USOO5395541A
United States Patent (19) 11 Patent Number: 5395.5419 9 Carpenter et al. 45 Date of Patent: Mar. 7, 1995
(54) CLEANING COMPOSITION CONTAINING A 4,925,796 5/1990 Bergh et al...... 435/97 TYPE ENDOGLYCOSDASE 4,939,123 7/1990 Neeser et al...... S14/8 a 4,963,491 10/1990 Hellgren et al...... 435/264 75) Inventors: Richard S. Carpenter, Cincinnati, 4,983,521 1/1991 Lingappa et al...... 435/172.3 Ohio; Irwin J. Goldstein, Ann Arbor, 4,994,390 2/1991 Wiatr ...... 435/264 X Mich.; Pushkaraj J. Lad, San Mateo, 5,041,236 8/1991 Carpenter et al...... 252/174.12 Calif.; Ann M. Wolff, Cincinnati, 5,041,376 8/1991 Gething et al...... 435/172.3 Ohio 5,089,409 2/1992. Howard et al...... 435/240.2 5,106,626 4/1992 Parsons et al...... 424/423 73) Assignees: The Procter & Gamble Company, 5,200,327 4/1993 Garvin et al...... 435/69.5 Cincinnati, Ohio; Genencor 5,208,321 5/1993 Hovanessian et al...... 530/350 International, Inc., Rochester, N.Y. 5,238,843 8/1993 Carpenter et al...... 435/264 5,258,304 il/1993 Carpenter et al...... 435/264 21) Appl. No.: 98,083 5,272,066 12/1993 Bergh et al...... 435/97 22 Filed: Jul. 26, 1993 Primary Examiner-David M. Naff 8 . A Assistant Examiner-Mike Meller Related U.S. Application Data Attorney, Agent, or Firm-Margaret A. Horn 62) Pyof Ser. No. 428,361, Oct. 27, 1989, Pat. No. 57 ABSTRACT 51 Int. Cl6 C11D 17/00; C12N 9/00; A cleaning composition is disclosed. The composition b Waw so o so were 88 48 0 & 0 & 0 & 0 0 to C12N 9/24: D06M 16/06 contains a first enzyme where the enzyme can be Endo 52 U.S. C. 252/174 12.252/DIG 12: D, Endo-H, Endo-L, Endo-C, Endo-CII, Endo-F-Gal saw was e s a sea e s we s a sea so a 435/183; e435/200; Laid 435/264• a as type, Endo-F and PNGaseF and a second enzyme 58 Field of Search ...... 435/200,264, 183; where the enzyme can be a protease, lipase, nuclease, 252/174.12, DIG. 12 glycosidase, an enzyme different from the first enzyme, and any combination of these. The composition also 56) References Cited contains a detergent surfactant and a builder. The com U.S. PATENT DOCUMENTS position can be used in a method for cleaning a surface 3,532,599 10/1970 Cooperman ...... 4564 on which is bound a glycoside-containing substance. 4,355,022 10/1982 Rabussay ...... 424/74 X The substance can be blood or components thereof, 4,521,254 6/1985 Anderson et al. ... sas w w & 134/26 fecal matter or components thereof or microorganisms. 4,566,985 l/1986 Bruno et al...... 435/264 The surface can be fabric, biological tissue, tooth 4,585,488 4/1986 Giefer ...... 35/2 enamel, contact lens, glass, ceramic, metal, metal alloy, 4,619,825 10/1986 Eigen et al. ... 424/49 plastic, plant, fruit and vegetable. 4,639,375 l/1987 Tsai ...... 426/49
4,710,313 12/1987 Miyajima et al. ... 252/105 4,749,511 6/1988 Lad et al...... 435/264 X 20 Claims, 28 Drawing Sheets U.S. Patent Mar. 7, 1995 Sheet 1 of 28 5,395,541
N-LINKED CORE STRUCTURE
Asn-GlcNAc-GlcNAc-Man
2.
D-LINKED CORESTRUCTURE
-GONAC-Neuac -GalNAc-Got
Ser- DR, -GotNAc-Got-Neuac Thr NeunAC -GalNAc-Gil --Fuc
Figure-1 U.S. Patent Mar. 7, 1995 Sheet 2 of 28 5,395,541
TYPE ENDDGLYCDSDASE SUBSTRATE X Glycopeptidase-F Asn-GlcNAc-GlcNAc-Mar?-v (PNGosef) 4. N CFuC) Mon-YN Z
X
Endo-H, F, D, C Asn-GlcNAc-GlcNAc-Mar?-vN v U Mon-Y N Z
Mon Endo-F-gal type Asn-GlcNAc-Go-Mo. M N Mon
Ser Enco-O-N- Or gotNAc-Gol Acetylgalactosarninidase Thr
Endo-B-N- Goactosidose R -GlcNAc-Gol-GlcNAc-R2
Figure-2 U.S. Patent 5,395,541
ç-eun614
U.S. Patent 5,395,541
ELIS350W//\\/ETIO #-eun61-I
U.S. Patent Mar. 7, 1995 Sheet 7 of 28 5,395,541
FIGURE - 6A U.S. Patent Mar. 7, 1995 Sheet 8 of 28 5,395,541
FIGURE-6B U.S. Patent Mar. 7, 1995 Sheet 9 of 28 5,395,541
FIGURE-7A U.S. Patent Mar. 7, 1995 Sheet 10 of 28 5,395,541
FIGURE-7B U.S. Patent Mar. 7, 1995 Sheet 11 of 28 5,395,541
FIGURE-7C U.S. Patent Mar. 7, 1995 Sheet 12 of 28 5,395,541
FIGURE-7D U.S. Patent Mar. 7, 1995 Sheet 13 of 28 5,395,541
FIGURE -7E U.S. Patent Mar. 7, 1995 Sheet 14 of 28 5,395,541
U.S. Patent Mar. 7, 1995 Sheet 15 of 28 5,395,541
FIGURE-7G U.S. Patent Mar. 7, 1995 Sheet 16 of 28 5,395,541
FIGURE-7H U.S. Patent Mar. 7, 1995 Sheet 17 of 28 5,395,541
H–opu3uddOwl(INº 3NICIIX3HNDTHOuddOy 3NICIIX3HNDTHOudd0+ 09 9–3-an513
02
U.S. Patent Mar. 7, 1995 Sheet 19 of 28 5,395,541
A LOG CFU E.coli VS, Endo-H)
O 40 BO 120 60 200 240 280 Figure-10AEndo-H) ppm
A LOG CFU E.coli VS, Endo-H) 3,53,6 200, 500, 1000 ppm Endo-H 3.4 3.3 3.2 3. 3.0 2.9 2,8 2.7 2.6 2.5 2.4 2.3 22 2.1: O 200 400 600 800 1000 (Endo-H) ppm Figure-10B U.S. Patent Mar. 7, 1995 Sheet 20 of 28 5,395,541
U.S. Patent Mar. 7, 1995 Sheet 21 of 28 5,395,541
×
pe) eeu) LION U.S. Patent Mar. 7, 1995 Sheet 22 of 28 5,395,541
ANTIMICROBIAL EFFECTS THROUGH THE WASH
LGS GROWTH
D5 = DETERGENT L = IRGASAN T = TRICLDCARBAN A = Endo-H 2 OR 1 =24 OR 1% RESPECTIVELY Figure-14 U.S. Patent Mar. 7, 1995.- v-wmum-- Sheet 23 of 28 5,395,541- - - -
w.
s -
E. -
.
FIGURE - 5A U.S. Patent Mar. 7, 1995 Sheet 24 of 28
FIGURE - 15B U.S. Patent Mar. 7, 1995 Sheet 25 of 28 5,395,541
FIGURE - 16A U.S. Patent Mar. 7, 1995 Sheet 26 of 28 5,395,541
t w 3 s s & t ty A.
Pessess
FIGURE - 16B U.S. Patent Mar. 7, 1995 Sheet 27 of 28 5,395,541
s
FIGURE - 17B U.S. Patent Mar. 7, 1995 Sheet 28 of 28 5,395,541
FIGURE 18A
FIGURE - 18B 5,395,541 1. 2 et al. (1977), Botanica Marina, 20, 13-17. As reported CLEANING COMPOSITON CONTAINING A therein, Pseudomonas species isolated from sea water TYPE ENDOGLYCOSIDASE was adhered to glass slides. Thereafter, the slides were treated with either pronase, trypsin, a-amylase (a Type This is a division of application Ser. No. 428,361, filed 5 I endoglycosidase), or lysozyme (also a Type I endo Oct. 27, 1989, now U.S. Pat. No. 5,238,843. glycosidase). In this report, treatment with the proteo lytic enzymes pronase and trypsin resulted in the release FIELD OF THE INVENTION of a portion of the population of adhered bacteria, The present invention relates to methods and formu whereas the cell degradative enzyme lysozyme showed lations for removing glycoside-containing substances, 10 diminished activity compared to the proteolytic en such as glycoproteins, from surfaces by treatment with zymes. The a-amylase reportedly had no effect at all. In Type II endoglycosidase alone or in combination with addition to the attachment of microorganisms to other enzymes and/or detergents. contact lenses, tooth enamel and glass surfaces, many other surfaces are subject to microbial attachment. See, BACKGROUND OF THE INVENTION 15 e.g., Marrie, T.J., et al. (1984), J. Clin. Microbiology, 19, The use of enzymes to remove stains comprising 991-914 (bacterial attachment to cardiac pacemaker proteins and/or carbohydrates, in combination with leads and powerpacks); Freimer, N. B., et al. (1978), various detergents, is well known in the art of detergent Acta, Path. Microbiol. Scand. Sectb, 86, 53–57 (binding formulations. Such enzyme formulations are designed of microorganisms to macrophages); and Mirelman, et to remove various types of stains from soft surfaces such 20 al. (1982), Tokai J. Exp. Clin. Med, 7, 77-183 (microbial as cloth and hard surfaces such as porcelain and metal. adherence to mammalian mucosal surfaces). Various Thus, for example, proteases such as trypsin, pancrea mechanisms have been proposed to describe the adhe tin, papain and bromelain have reportedly been used in sion of microorganisms, such as bacteria, to non-biolog detergent formulations to remove proteinaceous stains ical solid surfaces. See, e.g. Fletcher, M. (1987), Micro with variable degrees of success. Specific glycosidases 25 biological Sciences, 4, 133-136, and Duddridge, J. E., et such as cellulase, lysozyme, amylase and glucanase, on al. (1983), Factors Affecting the Adhesion of Bacteria to the other hand, have been formulated with various Surfaces in Microbial Corrosion, Delco Printing Co., detergents for removal of certain carbohydrate stains. Ltd., pp. 28-35. Although these references discuss mi Other detergent formulations have combined proteases crobial adherence to various surfaces and the factors and glycosidases for stain treatment. 30 which may be involved in such attachment, they do not Some of the glycosidases used in detergent formula discuss the control of microorganism growth on such tions, e.g., S-amylase, a-galactosidase and S-galactosi surfaces or their removal therefrom. dase, are exoglycosidases which cleave one or more Type II endoglycosidases, as used herein, are a cate terminal residues from an oligosaccharide or polysac gory of endoglycosidases which are capable of cleaving charide. Other glycosidases, e.g. cellulase and a-amy 35 specific internal glycosidic linkages found in glycopro lase are endoglycosidases which are reactive with spe teins. These endoglycosidases cleave all or part of the cific internal linkages within an oligo- or polysaccha carbohydrate moiety from a glycoprotein depending on ride substrate. Such endoglycosidases are referred to the location of the reactive glycosidic linkage in the herein as Type I endoglycosidases. Although formula glycoprotein. Examples include endo-E-N-acetyl tions of detergent with one or more proteases and/or glucosaminidases (Endo-D, Endo-H, Endo-L, Endo glycosidases (including Type I endoglycosidases) have CI, Endo-CII, Endo-F-Gal type and Endo-F), endo-a- greatly improved stain removal, many stains, e.g. blood, N-acetylgalactosaminidase and endo-3-N-galactosi fecal material and body soil stains, often leave a residual dases. See, e.g. Tarentino, A. L., et al. (1985), Biochem, stain after treatment. 24, 4665-4671; Arakawa, M., et al. (1974), J. Biochem., In the art of contact lens cleaning, similar en 45 76, 307-317; Plummer, T. H., et al. (1984), J. Biochem, zyme/detergent formulations have been used to clean 259, 10700-10704; Tarentino, A. L., et al. (1975), Bio and sterilize hard and soft contact lenses. In many cases, chem. and Biophys. Res. Comm., 67, 455-462; and Trim these formulations have been used to degrade the bi ble, R. B., et al. (1984), Anal. Biochem., 141, 515-522; ofilm which forms on the surface of contact lenses and and "Glycoprotein and Proteoglycan Techniques' which is used by various ophthalmic pathogens such as 50 (1985) by J. G. Beeley, Chapter 6, pp. 153-300, El Pseudomonas aeruginosa and Staphylococcus epidermidis sevier, Amsterdam, N.Y., Oxford. In addition to having to adhere to such lens. See, e.g., Duran, J. A., et al. a specificity for the internal glycosidic linkages of gly (1987), Arch. Ophthalmol, 105 106-109; Stern, G. A., et coproteins, at least one endoglycosidase (endo-3-N- al. (1987), Ophthalmology, 94, 115-119 (which reports acetylglucosaminidase H) has also demonstrated a spec the treatment of mucin coated contact lenses with vari 55 ificity which produces the cleavage of lipid-linked oli ous enzymes such as pancreatin, papain, trypsin and gosaccharides (Chalifour, R. J., et al. (1983), Archives of neuraminidase to inhibit Pseudomonas adherence); and Biochem, and Biophys, 229, 386-394) and reportedly Slucher, M. M., et al. (1987), Arch. Ophthalmol, 105, di-N-acetylchitobiose linkages in oligosaccharides and 110-115. glycoproteins (Tarention, A. L., et al. (1974), J. Biol. The use of biofilms for microbial adhesion is not Chem..., 249, 811-817). limited to contact lenses. Thus, Streptococcus mutans Such Type II endoglycosidases, in general, have been reportedly uses extracellular polysaccharides to adhere used primarily for analytical purposes, e.g. the determi to tooth enamel. EPO Publication No. 0195672 reports nation of protein or carbohydrate sequence and/or the the use of a-1,3-glucanase or a-1,6 glucanase to cleave structure and function of specific glycoproteins. See, the extracellular polysaccharides used by Streptococcus 65 e.g. Hsieh, P., et al. (1982), J. Biolchem, 258, 2555-2561, mutans to adhere to tooth enamel. and Geyar, R., et al. (1984), Eur. J. Biochem., 143, The effect of certain enzymes on cells adhered to 531-539. In a recent report, a Type II endoglycosidase glass surfaces has also been reported by Danielsson, A., was reportedly used to analyze a glycoprotein antigen 5,395,541 3 4. from Leishmania mexicana amazonensis. Chin Shen bound substance with a Type II endoglycosidase to Chang, et al. (1986), Mol. Biochem. Parasitol 18, cleave the distal portion of the substance from the prox 197-210. This glycoprotein antigen was first immuno imal portion thereby releasing the distal portion from logically bound to immunobeads. After reacting the the surface. If such releasing does not remove the distal immunologically bound glycoprotein with analytical 5 portion from the surface, such removal may be facili amounts of Endo-H, the immunobeads were washed tated by contacting the distal portion with a washing and boiled in buffer containing 1% SDS in preparation solution, a detergent formulation including those with for polyacrylamide gel electrophoresis. This analysis surfactants or a fluid containing a second enzyme such revealed a decrease in molecular weight attributed to as a different glycosidase, protease, lipase, nuclease or the cleavage of carbohydrate from the immunologically O combinations thereof. bound glycoprotein antigen. The invention further includes a method for releasing Type II endoglycosidases, however, have not been at least a portion of a glycoside-containing substance used to remove substances, including glycoproteins and from a surface to which it is bound by contacting the glycolipids, from surfaces of substances such as fabric, glycoside-containing substance with at least a first and a contact lenses, metals, ceramics, cells, tissue and the 15 like. Nor have they been used to control microorganism second enzyme to release a cleaved portion of the growth in suspension or on such surfaces. glycoside-containing substance from the surface. The Glycosidases have been used in combination with first enzyme is a Type II endoglycosidase reactive with other enzymes for removal of various materials. (3- a glycosidic linkage in the glycoside portion of the glycosidases are described as carbohydrate-metaboliz 20 substance. The second enzyme is reactive with a second ing enzymes in Anderson, et al. (1964), Biochen. J., 90, linkage in the glycoside-containing substance other than 30. Neuraminidase (N-acetylneuraminiate glycohydro the glycosidic linkage with which the first enzyme is lase) inhibitors are viewed as possible anti-viral, antibac reactive. Such second enzymes may include glycosi terial agents in Khorlin, et al. (1979), FEBS Letters, 8, dases (including a second Type II endoglycosidase), 17; and Haskell, et al. (1970), J. Med. Chem, 13, 48. 25 proteases, lipases and combinations thereof. The re Dextranase is described as catalyzing hydrolysis of moval of a portion of the glycoside-containing sub bacterial polysaccharide, dextran (a-1,6-glucan), to stance may be facilitated by contacting the cleaved isomaltose residues in Chalet, et al. (1970), Appl. Mi glycoside-containing substance with a detergent includ crobiol, 20, 421. Lysozyme (muramidase) is described as ing those containing a surfactant. hydrolyzing glycosidic linkages in the mucopolysac 30 The invention also includes a method for releasing charide cell wall structure of a variety of microbes in from a surface at least a portion of a glycoside-contain Chipman, et al. (1969), Science, 165, 454 and Montague ing substance having a carbohydrate portion, an agly (1964), Biochem. Biophys. Acta, 86, 588. Lastly, inhibi con portion and a glycosidic linkage at the juncture of tion of lysozyme by D-glucosamine derivatives is de the carbohydrate and aglycon portions wherein the scribed in Neuberger, et al. (1967), Nature, 215, 524. 35 glycoside containing substance is bound to a surface Type II endoglycosidases such as endo-g-N-acetyl through the carbohydrate portion. The method com glucosaminidase H, D, F and/or PNGase F have not, prises cleaving the glycosidic linkage with a Type II however, previously been combined with antimicrobial endoglycosidase to release the aglycon portion of the agents to form antimicrobial compositions. glycoside-containing substance from the surface. Re The references discussed above are provided solely 40 moval of the cleaved portion of the glycoside-contain for their disclosure prior to the filing date of the instant ing substance may, if necessary, be facilitated by treat case, and nothing herein is to be construed as an admis ment with a second enzyme, detergent and/or surfac sion that such references are prior art or that the inven tant. tors are not entitled to antedate such disclosure by vir In each of the above methods, a disulfide cleaving tue of prior invention or priority based on earlier filed 45 reagent may be employed to denature protein associ applications. ated with the glycoside-containing substance thereby facilitating cleavage by the Type II endoglycoside or SUMMARY OF THE INVENTION second enzyme or removal by detergent and/or surfac It is an object herein to provide methods utilizing tant. Type II endoglycosidases alone or in combination with 50 The invention also includes formulations. One com other enzymes, detergents, surfactants and/or disulfide position comprises at least a first and a second enzyme cleaving reagents to facilitate the removal of substances wherein the first enzyme is a Type II endoglycosidase. including glycoproteins and glycolipids from materials The second enzyme is selected from the group of en such as fabric, contact lenses, metallic surfaces, plastic zymes consisting of proteases, glycosidases, lipases and surfaces, ceramic surfaces, cell surfaces, tissue and the 55 combinations thereof. Such formulations may also in like. clude detergents and/or surfactants as well as disulfide It is a further object herein to provide formulations cleaving reagents. containing Type II endoglycosidases useful in practic Another composition includes Type II endoglycosi ing such methods. dase and detergent and/or surfactant. In accordance with the objects of the invention, 60 methods are provided for removing at least a portion of BRIEF EDESCRIPTION OF THE DRAWINGS a substance from a surface to which it is bound, other FIG. 1 depicts the common core structure of N than by an immunological bond. The substance has a linked and O-linked glycoproteins. proximal portion bound to a surface, a distal portion FIG. 2 depicts the substrates and known cleavage extending outwardly from the proximal portion, and a 65 sites for various Type II endoglycosidases. linkage disposed at the juncture of the proximal and FIG. 3 is a generic presentation of the protein amino distal portions which is reactive with a Type II endo acids, carbohydrate residues and cleavage sites of FIG. glycosidase. The method comprises contacting the 2. 5,395,541 5 6 FIG. 4 depicts the core structure of an N-linked gly sidic linkages between the amino acids serine, threonine coprotein, the cleavage site of a Type II endoglycosi or asparagine and the first carbohydrate residue and the dase and the relationship between the protein and car glycosidic linkages between at least the first, second and bohydrate units and the aglycon and carbohydrate por third carbohydrate residues. Although this core struc tions produced upon cleavage with a Type II endo ture will be described in more detail hereinafter in terms glycosidase. of the specific glycosidic linkages which exist in known FIGS. 5A-5E depict various mechanisms whereby a core structures, such specific linkages are not to be glycoside-containing substance, microorganisms or sub construed as limiting to this definition of Type II endo stances reactive with Type II endoglycosidase may be glycosidases. Accordingly, all possible glycosidic link released from a surface by treatment with Type II endo 10 ages between these amino acids and carbohydrate resi glycosidase alone or in combination with a second en dues define the core structure of N-and O-linked glyco zyme. protein used to identify Type II endoglycosidases. FIGS. 6A and 6B are electron micrographs (8100X) Type II endoglycosidases are not limited by the pres of nylon swatches stained with fecal matter and treated ent knowledge of the glycoprotein core structure and either with or without Endo-H. 15 the specificity of known endoglycosidases for such core FIGS. 7A through 7H are electron micrographs structures. A comparison of the core structures in FIG. (5000X) showing the effect of Endo-Hand other carbo 1 with the known substrates for Type II endoglycosi hydrase enzymes on cotton swatches stained with fecal dases in FIG. 2 indicates that Type II endoglycosidases natter. for each of the possible cleavage sites in the core struc FIGS. 8, 9 10A and 10B demonstrate the effect of 20 tures in FIG. 1, if they exist, have not yet been identi various concentrations of Endo-H and chlorhexidine, fied. Moreover, other core structures may also exist alone or in combination, on the viability of E. coli. which have not yet been identified. Endoglycosidases FIGS. 11 and 12 demonstrate that a detergent compo reactive with linkages in such, as yet, unknown core sition containing Endo-H is more effective in the re structures are also Type II endoglycosidases. Accord moval of S. aureaus from swine skin than a detergent 25 ingly, the glycosidic linkages in glycoproteins which composition not containing Endo-H. define Type II endoglycosidases are not limited to those FIG. 13 demonstrates that Endo-H is more effective located within the first three glycosidic linkages closest in removing mold from a shower curtain than water or to the protein unit of the glycoprotein but may extend a detergent composition. The center photograph is of a to more distant glycosidic linkages in the core structure, portion of the shower curtain. The other four photo 30 e.g. to the fourth or fifth glycosidic linkage from the graphs are enlargements of the corresponding quad protein unit depending on the core structure identified. rants of the center photograph. The specificity for the core structure of glycoproteins FIG, 14 demonstrates the antimicrobial effect of En provides a convenient definition of Type II endo do-H in combination with different antimicrobial glycosidases which distinguishes them from Type I agents. 35 endoglycosidases. Type I endoglycosidases cleave spe FIGS. 15A-B and 16A-B demonstrate the effect of cific linkages in oligo- or polysaccharides but generally Endo-H on different species of yeast. are not reactive with those core structure glycosidic FIGS. 17 and 18 demonstrate the enhanced removal linkages in glycoproteins which define Type II endo of fecal matter from diaper material by a detergent glycosidases. Examples of Type I endoglycosidases and composition containing Endo-H. 40 the linkages with which they are reactive are shown in Table I. DETALEED DESCRIPTION OF THE INVENTION TABLE I Type I Substrate Type II endoglycosidases and formulations employ Endoglycosidase oligo-or polysaccharide ing such endoglycosidases are used in the methods of 45 a-amylase o1-4 the present invention to release and/or remove sub (Glc-Glc) stances reactive with Type II endoglycosidases from a t surface. The mechanism of this reactivity is not known cellulase 31-4 with certainty. In some cases, such substances are (Glc-Glc) glycosides or glycoside-containing substances which 50 hyaluronidase g1-3 (31-4 g1-3 are believed to have glycosidic linkages that are known GlcA-GlcNAc-GlcA-GlcNAc cleavage sites for Type II endoglycosidases or linkages it which are closely related to such cleavage sites. lysozymes: B1-4 (31-4 (31-4 hen egg white GlcNAc-Murnac-GlcNAc-Murnac As used herein, “Type II endoglycosidases' are en lysozyme zymes which are capable of cleaving linkages at or near 55 T4 lysozyme the juncture of the protein and carbohydrate units of a mutanolysin glycoprotein. Preferably, such Type II endoglycosi pullulanase ol-4 oil-4 c.1-6 ot-4 Glc-(Glc-Glc-)Glc-Glc dases are capable of cleaving at least one glycosidic f linkage within about three glycosidic linkages of the GlcA is D-Glucuronic Acid protein-carbohydrate unit juncture (including the gly MurNAc is N-Acetylmuramic Acid cosidic linkage comprising the protein-carbohydrate Indicates cleavage site. junction). Most preferably, such glycosidic linkages are within about two glycosidic linkages of the protein-car Specific glycosidic linkages in glycoproteins which bohydrate unit juncture (see FIGS. 1, 2 and 3). define Type II endoglycosidases and which identify Type II endoglycosidases are also defined by their 65 preferred Type II endoglycosidases are shown in FIG. specificities for the particular glycosidic linkages shown 2. The cleavage sites are identified by a vertical arrow. in FIG. 1 for the known core structures of N-and O A generic presentation of the protein amino acids, car linked glycoproteins. These correspond to the glyco bohydrate residues and cleavage sites of FIG. 2 is 5,395,541 7 8 shown in FIG. 3. As can be seen, Type II endoglycosi aglycon portion containing one or more sugar residues dases preferably cleave the first, second or third glyco depending on the cleavage site of the Type II endo sidic linkages in N- or O-linked glycoproteins. These glycosidase. Further, the aglycon portion may be quite linkages comprise the glycosidic linkages (1) between complex as might be the case with peptidoglycans asparagine, serine or threonine in the protein unit and where crosslinked peptides can be found attached to a the first carbohydrate residue, (2) between carbohy matrix of carbohydrate. Thus, glycosides include glyco drate residues 1 and 2 and (3) between carbohydrate proteins, glycolipids, peptidoglycans and the like which residues 2 and 3, respectively. This specificity is defined upon treatment with a Type II endoglycosidase pro primarily by the carbohydrate sequence of the glyco duce a carbohydrate portion and aglycon portion protein with specificity and reactivity being influenced 10 wherein the carbohydrate portion and aglycon portion to some extent by the protein unit of the glycoprotein. are defined by the cleavage site of the Type II endo Thus, with regard to glycosidic linkages 2 and 3 (com glycosidase. This definition of glycoside will be appar prising glycosidic linkages between carbohydrate resi ent from the discussion which follows. dues only), Type II endoglycosidases may be reactive As used herein, "glycoprotein’ refers to a glycoside with identical or similar glycosidic linkages located in 15 which has one or more oligo- or polysaccharides cova other regions of a glycoprotein, perhaps quite distant lently attached to a peptide or protein. Oligo-and poly from the juncture of the protein and carbohydrate units saccharides are sometimes referred to herein as 'carbo of the glycoprotein. hydrate units'. Such carbohydrate units, however, may An application of the above definition to a particular be different from the "carbohydrate portion' of a gly glycoprotein is illustrative. Bovine thyroglobulin has 20 coside. As shown in FIG. 4, a carbohydrate unit com been analyzed using endo-S-N-acetylglucosaminidase prises the entire oligo- or polysaccharide attached to a H (Endo-H), a-mannosidase and 6-mannosidase. Taren second class of molecule, e.g., to a protein or peptide as tino, A. L. et al. (1973) J. Biol. Chem, 218,5547. The in a glycoprotein or to a lipid as in a glycolipid. If the Endo-H hydrolyzed the glycosidic linkage between the Type II endoglycosidase cleaves the carbohydrate unit two N-acetyl D-glucosamines, one of which was N 25 at its juncture with, for example, a protein then the linked to an asparagine in the protein unit of the thyro carbohydrate unit is synonymous with the carbohydrate globulin. The oligosaccharide or carbohydrate portion portion of a glycoside. If, however, the Type II endo of the thyroglobulin released upon treatment with En glycosidase cleaves the carbohydrate unit at a glyco do-H was also treated with a- and 3-mannosidase. Since sidic linkage within the carbohydrate unit, then the neither of these enzymes has a specificity for the sub 30 carbohydrate portion of the glycoside formed by such strates corresponding to those shown in FIGS. 1, 2 or 3, cleavage will be less than the entire carbohydrate unit. they are not Type II endoglycosidases and can be char This difference is shown in FIG. 4 for a Type II endo acterized as either an exoglycosidase or Type I endo glycosidase cleavage site indicated by the arrow. glycosidase. The specificity of the Endo-H is the same The carbohydrate units of a glycoprotein may be as that shown for Endo-H in FIG. 2 and Endo-H is 35 oligosaccharides containing 1 to 10 carbohydrate therefore a Type II endoglycosidase. This is of course a (sugar) residues or short polysaccharides which usually trivial application. But if a new endoglycosidase (e.g. contain between 10 to 25 carbohydrate residues. Many Endo-X) is discovered which also demonstrates this glycoproteins are produced by higher organisms such specificity or one or more of the other specificities in as eukaryotes including yeast and mammalian cells. The FIGS. 1, 2 or 3, that Endo-X would also be a Type II linkage between the carbohydrate unit and the peptide endoglycosidase. or protein unit of a glycoprotein is a glycosidic linkage This definition of a Type II endoglycosidase based on which results from a condensation reaction between an its specificity for glycoproteins, however, should not be amino acid side chain of the protein unit and the anom construed as a limitation on the mechanism utilized by eric carbon on the first residue of the carbohydrate unit. Type II endoglycosidases to release and/or remove a 45 Such glycosidic linkages in mammalian glycoproteins substance from a surface. Although it will be assumed in are either N-glycosidic linkages (carbohydrate linked to some instances that Type II endoglycosidases cleave at the amido nitrogen of asparagine) or O-glycosidic link least a part of a glycoside from a surface by reacting ages (carbohydrate linked to the hydroxy oxygen of with a glycosidic linkage in the glycoside, the invention serine or threonine). is not limited to such cleavage. Rather, the action of 50 The carbohydrate residues (monosaccharides) of a Type II endoglycosidases is defined functionally by carbohydrate unit (oligo or polysaccharide) may be their ability to cleave from a surface at least a part of joined together in many different ways. Thus, such any substance reactive with a Type II endoglycosidase. carbohydrate units may be unbranched, linear struc As used herein, the term "endoglycosidase' com tures or may be complex branched structures. In gen prises Type I and Type II endoglycosidases. 55 eral, however, each of the carbohydrate residues in the As used herein, "glycoside' refers to a polymer carbohydrate unit is linked by way of a glycosidic link which has one or more "carbohydrate portions' cova age wherein the anomeric carbon of one carbohydrate lently attached through a glycosidic linkage to an residue is condensed with the hydroxyl carbon in an "aglycon portion'. This definition of glycoside is de other carbohydrate residue. Such glycosidic bonds may rived from the common definition of glycoside which be either alpha or beta depending on the configuration refers to a compound that yields on hydrolysis a sugar of the anomeric carbon. The anomeric carbon of one and an aglycon, the aglycon being the non-sugar com residue may be combined with any of the hydroxyl pound resulting from such hydrolysis. As used herein, a carbons in another carbohydrate residue. Thus, the glycoside produces an aglycon and an oligo- or polysac complexity of many glycoproteins arises from the many charide carbohydrate portion when cleaved by a Type 65 different glycosidic linkages which are found in the II endoglycosidase. The aglycon unit, however, is not carbohydrate units of such molecules. limited to a non-sugar compound since Type II endo Many membrane glycoproteins carry asparagine glycosidases may hydrolyze a glycoside to produce an linked carbohydrate units (carbohydrate units linked to 5,395,541 9 10 asparagine in a peptide via an N-glycosidic linkage). muramic acid containing attached peptide side-chains. The structure of such asparagine linked glycoproteins As such, lysozymes are more appropriately character can be quite complex. See e.g., Schachterh (1984) Clini ized as a Type I endoglycosidase. Thus, even though cal Biochemistry 17, 3-14. The structure of many of lysozymes and Endo-H may have an overlap in reactiv these asparagine linked membranous glycoproteins ity with peptidoglycans, they are mutually exclusive, from a variety of sources (e.g., erythrocyte plasma for the most part, with regard to Endo-H's specificity membrane glycoproteins, viral envelope glycoproteins) for, and lysozyme's substantial lack of reactivity with, as well as the structure of non-membranous soluble the glycosidic linkages in glycoproteins which define glycoproteins indicate that the two types of glycopro Type II endoglycosidases. teins share many structural features. Id. at 3. The com 10 As used herein, a "glycoside containing substance' or mon core structure of such asparagine-linked glycopro "glycoprotein containing substance' is a glycoside or teins is shown in FIGS. 1 and 4, wherein GlcNAC is glycoprotein alone or a glycoside or glycoprotein com N-acetyl D-glucosamine and Man is mannose. The bined with another component. Thus, glycoside-con a 1-6, a 1-3 and 61-4 designations describe the type of taining substances include glycosides such as glycopro glycosidic linkage between the various carbohydrate 15 tein enzymes, e.g., alkaline phosphatase, bromelain, residues. This core linkage forms the basis of numerous carboxypeptidase-Y; glycoprotein hormones, e.g., cho glycoproteins having any of a number of carbohydrate rionic gonadotropin, erythropoietin; lectins, e.g., those residues attached to the core. Id. at 5. derived from potato and soybean; serum glycoproteins, O-linked glycoproteins contain a core structure e.g., IgG immunoglobulin, thyroglobulin, prothrombin wherein the protein unit of the glycoprotein is coupled 20 and the like and miscellaneous glycoproteins such as to the carbohydrate unit through the hydroxyl group of hemoglobin and interferon; and complex carbohy either serine or threonine. A common feature of this drates. Examples of glycosides combined with another core structure is the presence of N-acetyl D-galactosa component include glycoproteins comprising mem mine (GalNAc) linked to. serine or threonine. Other brane constituents, e.g., glycophorin contained by details of such glycoproteins are shown in FIG. 1 where 25 human erythrocytes, hemagglutinin contained by influ NeuAc is N-acetylneuraminic acid, Gal is Galactose enza virus, rhodopsin contained in bovine retina and and L-Fuc is L-Fucose. When Gal is the second carbo collagen contained by fibroblasts. Further glycoside hydrate residue the glycosidic linkage between Gal containing substances include viral envelope glycopro NAc and Gal is usually (31-3. For review of the struc teins and fecal matter which contains in part peptido ture biosynthesis and function of glycoproteins includ 30 glycans associated with intestinal bacteria. Thus, vi ing N- and O-linked glycoprotein, see Berger E. G. et ruses, fibroblasts, fecal matter etc. are considered glyco al. (1982) Experimentia, 38, 1229-1258. side-containing substances. Lower organisms such as prokaryotes, e.g., the bac As used herein, a “microorganism' (sometimes re teria E. coli, Pseudomonas species, Bacilis species and ferred to as a glycoside-containing microorganism) is the like, produce peptidoglycans rather than glycopro 35 one capable of being cleaved from the surface of a sub teins. Peptidoglycans are found in bacterial cell walls stance to which it is bound by a Type II endoglycosi and typically have a polysaccharide backbone of alter dase. Examples include the intestinal bacteria found in nating N-acetylglucosamine and N-acetylmuramic fecal matter and bacteria commonly contaminating acids. Peptide side chains are sometimes associated with contact lens. Other examples include fungi and algae the N-acetylmuramic acid residues with cross-linked which can be cleaved from a surface by Type II endo peptide bridges often being interposed between the glycosidase. peptide side chains. The cell wall of Gram-positive As used herein, the term "in vitro' refers to the envi bacteria typically comprises approximately 10% pepti ronment in which the processes and methods of the doglycan whereas the cell wall of Gram-negative bac invention are practiced. It is used only to distinguish teria typically have a peptidoglycan content of about 45 from the term “in vivo' which describes the environ 50%. ment in which Type II endoglycosidases are found Peptidoglycans, however, are not glycoproteins, at naturally, e.g. within organisms which naturally pro least to the extent that specific glycosidic linkages in duce Type II endoglycosidase. Accordingly, an in vitro glycoproteins are used to define the class of Type II method employing a Type II endoglycosidase is a endoglycosidase. Thus, Endo-H is a Type II endo 50 method or process which does not occur in nature. The glycosidase because it cleaves the glycosidic linkage term in vitro, however, is not to be construed as a limi between the two N-acetylglucosamine sugar residues tation of such methods to "in glass' or to exclude such found in some glycoproteins containing N-linked oligo methods from being practiced on or in a living organ saccharides. See FIG. 4. Endo-H, however, may also ism. The methods of the invention may be practiced on have an as yet undefined reactivity with peptidoglycan 55 a variety of surfaces other than glass including fabric, since it is capable of facilitating the removal of fecal contact lenses, metallic surfaces, ceramic surfaces, cell matter from a surface such as cloth swatches. Such fecal surfaces, plastic surfaces, tissue and the like. Further, matter is known to contain peptidoglycans associated such in vitro methods may be practiced for example in with intestinal bacteria. Lysozymes are enzymes which the human oral cavity as described in more detail here are reactive with peptidoglycan. Lysozymes, such as 60 inafter. hen egg white lysozyme, T4 lysozyme and mutanolysin Some known Type II endoglycosidases are listed in (Goodman, et al. (1981), J. Bacteriol, 146, 755), how Table II together with the natural biological source of ever, are not Type II endoglycosidases. This is because such enzymes. The cleavage sites for some Type II they do not have a substantial reactivity with the unique endoglycosidases are shown in FIG. 2. See "Glycopro glycosidic linkages found in N- and O-linked glycopro 65 tein and Proteoglycan Techniques' (1985) by J. G. teins used to define Type II endoglycosidases. They are, Beeley, Chapter 6, pp. 153-300, Elsevier, Amsterdam, however, reactive with peptidoglycans to produce di N.Y., Oxford. A Type II endoglycosidase not listed in saccharides of N-acetylglucosamine and N-acetyl Table II is Glycopeptidase Falso sometimes referred to 5,395,541 11 12 as PNGase F. PNGase F may be obtained from Flavo substrate core structure for Endo-F in FIG. 2 wherein bacterium meningosepticum. It is also commercially X and Y comprise NeuNAc-Gal-GlcNAc or similar available from Boehringer Mannheim Biochemical, structures and U comprises H or fucose. Indianapolis, Ind. Endo-L has a similar reactivity in cleaving the second TABLE II Enzyme Source Typical substrate endo-3-N- Acetylglycosaminidases D Diplococcus pneumoniae N-linked complex type (peripheral sugars removed) H Streptomyces plicatus N-linked hign-mannose (Streptomyces griseus) and hybrid types L Streptomyces plicatus N-linked low mol. wt. only CI Clostridium perfingens N-linked complex type (peripheral sugars removed) CII Clostridium perfingens N-linked high-mannose type F-Gal type Sporotricum N-linked complex type dimorphosphosphorum (biantennary only, requires terminal Gal) F Flavobacterium N-linked high-mannose and meminogosepticum complex types endo-o-N- Diplococcus pneumoniae O-linked, only Acetylgalactosaminidase Gal-ol-3GalNAc endo-g-N-Galactosidases Diplococcus pneumoniae Blood group A and B determinants Escherichia freundi Keratan sulphate and oligo Flavobacterium saccharides containing keratolyticus sequence R. GlcNAc-g1 3Gal-g1-4GlcNAc (or Glc)
As can be seen, Endo-H, F, D, CI and Endo-F-Gal glycosidic linkage in N-linked glycoproteins. It is spe type all cleave the second glycosidic linkage in a glyco cific for low molecular weight substrates comprising protein. In the case of Endo-F-Gal type, this glycosidic 30 Man-GlcNAc-GlcNAc-Asn. Endo-CII demonstrates a linkage is between GlcNAc and Gal. For Endo-H, F, specificity similar to Endo H. Endo-a-N-acetylgalac D, and CI, the cleavage is between two residues com tosaminidase hydrolyzes glycoprotein containing oligo prising GlcNAc, with specificity being defined by the saccharides O-linked to serine or threorine where substituents U, V, W, X, Y, and Z. GlcNAc and Gal are the first two carbohydrate resi Endo-H cleaves N-linked glycoproteins having a 35 dues. The specificity of endo-A-N-galactosidase is also high mannose content. Thus in FIG. 2, W comprises shown in FIG. 2 wherein R1 may be one of the man 2-150 mannose residues, Y comprises 1-2 mannose resi noses from which antennas in the carbohydrate unit dues and X,Z.V and U are H (hydrogen). Endo-H also may be formed. cleaves hybrid structures wherein W comprises 1-2 The Type II glycosidase glycopeptidase F (PNGase mannose residues and Y and/or Z comprise NeuNAc F) cleaves the first glycosidic linkage in N-linked glyco Gal-GlcNAc or similar structures and V comprises H proteins between asparagine and GlcNAc. It cleaves or GlcNAc. Endo-H is the preferred Type II endo high mannose structures wherein W, X and Y comprise glycosidase used in the formulations and methods of the one or more mannose residues and V and Z comprise H invention. with fucose being absent from the first carbohydrate Endo-D and Endo-CI have similar reactivities al 45 residue GlcNAc. It also cleaves hybrid structures though these enzymes are derived from different wherein W and X comprise mannose, Y and/or Z.com sources. Endo-D and Endo-CI are active on N-linked prise NeuNAc-Gal-GlcNAc or similar structure, V oligosaccharides of glycoproteins and cleave a high comprises H or GlcNAc with fucose typically being mannose structure containing more than a 5-mannose absent from the first carbohydrate residue. Complex carbohydrate residue in which case X comprises man 50 structures are also cleaved by glycopeptidase F. Such nose linked by way of an a 1-3 glycosidic bond to the structures comprise the core structure shown in FIG. 2 core structure, W comprises mannose linked by way of wherein Y and W comprise NeuNAc-Gal-GlcNAc or an oil-6 glycosidic bond to the core structure and the similar structure, X and Z comprise H, NeuNAc-Gal remaining substituents are H in FIG. 2. Endo-D also GlcNAc or similar structure, V comprises H or cleaves a core portion of a complex or hybrid structure 55 GlcNAc and fucose is sometimes present on the first after removal of most antennary residues with exo carbohydrate residue GlcNAc. glycosidases, in which case Y comprises H or GlcNAc Endo-S-N-galactosidase is known to cleave glycosy and U comprises H or fucose in FIG. 2. dic linkages within oligosaccharides on a glycoprotein The endoglycosidase Endo-F is active on N-linked or glycolipid. A typical glycoprotein substrate together glycoproteins having a high mannose content wherein with the cleavage site for Endo-3-N-galactosidase is in FIG. 2 X and Y are one or more mannose residues shown in FIG. 2 where R2 is protein, lipid or carbohy and the remaining substituents are H. Endo-F also drate, and R1 is a sugar residue or hydrogen. cleavesbiantennary hybrid structures wherein X and W Of course, the invention is not limited by the present comprise mannose linked to the core structure by way known specificity of endoglycosidases. Until recently, of a 1-3 and a 1-6 glycosidic linkages and Y comprises 65 the endoglycosidases which have been commercially NeuNAc-Gal-GlcNAc or similar structure and U com available have been expensive due to their relatively prises H or fucose. Biantennary complex structures are low levels of expression in their naturally occurring also cleaved by Endo-F. Such structures comprise the sources. Accordingly, the reactivity of such enzymes 5,395,541 13 14 has not been broadly investigated. However, with the One unit of Endo-H activity is the amount of enzyme advent of molecular cloning, greater amounts of endo required to release 1 umole of (H)-dansyl-Asn glycosidase have been or will be made available. To the GlcNAc from (H)-dansyl-Asn-(GlcNAc)4(Man)6 at extent that alternate reactivity and specificity may be pH 5.5 at 37 C. in one minute. Tarentino, A. et al. discovered for these or other endoglycosidases, such (1978) Methods in Enzymology, 50,574. The unit activity reactivity is intended to be within the scope of the in of other Type II endoglycosidases are similarly defined vention. by an appropriate substrate. Accordingly, as used herein, a "Type II endoglycosi Of course, other Type II endoglycosidases may exist dase-reactive substance' (also referred to as a "Type which have not yet been identified. Such Type II endo II-reactive substance' or a substance containing a 10 glycosidases as well as the ones described herein, in "Type II reactive linkage') is any substance which is cluding allelic variations and genetically engineered reactive with a Type II endoglycosidase. Included modifications of such endoglycosidases are within the within Type II reactive substances, of course, are glyco scope of the present invention. side-containing substances and glycoprotein. Also in Glycosides and glycoside-containing substances often cluded, however, are (1) other, as yet, unknown sub 15 strates reactive with Type II endoglycosidase at other become bound to a wide variety of surfaces. Thus, for than a glycosidic linkage, and (2) multicomponent ag example, glycoproteins, such as those associated with gregates containing components having Type II reac blood (e.g., glycosylated hemoglobin), can stain the tive linkages. surfaces of fabrics used for clothes, linen and the like. For example, microorganisms, such as bacteria, can Such stains have heretofore been highly resistant to be removed from surfaces by treatment with Endo-H. It complete removal by treatment with detergents or de is presently not known how this result occurs. Bacteria tergents in combination with various enzymes not com are not known to contain linkages which are normally prising the endoglycosidases utilized in the present in reactive with Endo-H and the details of their attach vention. A further glycoside-containing substance ment to surfaces, other microorganisms and other sub 25 which stains surfaces such as fabric and which is also stances is not well understood. Yet, bacteria removal by difficult to remove by known techniques comprises Endo-H has been observed. fecal matter. Such fecal stains include various glyco Further, other stains may involve complex aggre sides and glycoside-containing substances associated gates of substances some of which or all of which are with intestinal bacteria (e.g., peptidoglycans), catabolic reactive with Type II endoglycosidase. The term Type 30 excretions, including glycoproteins, and non-absorbed II reactive substance covers all such situations. Thus, nutrients and the like. uses of Type II endoglycosidase include (1) cleaning Other surfaces to which glycosides or glycoside-con surfaces containing Type II-reactive substances, (2) taining substances may be bound include the surfaces of treating Type II-reactive substances to prevent attach 35 hard and soft contact lenses. Soft contact lenses are ment to a surface, and (3) treating Type II-reactive typically hydrophilic cross-linked polymers having a substances such as microorganisms to produce an anti hydrogel structure or are made of silicon polymers. See, microbial effect. e.g., U.S. Pat. Nos. 3,403,393 and 2,976,576. Hard The Type II endoglycosidases used in the invention contact lenses, on the other hand, are typically made of can be obtained from the organisms listed in Table II methacrylate or methylmethacrylate polymers. Other according to methods known to those skilled in the art. surfaces include naturally occurring biofilms, cardiac Some of the Type II endoglycosidases in Table II, e.g., pacemaker leads and power packs, cellular and mucosal Endo-H from Streptomyces plicatus (initially classified as surfaces, tooth enamel, filters used to remove bacteria Streptomyces griseus) and produced in S. plicatus or S. and particulate material in processing foods; chemicals lividans and Endo-D from Diplococcus pneumoniae, are 45 and the like; air conditioning filters; the surfaces of commercially available from Boehringer Mannheim various structural components exposed to an aqueous Biochemical, Indianapolis, Ind. Besides the commer environment, e.g., boats, piers and the like; plastics and cially available preparations, Endo-H may be derived composites such as formica; and metals or metal alloys from E. coli transformed with a plasmid encoding the such as steel, aluminum, etc. Endo-H gene from Streptomyces plicatus and the pro 50 As will be shown in detail hereinafter, Type II endo moter from alkaline phosphatase (Oka, T., et al. (1985) glycosidases alone or in combination with a second Proc. Natl. Acad. Sci USA, 82, 7212-7216) by methods enzyme such as subtilisin, either with or without deter similar to that reported for the cloning and expression gent, effectively increases the removal of blood and of Endo-H from Streptomyces plicatus in E. coli (Rob fecal stains from cloth swatches. It is not known pre bins, et al. (1981) J. Biol. Chem. 256; 10 640). See also 55 Trumbly R. J. et al. (1985) J Biol. Chem..., 260, 5638. cisely how such stains adhere to such swatches. How Endo-H may also be derived from Streptomyces cells ever, the enhanced removal of such substances from engineered to express Endo-H derived from Streptomy these swatches by Type II endoglycosidase, alone or in ces plicatus (EPO Publication No. 0179449, Apr. 30, combination with other agents, suggests that at least one 1986). Alternatively, Endo-H may be produced by any glycosidic linkage is interposed between the fabric and appropriate host cell such as Bacillus subtilis using tech that part of the stain which is released upon treatment niques well known to those skilled in the art. The amino with Type II endoglycosidase. Based on these results, acid and DNA sequences of Endo-H for S. plicatus (S. the following are proposed mechanisms of the binding griseus) have been published. Robbins, P. W., et al. of glycoside-containing substances to a surface and the (1984).J. Biol. Chem, 159, 7577-7583. 65 release and/or removal of such substances by Type II The Endo-H used in the examples herein was ob endoglycosidase. These proposed mechanisms, how tained commercially or from E. coli or B. subtilis hosts ever, should not be considered as a limitation to the transformed to express Endo-H from S. plicatus. scope of the invention. 5,395,541 15 16 which may require further treatment with other en FIGURES zymes and/or detergent or surfactant. Thus, as shown in FIG. 5A, a glycoside-containing In FIG. 5E, a Type II endoglycosidase-reactive sub substance may be bound to a surface other than by an stance is shown bound to a surface. This Type II reac immunological bond. In this regard, an “immunological 5 tive substance has a proximal portion bound to the sur bond' is one which exists between an antigen and an face and a distal portion extending outwardly from the antibody, specific for that antigen (polyclonal or mono proximal portion. The proximal and distal portions are clonal). As shown in FIG. 5A, the glycoside-containing joined by a Type II reactive linkage which refers to a substance has a proximal portion bound to the surface linkage reactive with a Type II endoglycosidase. When and a distal portion extending outwardly from the prox O treated with Type II endoglycosidase, the distal portion imal portion. The proximal and distal portions are of the Type II reactive substance is "released' from the joined by a glycosidic linkage with which Type II en proximal portion of the Type II reactive substance. It is doglycosidase is reactive. As further shown in FIG.5A, to be understood that Type II reactive substances may when treated with Type II endoglycosidase, the distal comprise molecules, microorganisms or aggregates of portion of the glycoside-containing substance is 're 15 various components which may become attached to a leased' from the proximal portion of the glycoside-con surface. To the extent that the distal portion of the Type taining substance. To the extent that this distal portion is II reactive substance is not bound by other means to the not bound by other means to the surface, it is also surface, it is also readily “removed' from the surface readily “removed' from the surface and may be washed and may be washed away with a fluid. away with a fluid. The amount of Type II endoglycosidase used to pro In FIG. 5B, a glycoside-containing substance, in this duce the removal of the substances identified in the case a glycoprotein containing a carbohydrate unit and figures is defined functionally as an "amount effective' protein unit, is shown bound to a surface. This glyco for removal of the particular substance from a surface. side-containing substance further contains a carbohy This amount may vary depending on the substance and drate portion and an aglycon portion joined by a glyco 25 surface to be treated. Typical amounts are disclosed in sidic linkage which is reactive with Type II endo more detail herein with regard to the specific embodi glycosidase. In this particular case, the glycoside-con ments disclosed. taining substance (glycoprotein) is bound to the surface SECOND ENZYMES through the carbohydrate portion of the glycoside-con 30 "Second enzymes' include proteases, lipases, glycosi taining substance. When treated with Type II endo dases such as lysozyme and combinations thereof. Vari glycosidase, the aglycon portion is released from the ous proteases which may be combined with Type II carbohydrate portion of the glycoside-containing sub endoglycosidase include subtilisin, bromilain, papaine, stance. As in FIG. 5A, to the extent that the aglycon trypsin, chymotrypsin, pancreatin, lysozyme and com portion is not further bound to the surface by other 35 binations thereof. Such enzymes may be derived from means, the aglycon portion is also removed from the natural sources, e.g., subtilisin from Bacilius subtilis or surface. from genetically engineered clones, e.g., subtilisin and FIG.5C depicts the situation where a glycoside-con mutant subtilisins as described in EPO Publication No. taining substance is bound to a surface by way of at least 0130756. See also, Wells, J. A., et al. (1983) Nucleic two points of attachment. As indicated, a glycosidic Acids Res., 11, 7911-7915; Yang, M., et al. (1984) J. first linkage exists between the surface and the glyco Bacteriology, 160, 15-21; Estell, D. A., et al. (1985) J. side-containing substance. In addition, a second linkage Biological Chemistry, 260, 6518-6521. Many such en reactive with a second enzyme is also present between zymes, of course, are available from commercial the surface and the portion of the glycoside-containing SOCCS. substance to be removed. If treated only with Type II 45 In addition, Type II endoglycosidases may be com endoglycosidase, the portion of the glycoside contain bined with lipases such as bacterial, mammalian and ing substance distal from the first glycosidic linkage is fungal lipases and combinations thereof. released from the surface at least to the extent that it Glycosidases which may be used as a second enzyme was bound through the first glycosidic linkage. If con include exoglycosidases, a second Type II endoglycosi tacted with a second enzyme reactive with the second 50 dase and Type I endoglycosidases. Examples include a linkage shown, the portion of the glycoside-containing and 6-amylase, cellulase, pectinase, hemicellulase, dex substance as distal from the first glycosidic linkage and tranase, various glucanases, and the like and combina the second linkage is released from the surface. To the tions thereof. extent that this distal portion is not otherwise bound to Moreover, Type II endoglycosidase may be com the surface, i.e., by other contact points which may be 55 bined with more than one of the above classes of second reactive with other enzymes or susceptible to deter enzymes to facilitate the removal of a glycoside-con gents and/or surfactants, this distal portion is effec taining substance from a surface. tively removed from the surface. When a Type II endoglycosidase is combined with FIG. 5D shows a microorganism bound to a surface one or more second enzymes, the ratio of Type II endo through at least part of the glycoside portion of said glycosidase to second enzyme is preferably about 0.01 microorganism. The glycoside portion contains a glyco to 100 and most preferably 1 to 1. sidic linkage reactive with Type II endoglycosidase. A cleaved portion of the microorganism distal from the DSULFIDE CLEAVING REAGENTS glycosidic linkage is released from the surface when Type II endoglycosidases may also be used in combi treated with Type II endoglycosidase. To the extent 65 nation with detergents, either alone or in combination that this cleaved portion is not otherwise bound to the with one or more second enzymes and/or disulfide surface it is also removed from the surface. However, cleaving reagents to form a detergent formulation. Sub multiple points of contact may exist with the surface stances capable of cleaving disulfide bonds are varied, 5,395,541 17 18 but fall generally into three categories: oxidizing agents, hood laundromats, and professional laundry services, reducing agents, and miscellaneous addition substrates since in order to be commercially practical, the process such as those exemplified by fumaric acid and sodium needs to be conducted under conditions ordinarily sulfite. Suitable oxidizing agents include hydrogen per available to the user. oxide, performic acid, sodium perborate, and oxidizing 5 In another embodiment of the invention, conven bleaches. Effective reducing agents include dithio tional washing procedures using commercial detergents threitol (DTT), 3-mercaptoethanol (BME), sodium are used and the Type II endoglycosidase, second en borohydride, and the like. zyme and disulfide-cleaving substance are provided, Alternate disulfide cleavage reagents which are not either separately or together, as an additive, much in the easily classified include mercuric chloride, nitroprus 10 manner of the methods in which bleach is used. Thus, side, tributylphosphine, and phosphothiolate. A particu these may be added along with the detergent at the larly useful cleavage reagent is sodium sulfite, which beginning of the wash cycle or at some intermediate results in sulfitolysis of the disulfide according to the point, for example, after approximately half of the wash reaction: R-S-S-R--SO3-2 R-S-SO3-2-1-SR. cycle is completed. If handled in this way, assuming an The equilibrium of this reaction may be shifted by re 15 approximately 1:500 dilution of a solid or liquid deter moval of the thiol anion using heavy metal ions or oxi gent composition (approximately 2 mg/ml of the solid), dizing agents. The oxidizing power may be provided by arbitrary amounts of the Type II endoglycosidase, sec aeration or an oxidizing agent, such as CuSO4 or sodium ond enzyme and disulfide cleaving reagents may be perborate. added without the upper limit imposed by this dilution. The foregoing list of substances capable of cleaving 20 (If the Type II endoglycosidase, second enzyme and disulfides is not meant to be comprehensive, and con disulfide cleaving reagent had been added to the deter versely does include substances which are effective but gent composition originally, and if, for example, the not necessarily appropriate for a commercial product. disulfide cleaving reagent constituted 50% of the com In order to be successful commercially, the added sub position, only 1 mg/ml would result in the final wash stance must be relatively inexpensive and must not have 25 solution. However, if these materials are added sepa undesirable properties for its intended use. Thus, for rately, amounts most effective for the particular Type II example, while the use of mercuric chloride would be endoglycosidase, disulfide cleaving reagent and second workable in carrying out the process of the invention, it enzyme may be added.) would not be suitable for ordinary detergent products With respect to the Type II endoglycosidase and intended for commercial use. 3-mercaptoethanol and 30 second enzyme, only very small quantities are usually DTT are feasible commercially, except that they have required. Typically, the Type II endoglycosidase and mildly offensive odors. Particularly preferred sub second enzyme are added to a final concentration of stances, therefore, for commercial formulation, are so approximately 1-500 ug/ml of wash solution for each dium sulfite (preferably in combination with an oxidiz enzyme. In the case of the disulfide-cleaving reagent, ing agent) or hydrogen peroxide, which are inexpensive 35 however, larger amounts than would be permitted by and are relatively safe. Reviews of materials which are the dilution of the detergent may be desirable. For ex useful in the cleavage of disulfide bonds are found, for ample, cleavage of disulfide bonds using sodium boro example, in Chemical Modification of Proteins, Means, hydride may conveniently be carried out with concen G. E., et al., eds (1971), Holden-Day, Inc. San Fran trations as high as 0.2M reagent in the present of similar cisco, Calif., Ch 8; and Chemical Reagents for Protein quantities of buffer (Lundbald, R. L., et al., Chemical Modification, Lundbald, R. L. et al., eds (1984), CRC Reagents for Protein Modification, supra). Press, Inc., Boca Raton, Fla., Ch. 7. Although such high amounts are conventional, they Typically, the Type II endoglycosidase alone or in are not necessarily required, and lower concentrations combination with one or more second enzymes forms are workable. Sulfitolysis is ordinarily carried out in 0.01-3% wt/wt of the detergent compositions of the 45 sodium sulfite concentrations of the order of 0.1M, invention, and may include disulfide-cleaving reagents, although concentrations as low as 0.01M and lower can ranging from about 10-40% wt/wt thereof. The also be used. DTT is effective when supplied at concen amounts present depend, of course, on the nature of the trations of the order of 0.02-0.1M. In short, the disul endoglycosidase (and second enzyme, if used) and the fide-cleaving reagent concentration can be varied over disulfide cleavage reagent, the dilution of the detergent 50 a wide range for any of these reagents and effectiveness in the wash solution, and the conditions of the wash. maintained. The optimum concentration for a particular However, the ranges given are generally typical. application will, of course, depend on the nature of the In one embodiment of the invention, surfaces having stain and the nature of the reagent, as well as the condi glycoprotein containing substances bound thereto are tions of the wash procedure, including time, tempera treated with the combination (simultaneous or sequen 55 ture, and pH. tial) of a disulfide cleaving reagent, a Type II endo In an alternative and more convenient approach, the glycosidase and a second enzyme at suitable pH, tem Type II endoglycosidase, second enzyme and disulfide perature, for an appropriate period of time. These con cleaving substance are added to the original detergent ditions are, of course, variable according to conve composition, and the process is conducted as a standard nience, and the selection of the Type II endoglycosi 60 wash procedure using these modified detergents. Under dase, protease and the substance to cleave disulfides to these circumstances, the detergent composition will some extent depends on this selection. However, conve correspond to that described above, but the amount of nient conditions frequently encountered are pH values the composition can also be varied over the range of between 5 and 12. Temperatures of 20-55 C., particu approximately 0.5 mg/ml-10 mg/ml or greater of the larly around 40-55 C., and times of up to 20 minutes, 65 wash solution, depending, again, on the conditions of usually around 10-15 minutes are typical and preferred. the wash solution and procedure, and on the solubilities The preferred times and temperatures are those gener of the detergent components. In any case, the inclusion ally utilized in household washing machines, neighbor of the Type II endoglycosidase, disulfide-cleaving rea 5,395,541 19 20 gent and second enzyme in the detergent limits the In addition to the cleaning of fabrics using common concentrations of these components in accordance with cycles in washing machines, the cleaning compositions the dilution of the detergent. Thus, even if a 1:100 dilu herein may also be used for removing glycoside-con tion is used (10 mg/ml), and the disulfide-cleaving rea taining substances and/or microorganisms from other gent for example, is limited to 50% of the detergent surfaces such as metals and metal alloys such as found in composition, a maximum concentration of 5 mg/ml surgical instruments, pipelines, metal containers and the disulfide-cleaving reagent in the resulting wash solution like, and plastics and composite materials such as For is an upper limit. Typically, of course, the concentration mica and the surfaces of boats, piers and the like. De of disulfide-cleaving reagent in the detergent will be less pending upon the particular application, the composi than 50%, mandating even lower concentrations of the 10 tion may comprise Type II endoglycosidase alone or in disulfide-cleaving reagent. combination with a disulfide cleaving reagent, second The detergent compositions of the invention contain enzyme and/or detergent surfactant. mostly detergent active substances, relatively smaller Type II endoglycosidase may also be formulated in a amounts of disulfide-cleaving reagent, if used, and quite composition for removing glycoside-containing sub small amounts of Type II endoglycosidase and second 15 stances and/or microorganisms including yeast, fungi, enzyme, if used, which is especially desirable in view of algae and bacteria from “biological surfaces' such as the cost of enzymic components. Thus, in general, the surfaces of skin, skin pores, hair, hair follicles and tissue. preparation will contain 60-90% detergent active sub Thus, those skilled in the art of shampoo formulations, stances, including conventional commercial detergent conditioner formulations, soap formulations and the additives such as surfactant builders and whiteners, 20 medicinal arts can readily adapt the above disclosure 0.01-3% Type II endoglycosidase and second enzyme, for detergent formulations to employ Type II endo and approximately 0-40% disulfide cleavage reagent. glycosidase in such applications. When so formulated, Of course, it is also possible to add only one of these such compositions are useful in removing glycoside three additives to the original detergent and to supply containing substances which may adhere to such sur the other separately to the wash liquid. In particular, 25 faces. the Type II endoglycosidase may be added to a pre Type II endoglycosidase may also be formulated in a wash, followed by a detergent containing the second composition for removing glycoside-containing sub enzyme, or addition of the detergent containing endo stances and/or microorganisms, especially yeast and glycosidase may be followed or preceded by treatment fungus, from the surfaces of plants such as fruits and with the second enzyme. 30 vegetables. Such compositions preferably include non ionic surfactant. CLEANING COMPOSITIONS In addition, Type II endoglycosidase may be formu Endo D, F and H are preferred Type II endoglycosi lated in deodorant compositions in a manner known to dases for use in cleaning compositions. Endo-H is most those skilled in the art to provide endoglycosidase ac preferred. 35 tivity to remove glycoside-containing substances and For removal of glycoside-containing substances, the /or microorganisms responsible for undesirable odors. compositions herein preferably comprise from about 0.1 Such deodorant formulations employing Type II endo ppm (parts per million) to 1200 ppm, more preferably glycosidase may include modifications of formulations from about 1 ppm to 1000 ppm, most preferably from for stick, creams and aerosol deodorants known to those about 20 ppm to about 200 ppm, of Type II endo skilled in the art. glycosidase, depending on the type of composition. Further, Type II endoglycosidase may be formulated Cleaning compositions are preferred. Laundry deter for the treatment of acne which usually results from gent compositions are most preferred for use herein, inflammation, at least to the extent that glycoside-con and preferably comprise from about 0.1 ppm to 1200 45 taining substances and/or microorgansims responsible ppm of Type II endoglycosidase, preferably from about for or involved in such inflamation are bound to a sur 20 ppm to 200 ppm of Endo D, F or H, most preferably face. As with the above formulations, those skilled in from about 50 ppm to 125 ppm Endo H. the art are capable of modifying known acne formula When used to control or remove microorganisms, the tions to incorporate a Type II endoglycosidase alone or compositions preferably comprise from about 0.1 ppm 50 in combination with other enzymes, detergents and/or to 1200 ppm, more preferably from about 1 ppm to 1000 surfactants. ppm, most preferably from about 20 ppm to 400 ppm, of When used to treat contact lens, Type II endoglycosi Type II endoglycosidase, preferably Endo-H. Cleaning dase suitably is supplied at a concentration of about compositions are preferred and preferably comprise the 0.1-20 ug/ml in the cleaning compositions, and the same amounts of Type II endoglycosidase, preferably 55 concentration of a second enzyme such as a protease is Endo-H. in the same range if such second enzymes are utilized. Described below are suggested types of compositions Treatment times can vary from about five minutes to which comprise Type II endoglycosidase for removal about 15 hours, but a standard convenient cleaning time of glycoside-containing substances and/or microorgan is overnight, so that the wearer can allow the lenses to isms. The compositions can be made and used in any soak while he sleeps. A variety of protocols are suitable, way which does not destroy enzyme activity. They can but ones that are particularly preferred are the use of a be made up of any ingredients which do not unduly single solution containing Type II endoglycosidase and hinder the activity of the enzyme. The compositions can the second enzyme (if used) conducted from 10 minutes be laundry detergents, dishwashing detergents, hard to two hours or overnight at room temperature, or a surface cleaners, dental enamel cleaners, liquid and bar 65 10-minute to two-hour presoak in the presence of Type soaps, anti-acne compositions, antiperspirants, sham II endoglycosidase solution, followed by a similar over poos, face creams, fruit and vegetable surface preserva night treatment with a solution containing a second tives, or fabric softeners. enzyme. 5,395,541 21 22 Preferred general purpose second enzymes for 5. The cleaning solution contains 7 ug/ml subtilisin, 3 contact lens formulation include proteases such as pa ug/ml Type II endoglycosidase, 10 mM 2-mercaptoe pain, pancreatin and subtilisin. The preferred Type II thanol, and 2% sodium dodecyl sulfate (SDS). The endoglycosidase enzyme is Endo-H from Streptomyces lenses are soaked in this solution for 3 hours at 20° C. plicatus. A single second enzyme protease may be used, 5 6. The cleaning solution contains 4 ug/ml subtilisin, 2 or the composition may contain a mixture of second ug/ml trypsin, 10 g/ml Type II endoglycosidase, and enzymes. 2% SDS. The lenses are soaked in this solution for 7 In addition, the contact lens compositions may in hours at 20° C. - clude additional components which aid in the overall 7. Solution A contains 4 ug/ml subtilisin and 2 ug/ml enzymatic degradation. Particularly useful among these O trypsin in 2% SDS. Solution B contains 10 pg/ml Type are disulfide cleavage reagents such as 2-mercaptoe II endoglycosidase plus 10 mM 2-mercaptoethanol. The thanol, cysteine hydrochloride, dithiothreitol, dithioe lenses are immersed in solution B for 20 minutes at 30 rythritol, sodium bisulfate, sodium metabisulfite, thio C. and then in solution. A for 6 hours at 25 C. urea, and the like, generally preferred in a range of In all the foregoing examples, the lenses are thor about 0.01-5% by weight preferably 0.05-1% by 15 oughly rinsed in saline before being returned to the weight. In addition, detergents may be included in the wearer's eyes. composition to aid in the wetting of the lens with the The compositions herein can be formulated in a vari enzyme-containing solution. Suitable detergents include ety of physical forms, including liquids, gels, pastes and sodium dodecyl sulfate, sodium monolaurate, nonionic solid particles such as powders and granules. The com surfactants such as alcohol ethoxylates (e.g., polyethox 20 positions can be formulated as laundry detergents, such yethanol) anionic surfactants such as ether sulfonates, linear alkylbenzene sulfonates, sodium lauryl sulfate, as disclosed in U.S. Pat. Nos. 4,507,219, 4,318,818, and the like. 4,605,509 and 4,412,934; dishwashing detergents such as Suitable buffers and stabilizers for contact lens clean disclosed in U.S. Pat. Nos. 4,714,562, 3,630,923, ing may also be used and include sodium or potassium 25 4,133,779, 4,316,824 and 4,555,360; hard surfaces clean citrate, citric acid, boric acid, sodium EDTA, various ers such as disclosed in U.S. Pat. Nos. 4,414,128, mixed phosphate buffers and NaHCO3. Generally buff. 3,679,608, 3,985,668 and 4,005,027; fabric softeners such ers and stabilizers may be used in amounts ranging from as disclosed in U.S. Pat. Nos. 3,944,694, 4,073.996, about 0.001 to about 2.5% and preferably about 0.1 to 4,424,134 and 4,661,269; bar soaps such as disclosed in 1% by weight. It should be understood that the forego 30 U.S. Pat. Nos. 3,993,722 and 3,070,547; shampoos such ing description of the amounts of the various com as disclosed in U.S. Pat. Nos. 4,345,080, 4,704,272 and pounds which may be used in the present invention for 4,741,855; antiperspirants such as disclosed in U.S. Pat. cleaning contact lens are stated in percentage of ingredi No. 4,725,432; anti-acne products such as disclosed in ents in solution (wt/vol). The formulation may also take U.S. Pat. Nos. 4,318,907 and 4,608,370; and oral compo the form of one or more conventional solid dosage 35 sitions such as disclosed in U.S. Pat. No. 4,684,518. The forms such as tablets suitable for use in measured quan above patents are incorporated herein by reference. tity of a suitable solvent such as water. The percentage The compositions preferably have a pH from about 4 composition of the solid dosage forms is such that when to 10, more preferably from about 5 to 8 for good en dissolved in a specified volume of water, the solution zyme performance. will have the percentage composition within the ranges 40 Laboratory work on microorganism removal has set forth in the specification. If solid dosage forms are shown that, in order to obtain effective removal, the used, the formulation may include conventional lubri bathing of the surface holding the microorganisms in cants, binders, and excipients which include glycerol, some instances requires a physical or chemical action to sorbitol, boric acid, propylene glycol, polyethylene remove the microorganisms. Microorganisms tested glycols, dextran, methylcellulose, hydroxyethylcellu 45 include: lose, water soluble salts of carboxymethylcellulose, or Escherichia coli including Type 1 and 3 fimbriae naturally occurring hydrophilics such as gelatin, algi Staphylococcus aureus nates, tragacanth, pectin, acacia and soluble starches. Staphylococcus epidermidis Typical compositions and protocols useful in clean Serratia marcescens ing contact lens include the following: 50 Streptococcus mutans 1. The composition contains 1-100 pg/ml Type II Streptococcus sanguis endoglycosidase. The lenses are removed and placed in Bacillus sp. contact with the solution for a period of 12 hours at 22 Candida sp. C. The lenses are removed from the cleaning solution Aspergillus sp. and rinsed. 55 In the case of removal of bacteria such as E. coli, for 2. Solution A contains 10 ug/ml of Type II endo example, the surface-bound microorganisms may be glycosidase; solution B contains 5ug/ml subtilisin. The treated with Endo-H and then removed by chemical lenses are soaked in solution. A for 30 minutes at 25 C., action, such as by treatment with an antimicrobial removed, and immersed in solution B for 10 hours at 25 agent, or a physical action, such as by rinsing with C. water or hand wiping. It is preferred for liquid and bar 3. The cleaning solution contains 10 pg/ml of the soaps, dental enamel cleaners, antiperspirants, anti-odor protease pepsin and 10 ug/ml of Type II endoglycosi fabric softeners and anti-acne compositions that the dase. The lenses are soaked in this solution for 5 hours composition include an anti-microbial agent, such as at 20 C. Irgasan (E) (Ciba-Geigy) or chlorhexidine, in addition to 4. The cleaning solution contains 5 g/ml subtilisin, 5 65 the Endo-H. An antimicrobial agent is not required in pg/ml Type II endoglycosidase, and 10 mM 2-mercap the composition (for example a hard surface cleaner) toethanol. The lenses are immersed in this solution for 5 when physical action such as water rinsing or wiping by hours at 30° C. hand will occur. 5,395,541 23 24 Preferred herein are detergent cleaning compositions, Optional detergent composition components include especially granular and liquid laundry detergent com enzymes (e.g., proteases and amylases), peroxygen positions. These detergent cleaning compositions pref bleaches and bleach activators, halogen bleaches (e.g., erably comprise from about 1% to 90%, more prefera sodium and potassium dichloroisocyanurates), soil re bly from about 5% to 50%, by weight, of detergent lease agents (e.g., methylcellulose), soil suspending surfactants, most preferably from about 10% to 40% by agents (e.g., sodium carboxymethylcellulose), fabric weight. brighteners, enzyme stabilizing agents, color speckles, Surfactants useful in the detergent compositions Suds boosters or suds suppressors, anticorrosion agents, herein include well-known synthetic anionic, nonionic, dyes, fillers, germicides, pH adjusting agents, non amphoteric and zwitterionic surfactants. Typical of 10 builder alkalinity sources, and the like. these are the alkyl benzene sulfonates, alkyl- and al kylether sulfates, paraffin sulfonates, olefin sulfonates, ENDOGLYCOSIDASE PLUS ANTIMICROBIAL alkoxylated (especially ethoxylated) alcohols and alkyl AGENTS phenols, amine oxides, alpha-sulfonates of fatty acids Of the Type II endoglycosidases, endo-3-N-acetyl and of fatty acid esters, alkyl betaines, and the like, 5 glucosaminidase H, D, F and/or PNGase F are pre which are well known from the detergency art. In gen ferred for formulating antimicrobial compositions and eral, such detersive surfactants contain an alkyl group in for use in the antimicrobial methods herein. Endo-H is the C9-C18 range. The anionic detersive surfactants can most preferred. be used in the form of their sodium, potassium or trie When the Type II endoglycosidase is used alone, it is thanolammonium salts; and the nonionic surfactants formulated such that its concentration produces an generally contain from about 5 to about 17 ethylene antimicrobial effect. When the antimicrobial composi oxide groups. C11-C16 alkyl benzene sulfonates, tion comprises at least two different components, i.e. a C12-C18 paraffin-sulfonates and alkyl sulfates are espe Type II endoglycosidase and one or more antimicrobial cially preferred in the compositions of the present type. agents, each of the components are present at a concen A detailed listing of suitable surfactants for the com 25 tration sufficient to produce an antimicrobial effect. The positions herein can be found in U.S. Pat. No. 3,936,537, amount of at least one component in said compositions Baskerville, issued Feb. 3, 1976, incorporated by refer is generally less than the amount required for that com ence herein. Commercial sources of such surfactants ponent to produce the same antimicrobial effect if used can be found in McCutcheon's Emulsifiers and Deter alone in a similar composition. gents, North American Edition, 1984, McCutcheon 30 As used herein, an 'antimicrobial effect' includes the Division, MC Publishing Company, also incorporated removal, killing, inhibition of growth, change in gross herein by reference. morphology, protoplast formation and/or degradation Useful detergency builders for the detergent compo of the cell wall of a microorganism when contacted sitions herein include any of the conventional inorganic with a Type II endoglycosidase alone or in combination and organic water-soluble builder salts, as well as vari 35 with a second component comprising an antimicrobial ous water-insoluble and so-called "seeded' builders. agent. The instant laundry detergent compositions preferably As used herein, an "antimicrobial method' refers to a comprise from about 1% to 75%, more preferably from method which produced an antimicrobial effect. In one about 5% to 40%, most preferably from about 10% to aspect of the invention, the antimicrobial method causes 20%, by weight of detergent builders. These composi the killing of microorganisms, the inhibition of microor tions preferably have a pH of from about 6 to 10. ganism growth, and/or changes in the gross morphol Nonlimiting examples of suitable water-soluble, inor ogy of the microorganism. In another aspect of the ganic alkaline detergent builder salts include the alkali invention, the antimicrobial method causes the removal metal carbonates, borates, phosphates, polyphosphates, of a microorganism from a surface. In the antimicrobial tripolyphosphates, bicarbonates, silicates and sulfates. 45 methods to remove microorganisms from surfaces, it is Specific examples of such salts include the sodium and preferred that the surface be treated with the antimicro potassium tetraborates, bicarbonates, carbonates, tri bial agent and the Type II endoglycosidase simulta polyphosphates, pyrophosphates, and hexametaphos neously, rather than treating with the additional antimi phates. & crobial agent immediately after treating with Type II Examples of suitable organic alkaline detergenc 50 endoglycosidase. In some applications of the antimicro builder salts are: (1) water-soluble amino polyacetates, bial methods, a combined antimicrobial effect may be e.g., sodium and potassium ethylenediaminetetraace produced, e.g. killing and/or growth inhibition may tates, nitrilotriacetates, and N-(2-hydroxyethyl)nitrilod occur in combination with microorganism removal iacetates; (2) water-soluble salts of phytic acid, e.g., from a surface. sodium and potassium phytates; (3) water-soluble poly 55 As used herein, an "antimicrobial composition” refers phosphonates, including sodium, potassium and lithium to a composition containing at least two different com salts of ethane-1-hydroxy-1,1-diphosphonic acid, so ponents: a Type II endoglycosidase and a different con dium, potassium, and lithium salts of methylenediphos ponent comprising an antimicrobial agent. Such antimi phonic acid and the like. crobial compositions have variable antimicrobial effects Seeded builders include such materials as sodium depending upon the amount and choice of Type II carbonate or sodium silicate, seeded with calcium car endoglycosidase and antimicrobial agent. Observed bonate or barium sulfate. Hydrated sodium zeolite A antimicrobial effects include the killing of microorgan having a particle size less than about 5 microns is partic isms and/or inhibiting microorganism growth, the re ularly desirable. moval of microorganisms from a surface and the pre A detailed listing of suitable detergency builders can 65 vention of microorganism attachment to surfaces. be found in U.S. Pat. No. 3,936,537, incorporated herein As used herein, an "antimicrobial-effective concen by reference. Preferred builders are fatty acids, polycar tration' of Type II endoglycosidase generally refers to boxylates, polyphosphates and mixtures thereof. the final concentration of Type II endoglycosidase used 5,395,541 25 26 alone to contact a microorganism to produce an antini attachment to such surfaces. In other aspects, there is a crobial effect. negative effect on microorganism viability or microor As used herein, an "antimicrobial agent' is a second ganism morphology. different component of an antimicrobial composition. Surface treatment(s) with Type II endoglycosidase Such antimicrobial agents in general are antibiotics and and antimicrobial agent can be performed periodically include agents which kill microorganisms and those so as to prevent further growth or attachment or adhe which inhibit microorganism growth. Examples of such sion of microorganisms to the surfaces exposed to the antimicrobial agents include bacteriocides, fungicides treatment. and algicides each of which are capable of killing or Of the Type II endoglycosidases, Endo-H, D, F and inhibiting the growth of bacteria, fungi or algae, respec 10 /or PNGase F are preferred. Of these, Endo-H is most tively. Bacteriocides include compounds such as chlor preferred. In general, an antimicrobial-effective amount hexidine, 2,4,4-trichloro-2'-hydroxydiphenyl ether, of Type II endoglycosidases for use in combination Triclocarban (R), penicillins, tetracycline and Baci with antimicrobial agents is from about 1 to 1200 ppm tracin (R). Fungicides include Nystatin (R), Amphoteri Endo-H, D, F, and/or PNGase F, preferably from cin B (R), Benomyl (R), Captan (R) and Dichloran (R). 15 about 1 to 1200 ppm Endo-H, more preferably from Other examples of antimicrobial agents include surfact about 20 to 1000 ppm Endo-H, most preferably from ant-stable antimicrobial enzymes such as surfactant-sta about 50 to 400 ppm Endo-H. The amount used depends ble f8-1,3-glucanases, lysozymes, proteases and chiti upon the type of treatment and amount of exposure to nases, and detergent surfactants such as anionic, non the surface or microorganism to be treated. In general, ionic, zwitterionic, ampholytic and cationic surfactants 20 an effective amount of antimicrobial agent, which de known to those skilled in the art. The latter should be pends upon which agent is used, is from about 0.5 to employed in an amount sufficient to produce an antimi 1200 ppm, preferably 2 to 1200 ppm, most preferably crobial effect. The above antimicrobial agents identified from about 5 to 350 ppm chlorhexidine or 2,4,4-tri by generic name or trademark are compositions as iden chloro-2'-hydroxydiphenyl ether, or 0.5 to 100 ppm tified in the Merck Index, 10th Ed. (1983), Merck & Co., 25 Nystatin (R). Inc., Rahway, N.J. When Type II endoglycosidase is used alone to kill Type II endoglycosidases different from the first and/or inhibit microorganisms, the use of substantially component of the antimicrobial compositions may also more Type II endoglycosidase is generally required. be used as an antimicrobial agent. Thus, to the extent For example, about 100 ppm to 1000 ppm of Endo-H Type II endoglycosidases are themselves antimicrobial has been shown to substantially decrease the viability of agents (e.g. are capable of producing an antimicrobial yeast cells exposed to such concentrations. When yeast effect, such as morphological changes or protoplast is exposed to less than 100 ppm of Endo-H, however, a formation), they may be combined with a different significant decrease in viability has not been observed. Type II endoglycosidase to form an antimicrobial com Although the lower limit of Endo-H necessary to ad position. Antimicrobial compositions may therefore 35 versely affect yeast viability has not yet been deter comprise one or more different Type II endoglycosi mined, the lower limit of its antimicrobial-effective dase with or without one or more antimicrobial agents concentration is believed to be between 10 and 100 not comprising Type II endoglycosidase. ppm. Similar amounts of Endo-H are believed to be Preferred antimicrobial agents for use herein are useful to kill and/or inhibit other microorganisms such chlorhexidine, 2,4,4-trichloro-2'-hydroxydiphenyl as algae and fungi. The exact effect of Endo-H and ether, Triclocarban (R), Nystatin (R) Amphotericin B(R) other Type II endoglycosidases on these organisms and antibiotic, anionic and nonionic detergent surfactants. A others, e.g., bacteria, when not used in combination surfactant-stable antimicrobial lysozyme is disclosed in with antimicrobial agents has not yet been determined. U.S. Pat. No. 5,041,236 entitled Methods and Composi The range of antimicrobial-effective concentrations of tions Employing Certain Lysozymes and Endoglycosi 45 Type II endoglycosidase for use against such organ dases in the names of Richard S. Carpenter and Ann M. isms, however, can be routinely determined. Wolff, filed on even date as this application. Other lyso The antimicrobial methods and compositions of the zymes, e.g. hen egg white lysozyme, have been used in invention have a wide applicability and include antimi combination with Endo-H to produce antimicrobial crobial methods and compositions for personal care, effects albeit to a lesser extent and with variability in the 50 health care and household and industrial cleaning. results obtained. Thus, such methods and compositions may be used to The antimicrobial compositions and methods of the formulate and use antimicrobial mouthwash, dentifrice invention can produce an antimicrobial effect on a wide or denture cleaner, as well as antimicrobial liquid or range of microorganisms including Gram-positive and solid hand or body soaps, anti-acne medication, deodor negative bacteria, fungi, and algae. Such bacteria in 55 ant, shampoo and face creams and compositions for clude Escherichia coli, Streptococcus mutans, Staphylo cleansing wounds or suppressing infections. Typical coccus epidermidis, and Staphylococcus aureus. Such household applications include antimicrobial cleaning fungi include yeasts such as Candida and Saccharony products such as liquid soap, hard surface cleaners, and ces, and species and filamentous fungi such as Aspergil liquid and granular laundry detergents. Heavy duty lus, Sporobolomyces, Basidiobolus and Entomophthora. antimicrobial detergent compositions may also be for A specific advantage of combining a Type II endo mulated for industrial use. glycosidase (e.g. Endo-H, D, F and/or PNGase F) with Chlorhexidine is an effective oral antibacterial agent an antimicrobial agent is that less of the antimicrobial and is preferred for use in dental applications. 2,4,4-tri agent can be used to produce an antimicrobial effect. In chloro-2'-hydroxydiphenyl ether is available as Irgas some aspects of the invention, the antimicrobial agent 65 an(R) DP300 from Ciba-Geigy and is a broad-spectrum when used with a Type II endoglycosidase produces an antimicrobial effective in personal care and laundry antimicrobial effect comprising the removal of microor applications. Triclocarban (R) from Monsanto is a bacte ganisms attached to surfaces or the prevention of their riostat useful in bar soaps. Traditional antibiotics can 27 5,395,541 28 also be employed as the additional antimicrobial agent consisting of anionic, nonionic, zwitterionic, an herein. Lastly, surfactant-stable antimicrobial enzymes pholytic and cationic surfactants. The antimicrobial can be used in dental applications and for preservation hard surface cleaners herein optionally further comprise of shampoos and other surfactant-containing formula abrasive, builder, diluent, solvent, suspending agent tions. A preferred surfactant-stable antimicrobial en (such as clay, carboxymethylcellulose, and polyacryl zyme is the lysozyme disclosed in the previously identi ate), perfume, and/or colorant. fied copending application in the names of Carpenter The antimicrobial laundry detergent herein, in addi and Wolff. Surfactant-stability of antimicrobial enzymes tion to Type II endoglycosidase and antimicrobial can be gauged herein by retained activity in the pres agent, preferably comprises from about 1 to 99 weight ence of representative amounts of alkyl ether sulfate or 10 %, more preferably from about 5 to 60 weight %, most linear alkylbenzene sulfate, for example. preferably from about 10 to 40 weight % detergent The antimicrobial composition may be formulated as surfactant, preferably selected from the group consist an antimicrobial mouthwash, dentifrice, or denture ing of anionic, nonionic, zwitterionic, ampholytic and cleaner. The treatment of microorganisms to produce cationic surfactants. A preferred liquid or granular anti an antimicrobial effect (e.g. to remove or prevent mi 15 microbial laundry detergent comprises from about 2 to croorganism attachment to natural or synthetic soft 250 ppm Endo-H, from about 2.5 to 40 ppm 2,4,4'-tri and/or hard surfaces in the oral cavity or to kill micro chloro-2'-hydroxydiphenyl ether, and from about 1 to organisms or inhibit their growth in the oral cavity), 99 weight %, preferably from about 5 to 60 weight %, then, essentially comprises rinsing with an antimicrobial detergent surfactant. The antimicrobial laundry deter mouthwash, cleaning the teeth with an antimicrobial 20 gents herein optionally further comprise builder, per dentifrice, and/or cleaning dentures with an antimicro fume, bleach, diluent, suds suppressor, colorant, bright bial denture cleaner. The antimicrobial mouthwash, ener, soil suspending agent, antiredeposition aids, soft dentifrice and denture cleaners herein preferably com eners, and/or soil release agents. prise Endo-H, and chlorhexidine and/or surfactant sta The antimicrobial shampoo for use herein preferably ble antimicrobial enzyme as the antimicrobial agent. 25 comprises Endo-H, an antimicrobial agent, and from Where chlorhexidine is used, the antimicrobial mouth about 5 to 60 weight% detergent surfactant, preferably wash, dentifrice, or denture cleaner preferably com selected from the group consisting of lauryl sulfate, prises from about 50 to 1200 ppm Endo-H and from isoethionate, acyl amidobetaine, alkyl glyceryl ether about 50 to 350 ppm chlorhexidine. Where surfactant sulfonate, and alkyl ether sulfate. Optional ingredients stable antimicrobial enzyme is used, the antimicrobial 30 are suds booster, conditioner, dye, colorant, perfume mouthwash, dentifrice or denture cleaner preferably and/or anti-dandruff agent. comprises from about 50 to 150 ppm Endo-H and from The present antimicrobial compositions may also be about 50 to 1,000 ppm surfactant-stable antimicrobial in the form of a preservative or microorganism control enzyme. agent for treatment of plant surfaces. Preferred are a The antimicrobial composition may also be formu 35 preservative for the surfaces of fruits or vegetables or lated as antimicrobial personal care or household clean an antimicrobial product to be applied on crops for ing products. In such products, Endo-H is preferably microorganism control. The latter is preferably in the used at a concentration of from about 1 to 1200 ppm. form of a solution to be sprayed on crops such as corn, The antimicrobial agent for use in these products is citrus, wheat, tobacco, soybeans, tomatoes and straw preferably chlorhexidine, most preferably at a concen berries for control and prevention of microorganism tration of from about 150 to 1200 ppm, or 2,4,4-tri growth. chloro-2'-hydroxydiphenyl ether, most preferably at a The following is presented by way of example only concentration of from about 2 to 500 ppm. Preferred and is not to be construed as limiting the scope of the personal care or household cleaning products are liquid invention. hand soaps, hard surface cleaners, laundry detergents 45 and shampoo (described below). EXAMPLE 1. A preferred antimicrobial liquid hand soap comprises from about 50 to 400 ppm Endo-H, from about 5 to 100 Removal of Blood and Fecal Matter from Fabric ppm 2,4,4'-trichloro-2'-hydroxydiphenyl ether, and Separate blood and fecal matter stained (cotton fab preferably from about 1 to 40 weight % to detergent 50 ric) swatches were washed with commercial detergents surfactant. Preferably from about 2 to 20 weight %, in an automatic washing machine using a warm (ap most preferably from about 3 to 10 weight %, detergent proximately 37 C.) wash cycle. The swatches were surfactant is employed, preferably selected from the then rinsed and air dried. They were then incubated group consisting of anionic, nonionic, zwitterionic, an with various amounts and types of endoglycosidase pholytic and cationic surfactants. The liquid hand soap 55 (0.005 U of Endo-D (Boehringer Mannheim Biochemi can further comprise emollient (up to about 30 weight cal), or Endo-H (Boehringer Mannheim Biochemical %) and minor amounts of perfume, colorant, solvent, from S. griseus, Catalog No. 752 967), and 0.25U N and opacifier. glycanase (PNGase F or peptide endoglycosidase F) The antimicrobial hard surface cleaners herein can be Genzyme, Boston, Mass. in 0.75 ml of 50 mM Tris glass cleaners, abrasive hard surface cleaners, scouring 60 HCl, pH 7.0 at 37° C. for 30 minutes in a test tube. The cleansers, or toilet bowel cleaners. These should be control contained buffer but no endoglycosidase. At the substantially free of hypochlorite-generating bleaches, end of the incubation period, 0.25 ml of detergent solu and other endoglycosidase-incompatible ingredients. A tion (1:125 dilution of a commercial liquid detergent preferred hard surface cleaner comprises from about composition which did not contain dyes, perfumes, 100 to 1000 ppm Endo-H, and antimicrobial agent, and 65 enzymes or brighteners in 1M Tris-HCl, pH 7.5) con from about 0.1 to 20 weight % detergent surfactant. taining 80 ug of subtilisin BPN'/ml was added to the From about 2 to 10 weight %, detergent surfactant is control and enzyme containing samples and incubated most preferred, preferably selected from the group for an additional 20 minutes. At the end of this treat 5,395,541 29 30 ment, the tubes were centrifuged and the protein con bance of each was measured at 280 mm. Change in tent in the supernatants were determined by measuring A280 was determined by subtracting appropriate blanks absorbance at 280 nm. For each treatment, a reaction (see Example 1). Higher absorbance includes the in blank was prepared which contained no swatch during crease in the amount of protein or material absorbing at the assay. The blank values were subtracted from the 5 280 mm released from the swatches. For the controls, absorbance of treated samples to determine the release the average change in A280 was 0.93. For swatches of 280 nm absorbing material during incubation. Higher treated with Endo-F the average change in A280 was absorbance represents increased release of protein from 1.05. This indicated that Endo-F increases the effi fibers. The results are shown in Table III. ciency of fecal stain removal. TABLE III 10 B. Swatches were incubated in 0.75 10 mM sodium acetate buffer pH 5.5 with or without Endo-F (0.15 Absorbance at 280 nm units) for 15 minutes at 37° C. At the end of this treat Treatment Blood Stain Fecal Matter Stain ment, 0.25 ml of detergent solution (in 0.1M Tris-HCl, Control 0.79 2.07 Endo-D 0.84 2.14 pH 7.5) containing 10 ug of the protease subtilisin BPN' Endo-H 0.83 2.12 15 was added and the tubes were incubated at 37 C. for N-Glycanase 0.78 2.10 another 15 minutes. At the end, tubes were centrifuged, supernatants were removed and absorbance at 280 nm. These results suggested that the endoglycosidases, was measured. In the case of the control (no Endo-F), Endo-D and Endo-H, in combination with the second the average change in A280 was 1.08 whereas the sam enzyme subtilisin increased the release of 280 nm ab ple treated with Endo-F showed a change in A280 of sorbing material from the blood stained swatches as 1.36. This indicated that the effect of Endo-F was en compared to the control. In addition, Endo-D, Endo-H hanced by the presence of the detergent. and N-glycanase all showed an increase in the release of C. An experiment similar to "B” was performed ex 280 nm absorbing material from the fecal stained 2 cept the detergent solution contained 10 mM 2-mercap swatches. toethanol instead of subtilisin. The average change in A280 for the control was 1.05 whereas the sample EXAMPLE 2 treated with Endo-F produced a change in A280 of Effect of Endo-H on Removal of Fecal Matter Stain 1.24. These results demonstrated the ability of Endo-F in the presence of disulfide cleaving reagents to remove This example is similar to Example 1 but was per 30 fecal stains. formed by using fecal matter stained swatches made of D. An experiment similar to “B” was performed nylon fabric. The swatches were washed in detergent except that the detergent solution contained 10 mM solution, rinsed and dried. The detergent consisted of 2-mercaptoethanol and 10 ug subtilisin BPN'. The aver liquid commercial detergent which did not contain age change in A280 for the control was 1.14 whereas enzymes, brighteners, dyes or perfumes. One set of 35 swatches was kept aside and referred as "untreated the Endo-F treated sample had a change in A280 of control'. These swatches were treated the same as the 1.29. These results indicate that Endo-F is capable of sample swatches except that they were not treated with removing fecal matter in the presence of detergent, a Endo-H. The sample swatches were incubated with protease and a disulfide cleaving reagent (2-mercaptoe 0.01U Endo-H (Boehringer Mannheim Biochemical thanol). -- Catalog No. 752967) in buffer (10 mM Na-acetate, pH EXAMPLE 4 6.0) at 37° C. for 15 minutes. Then 0.25 ml of detergent solution (1:125 dilution in 1.0M Tris-HCl, pH 7.5) was Comparison of Endo-H with Other Enzymes added and incubated for an additional 15 minutes. At Experiments similar to those described in part B of the end, tubes were centrifuged and the supernatants 45 Example 3 were repeated with Endo-H (Boehringer removed by suction. The swatches were air dried. Fi Mannheim Biochemical Catalog No. 100 119) and other bers from the swatches were examined by scanning carbohydrase enzymes except that no protease such as electron microscopy following critical point drying. An subtilisin was used. Changes in A280 were monitored electron micrograph of a detergent-washed swatch and fibers were examined by scanning electron micros stained with fecal matter is shown in FIG. 6A. As can 50 copy. Removal of particulate and bacterial debris from be seen, rod like bacteria and particulate matter are fabric was seen with Endo-Hand "Lysing Enzymes' (a found on the surface of the fabric. FIG. 6B shows a mix of proteases and glyconases obtained from Sigma swatch treated with Endo-Hand detergent. This figure Chemical Company). However, the enzymes, lyso shows a smooth clean fabric which demonstrates that zyme, a-glycosidase, g-glucosidase and 3-glucorina Endo-Hand detergents facilitates the removal of partic 55 dase, showed little or no benefit. (Results not shown.) ulate material and bacterial debris. The results of electron microscopy for this experiment EXAMPLE 3 for treatment with or without the above enzymes are shown in FIGS. 7A through 7H. FIG. 7A is a control Effect of Endo-F on Fecal Matter Stain which was not treated with endoglycosidase. FIG. 7B is Swatches stained with fecal matter (1 inch diameter) an electron micrograph of a swatch treated with lyso were washed in detergent solution, rinsed and dried. zyme; FIG. 7C is a swatch treated with Endo-H; FIG. Swatches were cut into quarters and used in the follow 7D is a swatch treated with a-glucosidase; FIG. 7E is a ing experiments. swatch treated with 6-glucosidase; FIG. 7F is an elec A. Swatches were incubated in 1 ml 10 nM sodium tron micrograph of a fiber treated with "Lysing En acetate buffer, pH 5.5 with or without Endo-F (Boehr 65 zymes'; FIG. 7G is an electron micrograph of a swatch inger Mannheim Biochemical) (0.15 units) for 30 min treated with 6-glucorinadase; and FIG. 7H is an elec utes at 37° C. The tubes were then centrifuged for eight tron micrograph of a swatch treated with chitinase. As minutes. Supernatants were removed and the absor can be seen, the swatch treated with Endo-H (FIG. 7C) 5,395,541 31 32 has been thoroughly cleansed of the fecal matter stain. Similar results were obtained for the swatches treated EXAMPLE 6 with "Lysing Enzymes' as shown in FIG. 7F. Removal of Bacteria from a Solid Surface EXAMPLE 5 An experiment similar to Example 5 was performed with two slime-producing Staphylococcus aureus cul Removal of Bacteria from a Solid Surface tures (determined by their abilities to bind to polysty To test the effect of Endo-H on removal of bacteria rene tubes). Microscope slides were modified by form from solid surfaces (glass),the following protocol was ing two rings (e1.7 cm diameter) with nail polish. Over used. Trypticase soy broth (TSB) (10 ml) was inocu O night culture of the organisms were diluted 1:10 with lated with a microbial species (Staphylococcus aureus 1% peptone solution. Diluted culture (100 ul) was put ATCC culture #6538 or Escherichia coli ATCC culture in rings. Slides were put in 150 cm petri dishes and #10536) from a stock culture slant and incubated over incubated at 37 C. After two hours incubation, slides night at 37° C. A suspension of about 108 cells/ml TSB were rinsed with distilled water and treated with three was prepared and 100 ul of this suspension was placed 15 different conditions (A. Na-acetate buffer, B. detergent, within the etched ring on a glass slide. Each slide was and C. detergent plus 1 ug Endo-H/ml) as in Example incubated for 5 minutes at 37 C. in a dry incubator oven 5. The Endo-H was obtained from E. coli transformed after which excess microbial solution was tapped off. to produce Endo-H from S. plicatus. At end of 15 min The slides were then rinsed with 100 ul of sterile dis utes, incubation slides were rinsed with distilled water tilled water. The excess solution and loose organisms 20 and Gram stained. The number of bacteria was counted were then tapped off. under microscope per 100X field for 20 fields. The After the bacteria were adhered to the glass slides (2 results are expressed as the average number of cells per or more hours at 37° C), 100 ul of the following solu field. tions were applied to separate slides: (a) 10 mM acetate buffer, pH 5.5, (b) 10 mMacetate buffer, pH 5.5-1 ppm 25 Endo-H (Boehringer Mannheim Biochemical Catalog Condition Culture Culture II No. 100 119), (c) detergent solution, (d) detergent solu A. Control 23 202 tion--1 ppm Endo-H. A set of slides were kept aside as B. Detergent 9 58 untreated controls and were not treated with any solu C. Detergent -- Endo-H 2 33 tions. The non-control slides were then incubated for 15 30 minutes at 37 C. At the end of the incubation, the solu EXAMPLE 7 tions were tapped off. The slides were then rinsed with 100 ul of sterile distilled water and air dried at room Removal of Bacteria from a Cloth Surface temperature. The bacteria which remained after this To test the effect of Endo-H on the removal of bac treatment were heat fixed and stained by a standard 35 teria from a cloth surface, the following protocol was Gram staining method. The slides were then examined used. Staphylococcus aureus (ATCC 6538) and Staphyllo by a light microscope (bright field illumination, 125X coccus epidermidis (ATCC 155) were separately cul magnification) and the number of organisms/field was tured in 5 ml of Luria's broth and allowed to grow at determined. Twenty fields were counted for each slide 37 C. for 12 hours. The cultures were then added to 30 from which the average organisms/field was calcu ml of 0.2M. NaCitrate, pH 5.5 buffer at about 103 lated. cells/ml, in two 100 ml shake flasks. Twelve cloth The following results were obtained: swatches (0.5x0.5 inch cotton swatches) were also added to the flasks after inoculation. After incubation at A. For Staphylococcus aureus 45 37 C. for two hours with gentle rotation (150 rpm), the swatches were transferred to sterile tubes and washed i) No treatment >100 organisms/field ii) Buffer > 100 organisms/field 3x with buffer comprising 200 mM. NaCitrate, pH 5.5 iii) Buffer -- Endo-H <10 organisms/field which had been previously sterilized by 0.22 micron iv) Detergent solution >100 organisms/field filtration. Six swatches were then added to a shake flask v) Detergent -- Endo-H <10 organisms/field 50 containing 0.5 mg/ml Endo-H in 30 ml citrate buffer, and six swatches were added to a shake flask containing These results indicate that Endo-H buffer alone or in only citrate buffer as the control. The Endo-H was combination with detergent reduced the number of S. obtained from E. coli producing S. plicatus Endo-H. aureus bacteria retained on the glass slides 10 fold as After incubation at 37 C. for 1.5 hours with gentle compared to treatment with detergent alone. 55 rotation (100 rpm), the swatches were transferred to sterile tubes and washed as previously described. Swatches were then plated carefully on trypticase soy B. For Escherichia coli agar plates and overlaid with enough liquid trypticase i) No treatment > 100 organisms/field soy agar to cover the swatches. After the plates were i) Buffer >100 organisms/field dry, they were incubated at 37 C. for 18 hours, and iii) Buffer -- Endo H > 100 organisms/field colonies of Staphylococcus aureus and Staphylococcus iv) Detergent >100 organisms/field epidermidis on the cloth surface were counted using a v) Detergent -- Endo H <10 organisms/field dissecting scope. The following results were obtained: These results indicate that Endo-H in combination 65 with a detergent reduced the number of E. coli retained on the glass slide 10-fold as compared to treatment with A. For Staphylococcus aureus detergent alone. Control 103 +/- 24 colonies per swatch 5,395,541 33 34 -continued bated for 30 minutes at room temperature. The tubes were again centrifuged and washed as previously de A. For Staphylococcus aureus scribed. Detection of Endo-H binding to the bacterial Endo-H 53 --/- 18 colonies per swatch cells was determined by the detection of HRP-strep tavidin, which will bind very tightly to the biotinylated 49% decrease in bacterial colonies by Endo-H treat Endo-H bound to the cells. HRP detection was deter ment mined by adding 0.5 ml of the HRP substrate OPD B. For Staphylococcus epidermidis (O-phenylenediamine) diluted in citrate phosphate buffer solution containing hydrogen peroxide. The 10 B. For Staphylococcus epidermidis chromogen generation was quenched with 2M H2SO4 Control 57 --/- 11 colonies per swatch one minute after adding OPD. The cells were centri Endo-H 16 +/- 10 colonies per swatch fuged and the supernatant was read at 490 nm. The following results were obtained: 72% decrease in bacterial colonies by Endo-H treat- 15 net. OD 490. In These results indicate that Endo-H treatment signifi cantly reduces the number of bacteria adhered to a For Staphylococcus Aureus cloth surface. Control 0.13 Endo-H, 2 minutes 1.89 EXAMPLE 8 2O Endo-H, 30 minutes 1.90 For Streptococcus mutans Binding of Endo-H to Bacteria Control 0.8 The following experiment was conducted to deter Endo-H, 2 minutes 3.76 mine if the Type II endoglycosidase, Endo-H, interacts 25 Endo-H, 30 minutes 3.80 with a surface component on the bacteria Staphylococ cus aureus and Streptococcus mutans. Such an interaction These results indicate that there is binding of Endo-H was detected. Although not completely characterized to the bacteria Staphylococcus aureus and Streptococcus herein, this interaction was not previously known and mutans. The data show that the majority of Endo-H may form the basis of the above described ability of that binds occurs in the first two minutes or less after Endo-H to remove such bacteria from a surface. 30 contact with the cells. The higher absorbance obtained Endo-H from transformed E. coli and purified by with Streptococcus mutans may indicate a higher level of modifying the methods described by Trimble R.J. et al. Endo-H binding. (1985), J. Biol. Chem., 260,5638-5690, was labelled with biotin according to the procedure described by Updyke, EXAMPLE 9 T. V. and Nicolson, G. L. (1986), Methods in Enzymol-35 A heavy duty liquid laundry detergent composition ogy, 121, 717-725. After such labelling, the Endo-H retained most of its reactivity with the glycoprotein of the present invention is as follows: ovalbumin. Overnight cultures of Staphylococcus aureus (ATCC Active 6538) grown in Luria's broth, and Streptococcus mutans 40 Component Weight % (ATCC 27607) grown in Difco Brain Heart Infusion C13 linear alkylbenzene sulfonic acid 8.00 media, were centrifuged and washed three times with C14-15 alkyl polyethoxylate (2.25) 12.00 200 mM. NaCitrate pH 5.5 buffer and suspended in the sulfonic acid same buffer to a concentration of about 109 cells/mi. 1,2 propanediol 3.50 Sodium diethylenetrianine pentaacetate 0.30 Aliquots of 0.5 ml were placed in 31.5 ml Eppendorf 45 Monoethanolamine 2.00 tubes and incubated under various conditions and times. C12-13 alcohol polyethoxylate (6.5) 5.00 Ethanol 8.50 Potassium hydroxide 1.80 Sodium hydroxide 3.85 Incu C12-14 fatty acid 10.00 Biotiny- 0.2M bation 50 Citric acid 4.00 lated NaCitrate Tine Calcium formate 0.12 Tube Cells 2% BSA Endo- pH 5.5 (min.) Sodium formate 0.86 1 0.5 m 5ul 0.5 ml 30 C2 alkyltrimethylammonium chloride 0.50 2 0.5 m 5ul 0.5 m 2 Tetraethylene pentamine ethoxylate (15-18) 2.00 3 0.5 m 5ul 0.5 ml 30 55 Water 35.2 Dye 0.08 Perfume 0.25 Incubation was done at room temperature using a Protease 0.12S slow speed rocker for either two or 30 minutes. BSA Endoglycosidase H 2000 ppm (bovine serum albumin), diluted in tris-buffered saline Notes was used as a control solution in order to prevent any 60 (). Alcohol and monoethoxylated alcohol removed. non-specific protein binding to the cells. After incuba () mg active enzyme/g (G34 mg active enzyme/g stock) tion, the tubes were centrifuged and the supernatants were discarded. Two cell washes with 2% BSA solu The ingredients listed above are added to a mixing tion were done by adding 1.0 ml BSA to the cells, Vor tank with a single agitator in the order in which they texing well, centrifuging and discarding the superna- 65 appear. Before the protease enzyme, dye and perfume tant. To the washed cells, 0.5 ml of streptavidin-HRP are added, the pH of the mix is adjusted so that a 10% (streptavidin-labeled horse radish peroxidase, Kirkeg by weight solution in water at 20° C. has a pH of about aard and Perry Laboratories, Inc.) was used and incu 8.5. 5,395,541 35 36 This composition provides superior cleaning of car bohydrate-containing stains, even compared to pro EXAMPLE 12 tease-containing and/or amylase-containing detergents. A hard surface scouring cleanser of the present inven tion is as follows: EXAMPLE 10 A heavy duty liquid laundry detergent composition of the present invention is as follows: Component Weight % False Body Fluid Phase 93.5 (Specific Gravity 1.1) Active Barasum NAS-100 4.25 Component Weight % O (Sodium saponite clay) Tetrapotassium pyrophosphate 6.00 C3 linear alkylbenzene sulfonic acid 8.00 Tripotassium phosphate 2.00 C14-15 alkyl polyethoxylate (2.25) 2.00 Sodium hypochlorite bleach 0.90 sulfonic acid Sodium lauryl alkyl sulfate 0.25 1,2 Propanediol 3.50 Surfactant Sodium diethylenetrianine pentaacetate 0.30 15 Dye and Perfume 0.26 Monoethanolamine 2.00 Endoglycosidase H 1000 ppm C12-13 alcohol polyethoxylate (6.5) 5.00 Soft Water 78.86 Ethanol 8.50 Abrasive 5.0 Potassium hydroxide 80 (Expanded Perlite-Specific Gravity 2.0 Sodium hydroxide 3.85 Average Particle Diameter 50 microns) C2-14 fatty acid 10,00 20 Hercofat 35 Fler 1.50 Citric acid - 4.00 (powdered polypro-pylene, Calcium formate 0.2 Sodium formate 0.86 Specific Gravity 0.9 Cl2 alkyltrimethylammonium chloride 0.50 Average Particle Diameter 35 microns) Tetraethylene pentamine ethoxylate (15-18) 2.00 Ratio Average Particle Water 37.12 Diameter Abrasive/Filler = .43: Dye 0.08 25 Perfune 0.25 Protease 0.125 The composition is prepared by mixing tetrapotas Endoglycosidase H 125 ppm sium pyrophosphate, tripotassium phosphate, sodium Notes saponite clay, dye, perfume and deionized water using () Alcohol and monoethoxylated alcohol removed. relatively high shear agitation to the extent necessary to () mg active enzyme/g (G34 mg active enzyme/g stock) 30 form a false body fluid phase. The alkyl sulfate surfac tant is then blended into this mixture followed by the The ingredients listed above are added to a mixing polypropylene filler material. A separate aqueous slurry tank with a single agitator in the order in which they appear. Before the protease enzyme, dye and perfume of sodium hypochlorite and perlite abrasive is prepared are added, the pH of the mix is adjusted so that a 10% 35 and then blended into the false body fluid phase while it by weight solution in water at 20° C. has a pH of about is being liquified under moderate shear agitation. The 8.5. resulting scouring composition is false bodied, i.e., gel This composition provides superior cleaning of car like in its quiescent state but easily fluidized by applica bohydrate-containing stains, particularly fecal stains. tion of shear stress. Such a composition is especially Other compositions of the present invention are ob effective for removal of stains and soil from hard sur tained when the Endo Hlevel is reduced to 0.40 mg/ml, faces. water is decreased to 35.72, and 1% Irgasan (a Ciba EXAMPLE 13 Geigy antibacterial) is added. A shampoo composition of the present invention is as EXAMPLE 11 45 follows: A liquid soap composition of the present invention is as follows: Component Level Ammoniun alkyl sulfate 55.25% (29% Aqueous solution) Active 50 Zinc pyridinethione crystals of 2.0 Component Weight % Ex. I of U.S. Pat. No. 4,345,080 Ammonium lauryl sulfate 6.0 Coconut monoethanolamide 3.0 Sodium lauryl sarcosinate 5.7 Ethylene glycol distearate 5.0 Cocamidopropyl betaine 6.3 Sodium citrate 0.5 Coconut fatty acid .0 Citric acid 0.2 Quaternary amine 0.3 55 Color solution O. Ethylenediamine tetraacetic acid 0.2 Perfume 0.5 Ammonium sulfate 0.4 Endoglycosidase H 1000 ppm Perfume 0.25 Water qs. 100.00% Kathon 5 ppm Water 720 Endoglycosidase H 1000 ppm Triclocarban 150 EXAMPLE 14 An antiperspirant stick of the present invention is The ingredients listed above are added to a mixing made utilizing the following components: tank with a single agitator in the order in which they appear below. 65 Component Level This composition provides antibacterial action for Cyclomethicone 42.55 removal of common skin flora, even when compared to Fluid AP 4.99 non-glycosidase containing, antibacterial soaps. Stearyl alcohol 49 5,395,541 37 38 -continued Component Level Active Castor wax 4.99 Componen t Weieight % Talc 6.99 5 Water 96.4 Zirconium/aluminum/glycine complex 26.67 C12-13 alcohol polyethoxylate (6.5) 0. Fragrance masking agent 0.80 Endo-H 3500 ppm C20 alcohol 0.2 EndoglycosidasePyridoxal phosphate H 500 1.00ppm This composition is prepared by mixing the alcohol O polyethoxylate and Endo-H in water at their respective levels and adjusting the final pH to between 6-7. The final composition, when sprayed on plant surfaces such EXAMPLE 15 as whole fruit or vegetables, is useful in preventing A liquid soap composition of the present invention is microbial growth on said surfaces. as follows: 15 EXAMPLE 8
Active Potentiation of Bacteriocidal Effect of Antimicrobial by Component Weight % Endo-H Ammonium lauryl sulfate 6.0 An overnight culture of Escherichia coli was diluted Sodium alkyl sarcosinate 5.7 20 into fresh nutrient broth and grown for four hours at 37 Cocamidopropyl betaine M 6.3 C. Cells were obtained by centrifugation and washed in Coconut fatty acid 10 0.2M Na-citrate buffer (SCB) pH 5.5. After centrifug Ethylenediamine tetraacetic acid 0.2 Ammonium sulfate 0.4 ing, cells were resuspended in SCB. The following Perfume 0.25 25 tubes (in duplicate) were prepared: Dye 5 ppm Water 80.15 Endo-H 50 ppm 1000 ppm 5000 pm 2,4,4-trichloro-2'-hydroxydiphenyl ether 100 ppm Condition Endo-H Chlorhexidine SCB Water Control 0 ul 0 ul 200 ul 10 ul 30 Chlorhexidine Oul 10 pull 200 pil 10 ul The ingredients listed above are added to a mixing Endo-H 200 ul 0 pull 0 ul 10 ul tank with a single agitator in the order in which they Endo-H 200 li 10 pil 0 ul -0 ul appear above. Before the dye and perfume are added, -- the pH of the mix is adjusted so that a 10% by weight Chlorhexidine solution in water at 20° C. has a pH of about 6.5. This composition provides antibacterial action for the 35 The Endo-H was from E. coli producing S. plicatus removal of common skin flora. Endo-H. To each tube, 790 ul of cell suspension added (final volume now 1 ml) and 10 ul samples were taken EXAMPLE 16 out as a 0 min control. Tubes were incubated at 37 C., on a rotary shaker and 10 ul samples were removed at A hard surface cleanser of the present invention is as 40 1 and 3 hours. The 10 ul aliquots were mixed with 990 follows: ul of PBS (Phosphate buffered saline) (10-2 dilution) and diluted further sequentially (1:10) in PBS (100 ul in Active 900 ul of PBS). 10 ul of each diluted solution was plated Component Weight 2% 45 on Luria-Bertaniagar plates. The plates were incubated Sodium lauryl alkyl sulfate 0.5 at 37° C. overnight and colonies were counted. Number Sodium alkyl sulfate 0.5 of colony forming bacteria in tubes were calculated Butyl carbitol 4.0 according to dilutions made and the logarithm of this Sodium bicarbonate 0.5 Citric acid 0.04 number used for further graphs and calculations. Formaldehyde 0.03 SO Perfume 0.05 Oninute 1 hour 3 hours Tartrate mono/disuccinate 5.0 Condition Control (log kill) (log kill) Endo-H 1000 ppm Water 88.4 Control 8.62 8.57 (05) 8.53 (09) 200 ppm 8.64 8.55 (09) 8.55 (0.9) Endo-H 55 50 ppm 8.60 4.42 (4.15) 2.44 (6.59) The ingredients listed above are added to a mixing Chlorhexidine tank with a single agitator in the order in which they 200 ppm 8.6 2.40 (6.17) 2.00 (>6.53) appear above. Before the perfume is added, the pH of Endo-H -- the mix is adjusted so that a 10% by weight solution in Chlorihexidine water at 20 C. has a pH of about 7. 60 This composition is effective for the removal of soap These results are plotted in FIG. 8. As can be seen, scum and mold from hard surfaces, and is more effica 200 ppm Endo-Henhances the bacteriocidal effect of 50 cious than a cleanser without the endoglycosidase. ppm chlorhexidine. EXAMPLE 17 Similar results were obtained for slightly different 65 concentrations of chlorhexidine and Endo-H as mea A composition used for the cleaning and/or preserva sured over a one hour time period. These results are tion of whole fruit, vegetables or other plant surfaces is depicted in FIG. 9. As can be seen, 140 ppm of Endo-H as follows: enhances the efficacy of 40 ppm chlorhexidine. 5,395,541 39 40 To further investigate this effect, a similar experiment was conducted using 20 ppm chlorhexidine (final con EXAMPLE 20 centration) with varying concentrations of Endo-H. Antimicrobial Effect of Endo-H Alone or in The results are shown in FIGS. 10A and 10B. These Combination with a Lysozyme plots represent the change in the log of colony forming A 48-hour subculture of E. coli (ATCC 31617) was units (CFU). As can be seen, a relatively linear relation used to test the effect of the lysozyme mutanolysin ship exists between the amount of Endo-H added (Sigma Chemical Co.) alone or in combination with through about 280 ppm Endo-H. Further increases in detergent and/or Endo-H. The Endo-H was from E. Endo-H concentration enhance the adverse effect on coli transformed to produce Endo-H from S. plicatus. bacterial viability through at least 1000 ppm Endo-H in 10 The following protocol and results were obtained after combination with 20 ppm chlorhexidine. treatment for two hours at 37 C.: EXAMPLE 19 Na Citrate Effect of Endo-H Alone and in Combination with 5 Mutano- pH Tide Endo-H Antimicrobial on Viability of Fungi lysin 5.5 7.0 200 ppm 200 ppm Results A log phase culture of Candida albicans was grown, 1 Control Fimbriae, tight cell wall diluted into fresh growth medium, and treated with 0, 1, 2 200 ppm -- Fimbriae, tight 10, 100 and 1000 ppm Endo-H (final concentration) for 20 cell wall 4 hours while incubating with agitation at 37 C. The 3 200 ppm -- Fimbriae, tight Endo-H was from Bacillus subtilis transformed to pro cell wall 4 200 ppm -- -- Fimbriae, tight duce Endo-H from S. plicatus. One, ten and one hun cell wall dred fold dilutions were made and plated to give viable 5 200 ppm -- -- Fimbriae, cell cell counts. Zero through 10 ppm Endo-H did not sig 25 condensation 6 200 ppm -- -- Loss of fimbriae nificantly reduce cell viability, although in one case 10 7 200 ppm -- -- Few cells, some ppm Endo-H reduced viability by about 36% after 18 ghosts, cell wall hours of incubation. However, 100 ppm to 1000 ppm disintegration Endo-H reduced the number of viable cells recovered 8 200 ppm ------Some fimbriae 9 200 ppm ------Cells in bad by about 50% to 88%, respectively, compared to the 30 shape (con control not treated with Endo-H when treated for four densed) but hours. still present In a separate experiment, a culture of Candida albi cans was grown, diluted into fresh medium, and treated As can be seen, the gross morphology of the bacteria with 2.5 g/ml Nystatin (R) in addition to either 0, 1, 10, 35 exposed to Endo-H and mutanolysin either with or 100 or 1000 ppm Endo-H (final concentration) for 18 without detergent at various pH, was significantly mod hours, while incubating with agitation at 37 C. One, ified. The most dramatic effects occurred at pH 7 when ten, one hundred and one thousand fold dilutions were Endo-H was used alone or in combination with deter made and plated to give viable cell counts. Endo-H gent. Cell viability, however, was apparently not ef reduced viable cells recovered as follows as compared fected. Endo-H and mutanolysin did not reduce the to that obtained with Nystatin (R) alone: number of colonies obtained in a plating experiment as compared to a buffer control.
ppm Endo-H % Reduction EXAMPLE 21 45 O 0% Bacterial Removal from Glass Surfaces by Endo-Hand 1 ppm 69% PNGase F 10 ppm 93% Escherichia coli (ATCC 31617) and Staphylococcus 100 ppm 99% epidermidis (ATCC 155) were used to inoculate glass 50 slides. Each slide contained two etched circles and each As can be seen, as little as 1 ppm Endo-Hsignificantly was inoculated with E. coli or S. epidermidis. enhances the mycocidal effect of Nystatin (R) whereas The bacteria were allowed to incubate at 37 C. for 10 ppm and 100 ppm Endo-H kill almost all of the fungi two hours. surviving Nystatin (R) treatment alone. After rinsing with distilled water, the slides were A similar experiment was conducted using Ampho 55 treated with either 1) PBS buffer, 2) Endo-H (100 ppm) tericin B (R) at a concentration of 0.5 micrograms per ml in PBS buffer, or 3) PNGase F (100 ppm) in PBS buffer. for three hours. The results were as follows: The Endo-H was derived from E. coli producing S. plicatus Endo-H. After 30 minutes at 37 C. the slides were rinsed in distilled water. After Gran staining, the ppm Endo-H % reduction in viability 60 slides were read with bright field optics on a light mi O 0 croscope. 1 17% In the case of the buffer control, the number of bac O 5% teria remaining on the slide was greater than 100 per 100 96% field. The slides treated with Endo-H contained far 1000 94% 65 fewer bacteria. In the case of S. epidermidis, only about 1 to 3 bacteria were observed perfield. In the case of E. As can be seen, 100 ppm of Endo-Henhances the coli, about 5 to 10 were observed per field. For those mycocidal effect of Amphotericin B (R). slides treated with PNGase F, moderate numbers of 5,395,541 41 42 bacteria were observed for both S. epidermidis and E. coli (approximately 20 per field). These results indicated that PNGase F is capable of Association (CTFA) name: removing bacteria from glass surfaces albeit not as effi Ingredient Weight % ciently as Endo-H. 5 Water Phase: Methylparaben (preservative) 0.20 EXAMPLE 22 Pantethine (moisturizer) 0.10 Carbomer 934 (thickener) 0.08 Tablet Denture Cleaner with Endo-H Sodium hydroxide, 10% (neutralizer) 1.00 Endo-H 100 ppm Sodium bicarbonate, sodium perborate monohydrate, 10 Purified water, q.s. to 100% tartaric acid, sodium tripolyphosphate, sulphamic acid, Oil Phase: polyethylene glycol (20,000 m.wt.) and ethylene di Heavy mineral oil 4.00 amine tetraacetate are separately granulated by fluidiz Stearic acid, double pressed 3.00 ing in a hot air bed at 60-65 C. for 30 minutes. Such (anionic emulsifier) Cholesterol (auxiliary emulsifier) 100 granulates are then tumble mixed with the other ingre 15 Cetyl alcohol (auxiliary emulsifier) 1.80 dients to produce a "first layer” mixture and a "second Castor oil (emollient) 100 layer' mixture, wherein the "first layer' mixture has the Cetyl palmitate (emolient) 120 following composition: Octyl dimethyl PABA (U.V.-absorber) 1.40 Propylparaben (preservative) 0.10 % by Weight 20 In a mixing vessel equipped with a mechanical stirrer, Sodium bicarbonate 3000 Tartaric acid 23,00 water and the water phase ingredients other than the Potassium monopersulphate 16.00 sodium hydroxide and Endo-Haqueous solution are Sulphamic acid 1.00 added and mixed with heating to about 75-80 C. to Oisodium pyrophosphate 8.20 form a uniform aqueous dispersion. The sodium hydrox Sodium carbonate 3.90 25 Polyethylene glycol 2.60 ide solution is then added and mixed into the aqueous Sodium sulphate 2.00 phase to neutralize the acidic Carbomer thickener. Peppermint powder 2.50 In a separate mixing vessel, the mineral oil and oil Silicon dioxide 1.30 phase ingredients are added and mixed with heating to Sodium dodecyl benzene sulphonate 0.50 about 80-82 C. to form a uniform oil phase. The 30 heated oil phase is slowly added to the heated water and the 'second layer' mixture has the following com phase using high speed mechanical dispersing means. position: Mixing is continued until a homogeneous oil/water emulsion is obtained. The emulsion is cooled to room temperature. If desired, optional colorants such as % by Weight 35 water-soluble dyes are preferably mixed into the emul Sodium perborate monohydrate 30.00 sion at about 45-50° C. and fragrant oils are preferably Potassium monopersulphate 28.00 added at about 35-40 C. Endo-H is mixed into the Sodium bicarbonate 13.34 Sodium tripolyphosphate 10.00 emulsion at about 35-40 C. Sodium bicarbonate/colour 4.00 Trilon B 3.00 EXAMPLE 24 Sodium carbonate 3.00 Polyethylene glycol 2.50 Removal of S. aureus from Pig Skin Silicone dioxide 2.00 Peppermint powder 150 Pigskin was inoculated with S. aureus (1.2X107 colo Wasag ester 7 0.70 nies/ml) by spreading 0.1 cc of the culture on the skin Wasag ester 15 0.70 45 surface. The organisms were allowed to set on the skin Hardened triglycerides 0.50 for two hours at room temperature. Duplicate pieces of Sodium dodecyl benzene sulphonate 0.40 skin were then treated for 30 seconds with: Succinate detergent 0.30 Blue Lake No. 1 0.06 1) untreated control Endo-H 100 ppm 2) water alone 50 3) 10% soap solution 4) #3--Endo-H (20 ppm) A tablet is produced by compressing in a HORN 5) 20 ppm Endo-H in buffer rotary tableting press of 39 stations. Compressing is in The Endo-H was obtained from E. coli transformed two stages: Initially the 'second layer', blue mixture is to produce Endo-H from S. plicatus. After treatment the compressed to very low pressure (10 kN per tablet) by 55 samples were rinsed in distilled water and placed in 2% way of tamping. The "first layer', white mixture is then osmium tetroxide followed by fixation in Ryter-Kellen instilled and pressed to 70 kN per tablet. In this way a berger fixative. The samples were then processed alter tablet of 4 grams is produced being 2.7 grams blue and natively in osmium and thiosemicarbizone. After criti 1.3 grams white. cal point drying, all samples were examined on the Tablets are dissolved in water by the consumer to 60 SEM. Photomicrographs were taken. clean dentures placed in the water. S. aureus colonies were found in abundance on the EXAMPLE 23 untreated, water treated, or plain soap treated samples. See, e.g. FIG. 11 which demonstrates the effect of treat Light Cream with Endo-H ment with liquid hand soap. The Endo-H-treated sam An oil-in-water sunscreen emulsion base is made from 65 ples demonstrated a significant loss of organisms. See, the following ingredients, which are indicated by their e.g. FIG. 12 which demonstrates the removal of S. chemical or Cosmetic, Toiletry and Fragrance Associa aureus from swine skin when treated with liquid hand tion (CTFA) name: soap plus Endo-H. 5,395,541 43 44 glass beads in a 250 ml Erlenmeyer flask (sterile) at EXAMPLE 25 room temperature for two minutes with shaking. Mold Removal from Shower Curtain The fabric swatches were then placed in petri dishes A plastic shower curtain was moistened with tap and overlaid with 3 mls of one-half strength trypticase water and placed in the dark for 3 weeks. At the end of soy agar with tetrazolium. After incubation for 48 that time, a small sample of the curtain that was covered hours, the colonies were counted. with mold was treated with: The results are shown in FIG. 15. These results indi 1) distilled water cate that 2% Irgasan plus Liquid Tide provide a two log 2) --2000 ppm Joy detergent decrease in bacterial growth as compared to Tide alone. 3) -- 1000 ppm Endo-H 10 The addition of 40 ppm Endo-H, however, reduces 4) untreated bacterial growth another log unit. The Endo-H was obtained from E. coli transformed to produce Endo-H from S. plicatus. The treatments EXAMPLE 27 lasted 10-15 seconds at room temperature. The shower Effect of Endo-H on Yeast curtain was wiped off after treatment with a cotton 15 swab. Broth cultures (18 hour) of Candida albicans and FIG. 13 depicts the results obtained. The non-treated Sacchromyces cerevisiae were treated with: control (lower right photograph-lower right quadrant 1) 0.2M Na citrate buffer, pH 5.5 of center photograph) showed abundant mold and mil 2) #1 plus 200 ppm Endo-H (from E. coli producing dew particles both macro and microscopically. 20 S. plicatus Endo-H) The distilled water control (upper right photogra The treatments lasted 2 hours at 37 C. ph-upper right quadrant of center photograph) After treatment, an aliquot of each was placed on a showed less organisms, although particles still remained formvar-coated 200 mesh copper grid, and examined by and discoloration was evident. TEM. Photomicrographs of the examinations were The Joy-treated control (lower left photograph 25 taken and are presented in FIGS. 15 and 16. lower left quadrant of center photograph) showed less As can be seen in FIG. 15A, Candida treated with organisms than the water treated sample, but discolor buffer alone was in good morphological condition. As ation was still evident. indicated in FIG. 15B, Candida treated with Endo-H The Endo-H treated sample (upper left photogra leaked material at a rapid rate and lost structural integ ph-upper left quadrant of center photograph) was free 30 rity. of both organisms and any discolorations. Sacchromyces treated with buffer alone was in good EXAMPLE 26 morphological condition as can be seen in FIG. 16A. When treated with Endo-H, however, all that remained Bacterial Removal from Fabric were very limited pieces of membranous material. See Fabric swatches were cut to the size of a petri dish. 35 FIG. 16B. Additional fabric was added to reach a 5% fabric load (which was not inoculated). The swatches were steril EXAMPLE 28 ized in an autoclave for 15 minutes at 15 lbs. 121 C. One Effect of Endo-Hand Lysozyme on Viability of E. Coli fabric load is needed for each treatment. Glass beads (40 g) and 100 mls 0.2M pH 7.0 citrate buffer was placed 40 A culture of E. coli K12 grown overnight in Laurie into 250 ml Erlenmeyer flasks. The flasks were plugged Broth (LB), was diluted 1:1000 in LB and regrown for with rubber stoppers and aluminum foil and sterilized in 4 hours at 37 C. Cells were centrifuged, washed and an autoclave. E. coli subcultured into fresh nutrient resuspended in 0-1M NA-acetate pH 5.5 (NA) buffer. broth and allowed to incubate for 48 hours at 37 C. Eight tubes were set up as follows: Half strength trypticase soy agar plates (10 g/500 mls) 45 were prepared and sterilized. After cooling, tetrazolium Tube Number (1 ml/liter) was added. 12 3,4 5,6 7,8 The agar plates were inoculated as follows: ul cells 800 800 800 800 1) serial dilutions from the 48-hour culture were pre ul NA buffer 200 w 200 pared (1:10, and 10 fold dilutions through three more SO ul Endo-H (1 mg/ml) 200 200 tubes in peptone water); 2) Thereafter, each swatch was inoculated with 2 mls of the last dilution (10). The Endo-H was from B. subtilis transformed to pro 3) The swatches were then incubated at 37 C. for duce Endo-H from S. plicatus. Tubes were incubated for two hours (two swatches/treatment). 55 one hour at 37 C. Tubes were centrifuged, washed and After incubation, the swatches were laundered as resuspended in 8.00 pil of 0.1M Na-phosphate, pH 7.2 follows: (NP) buffer containing 0.1M EDTA. Buffer or hen egg Wash white lysozyme solution was added to tubes as follows: 100 mls sterile 0.2M pH 7.0 citrate buffer-40 g glass beads--the treatment described in FIG. 14 (where 60 AWA is Endo-H from E. coli transformed to produce Endo-H from S. plicatus) in a 250 ml Erlenmeyer flask Tube Number (sterile). Two inoculated swatches--sterile fabric to 1,2 34, 56, 7,8 make 5% fabric load were washed at 95 F. for 12 min ul NP buffer 200 200 utes with shaking. 65 ul lysozyme (1 mg/ml) m 200 200 Rinse After washing, the swatches were rinsed by adding Aliquots were taken at this time to determine colony 100 mls sterile doubly distilled/deionized water-40 g forming units (CFU) (Column A). After incubation for 5,395,541 45 46 one hour at 37 C. aliquots were used to determine of the present invention are obtained when Endo-D or CFUs (Column B). The log of colony forming units For PNGase F are substituted for Endo-H in the Exam were calculated. The decrease in log CFUs was deter ples. mined by subtracting B from A. The results are shown All references cited herein are expressly incorporated below: by reference. What is claimed is: Log CFUs Change 1. A cleaning composition comprising: a) a first enzyme and a second enzyme, wherein said Condition A B in log CFUs first enzyme is a Type II endoglycosidase selected Control 7.89 7.90 --0.01 O Endo-H (200 ppm) 8.21 7.92 -0.29 from the group consisting of Endo-D, Endo-H, Lysozyme (200 ppm) 7.87 7.68 -0.9 Endo-L, Endo-C, Endo-CII, Endo-F-Gal type, Endo-H -- lysozyme 8.17 7.53 -0.64 Endo-F and PNGaseF, and said second enzyme is selected from the group consisting of proteases, These results indicate that the combination of Endo lipases, nucleases, glycosidases different from said H and lysozyme decreases the viability of E. coli as 15 first enzyme, and combinations thereof; compared to Endo-H or lysozyme alone. b) a detergent surfactant; and c) a detergent builder. EXAMPLE 29 2. The composition of claim 1 wherein said Type II Comparison of Endo-H with T-4 or Hen Egg White endoglycosidase is Endo-H. Lysozyme on Viability of E. coli 20 3. A cleaning composition of claim 1 wherein the E. coli cells were washed and suspended in 0.1M Type II endoglycosidase is present in an amount effec Na-acetate pH 5.5 buffer. Cells were aliquoted (10 ml) tive for removal of glycosidase-containing substances in two tubes. To one tube, only buffer was added (con from surfaces. trol) and to another Endo-H was added (treated). The 25 4. The cleaning composition of claim 1 wherein said Endo-H was from B. subtilis transformed to produce second enzyme is a protease. Endo-H from S. plicatus. Cells were incubated for one 5. The cleaning composition of claim 1 wherein said hour at 37 C. Cells were centrifuged, washed and re first enzyme is Endo-H and said second enzyme is a suspended in 0.1M Na-phosphate (pH 7.2) buffer. Cells protease. were aliquoted equally and incubated either with buffer 30 6. The cleaning composition of claim 5 wherein said or lysozyme. Hen egg white (HL) and T4 (TL) lyso second enzyme is a subtilisin or a mutant subtilisin. zymes were compared in this experiment. Tubes were 7. The composition of claim 1 wherein said different incubated for 1.5 hours. Samples were diluted and glycosidase is a Type II endoglycosidase. plated for CFU determination before (A) and after (B) 8. The composition of claim 1 wherein said different incubation. The log of CFUs were determined. The 35 glycosidase is an exoglycosidase. following results were obtained. 9. The composition of claim 1 further comprising a surfactant selected from the group consisting of anionic, Incubation Condition Log CFUs Change in nonionic, ampholytic, zwitterionic and cationic surfac First Second A. B log CFUs tantS. Endo-H (300 ppm) - 7.60 7.23 -0.37 10. The composition of claim 1 further comprising a - HEWL (445 ppm) 6.69 6.73 -0.23 disulfide cleaving reagent. Endo-H (300 ppm) HEWL (445 ppm) 7.41 6.81 -0.60 - TL (445 ppm) 4.98 4.53 -0.45 11. The composition of claim 1 wherein said second Endo-H (300 ppm) TL (445 ppm) 5.27 4.30 -0.93 enzyme is a subtilisin or mutant subtilisin. 12. A cleaning composition according to claim 3 45 having a pH of from 4 to 10. These results indicate that T4 lysozyme is also effec 13. A cleaning composition according to claim 3 tive in reducing the viability of E. coli in combination comprising a Type II endoglycosidase selected from the with Endo-H. group consisting of Endo-H, Endo-F and Endo-D and EXAMPLE 30 having a pH of from 5 to 8. 50 14. A cleaning composition according to claim 3 Treatment of Soiled Diaper Material with Endo-H further comprising a protease enzyme. Samples were obtained from a soiled diaper. Each 15. A cleaning composition according to claim 13 sample was divided. The left side of the sample was further comprising from about 1% to 90% by weight of washed in 2000 ppm Tide and 1 ppm BPN' (subtilisin protease from Bacillus amyloliquifaciens). The right side 55 detergent surfactant. was washed in 2000 ppm Tide, 1 ppm BPN' and 40 ppm 16. A cleaning composition according to claim 15 Endo-H (Boehringer Mannheim Biochemical Catalog comprising from about 5% to 50% by weight of deter No. 100 119). Each sample was washed for 12 minutes gent surfactant. at 95° F. The results of two experiments are shown in 17. A cleaning composition according to claim 3 FIGS. 17 and 18. As can be seen, the diaper material on further comprising from about 1% to 75% by weight of the right side of FIGS. 17 and 18 contains substantially detergent builder. less fecal stain as compared to the Tide-protease treated 18. A cleaning composition according to claim 17 diaper shown on the left of FIGS. 17 and 18. comprising Type II endoglycosidase selected from the Having described the preferred embodiments of the group consisting of Endo-H, Endo-F and Endo-D, and present invention, it will appear to those of ordinary 65 mixtures thereof, having a pH of from 6 to 10. skill in the art that various modifications may be made 19. A cleaning composition according to claim 18 and that such modifications are intended to be within comprising from about 5% to about 40% by weight of the scope of the present invention. Other compositions detergent builder selected from the group consisting of 5,395,541 47 48 fatty acids, polycarboxylates, polyphosphates, and mix- about 10% to about 20% by weight of detergent tures thereof. 20. A cleaning composition according to claim 19 builder, and from about 20 ppm to 200 ppm of Endo-H, formulated as a liquid comprising from about 10% to Endo-F or Endo-D. about 40% by weight of detergent surfactant, and from 5
O
15
20
25
30
35
40
45
50
55
60
65