whole soybean meal, and 0.5 calcium carbonate would support both good growth and maximum proteinase production by members of the A. Jlavus-oryzae group. Course of Proteinme Production

Six strains belonging to the A. jlavus-oryzae group were selected for studies on the course of proteinase production in the survey medium. Each strain was used to inoculate eight flasks of medium, and one flask was analyzed each day for enzyme content. Maximum proteinase TABLE !l’he h’flecl

of L)ialiElers’

Solubles,

Casein

I Per cent

in medium”

Distillers’

III

Corn Steep Liquor Survey Medium digestion

Corn

unils

0 ?5 1 2 3 5

64

71 77 76 70 64

Meal

in the

pH 7.P

solubles

I %

at

and Soybean

steep liquor

Soybean

meal

II unils ~

63 70 75 78 72 69

WifX

pi I 75 72 69

units

64 76 77 75 71 66

o Adjunct as indicated plus 2% whole corn m‘eal and 0.5% CaCOa. b Analyses of -4. fEaa,ts-orwzae NRRL strain 491 after 5 days’ incubation.

,

production was attained between the third and fifth day. Gelatin liquefaction and amino nitrogen formation activities remained high through the eighth day, while casein digestion and milk clotting activities decreasedgradually after the fourth or fifth day. The pH of the fermenting media rose until the sixth day, and thereafter tended to remain constant. On the basis of these results, the first 200 mold strains were analyzed for proteinase and amylase on the third and fifth days of incubation. The data obtained indicated that a single analysis on either day would have yielded, without exception, the same results in selecting good molds for further work. Therefore, to expedite the survey, the cultures were subsequently analyzed only on the fourth day.

56

DWORSCHACK, KOEPSELL AND LAGODA RESULTS AND DISCUSSION

The preceding survey procedure for proteinase production under submerged culture conditions was applied to 491 mold cultures, consisting of 360 strains of either Aspergillus flavus Link or Aspergillus oryzae (Ahlburg) Cohn, 59 strains of Aspergillus tamarii Kita, 37 strains of Aspergillus went&i, and 35 other strains which Thorn and Raper (19) regard as belonging to closely related species. All of the molds produced proteinases in at least small amounts. Eighty strains produced sufficient enzymes to warrant further study. Over-all results of the survey are shown graphically in Fig. 2. In general, greatest enzyme activity was observed at pH 7.5. Somewhat less activity occurred at pH 5.0, and essentially no activity was found at pH 2.5. Of the types of proteolysis tested, high liquefaction activity and amino nitrogen liberation from gelatin were found most frequently. In contrast, ability to digest casein rapidly occurred much less frequently, and high milk-clotting activity was rare. Mold strains showing greatest proteolytic potency, as measured by each of the various assays, are listed in Table IV. Examination of the table indicates that individual strains differed in proteolytic behavior. The differences were of several types. Variations in attack on the same protein substrate were noted. For example, A. jlavus NRRL strains 453 and 2214 possess comparable gelatin liquefaction ability (73 units) at pH 7.5, but vary in amino nitrogen formation (3.7 and 1.4 units, respectively). Differences in ability to attack different protein substrates were also observed. Thus A. ovzae NRRL strain 2220 and A. jlavus NRRL strain 485 have similar gelatin liquefaction activities (74 units) at pH 7.5,~but differ in casein digestion activity (39 and 73 units, respectively). High milk-clotting activity, when found, was frequently not associated with high casein-digesting ability. These dissimilarities in activity suggest differences in the character and composition of the proteolytic enzyme systems elaborated by the various mold strains. The culture filtrates were more active in proteolysis at pH 7.5 than at pH 5.0. It may be noted from Table IV that the pH range for activity appears to vary among types of proteolysis. Whereas gelatin liquefaction at pH 5.0 frequently approximated that found at pH 7.5, amino nitrogen production and casein digestion were generally much less at the lower pH value. Mold strains exhibiting good proteolytic activity had little amylolytic activity. High activities of both types did not occur together in any of

PROTEOLYTIC

ENZYMES

57

OF MICROORGANISMS

the filtrates. Only five strains produced sufficient amylase to warrant interest as a source for this enzyme, and these strains have a previous history for good amylase production when grown in surface culture. 476 410

a 66 0 50 60 70 80

IIlL 0.5

5 I

0 2

n

0

0

0

4

0 20 40 60 80

4

0 20 40 80 80

270 168

50 0 50 60 70 80

Ill 0.5

3 I

2

320

152148'~~

146'56 110 PH

80

59 53 59

31 7.5 all 0 50 60 70 80 6elatin Llqusfoctlon

OIL 0.5

I

2

Amino Nitrogen Libwotlon

4

I, 0 20 40 60 80 CoreIn Oig0otion

FIG. 2. Distribution of various types of proteolytic activity. In each type of activity the mold strains were arbitrarily divided into four groups, shown as vertical bars, representing poor, fair, good, and excellent activity levels. The range of units is given at the base of each bar. The number of strains in this range is indicated by the height of the bar and by the number at the top.

It is possible to concentrate culture filtrates in a vacuum evaporator to a solution containing 3-5% solids and then dry them to a powder by the lyophilization process. Approximately 80% or 28,000 units/g.

Groups

and species

of Aspergillus

tlavus,

orljzae

of

7 1246

133

227

strains tested

lumber

A. oryeae

2211 1946 627 492 2216 485 453 2210 550 2214 2160 2217 2218 2220 470 466 502

NRRL numbers of selected strains

and Related

-

77.6 76.1 74.0 72.5 73.7 73.5 72.7 77.5 76.Y 74.8 74.4 <50.0 64.5 68.6

78.4

78.8

79.8

unifs

70.5 65.5 67.0 72.8 73.4 68.4 70.5 70.3 66.3 66.9 76.0 72.3 70.4 74.5 <:50.0 62.8 65.6

unds

_-

-

1.5

3.8 2.9 2.9 9.8 1.9 2.1 3.7 1.4 0.9 1.4 2.4 3.0 1.6 1.6 0.5 1.3 8.0

mg.

2.5 1.0 1.8 1.8 1.1 0.8 2.2 1.1 1.2 1.0 0.7‘ 1.5 1.2 0.9 0.2 0.6 1.5

m.

5.0

Analytical

73 39 78 64 42 73 41 75 77 76 60 37 42 39 0 0 37

w.

5.0

29 76 42 31 41 25 66 20 56 37 36 32 30 10 I4 37

71

-

7.2 9.5 20.7 6.9 27.3 14.8 8.2 17.7 18.1 16.0 10.6 <2.0 <2.0 7.4

21.5

6.1

10.7

Milk clotting

in Submerged

m-.

I-

-

Casein digestion

Yields

7.5

data

_-/

-

Proteinase

Amino nitrogen

Superior

8 t L4 F $ r $

0 0 0.0 0.0 0.7 1.0 1.2 IS.4 11.5 0.1

“k

$

I!

g

0.1 0.0 0.1 0.1 0.4

0.2 0.1

0.0

units

4.8

-Amylase

Culture

7, A. e#wus 5, A. luteo-virescens 4, A. terricola wr. a ,,eri2, A. avenacens 1, A. ostian,trs 1, and A, terricola 1.

i.

5.0

Producing

Gelatin liquefaction

7.5

Species = I

= Exceptional activity of each type is shown in italics. * This group includes A. tamarii 59, A. we&ii 37, A. alliaceus cana 3. A. luiescens 2, A. micro viridocitrinus 2, A. panamensis

A. parasiticws 2. Miscellaneous

A

1. Aspergillus jlavus-oryrae group Aspergillus jeaous

Strains

PROTEOLYTIC

ENZYMES

OF MICROORGANISMS

59

of casein digestion activity at pH 7.5 was recovered. For comparison, the enzyme potency of 12 commercial preparations produced from animal tissues and microorganisms was determined. They contained various L’fillers,” unfermented constituents of the medium, precipitating and stabilizing agents, etc. They were found to vary from 100 to 15,000 units/g. of casein digestion at pH 7.5, the majority of samples ranging from 100 to 2000 units. It has thus been shown that proteinase production by submerged cultivation of certain members of the A. jh~usoryzae group and related species of molds is feasible. By the selection of particular mold strains and culture conditions it may be possible to favor some types of proteolytic activity over others. The survey procedure described here is now being extended to other groups of molds. SUMMARY

A procedure for surveying mold strains for proteinase production under submerged culture conditions is described. Four hundred and ninety-one strains of the Aspergillus jlavus-oryzae group were tested. Eighty strains produced sufficient proteinase activity to warrant further study. On an over-all basis, greatest activity was found at pH 7.5, much less at pH 5.0, and no appreciable activity at pH 2.5. Ability to hydrolyze gelatin occurred frequently, and milk-clotting activity was rare. High a-amylase activity occurred infrequently and was not associated with high proteinase activity. Individual mold strains varied in their ability to attack various substrates. Seventeen strains showing superior proteinase or a-amylase activity are listed with quantitative data regarding their enzyme activities. REFERENCES 1. AYRES; G. B., AND TOBIE, W. C., J. Bact. 46,lS (1943). 2. BERCER, J., JOHNSON, M. J., AND PETERSON, W. H., J. Biol. Chem. 117, 429 (1937). 3. DION, W. M., Can. J. Research (X8,577 (1950). 4. Dox, A. W., U. 8. Dept. Agr., Bur. Animal Ind., Tech. Bull. No. 120 (1910). 5. HAINES, R. B., Biol. Revs. Biol. Proc. Cambridge Phil. Sot. 9, 235 (1934). 6. QSHIMA, K., AND CHURCH, M., Ind. Eng. Chem. 16,67 (1923). 7. WAKSMAN, S. A., J. Bact. 3, 509 (1918). 8. WAKSMAN, S. A., Abstracts Bact. 6, 265, 331 (1922). 9. WALLERSTEIN, L., Ind. Eng. Chem. 31.1218 (1939). 10. WEHMER, C., Lafar’s Handbuch der technischen Mykologie 4, 239, 596 (1907). 11. LEMENSE, E. H., CORMAN, J., VANLANEN, J. M., AND LANGLYKKE, A. F., J. Bact. 64, 149 (1947).

60 12. 13. 14. 15. 16. 17. 18. 19.

DWORSCHACK,

KOEPSELL

AND

LAGODA

LENNOX, F. G., AND ELLIS, W. J., Biochem. J. 39, 465 (1945). LANDIS, Q., AND REDFERN, S., Cereal Chem. 24, 157 (1947). LENNOX, F. G., J. Council Sci. Ind. Research 16, 155 (1943). SORENSEN, S. P. L., Biochem. 2.7.45 (1908). KRIGGSMAN, B. J., Z. physiol. Chem. 228, 256 (1934). BALLS, A. K., AND HOOVER, S., J. Biol. Chem. 121, 737 (1937). SANDSTEDT, R. M., KNEEN, E., AND BLISH, M. J., Cereal Chem. 16,712 (1939). THOM, C., AND RAPER, K. B., A Manual of the Aspergilli. The Williams and Wilkins Co., Baltimore, Md., 1945.