Method for the preparation of a hydrophobic enzyme-containing composition and the composition produced thereby

A enzyme-containing solid composition is prepared by a process comprising removing the solvent from a mixture of a water-soluble enzyme, a water-insoluble metal salt of a fatty acid and an organic solvent.

FIELD OF THE INVENTION 
The invention relates to a method for the preparation of a composition 
which comprises a hydrophilic enzyme which is modified to impart 
hydrophobic characteristics thereto. The enzyme-containing composition is 
useful for the enzymatic modification of a hydrophobic substrate in an 
aqueous environment. 
BACKGROUND OF THE INVENTION 
Enzymes are proteins which catalyze a wide variety of chemical reactions, 
many of great commercial importance. Enzymes are generally classified 
according to the type of reaction which they catalyze, e.g., hydrolases 
are enzymes that catalyze the addition of the elements of water across the 
bond that is cleaved, e.q., an ester or peptide linkage. 
Commercially-important hydrolases include proteases which are employed in 
laundry detergents, polysaccharidases which control slime in industrial 
process waters, and lipases which are employed to transesterify fats and 
oils. Cellulases and ligninolases can be employed in wood fiber production 
and bleaching. 
The hydrophilicity, or high water-solubility, of many of these enzymes 
often reduces their utility in hydrophobic (lipophilic) media and their 
reactivity with hydrophobic substrates. The biocatalytic reaction is 
inefficient due to phase separation between the two reactants. Either the 
enzyme is introduced into an aqueous phase in which the hydrophobic 
substrate is insoluble, or neither the enzyme nor the hydrophilic 
substrate can dissolve or disperse in the hydrophobic medium. For example, 
the removal of oily or fatty soils from food processing equipment cannot 
be readily accomplished with aqueous lipase solutions due to the lack of 
affinity of the enzyme for the hydrophobic residues. Furthermore, even in 
the presence of stabilizers, the activity of free enzymes often decreases 
rapidly in aqueous media. 
One attempt to partly circumvent these problems involves contacting aqueous 
dispersions or solutions of the target substrate with enzymes which have 
been immobilized by physical adsorption or covalent bonding to 
water-insoluble carriers such as cellulose fibers or silica beads. Enzymes 
have also been immobilized by entrapping them in polymeric matrices. See 
K. Yokozeki et al., European J. Appl. Microbiol. Biotechnol., 14, 1, 
(1982). Circulation of a stream of the substrate or a dispersion thereof 
through a zone containing the immobilized enzyme can reduce losses due to 
the addition of free enzymes to a process water stream. However, 
immobilization or entrapment of enzymes can reduce their activity. 
Furthermore, the substrate matrix can further reduce the efficiency of 
enzyme-substrate contact. 
Therefore, a need exists for a method to selectively decrease the 
water-solubility of hydrophilic enzymes such as hydrolases, and thereby to 
increase the efficiency of enzymatic interaction with hydrophobic 
substrates in aqueous media. 
SUMMARY OF THE INVENTION 
The present invention provides a method for increasing the lipophilicity of 
a water-soluble enzyme, such as a hydrolase, comprising mixing the enzyme 
with the metal salt of a water-insoluble fatty acid in the presence of an 
organic solvent and removing the solvent. A dry solid composition results 
which exhibits reduced hydrophilicity while retaining essentially all of 
its initial enzymatic activity. 
Although the hydrophile-lipophile balance number (HLB) exhibited by the 
present composition is a function of the type and relative amount of the 
fatty acid salt or salts which are combined with the enzyme, the 
compositions are immiscible in water. In the presence of an excess of 
water, the enzyme gradually dissociates from the composition, e.g., over 
about 0.25-3.0 hrs. Although the enzyme composition can be physically 
dispersed in aqueous systems to a limited extent, the dispersion of the 
composition and the release of the enzyme therefrom can be enhanced and 
controlled by incorporating suitable surfactants into the composition. 
Therefore, the composition of the present invention entrains the enzyme in 
an environment which can adjust its HLB so that it is compatible with a 
wide variety of hydrophobic media and/or subtrates. The present invention 
provides a composition that will associate and enzymatically react with 
hydrophobic substrates in aqueous solution, or that will react with 
hydrophilic substrates (starches, celluloses and proteins) entrapped by or 
dispersed in hydrophobic substances in aqueous solution. 
The modified enzymes of the invention are stable, non-toxic and inexpensive 
to prepare. Furthermore, the changes in the molecular structure of enzymes 
which are necessarily involved in immobilization of enzymes by covalent 
bonding are avoided. 
In reference to the present invention, all percentages and parts are by 
weight unless otherwise noted. The term "water insoluble" is defined 
herein to include materials which are essentially or practically 
water-insoluble, e.g., which exhibit only slight water-solubility. 
DETAILED DESCRIPTION OF THE INVENTION 
The present compositions are prepared by mixing one or more hydrophilic 
enzymes with one or more water-insoluble fatty acid metal salts in the 
presence of an organic solvent. Although not intending to be bound by any 
theory of action, it is believed that the solvent causes the carbon chain 
of the fatty acid to unfold. When the solvent is removed from the mixture, 
the chain refolds and intertwines with the enzyme. This interaction may 
act to physically complex the enzyme and the fatty acid, so that the 
fatty acid acts as a hydrophobic carrier for the enzyme. As further 
described hereinbelow, the effective HLB of the resultant solid 
composition can be varied by changing the ratio of enzyme to the fatty 
acid salt, by varying the fatty acid salt carrier, or by optionally 
incorporating various amounts of surfactant into the composition. 
Enzyme 
Any hydrophilic, or water-soluble, enzymes can be employed in the 
composition and method of the present invention, including hydrolases, 
oxidoreductases (glucose oxidase, xanthic oxidase, amino acid oxidase), 
transferases (transglycosidases, transphosphorylases, phosphomutases, 
transaminases, transmethylases, transacetylases), desmolases (ligases, 
lyases) and isomerases (racemases, cis-trans isomerases) and the like. Of 
these enzymes, the hydrolases are preferred for use in the present 
compositions. Hydrolases catalyze a wide variety of hydrolytic reactions, 
including (a) the cleavage of ester linkages (esterases such as lipases, 
phosphoric mono- and di-esterhydrolases such as phosphatases), (b) the 
cleavage of glycosides (carbohydrases such as polysaccharidases, e.g., 
levan hydrolase, cellulase, amylase, ligninolase and the like). (c) the 
cleavage of peptide linkages (proteases such as alpha-aminopeptide amino 
acid hydrolases, alpha-carboxypeptide amino acid hydrolases) and the 
cleavage of nucleic acids (nucleases). 
The hydrolases catalyze the addition of water to the substrate, i.e., the 
soils with which they interact, and thus, generally, cause a breakdown or 
degradation of such a substrate. This is particularly valuable in cleaning 
procedures. Particularly preferred hydrolases are the proteases, 
esterases, carbohydrases and nucleases, with the proteases having the 
broadest range of soil degradation capability. Mixtures of the enzymes may 
be used if desired. 
The proteases catalyzes the hydrolysis of the peptide linkage of proteins, 
polypeptides and related compounds to free amino and carboxyl groups and 
thus break down the protein structure in soil. Specific examples of 
proteases suitable for use in this invention are pepsin, trypsin, 
chymotrypsin, collaqenase, keratinase, elastase, subtilisin, BPN' (a 
bacterial protese derived from Bacillus Subtilis N'), papalin, bromelin, 
carboxy peptidase A and B, amino peptidase, asperqillopeptidase A and 
aspergillopeptidase B. Preferred proteases are serine proteases which are 
active in the neutral to alkaline pH range and are produced from 
microorganisms such as bacteria, fungi or mold. The serine proteases which 
are procured by mammalian systems, e.q., pancreatin, are useful in acidic 
media. 
Esterases catalyze the hydrolysis of an ester, such as lipid soil, to an 
acid and an alcohol. Specific examples of the esterases are gastric 
lipase, pancreatic lipase, plant lipases, phospholipases, cholinesterases 
and phosphatases. Esterases function primarily in acidic systems. 
Carbohydrases catalyze the breakdown of carbohydrate soil. Specific 
examples of this class of enzymes are maltase, saccharase, amylases such 
as alpha-amylase and amyloglucosidase, cellulase, pectinase, lysozyme, 
.alpha.-glycol-sidase and .beta.-glycosidase. They function primarily in 
acidic to neutral systems. 
The commercially-available enzyme products are useful and are generally dry 
powdered products comprising 2% to 80% of active enzymes in combination 
with an inert powdered vehicle such as sodium, ammonium or calcium 
sulphate or sodium chloride, clay or starch as the remaining 98-20%. 
Active enzyme content of a commercial product is a result of manufacturing 
methods employed and is not critical herein so long as the final complex 
has the desired enzymatic activity. For an extensive listing of 
commercially-available hydrolases, see Sigma Chemical Company Catalog of 
Biochemical and Organic Compounds, St. Louis, MO (February, 1986) at pages 
33-34, the disclosure of which is incorporated by reference herein. 
Preferred enzymes for incorporation in the present compositions include 
esterases, carbohydrases or mixtures thereof. 
Fatty Acid Salt 
The water-insoluble fatty acid metal salts useful in the present invention 
can be represented by the general formula: 
EQU (RCO.sub.2.sup.-).sub.y (.sup.- OH).sub.x (M.sup.x+v) 
wherein R is an about C.sub.6 -C.sub.30 alkyl group, preferably a C.sub.8 
-C.sub.22 alkyl group, wherein about 0-3 double bonds are present in the 
alkyl group; x is 0 or a natural number, y is a natural number and x+y is 
the valency of the metal (M). Preferably, x is 0-2 and y is 1-3, most 
preferably x is 0-1. 
Therefore, a suitable fatty acid anion group (RCO.sub.2.sup.-) is 
palmitate, strerate, oleate, myristate, cocoate, laurate, caprylate, 
undecylenate, myristolenate, palmitolenate, petroselate, erucate, 
grassidate, geranate, linoleate, linolenate and the like. For other useful 
monobasic aliphatic fatty acids, see Organic Chemistry, F. C. Whitmore, 
ed., Dover Pubs., Inc., NY, (2d ed., 1951), the disclosure of which is 
incorporated by reference herein. 
Any metal cation can be employed in the salt as long as the cation is 
chosen so that the metal salt of the fatty acid is water-insoluble. 
Examples of the metal cations that may be employed are the alkaline earth 
metal cations such as Ca.sup.+2 and Mq.sup.+2, and Al.sup.+3. Of these 
three, Al.sup.+3 is preferred because its fatty acid salts are more 
hydrophobic than the fatty acid salts of Ca.sup.+2 and Mq.sup.+2. 
Commercially-available fatty acid salts of these metals include magnesium 
oleate and stearate, aluminum stearate, palmitate and oleate, and calcium 
stearate, palmitate and oleate, as well as the hydroxylated derivatives 
thereof. More particularly, it has been found that Al(OH) 
(stearate).sub.2, hereinafter referred to as aluminum distearate, and 
Al(OH) (oleate).sub.2, hereinafter referred to as aluminum dioleate, may 
be very advantageously employed in the method and composition of the 
present invention. 
Organic Solvent 
The organic solvent must be chosen so that (1) it will not appreciably 
denature the enzyme and (2) it will substantially dissolve the fatty acid 
salt. The enzyme compatibility with a driven solvent can be readily 
determined by one skilled in the art without undue experimentation by 
adding a portion of solvent to the medium during an enzyme assay, such as 
the assays described hereinbelow, and measuring the decrease, if any, in 
enzymatic activity as compared to the activity determined by an assay 
performed without the addition of the solvent. 
More particularly, the organic solvent may be selected from the group 
consisting of alcohols, ketones, aromatics, alkyl halides and ethers. 
Useful alcohols include 3-methyl-3-hexanol, 2-octanol, tert-butanol, 
n-butanol, 2-methylcyclopentanol, n-propanol, isopropanol, ethanol, 
geraniol, n-hexadecanol, n-decanol, or n-heptanol. Useful ketones include 
methyl ethyl ketone or acetone. Useful aromatic solvents include benzene, 
pyridine, aniline, or toluene; useful alkyl halides include carbon 
tetrachloride, chloroform, or methylene chloride; and useful ethers 
include diethyl ether, methyl ethyl ether, diphenyl ether, or anisole. 
Surfactant 
A surfactant or wetting agent may be included in the composition of the 
present invention in a weight amount from 0% to about 10% based on the 
combined weight of the enzyme and fatty acid salt. The optional surfactant 
must be chosen so that it will not appreciably denature the enzyme. 
Whether or not it exhibits an inhibitory effect on the enzyme can be 
easily determined by one skilled in the art without undue experimentation. 
The surfactant counteracts the water repellency of the fatty acid salt. 
Thus, the degree of water dispersibility of the complex can be varied 
depending on the amount of surfactant which is incorporated therein. 
Furthermore, when a surfactant is incorporated in the composition, the 
dissociation rate of the complex is increased and thus the rate of 
solubility of the enzyme in the water increases. A complex without any 
surfactant incorporated therein may take up to an hour or more for about 
half of the enzyme to leach therefrom when the complex is quiescent in 
water. On the other hand, incorporation of as little as about 1-2% by 
weight of a surfactant based on the combined weight of enzyme and fatty 
acid salt may cause essentially all of the enzyme to leach from the 
complex into the water in about one hour or less. 
Of the various classes of surfactants, nonionic and anionic surfactants or 
mixtures thereof are preferred for use in the present invention. 
Preferred nonionic surfactants include the condensation products of 
ethylene oxide with a hydrophobic polyoxyalkylene base formed by the 
condensation of propylene oxide with propylene glycol. The hydrophobic 
portion of these compounds has a molecular weight sufficiently high so as 
to render it water-insoluble. The addition of polyoxyethylene moieties to 
this hydrophobic portion increases the water-solubility of the molecule as 
a whole, and the liquid character of the product is retained up to the 
point where the polyoxyethylene content is about 50% of the total weight 
of the condensation product. Examples of compounds of this type include 
certain of the commercially-available Pluronic.TM. surfactants (BASF 
Wyandotte Corp., Parsippany, NJ), especially those in which the 
polyoxypropylene ether has a molecular weight of about 1500-3000 and the 
polyoxyethylene content is about 35-55% of the molecule by weight, i.e., 
Pluronic.TM. L-62. 
Other preferred nonionic surfactants include the condensation products of 
C.sub.8 -C.sub.22 alkyl alcohols with 2-50 moles of ethylene oxide per 
mole of alcohol. Examples of compounds of this type include the 
condensation products of C.sub.11 -C.sub.15 fatty alkyl alcohols with 
about 3-45 moles of ethylene oxide per mole of alcohol which are 
commercially-available as the Poly-Tergent.TM. SLF series from Olin 
Chemicals or the Tergitol.TM. series from Union Carbide, i.e., 
Tergitol.TM. 15-S-20, 15-S-12, and 15-S-15, which are formed by condensing 
a C.sub.11 -C.sub.15 -fatty alcohol mixture with an average of 20, 12 and 
15 moles of ethylene oxide, respectively. These compounds are also 
available from Shell Chemical Co. as Neodol.TM. 25-3, 25-7 and 25-9, which 
are the condensation products of C.sub.12 -C.sub.15 fatty alkyl alcohols 
with about 3, 7 and 9 moles of ethylene oxide, respectively. 
Other nonionic surfactants which may be employed include the ethylene oxide 
esters of C.sub.6 -C.sub.12 alkyl phenols such as 
(nonylphenoxy)polyoxyethylene ether. Particularly useful are the esters 
prepared by condensing about 8-12 moles of ethylene oxide with 
nonylphenol, i.e. the Igepal.TM. CO series (GAF Corp., New York, NY). 
Another useful class of nonionic surfactant is the silicone-glycol 
copolymers. These surfactants are prepared by adding poly(lower)alkylenoxy 
chains to the free hydroxyl groups of dimethylpolysilioxanols and are 
available from the Dow Corning Corp. as Dow Corning 190 and 193 
surfactants (CTFA name: dimethicone copolyol). 
Other useful nonionics include the ethylene oxide esters of alkyl 
mercaptans such as dodecyl mercaptan polyoxyethylene thioether, the 
ethylene oxide esters of fatty acids such as the lauric ester of 
polyethylene glycol and the lauric ester of methoxypolyethylene glycol, 
the ethylene oxide ethers of fatty acid amides, the condensation products 
of ethylene oxide with partial fatty acid esters of sorbitol such as the 
lauric ester of sorbitan polyethylene glycol ether, and other similar 
materials, wherein the mole ratio of ethylene oxide to the acid, phenol, 
amide or alcohol is about 5-50:1. 
Useful anionic surfactants include the ammonium and alkali metal salts of 
sulfated ethylenoxy fatty alcohols (the sodium or ammonium sulfates of the 
condensation products of about 1-4 moles of ethylene oxide with a C.sub.8 
-C.sub.22 fatty alcohol, such as a C.sub.12 -C.sub.15 n-alkanol, i.e., the 
Neodol.TM. ethoxysulfates, such as Neodol.TM. 25-3S, Shell Chemical Co.; 
n-C.sub.12 -C.sub.15 -alkyl(OEt).sub.3 OSO.sub.3 Na; and Neodol.TM. 25-3A, 
the corresponding ammonium salt. 
Another useful class of anionic surfactants encompasses the water-soluble 
sulfated and sulfonated anionic ammonium, alkali-metal and alkaline earth 
metal detergent salts containing a hydrophobic higher alkyl moiety 
(typically containing from about 1 to 22 carbon atoms), such as salts of 
alkyl mono or polynuclear aryl sulfonates having from about 1 to 16 carbon 
atoms in the alkyl group (e.g., sodium toluene sulfonate, sodium xylene 
sulfonate, sodium dodecylbenzenesulfonate, magnesium 
tridecylbenzenesulfonate, lithium or potassium 
pentapropylenebenzenesulfonate). These compounds are available as 
Nacconol.TM. 35 SL (Stephan Chemical Co., Northfield, IL, sodium 
dodecylbenzene sulfonate) or as Stephanate.TM. X (sodium xylene sulfonate) 
or Stephanate.TM. AM (ammonium xylene sulfonate, Stephan Chemical Co.). 
The alkali metal salts of alkyl naphthalene sulfonic acids (methyl 
naphthalene sulfonates) are available as Petro.TM. AA. Petrochemical 
Corporation. 
Also useful are the sulfated higher fatty acid monoglycerides such as the 
sodium salt of the sulfated monoglyceride of coconut oil fatty acids and 
the potassium salt of the sulfated monoglyceride of tallow fatty acids; 
alkali metal salts of sulfated fatty alcohols containing from about 10 to 
18 carbon atoms (e.g., sodium lauryl sulfate and sodium stearyl sulfate); 
sodium C.sub.14 -C.sub.16 -alpha-olefin sulfonates such as the 
Bio-Terge.TM. series (Stephan Chemical Co.); alkali metal salts of higher 
fatty esters of low molecular weight alkyl sulfonic acids, e.g., fatty 
acid esters of the sodium salt of isethionic acid; the fatty ethanolamide 
sulfates; the fatty acid amides of amino alkyl sulfonic acids, e.g., 
lauric acid amide of taurine, and the alkali metal salts of sulfosuccinic 
acid esters, e.g., dioctyl sodium sulfosuccinate (Monawet.TM. series, Mona 
Industries, Inc., Patterson, NJ). 
Preparation 
The method of the present invention may be accomplished by mixing, in any 
order, the enzyme, the fatty acid salt, the solvent, and the surfactant if 
any, followed by the removal of the solvent. In the preferred method of 
the present invention, in order to ensure homogeneous mixing, one or more 
enzymes are first dry blended with the fatty acid salt, and then the dry 
blend is dispersed in the solvent. The dry blending may be achieved using 
commercially-available equipment. An example of such an apparatus in the 
P-K Twin Shell.TM. laboratory shaker manufactured by Patterson-Kelley Co., 
Inc., East Stroudsburg, Pa. There is no particular length of time required 
for conducting the mixing so long as the enzyme and the fatty acid salt 
components are thoroughly blended. Typically, the mixing is conducted from 
about 5 minutes to about 60 minutes, and preferably from about 15 minutes 
to about 45 minutes. 
Preferably the weight ratio of enzyme component to fatty acid salt in the 
mixture is about 10-0.1:1, most preferably about 20-0.5:1. For example, an 
enzyme:fatty acid salt ratio of a 1.0-0.5:1.0 is preferred when aluminum 
dioleate is employed as the fatty acid salt, and a ratio of 1.5-1.0:1 is 
preferred when aluminum distearate is employed. 
The volume of solvent is determined on a v/w (volume/weight) ratio based on 
the combined weight of the enzyme and the fatty acid salt. Sufficient 
solvent must be employed to wet the enzyme and fatty acid salt. This can 
require a solvent volume/weight ratio of at least about 1:1 v/w. or more, 
e.g., about 10-1:1, preferably about 5-1:1. For example, when the solvent 
is acetone and the fatty acid salt is aluminum dioleate, an about 2-1:1 
v/w ratio of solvent to enzyme and salt can be employed. On the other 
hand, when aluminum distearate is employed with acetone, an about 
2.5-1.5:1 v/w ratio of solvent to enzyme and salt can be employed. 
In order to ensure homogeneous distribution of the optional surfactant 
throughout the complex, the surfactant may be premixed in the solvent so 
that the solvent contains surfactant in an amount of up to about 10% by 
weight based on the combined weight of enzyme and fatty acid salt 
components. The surfactant may also be added during or after the mixing of 
the enzyme, the fatty acid salt, and the solvent. 
Preferably, the surfactant is employed in an amount from about 0.1-5% by 
weight of the enzyme-fatty acid salt mixture, most preferably about 
0.5-5%. For example, it has been found that when aluminum dioleate is 
employed, about 1% to 2% surfactant is preferably employed, whereas when 
the fatty acid salt is aluminum distearate, about 1% to 4% surfactant is 
preferably employed. 
Following admixture of the enzyme, fatty acid salt, solvent and any 
surfactants, the solvent is removed. For example, solvent can be 
evaporated via a rotary vacuum dryer equipped with an internal scraper. 
Preferably, the solvent removal step is accomplished at ambient 
temperatures, in order to avoid thermal deactivation of the enzyme. 
Removal of the solvent yields a solid which may be ground in a general 
purpose mill to yield a finer granulation. No particular particle size is 
necessary for the finished composition, as it is only desirable to provide 
a substantially uniform particle size. 
The invention will be further described by reference to the following 
detailed examples, wherein the enzyme mixture used in Examples I-IV was a 
1:1 mixture of New Sumyzyme.TM. and Lipase-MY.TM.. New Sumyzyme.TM. (Shin 
Nihon Chemical Company, Japan) is a dry granulation of amyloglucosidase 
(AG) produced by Rizopus species. Lipase-MY.TM.. (Meito Sangyo Company, 
Japan) is a dry granulation of lipase produced by Candida cylindracea. 
Equal amounts by weight of the two enzymes were mixed for 30 minutes using 
a P-K Twin Shell.TM. Laboratory shaker apparatus (manufactured by 
Patterson-Kelley Co., Inc., East Stroudsburg, Pa.). 
The aluminum dioleate (Alumagel.TM.) and aluminum distearate (Aluminum 
Stearate #22.TM.) used in the Examples hereinbelow were supplied by Witco 
Chemical Corporation Organics Division, Chicago, Ill. 
In the Examples hereinbelow, the activity of the amyloglucosidase (AG) was 
measured using the Diazyme.TM. Assay Method, Miles Laboratories, Inc. 
Technical Bulletin No. L-1042. This assay is based on the Schoorl method, 
a copper reduction method employing Fehling solution. One Diazyme.TM. Unit 
(DU) is that amount of amyloglucosidase that will liberate 1 g of reducing 
sugar as glucose per hour under the conditions of the assay. The activity 
was calculated as DU/g of amyloglucosidase unless otherwise indicated. 
The activity of the lipase was measured using the Esterase Assay Method, 
Miles Laboratories, Inc., Technical Bulletin No. MM-800.17, which is based 
on the method described in Food Chemicals Codex, 3rd ed., National 
Academic Press, Washington, D.C. (1981). One Esterase Unit (EU) is defined 
as the activity that releases 1.25 micromoles of butyric acid per minute 
under the conditions of the assay. The activity was calculated as EU/g of 
lipase unless otherwise indicated.

EXAMPLE I 
Amyloglucosidase/Lipase Mixture Modified with Aluminum Dioleate 
A mixture of 75 g of New Sumyzyme.TM. amyloglucosidase (AG) and 
Lipase-MY.TM. lipase in a weight ratio of 1:1 was dry blended with 100 g 
of aluminum dioleate employing a P-K Twin Shell shaker for 30 minutes. 
Acetone (263 ml) was added to the resultant dry blend with stirring. The 
resultant slurry was then dried in a rotary vacuum dryer equipped with an 
internal scraper at room temperature for 60 minutes. The acetone solvent 
evaporated and was recovered by a condenser that had been attached to the 
vacuum line of the dryer. 
The dried enzyme-aluminum dioleate complex was then around in a general 
purpose mill to a particle size of about 20 mesh on the U.S. mesh series 
(about 850 micometers) to rid the product of lumps and provide a fine 
granulation. About 175 g of the finished composition were recovered. 
The resultant granules exhibited an activity of: 
Amyloglucosidase--55 DU/g of granules, 
Lipase--320 EU/g of granules. 
EXAMPLE II 
Amyloglucosidase/Lipase Mixture Modified with Aluminum Dioleate and 
Surfactant 
Example I was repeated except that Neodol.TM. 25-3, an ethoxylated nonionic 
surfactant supplied by Shell Chemical Company, was added to the acetone by 
thoroughly mixing 1.75 g of the surfactant and 263 ml of acetone. The 
amount of the surfactant used was 1% of the 175 g weight of dry blend of 
enzymes and aluminum dioleate. The surfactant-containing acetone was used 
for slurrying the dry blend. Evaporation of the acetone provided a dry 
composition including the enzymes, aluminum dioleate and surfactant. 
After grinding the composition on the general purpose mill to rid it of 
lumps, about 175 g of fine dry granules were recovered. The resultant 
granules exhibited an activity of: 
Amyloglucosidase--55 DU/g of granules, 
Lipase--320 EU/g of granules. 
EXAMPLE III 
Amyloglucosidase/Lipase Mixture Modified with Aluminum Distearate 
The procedure of Example I was repeated, wherein the enzyme mixture of New 
Sumyzyme.TM. and Lipase-MY.TM. was dry blended on the P-K Twin Shell 
shaker with aluminum distearate on a 1.0:0.75 dry weight/weight ratio for 
30 minutes. Thus, 100 g of enzyme mixture and 75 g aluminum distearate 
were combined to yield 175 g of dry blend which was then slurried in 350 
ml of acetone. After slurrying, the acetone was evaporated. 
After grinding the composition on the general purpose mill to rid it of 
lumps, fine dry granules of product were recovered. The resultant granules 
had an activity of: 
Amyloglucosidase--74 DU/g of granules, 
Lipase--425 EU/g of granules. 
EXAMPLE IV 
Amyloglucosidase/Lipase Mixture Modified with Aluminum Distearate and 
Surfactant 
The procedure of Example III was repeated except that the 175 g dry blend 
of enzymes-aluminum distearate was slurried in 350 ml of a solution of 3.5 
g of Neodol.TM. 25-3 in 350 ml of acetone. The amount of surfactant used 
was 2% of the 175 g weight of the dry blend of aluminum distearate and 
enzymes. After slurrying, the acetone was evaporated to provide a dry 
solid which was milled to afford about 175 g of fine granules of product. 
The resultant granules had an activity of: 
Amyloglucosidase--74 DU/g of granules, 
Lipase--425 EU/g of granules. 
EXAMPLE V 
Comparative Enzyme Release 
A. Amyloglucosidase Modified with Aluminum Dioleate and 0 to 2% Surfactant 
Five portions of a dry blend of 175 g of New Sumyzyme.TM. (AG) and aluminum 
dioleate, on a 1.0:0.75 w/w ratio basis of enzyme to fatty acid salt were 
each slurried in 263 ml portions of acetone. The amount of Neodol.TM. 
surfactant pre-mixed into the five 263 ml aliquots of acetone was 0, 0.25, 
0.5, 1.0 and 2.0% of the weight of the dry blend. 
The enzymatic activity of samples of each of the resultant granular 
products was measured by adding eight 0.4 g samples of product to 9.6 ml 
portions of water. Samples 1-4 were left quiescent for 10, 20, 40 and 60 
minutes, respectively, and samples 5-8 were mixed by shaking for 10 
minutes on a Vortex-Genie.TM. apparatus (Scientific Industries, Inc., 
Bohemia, NY) at increasing speeds represented by settings No. 2, No. 4, 
No. 6 and No. 8 of the apparatus, respectively. 
After the specified contact time, each of the eight samples was then 
filtered using Sharkskin.TM. analytical filter paper (Schleicher and 
Schuell, Inc., Keene, NH). The variance among the samples in the rate of 
extraction or leaching of the enzyme from the complex into the water was 
ascertained by measuring the activity of each filtrate, using the Diazyme 
Assay Method. The results are summarized on Table A, below. 
TABLE A 
______________________________________ 
Enzymatic Activity of Aluminum Dioleate-Modified AG 
Compositon at Various Levels of Surfactant 
Static 
Sample Contact Neodol .TM. 25-3 Surfactant 
No. Time 0% 0.25% 0.5% 1.0% 2.0% 
______________________________________ 
1 10 min. 70.6* 133.7 203.9 
212.8 
237.8 
2 20 min. 94.9 158.0 214.7 
232.8 
247.5 
3 40 min. 112.5 176.9 221.7 
242.9 
259.9 
4 60 min. 126.5 195.8 240.5 
254.5 
266.0 
Mixing 
(10 min.) 
at Speed 
(No.) 
5 2 186.0 197.2 240.1 
248.3 
268.1 
6 4 198.3 207.9 248.3 
257.4 
277.4 
7 6 207.4 228.4 259.8 
271.8 
286.7 
8 8 234.9 240.5 273.9 
277.7 
295.4 
AG Control 
-- 260.0 -- -- -- -- 
Solution 
______________________________________ 
*Activity of the composition (DU/q of AG) at various levels of surfactant 
was measured by determining the activity of the AG solubilized from the 
compositions at the given solubilization conditions. 
The data summarized on Table A establish that the aluminum dioleate 
substantially retards the rate of solubilization of the AG enzyme into an 
aqueous medium. For example, a sample of complex which did not include 
surfactant yielded an aqueous phase having only about 27% of the enzymatic 
activity of a solution of the same amount of the free enzyme. 
Furthermore, the rate of enzyme solubilization increased if either the 
mixing speed or the percentage of Neodol.TM. surfactant is increased. For 
instance, the activity of the sample with no Neodol.TM. mixed at a fast 
speed (setting No. 8) was about the same as the activity of the sample 
with 1% Neodol.TM. which was exposed to water with no agitation for 20 
minutes. 
Although the enzymatic activity of the aqueous phase could be increased by 
stirring the complex, 10 minutes of stirring at setting No. 8 was required 
to increase the activity of the aqueous phase to 90% of the activity 
exhibited by the control solution. 
Addition of the surfactant substantially increased the rate of release of 
enzyme from the complex. For example, the addition of about 0.25-2.0% of 
Neodol.TM. 25-3 resulted in release of a major proportion of the activity 
after 10 minutes, whether or not the samples were agitated. Even static 
contact resulted in the substantially complete release of the enzymatic 
activity after 0.6-1.0 hour in the case of samples containing 1-2% of 
surfactant. 
Thus, on a large industrial scale where physical mixing to increase the 
rate of dispersion of the enzyme may not be feasible, employing a 
surfactant in the present compositions with no mixing can achieve 
substantially the same result as employing the surfactant-free composition 
in an agitated aqueous medium. 
B. Amyloglucosidase Modified with Aluminum Distearate and 0 to 4% 
Surfactant 
The procedure of Example V(A) was repeated employing New Sumyzume.TM. (AG) 
and aluminum distearate in a 1.0:0.75 w/w ratio basis of enzyme to fatty 
acid salt. Thus, to prepare each sample, 175 g of the 
amyloglucosidase-aluminum distearate mixture were slurried in 350 ml 
aliquots of acetone. Also, the amount of Neodol.TM. 25-3 surfactant 
premixed into the acetone was varied incrementaly from 0% to 4% of the 
weight of dry blend of AG and aluminum stearate. As in Example V, the 
activity of each filtrate was measured by the Diazyme Assay Method. 
The results are summarized in Table B below. 
TABLE B 
______________________________________ 
Enzymatic Activity of Aluminum Distearate-Modified AG 
Composition at Various Levels of Surfactant 
Sam- Static 
ple Contact Neodol .TM. 25-3 Surfactant 
No. Time 0% 0.25% 0.5% 1.0% 2.0% 3.0% 4.0% 
______________________________________ 
1 10 min. 32.0* 41.8 31.5 50.4 153.5 
184.3 
218.9 
2 20 min. 38.3 47.9 40.4 56.5 177.3 
207.7 
231.0 
3 40 min. 43.8 51.6 48.7 61.1 239.6 
249.2 
250.6 
4 60 min. 26.5 61.3 57.1 66.3 270.4 
267.9 
260.1 
Mixing (10 min.) at Speed (No.) 
5 2 77.5 68.1 94.2 
101.3 
278.6 
270.2 
278.9 
6 4 127.9 137.2 137.0 
144.4 
289.1 
281.4 
288.6 
7 6 142.6 184.6 186.0 
173.8 
295.1 
290.2 
298.2 
8 8 195.1 229.4 231.0 
244.0 
301.5 
301.8 
298.7 
______________________________________ 
*Activity of the composition (DU/g of AG) at different levels of 
surfactant was measured by determining the activity of the AG solubilized 
from the composition at the given solubilization conditions. 
The data summarized in Table B also demonstrate that the rate of enzymatic 
release increased as the percentage of surfactant increased and/or the ten 
minute mixing speed increased. However, the aluminum distearate-containing 
samples prepared in accordance with this example required more Neodol.TM. 
surfactant than did the aluminum dioleate-containing samples assayed in 
Example V(A) (2% Neodol.TM. surfactant for aluminum distearate modified AG 
versus 1% Neodol.TM. surfactant for aluminum dioleate modified AG) to 
obtain an equivalent degree of solubility. 
EXAMPLE VII 
Properties of AG/Lipase Modified with Aluminum Dioleate 
The compositions of Example I are evaluated with respect to their ability 
to degrade greasy soil as follows. First, 20 grams of greasy material from 
a grease trap is placed in 100 ml of 30.degree. C. water in a 200 ml 
beaker. Then, 0.25 g of the composition of Example I are added thereto 
with stirring. For comparison, a sample containing 20 grams of the greasy 
substance and 0.11 g of unmodified AG/lipase (0.11 g is used since the 
0.25 g of modified AG/lipase comprises about 0.11 g of enzymes and about 
0.14 g of salt) in 100 ml of 30.degree. C. water is also prepared. The 
test mixtures and the control mixture (no enzyme) are maintained at 
30.degree. C. In the sample beakers, the modified enzyme-containing 
granules immediately associate with the layer of the oil, while the free 
enzymes dissolved in the water. A lipolytic reaction in the sample beakers 
is noticeable in about 24 hours and thus both beakers (modified and free 
enzyme) are visually evaluated after 1 day and 2 days. The results of this 
study are summarized in Table C below. 
TABLE C 
______________________________________ 
Comparative Degradation of Oil 
Sample 24 Hours Two Days 
______________________________________ 
ControI a. All the greasy a. Same 
substance is 
settled on bottom 
of beaker. 
b. Supernatant is b. Same 
clear. 
Free a. Some sediment is 
a. Less sediment is 
Enzyme- on bottom of on bottom of 
Containing beaker. beaker. 
Sample 
b. There is a loosely 
b. There is some 
packed surface packing of upper 
layer of greasy layer with more 
substance having gas bubbles. 
small gas bubbles. 
c. Supernatant is c. Supernatant is 
slightly turbid. turbid. 
Lipophilic 
a. Some sediment is 
a. Less sediment is 
Enzyme- on bottom of on bottom of 
Containing beaker. beaker. 
Composition 
b. There is a packed 
b. There is more 
surface layer of packing of upper 
greasy substance layer with more 
having gas bubbles. 
gas bubbles. 
c. Supernatant is c. Supernatant is 
turbid. more turbid. 
______________________________________ 
The data summarized on Table C indicate that the lipase-containng 
composition of the present invention can substantially degrade soil 
comprising fat and grease in aqueous media under static conditions, and 
can do so more effectively than an equivalent amount of enzymes which are 
simply dissolved in the medium. It is believed that this result is due 
both to the enhanced association between the hydrophobic fatty-acid enzyme 
complex and the gradual release of the enzyme therefrom, which effectively 
enhances its stability. 
While certain representative embodiments of the invention have been 
described herein for purposes of illustration, it will be apparent to 
those skilled in the art that modifications therein may be made without 
departing from the spirit and scope of the invention.