SYMPOSIUM: DETERGENT ADDITIVES conducted by The American Oil Chemists' Society at the AOCS 42nd Fall Meeting, New Yorl , N.Y. October 20-23, 1968 G. D. M O U L T O N ,

Program Chairman

Enzymes m Detergents R. L. LISS and R. P. L A N G G U T H , Monsanto Company,

Inorganic Chemicals Division, Research and Development Department, St. Louis, Missouri 63166 I n simple terms, an enzyme can be defined as a catalyst produced by living cells. While the spectrum of reactions catalyzed b y enzymes is very broad, the catalytic action of an enzyme is usually quite specific. E n z y m a t i c reactions under optimum conditions are very rapid and efficient, proceeding 10 s to 1011 times more rapidly t h a n the corresponding nonenzymatie reaction. E n z y m e s are complex proteins of high molecular weights consisting of hundreds of amino acids combined in a characteristic sterically oriented structure. This structure m a y contain metal ions and other linking agents to provide its tridimensional sterie p a t t e r n . F u r t h e r , appended to these complex structures are m a n y reactive groups which are quite vulnerable to modification b y hydrolysis, heat, p H , ionic effects, sequestration, oxidation and most of other types of chemical reactions. Since this is true, it is i m p o r t a n t to know the effects of the various l a u n d r y conditions and detergent compositions on

Abstract Factors affecting the p e r f o r m a n c e of proteolytic and amylolytic enzymes in an anionic and nonionic detergent formulation have been studied using stain removal f r o m E M P A blood-milk-ink and cocoa-milk-sugar soil test cloths as a measure of enzyme activity in the detergent solution. Factors considered include enzyme concentration, and t e m p e r a t u r e and p H of the wash solution. Results on stability of these enzymes in the two detergent formulations u n d e r accelerated storage conditions are also given. Introduction I n recent months there has been a n u m b e r of papers and articles in technical journals a n d trade magazines on enzymes as related to detergent products (1-6). I n fact, their n u m b e r closely parallels the large n u m b e r o f enzyme-containing detergent products c u r r e n t l y in p r o p r i e t a r y markets or test markets (6). W h y the sudden p o p u l a r i t y of enzymes and enzyme products? I t appears t h a t with the incorporation of enzymatic activity into household detergent products the housewife is able to see a demonstratable p e r f o r m a n c e benefit in the removal of certain f o r m e r l y stubborn stains and soils (7). M a n y housewives ascribe a more delicate cleaning operation to the new "easy care" fabrics and g a r m e n t s : this has made her more conscious of a need for yet another product in her arsenal of cleaning materials. W i t h the new enzyme products she a p p a r e n t l y visualizes a subtle, gentle, specific and safe removal of soils and stains without resort to more harsh treatment. F u r t h e r , a widespread success experience in E u r o p e has given domestic detergent producers the background and confidence for a r a p i d and aggressive product introduction p r o g r a m in the United States.

INDEX 507-510 ENZYMES IN DETERGENTS,by 1~. J~. Liss and R. P. Langguth 511-514

515-519

]~VALUATION 0E ]~NZYMES FOR LAUNDRY PRODUCTS,

520-522

DEVELOPMENT OF SCREENING TESTS FOR HARD SURFACE CLEANERS: I . ARTIFICIAL SOIL REMOVAL F R O ~

523-528

TABLE I

U n i t s / g r a m o~ enzyme

Protease at p H 10.3 Protease at p H 7.0 a-Amylase at 1)H 6.0

330,000 200,000 7.000

SOME THEORETICAL AND PRACTICAL ASPECTS OF POLYESTER WHITENING FRO~/[ THE W A S H BATH, b y

R. Anliker, H. Heft~, H. Kasperl and B. Milicevie 529-531

A

532-536

ROLE OF VIRUCIDES IN SEMINATION BY FABRICS,

Activity Values for Enzymes Considered in This study

Enzyme A

by Theodore Cayle

LINOLEUM Sm~FACES, by R. R. Adler, T. B. A]bin and B. M. Finger

1 Presented at the A O C S ZIeeting, New York, October, 1968.

]~nzyme

AUTO,fATED :BIOASSAY OF PROTEOLYTIC ENZYI~ES IN

DETERGENTS,by L. M. Paixao, S. ~r. Babulak, S. M. Barkin, D. K. Shumway and S. D. Friedman

Enzyme B

TLC

METHOD

Glen J. Dixon

320.000 1,340,000 310,000

507

FOR IDENTIFICATION

OF GERMICIDES

IN PERSONAL CARE PRODUCTS, by M. B. Graber, I. I. Domsky and M. E. Ginn CONTROLLING VIRUS

DIS-

by Robert W. Sidwell and

5O8

JOURNAL

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T A B L E II Composition of Detergents Used to Study Enzyme

THE

AMERICAN

OIL

CHEMISTS'

SOCIETY

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45

Performance

P e r Cent by W e i g h t

Ingredient

Anionic Sodium tripolyphosphate Alkylbenzene sulfonate Alcohol ethoxylate Sodium metasilicate Carboxymethylcellulose Optical b r i g h t e n e r s Sodium sulfate

40.0 18.0 ...... 6.0 0.7 0.4 26.9 8.0

Water

Nenionic

~.

4O

~

35

.E N

30

40.0 10,0 6.0 0.7 0.4 34,9 8.0

these enzymes, so t h a t m a x i m u m benefits can be obtained f r o m their use. This p a p e r presents the results of studies to determine the effect of certain of these factors on proteolytic and amylolytic enzyme performance. Materials E n z y m e s U s e d in S t u d y

and

25

2o

,

80

,

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TABLE Protease

I

I00 120 140 Wash Temperature, OF. on stain removal

,

,

160

180

from

III

or a-Amylase C o n c e n t r a t i o n From E~IPA-Stained Fabric

on

Stain

Removal

S t a i n removal, AF~d u n i t s Anionic formulation

Enzyme concentration

Enzyme A Total Al~d

Nonionic f o r m u l a t i o n Enzyme B

Al~d due to enzyme

Enzyme A

Enzyme B

Total A R d

ARd due to enzyme

Total A R d

ARd due to enzyme

Total A!~d

ARd due to enzyme

0 10 14 16 17

28 37 42 44 45

0 9 14 16 17

25 36 39 41 42

0 11 14 16 17

25 35 40 41 43

0 10 15 16 18

0 2 5 6

12 22 26 28 30

0 10 14 16 18

7 8 11 12 . . . . . . . .

0 1 4 5

7 16 20 24 26

0 9 13 17 19

Alkaline proteasecasein 0 550 1100 2200 3300

28 38 42 44 45

a-Amylasestarch

units/liter water 0 500 1250 2500 3750

12 14 17 18 . . . . . . . .

EMPA

The most commonly used standard stains for evaluating the functional performance of enzymes in detergent products are the E M P A blood-milk-ink and cocoa-milk-sugar soil test cloths. The blood-milk-ink soil is used to evaluate proteolytic enzymes, whereas the cocoa-milk-sugar stain is responsive to carbohydrase or amylolytic activity. These standard stains, like the standard soil cloths used by m a n y laboratories for detergency testing, do not necessarily give practical results, but rather are used p r i m a r i l y for screening purposes to indicate enzyme activity in a detergent solution. They have been used in this p a p e r to illustrate the effect of washing and formulation variables on enzyme performance. To determine the effect of the variables considered in this study the E M P A - s t a i n e d fabric was washed in solutions containing the detergent u n d e r investigation both with and without the enzyme. The difference in results then is the performance attributable to the enzyme. The specific conditions used in these tests were the following: Terg-O-Tometer speed, 90 r p m ; volume wash solution, 1 liter; wash tinle, 10 min; temperature, 1 2 0 F ; water hardness (3/2 C a / m g ) , 150 p p m ; detergent concentration, 1.5 g / l i t e r ; enzyme concentration, 1100 casein units/liter or 1250 m-amylase units/liter. Stained fabric characteristics were: E M P A No. 116 or No. 112, 3 × 41fi2 in. F o u r swatches were used per wash. These conditions were held constant unless they were one of the variables under study, in which case the values are so indicated.

The compositions of the anionic and nonionic detergents used in this s t u d y are given in Table If. These represent typical l a u n d r y detergent formulations for each of these actives. The anionic active was an average C~3 linear alkylbenzene sulfonate and the nonionic was a C~4-~5 linear alcohol with 12 moles of ethylene oxide.

of

v

Method for Determining Enzyme Performance

Detergent Compositions

Effect

.

60

F I G . 1. E f f e c t o f t e m p e r a t u r e blood-milk-ink stain.

Methods

Protease enzymes used in detergent products are produced generally in fermentation processes with a Bacillus subtilis organism. The two protease enzymes used in these studies were produced f r o m this type of organism. Assay data based on a modified Kunitz casein method for protease activity and a starch digestion method for a-amylase are given in Table I. E n z y m e A is a typical alkaline protease with 330,000 casein units per g r a m when assayed at an alkaline p H of 10.3. The lower activity of 200,000 u n i t s / g at p H 7 indicates the absence of any neutral protease in the enzyme. The low a-amylase value is typical of alkaline protease enzymes of this class. E n z y m e B differs f r o m Enzyme A in that it contains neutral protease and a higher level of a-amylase in addition to a p p r o x i m a t e l y the same amount of alkaline protease. The neutral protease activity of this enzyme can be a p p r o x i m a t e d by subtracting the alkaline protease activity observed at p H 10.3 f r o m the total protease activity of the enzyme at p H 7. Hence, for E n z y m e B the neutral protease activity is approximately 1,000,000 casein units per gram.

units/liter water

46

O C T O B E R 1969

LISS

AND LANGGUTH:

ENZYMES

IN

DETERGENTS

509

16

20
15

o

12

8

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4

0

I

|

6o

8o

1~o

1~o

145

1~'o

I

O

18o

I

.I

7,5

I

80

Wash Temperature, OF.

FIG. 2. E f f e c t of t e m p e r a t u r e on s t a i n r e m o v a l f r o m E M P A cocoa-milk-sugar stain.

Performance Results ~.nzyme Activity

The relationship between proteolytic activity or amylolytic activity per liter of wash water and stain removal (A Rd) from the EMPA-stained fabrics is shown in Table I I I for both the anionic and nonionic detergent formulations. The first column of numbers for each enzyme gives the total stain removal for the detergent and the protease or amylase concentration, i.e., the stain removal due to detergent plus enzyme activity. Considering these results one finds better results are obtained with the anionic formulations for both kinds of enzymes. When the detergent effect (zero enzyme concentration) is subtracted from the total ± Rd, we find the stain removal is essentially the same at any protease or amylase concentration for the two types of detergents. This means that any effect of the surfactant on the enzyme under these wash conditions is the same in each case. Enzyme A, which has a low level of a-amylase compared to Enzyme B, gave a much poorer performance than would be expected when compared to Enzyme B at an equivalent a-amylase activity. Wash Temperature

The effect of wash temperature on milk-ink stain removal performance or Enzyme B in an anionic detergent shown in F i g u r e 1. Both enzymes

E M F A bloodof Enzyme A formulation is had the same

9.10

i

i

8.5 g. 5 10.0 pH Value of Detergent Solution at 120 F.

i

10.5

F l a . 4. E f f e c t of p t I v a l u e o f d e t e r g e n t solution oll perf o r m a n c e of E n z y m e A a n d E n z y m e B on E M P A bloodmilk-ink stain.

performance under these conditions. These data show a large temperature dependency for the protease enzyme. The greatest benefit from the protease enzyme is realized at wash temperatures around 120 F, which is near the average wash temperature used in the home. A t cold water wash temperatures (6070 F ) , the enzyme produces only slightly better stain removal than the detergent alone. Wash temperatures higher than 120 F cause degradation of the enzyme in the presence of the detergent as indicated by the fall-off in enzyme performance. The coincidence of the data points at 160 F indicates that this is the wash temperature at which there is essentially complete inactivation of the enzyme. The effect of wash temperatures on E M P A cocoamilk-sugar stain removal performance of an anionic formulation with and without Enzyme B is given in F i g u r e 2. These curves show, first of all, that removal of the cocoa-milk-sugar stain improves with increasing temperatures up to 120 F. However, the amount of stain removal, primarily attributable to a-amylase activity, is nearly the same over the temperature range from 7 0 - 1 4 0 F . At 160F, there is evidence of some a-amylase degradation. Comparing these results with those in F i g u r e 1 on alkaline protease, we find the a-amylase activity is more stable at the higher wash temperatures than alkaline protease activity and less temperature-dependent.

20

20

16

16

•

~0

,=, 12 o

g

N 8

8

7: 0

I

7.5

I

8.0

i

8.5

~

9.0

i

9.5

i

lO.O

I

10.5

pH Value of Detergent Solution at 120 F.

Fin. 3. E f f e c t of p H value of d e t e r g e n t solution on E n z y m e A p e r f o r m a n c e on E M P A blood-milk-ink stain.

I

i

7.5

8.0

I

L

I

I

8,5 9, 0 g, 5 IO.O pH Value of Detergent Solution at 120 F

i

,

10.5

Fro. 5. E f f e c t of p i t value of d e t e r g e n t solution on E n z y m e B p e r f o r m a n c e on E M P A cocoa-milk-sugar stain.

JOURNAL

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100

i00

Nonionic Detergent

~c 8o

¥OL. 46

SOCIETY

~

nionic

Nonionic

Anionic Detergent

~ 69 N

6(3

4o

I

I

2

4

"~

4C

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2

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Storage rime in Weeks

Storage Time in Weeks

Fro. 6. Storage stability of Enzyme A in an anionic and nonionie detergent formulation.

FIG. 7. S t o r a g e s t a b i l i t y o f a - a m y l a s e i n E n z y m e anionic and nonionic detergent formulation.

Wash Solution pH

formulation may have an inactivation or inhibition effect on a-amylase at p H values below about 9.

The effect of p H on the performance of Enzyme A, an alkaline protease, in an anionic and nonionic detergent formulation is shown in F i g u r e 3. The p H of the detergent solution was adjusted with either sodium hydroxide or sulfuric acid to obtain these data. They show that the enzyme has an apparent greater tolerance to p H in the presence of an anionic detergent. In both instances, the stain removal performance falls off quickly and substantially at p H values greater than 10. An example of the difference in enzyme performance between an alkaline protease (Enzyme A) and an enzyme containing both an alkaline and neutral protease (Enzyme B) at different p H values is given in F i g u r e 4. The data show similar performance for the two enzymes in the p H range of 9.5-10.5, but at lower wash p H values the enzyme containing the neutral protease outperforms the alkaline protease by a large margin. Figure 5 shows the effect of p H on the a-amylase performance in Enzyme B in both an anionic and nonionic detergent. These data show that a greater performance is obtained from the a-amylase in the nonionie detergent formulation than in the anionic formulation over the p H range of 7.5-10.5. The maximum a-amylase performance is obtained at a p H of 8.5 for the nonionic detergent and p H 9.5 for the anionic detergent. Furthermore, the data illustrate that the a-amylase is almost completely inactivated at a p i t of 7.5 in an anionic detergent. This is a surprising result considering that the maximum activity of the a-amylase in the absence of detergent ingredients occurs at a p H value of about 6. Thus, one may conclude that the LAS surfactant in this

B in an

Storage Stability

The storage stability of Enzyme A, an alkaline protease, in an anionic and nonionic detergent formulation stored under accelerated conditions of temperature ( 9 0 F ) and relative humidity (85%) is given in F i g u r e 6. The enzyme was admixed with the detergents to give 1500 casein units of proteolytie activity per gram of detergent. Results of these tests show the alkaline protease to be slightly more stable in the nonionie formulation. However, the loss of proteolytic activity in both cases is not great under the highly exaggerated and aggressive storage conditions, suggesting there should be no problem with stability under ambient storage conditions. The storage stability of the a-amylase portion of Enzyme B m an anionic and nonionic detergent formulation stored under accelerated conditions of temperature and relative hunlidity is shown in Figure 7. The enzyme was d r y mixed with the detergents to give 1600 units of a-amylase activity per gram of detergent. The data show the a-amylase to be more stable in the presence of the anionic detergent, although the loss of activity is small in either case for such aggressive storage conditions. REFERENCES 1. " T h e Enzyme Explosion," Detergent Age 4, 4 7 - 4 9 ( 1 1 9 6 7 ) . 2. Hoogerheide, J. C., " E n z y m e s as Additives to L a u n d r y Detergents," AOCS Meeting, Chicago, October 5967. 3. " E n z y m e s ' Big Change to Clean U p , " Chem. Week, J u n e 22, 89-90 (1968). 4. ~rorne, It. E., Detergent Age 5, 1 9 - 2 2 ( 1 9 6 8 ) . 5. "Will Enzymes Trigger a Detergent Revolution?." Chem. Eng., Sept. 23, 1 0 8 - 1 1 0 ( 1 9 6 8 ) . 6. " B a t t l e of O m a h a Beach," Newsweek, Oct. 7, 8 9 - 9 0 (1968). 7. "Fmzyme-Active L a u n d r y l~roduets, '' Consumer Bull., Oat., 4, 39, 40 ( 1 9 6 8 ) . [Received

March

4, 19691