Cleaning composition comprising microbial lipase SD2, sodium dodecylbenzene sulfonate and gelatin

This invention is directed to a detergent composition comprising the microbial lipase SD2, dodecylbenzene sulfonate, and gelatin. In the detergent composition, the lipase SD2 is characterized by having (i) optimum pH for washing activity of about 8.+-.0.5; (ii) an optimum temperature for activity of about 30+ to 55.degree. C. and (iii) a molecular weight as measured by gel permeation chromatography of about 8.8.times.10.sup.4.

The invention herein described relates generally to a new detergent 
composition, and more particularly a composition suitable for use in 
laundry and/or dishwashing applications. 
By way of background, dodecylbenzene sulfonate ("DBS") is a commonly used 
surfactant employed in household detergents. It is considered low-cost, 
safe and effective. Because of dodecylbenzene sulfonate's wide-spread 
usage in cleaning products, compatibility with and efficacy in the 
presence of this surfactant is an important consideration in the 
evaluation of new detergent additives. 
Recently, lipases have become of interest as laundry detergent additives. 
By way of illustration, Novo Industri A/S has recently introduced into the 
marketplace a lipase referred to as LIPOLASE. However, the present 
inventors have found that LIPOLASE is not as effective as might be desired 
in performing its function of breaking down lipids into fatty acids, 
particularly in the presence of DBS when formulated into dodecylbenzene 
sulfonate-containing laundering formulations. 
In view of the above, new lipase compositions exhibiting enhanced cleaning 
efficacy and/or lipase stability in the presence of dodecylbenzene 
sulfonate would be highly desired by the detergent manufacturing 
community. 
In one aspect, the present invention relates to a detergent composition 
comprising the microbial lipase SD2, sodium dodecylbenzene sulfonate, and 
gelatin. In the detergent, the lipase is characterized by having (i) an 
optimum pH for washing activity of about 8.+-.0.5; (ii) an optimum 
temperature for activity of about 30.degree. to 55.degree. C. and (iii) a 
molecular weight as measured by gel permeation chromatography of about 
8.8.times.10.sup.4. This and other aspects will become apparent from a 
reading of the following detailed specification. 
The present inventors have isolated a biologically pure culture of a 
previously undescribed strain of Pseudomonas alcaligenes, strain SD2, as 
disclosed and claimed in co-pending, commonly-assigned U.S. application 
Ser. No. 324,062, incorporated herein by reference in its entirety. The 
organism is a natural isolate and has been deposited with the American 
Type Culture Collection (ATCC), having been assigned the accession number 
ATCC 53877. This novel strain SD2 was found to produce a novel lipase. 
The microorganism, P. alcaligenes, strain SD2, was isolated from a shower 
drain by direct isolation on a Tryptone-Soytone-Olive oil isolation 
medium. The isolation medium employed is more fully described in Table I 
below. 
TABLE I 
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Isolation Medium 
Percent by Weight 
______________________________________ 
Ammonium sulfate 0.5 
Potassium phosphate, dibasic 
0.05 
Magnesium sulfate, heptahydrate 
0.025 
Tryptone (Difco) 1.7 
Soytone (Difco) 0.3 
Olive oil 1.0 
Rhodamine B 0.001 
Agar 1.5 
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The Rhodamine B dye in the isolation medium causes lipase-producing 
bacterial colonies to fluoresce an orange color when irradiated with long 
wavelength ultraviolet light (Kouker, G. and K.-E. Jaeger, 1987, Appl. 
Environ. Microbiol., 53 211-3). This fluorescence permits the easy 
identification of lipase-producers. Colonies so identified were purified 
by restreaking onto similar media. Stock cultures were maintained on Difco 
TSA slants. 
The bacterial isolate was identified using standard taxonomic procedures 
from Bergey's Manual of Systematic Bacteriology (Williams & Wilkins, 
Baltimore, 1984). The results of applicable physiological characterization 
tests of P. alcaligenes strain SD2 are presented in Table II and compared 
with characteristics of P. alcaligenes and P. pseudoalicaligenes published 
in Bergey's Manual. 
TABLE II 
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Substrate Utilization of P. alcaligenes 
Strain SD2, P. alcaligenes, and P. pseudoalicaligenes 
Strain* 
SD2 P. alcaligenes 
P. pseudoalicaligenes 
______________________________________ 
Fructose - - + 
L-aspartate 
+ - - 
L-glutamate 
- + + 
D-gluconate 
- - d 
L-Histidine 
- d d 
Ethanolamine 
- - + 
n-Butanol 
- d + 
Isobutanol 
+ d - 
Citrate - d d 
Betaine - - + 
Glycerol - - d 
Sorbitol - - d 
Itaconate 
- - d 
______________________________________ 
Abbreviation: d (11-80 percent of strains positive); + (strain was able t 
utilize the indicated chemical for growth); - (strain did not utilize the 
chemical for growth). 
*Data for P. alcaligenes and P. pseudoalicaligenes are from Bergey's 
Manual of Systematic Bacteriology (Williams & Wilkins [Baltimore, 1984]). 
Compounds utilized by all strains include: DLlactate, succinate, fumarate 
acetate, Larginine, caprate, and Lmalate. 
Compounds not utilized by any strain include: Dglucose, Larabinose, 
Dmannose, Dmannitol, Lrhamnose, D(+)galactose, D(-)ribose, minositol, 
Lthreonine, mtartrate, adipate, phenylacetate, nicotinate, sebacate, 
suberate, benzoate, and pimelate. 
This table illustrates nutritional capabilities of the indicated strains 
and further illustrates their differences. 
Several lipase-producing strains of P. pseudoalicaligenes are disclosed in 
International Publication No. WO87/00859 published under the Patent 
Cooperation Treaty. Table III presents certain morphological and 
physiological characteristics of P. alcaligenes strain SD2, as compared to 
the characteristics of four strains of P. pseudoalicaligenes disclosed in 
International Publication No. WO 87/00859. Differences between the SD2 
strain of the present invention and the other strains are readily 
apparent. For example, SD2 utilized L-aspartate, while the two other 
Psudomonas species did not, as noted noted in Table II. 
TABLE III 
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Characteristics of P. alcaligenes Strain SD2 and 
Selected Lipase-Producing Strains of P. pseudoalicaligenes. 
(The CBS Strain Accession Numbers Correspond to Those 
Referenced in International Publication No. WO 87/00859) 
Strain 
of Comparison Strains 
Invention CBS CBS CBS CBS 
Characteristic 
SD2 467.85 468.85 471.85 
473.85 
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Cell shape 
rod rod rod rod rod 
Motility + + + + + 
Spores - - - - - 
Gram strain 
- - - - - 
Oxidase + + + + + 
Anaerobic 
- - - - - 
glucose 
Aerobic - - - - - 
glucose 
Aerobic - - - - - 
maltose 
Aerobic - - - - - 
sucrose 
Aerobic - - - - + 
D-xylose 
Arginine + + + - + 
dyhydrolase 
Gelatin - - - - - 
hydrolysis 
Starch - - - - - 
hydrolysis 
NO.sub.3 NO.sub.2 
+ + + + + 
NO.sub.3 N.sub.2 
+ - - - - 
Citrate - + + + + 
Utilization 
Catalase + + + + + 
Growth at 
+ + + + + 
41.degree. C. 
______________________________________ 
Strain SD2 of the present invention can be grown in various types of 
culture media under conditions suitable for growth of pseudomonads. 
Typically, such media contain assimilable sources of carbon, nitrogen, and 
various inorganic mineral nutrients. By way of illustration, P. 
alcaligenes strain SD2 was grown in L-Aspartate Medium having the 
formulation as shown in Table IV. 
TABLE IV 
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Culture Medium 
Ingredient Percent by Weight 
______________________________________ 
Ammonium sulfate 0.5 
Potassium phosphate, dibasic 
0.05 
Magnesium sulfate, heptahydrate 
0.025 
Tris(hydroxymethyl)aminomethane 
1.21 
L-Aspartic acid 2.0 
Brij .RTM.58 1.0 mM 
FeCl.sub.3 1.0 uM 
______________________________________ 
The medium is adjusted to pH 7.5-8.0 with potassium hydroxide prior to 
sterilization. The advantage of this medium over the Tryptone medium 
referred to in U.S. application Ser. No. 324,062 is that a white product 
is obtained, free of colored high molecular weight metabolites typically 
found in Tryptone medium. 
The lipase of the invention is found in culture media, Preferably liquid 
media, containing P. alcaligenes strain SD2. Quantities of this enzyme can 
be obtained by culturing P. alcaligenes strain SD2 in liquid culture and 
under culture conditions suitable for growth of organisms of this type. 
For example, an actively growinq broth culture of P. alcaligenes strain 
SD2 is suitably used as an inoculum and introduced into Erlenmeyer flasks 
containing L-Aspartate medium (C.F. Table IV). In addition, the inclusion 
of the non-ionic surfactant BRIJ.RTM. 58 [polyoxyethylene (20) cetyl 
ether] in liquid growth medium containing P. alcaligenes strain SD2 at a 
1-10 mM concentration, preferably 1 mM, increased the yield of the lipase 
by a factor of two-fold or more in contrast to control cultures without 
this surfactant. Cultures are incubated with shaking for about 24 hours at 
a temperature of about 30.degree. C. Following this culture growth period, 
the bacterial cells are removed by centrifugation or filtration or other 
suitable techniques. The lipase which is found in the resultant clarified 
culture liquor is then generally concentrated prior to use. Several 
methods may be used to concentrate this enzyme, including ultrafiltration 
as discussed in Example 1. 
It is desirable that lipases intended for commercial utilization be stable 
in the presence of various surfactants commonly found in cleaning product 
formulations. Advantageously, the lipase of P. alcaligenes strain SD2 was 
found to be functional in the presence of commercial surfactants such as 
dodecylbenzene sulfonate and fatty alcohol ethoxysulfates. 
In accordance with an important aspect of the present invention, it has now 
been surprisingly found that the incorporation of gelatin into a 
composition containing the lipase SD2 and dodecylbenzene sulfonate 
provides enhanced lipase SD2 stability at higher loadings of 
dodecylbenzene sulfonate than would otherwise be possible without 
destabilizing the lipase. In a laundry detergent composition the lipase 
strain SD2 is employed in an amount of between about one million and about 
100 million, preferably between about 5 and about 10 million lipase units 
per kilogram of DBS in the detergent. The gelatin is employed in the 
laundry detergent composition in an amount of between about 0.1 and about 
100 kilograms, preferably between about 0.5 and about 10 kilograms per 
kilogram of DBS in the detergent. 
Upon dilution of the detergent composition with water to form a wash 
solution, the lipase SD2 is generally present in an amount of between 
about one and about 500, preferably between about 3 and about 5 lipase 
units per milliliter of laundry wash solution. The term "lipase unit" is 
defined in Table V, footnote (1). In the wash solution, the gelatin is 
employed in an amount of between about 0.01 and about 10 weight percent, 
preferably between about 0.05 and about 1 weight percent, based upon the 
total volume of the laundry wash solution. 
Regarding the stability of the lipase produced by P. alcaligenes strain 
SD2, this enzyme loses activity during storage at a rate that is directly 
proportional to temperature. For example, during accelerated aqing tests 
conducted at a temperature of 37.degree. C. and a pH of 7.0, the lipase 
useful in this invention demonstrated a half-life of about 5 days in the 
absence of surfactants. The addition of calcium, in the form of 
CaCl.sub.2, stabilized the SD2 lipase and increased its half-life to over 
45 days at suitable CaCl.sub.2 concentrations. The concentration of 
CaCl.sub.2 required to enhance such enzyme longevity is related to the 
particular lipase formulation. For example, in simple buffered enzyme 
solutions lacking surfactants, where the buffer is, for example, 50 mM BES 
[N, N-bis (2-hydroxyethyl)-2-aminoethanesulfonic acid] at pH 7.0, the 
addition of 5 mM CaCl.sub.2, preferably 10 mM, is sufficient. The optimum 
concentration of CaCl.sub.2 in the presence of preferred surfactants is 
about 25 mM or more. In formulations of the lipase of P. alcaligenes 
strain SD2, various surfactants can be used in view of this lipase's 
stability in the presence of surfactants. Examples of preferred 
surfactants include the non-ionic surfactant BRIJ(R).RTM. 35 
[polyoxyethylene (23) lauryl ether] and the anionic surfactant 
SANDOPAN.RTM. DTC gel (sodium trideceth-7-carboxylate). Preferred 
non-ionic surfactants are those having a hydrophobic end containing 12-16 
carbon units, and a polyoxyethylene chain size of about 20-23 ethylene 
oxide units. In qeneral, anionic surfactants of the carboxylated type are 
preferred and are most compatible with the novel lipase of P. alcaligenes 
strain SD2.

While the invention has been described above with reference to specific 
embodiments thereof, it is apparent that many changes, modifications and 
variations can be made without departing from the inventive concept 
disclosed herein. Accordingly, it is intended to embrace all such changes, 
modifications and variations that fall within the spirit and broad scope 
of the appended claims. All Patent applications, patents and other 
publications cited herein are incorporated by reference in their entirety. 
EXAMPLE 1 
Part (A)- Preparation of lipase From Pseudomonas alcaligenes Strain SD2 
The microorganism of the invention, P. alcaligenes SD2, was conveniently 
grown in the culture medium Previously presented in Table IV. 
A 50 mL starter culture of P. alcaligenes SD2 in a 250 mL Erlenmeyer flask 
was grown for about 16 hours at a temperature of 30.degree. C. at 175 rpm 
on a gyratory shaker. This starter culture was then used to inoculate 8 
liters of culture medium divided among 4 and 6 L fluted Erlenmeyer flasks 
such that no individual flask contained more than 25 percent flask 
capacity as liquid. The culture flasks thus prepared were incubated for 24 
hours at a temperature of 30.degree. C. with gyratory shaking at 150 rpm. 
Following the culture period, the lipase of the invention was harvested and 
concentrated by first removing the bacterial cells from the 8 liters of 
liquid culture by tangential flow filtration using Pharmacia 10.sup.6 
(NMWC) Omega membrane cassettes. The resultant cell-free filtrate was then 
concentrated by tangential flow ultrafiltration using Pharmacia 30,000 
(NMWC) Omega membrane cassettes. Thereafter, the concentrate was 
diafiltered at 3.degree. C. with about 10 volumes of 50 mM BES, pH 7.0, 
supplemented with 10 mM CaCl.sub.2 in order to eliminate all low molecular 
weight contaminants (those with molecular weights less than or equal to 
30,000), and to change the lipase solvent to one with buffer and 
stabilizing CaCl.sub.2. The yields of enzyme from three separate batch 
cultures are presented in Table V. 
TABLE V 
______________________________________ 
Yields of Lipase Produced by Cultures 
of P. alcaligenes Strain SD2 
Batch No. Units/mL.sup.(1) 
Total Units 
______________________________________ 
20 39.15 10,571 
21 34.69 7,840 
22 37.41 6,172 
______________________________________ 
.sup.(1) One unit is the amount of lipase which produces one 
microequivalent of fatty acid from olive oil per minute at 37.degree. C. 
and at pH 10. 
Part (B)--Production of the lipase P. alcaligenes Strain SD2 and Molecular 
Weight Measurement 
Quantities of the lipase of P. alcaligenes strain SD2 were obtained by 
culturing of the organism in the medium of Table IV, removing the 
bacterial cells by filtration, concentrating the enzyme by ultrafiltration 
as already described. Lipolytic activity was assayed using the following 
standard composition: (i) 2.5 mL substrate [10 percent (w/v) olive oil 
emulsified in 10 percent (w/v) gum arabic]; (ii) 2.0 mL buffer [1.0M CHES 
(2[N-cyclohexylamino]-ethane sulfonic acid), pH 10.0]; (iii) enzyme; and 
(iv) distilled water added for a final volume of 6.0 mL. Enzymatic assays 
were conducted at a temperature of 37.degree. C. The fatty acids formed 
during the hydrolytic enzymatic reaction were extracted with an organic 
solvent and titrated following the procedure described in U.S. Pat. No. 
4,283,494. 
A quantity of the lipase of the invention was used to determine its 
molecular weight. The molecular weight of the lipase of P. alcaligenes was 
found to be about 88,000 using gel filtration chromatography and comparing 
the retention time of the lipase with molecular weight calibration 
standards. 
Part (C)--Laundering Effectiveness of P. alcaligenes SD2 And Dodecylbenzene 
Sulfonate 
Laundering effectiveness of SD2 lipase was evaluated in a standardized 
procedure adapted from one disclosed in European Patent Application 
0214761 #86306091.9 (6/8/86), incorporated herein by reference in its 
entirety. The ingredients in the laundering solution and the procedure 
followed in the laundering protocol are described below. 
Laundering Solution 
(a) 0.2M Tris HCl, pH 8.5 
(b) Lipase, 5 units/mL (determined at pH 10.0, 37.degree. C.) 
(c) Surfactant, 0.05% (w/v) sodium dodecylbenzene sulfonate based upon the 
volume of the laundering solution 
(d) Water added to provide a final solution volume of 10.0 mL 
Laundering Protocol 
The tests were conducted at 45.degree. C. for the times indicated. Cleaning 
efficacy was determined by measuring the amount of fatty acids produced as 
a result of triglyceride hydrolysis, expressed as a percentage of 
available fatty acids added as triglyceride to the fabric. Triglyceride 
stained fabric is Prepared by adding 0.5 mL of a 3.0% (v/v) lard oil in 
chloroform solution to each side of a 2".times.3" swatch of No. 400M 
Mercerized white cotton fabric (Testfabrics, Inc., Middlesex, N.J.). 
The stained cloths are cut into 8 similar size pieces then Placed in 125 ml 
erlenmeyer flasks containing the laundering solution. The laundering 
Proceeds for 60 minutes. The flasks are continuously agitated in a shaking 
water bath set at 250 rpm for the duration of the test. After completion 
of the laundering period, samples of the laundering solution are taken for 
fatty acid analysis which is Performed using the NEFA-C test kit produced 
by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Previous tests have 
shown this test kit procedure is not interfered with by the ingredients of 
the laundering solution at the concentrations used. 
The results in terms of cleaning efficacy for two trials, using two 
different batches of SD2 lipase, were compared against the results 
obtained using similar amounts of NOVO Lipolase.TM., a commercial lipase 
intended for use as a laundering detergent additive. With SD2 lipase, 
cleaning increased with time of surfactant exposure. In contrast, cleaning 
decreased with exposure to the surfactant in the case of NOVO Lipolase. 
Percent cleaning ranged from 22% to 35% using SD2 lipase. By contrast, 
NOVO Lipolase exhibited no better than 2% to 3% cleaning. 
It is clear that SD2 lipase is effective in removing triglyceride stains 
from cotton fabric in the presence of the commonly used dodecylbenzene 
sulfonate surfactant at a concentration of the latter that is commonly 
encountered in laundering. SD2 lipase is clearly superior to NOVO Lipolase 
in these comparisons and, on the basis of these results, would be expected 
to outperform NOVO Lipolase in formulations containing this anionic 
surfactant. 
Part (D)--Laundering Effectiveness of P. alcaligenes SD2, Dodecylbenzene 
Sulfonate, and Gelatin 
Following the Laundering Protocol described in Part (C) above, measurements 
were made of the cleaning efficacy of a cleaning composition which was a 
modified version of the Laundering Solution of Part (C) above, modified to 
incorporate gelatin in an amount of 0.5% on a weight/volume basis of the 
laundering solution at various levels of dodecylbenzene sulfonate ranging 
from 0.02% up to 0.2% on a weight/volume basis of the laundering solution. 
Control laundering solutions were also prepared using identical 
formulations but without gelatin and without dodecylbenzene sulfonate. 
Cloths stained in accordance with the procedure described in Part (C) 
above were laundered and samples of the resulting laundering solution were 
subjected to fatty acid analysis as described above. 
The results in terms of cleaning efficacy for the laundering solutions 
containing gelatin at various levels of dodecylbenzene sulfonate were 
compared against otherwise identical laundering solutions which did not 
contain gelatin, as well as a single control which did not contain either 
gelatin or dodecylbenzene sulfonate. The results indicated that the 
gelatin-containing formulations provided an enhanced percent fatty acid 
recovery at each level of dodecylbenzene sulfonate tested. For example, at 
0.1% dodecylbenzene sulfonate, the gelatin containing composition provided 
a 35% fatty acid recovery, whereas the comparison without gelatin provided 
only a 20% fatty acid recovery. The control cleaning composition, which 
contained no gelatin and no dodecylbenzenesulfonate, provided a 27% fatty 
acid recovery. These results indicate that the presence of gelatin in the 
cleaning composition enhances the cleaning efficacy of the composition as 
compared to an otherwise identical gelatin-free composition. 
At higher loadings of dodecylbenzene sulfonate of 0.2%, the 
gelatin-containing composition maintained a 30% fatty acid recovery, 
whereas the otherwise identical gelatin-free composition provided a fatty 
acid recovery of only 5%. Without wishing to be bound by any particular 
theory, these results suggest that the gelatin helps to enhance the 
stability and efficacy of the lipase SD2 enzyme. In contrast, when an 
analogous attempt was made to maintain a fatty acid recovery using gelatin 
in an analogous comparison formulation containing Novo's Lipolase as the 
lipase, the Percent fatty acid recovery was not maintained above an 
otherwise identical gelatin free composition. 
Based upon the results, it is clear that gelatin enhances the efficacy of a 
laundering composition containing the lipase SD2.