||||||||||||I|| US005356803A United States Patent (19) 11 Patent Number: 5,356,803 Carpenter et al. (45) Date of Patent: ck Oct. 18, 1994

(54). ANTIMICROBIAL COMPOSITION FOREIGN PATENT DOCUMENTS ESSESSASEAND 0179449 4/1986 European Pat. Off. . 192401 8/1986 European Pat. Off. . ANTMICROBAL AGENT 0197622 10/1986 European Pat. Off. . 75 Inventors: Richard S. Carpenter, Cincinnati, 8:22 A3, E. Ea. g Ohio; Pushkaraj J. Lad, San Mateo uropean Pat. Off. . VV, 2937964. 11/1982 Fed. Rep. of Germany. Calif.; Ann M. Wolff, Cincinnati, 2122301 of 0000 France . Ohio 24483.51 9/1980 France. 73 Assignees: Genencor International, Inc., So. San 4. 8609459 12/198733, Ene Francisco, Calif.; The Procter & 55-153709 11/1980 Japan. Gamble Company, Cincinnati, Ohio 57-075926 5/1982 Japan. * Notice: The portion of the term of this patent 59-088086215498 12/19855/1984 JE. subsequent to Aug. 24, 2010 has been 61-015827 1/1986 Japan. disclaimed. 62-044-180 2/1987 Japan. 62-248487 10/1987 Japan . 21 Appl. No.: 869,356 (List continued on next page.) 22) Filed: Mar. 30, 1992 l OTHER PUBLICATIONS Related U.S. Application Data Hughes, R. C., 1983 Glycoproteins, pp. 23-26 (Chap (63) continuationdoned. of ser, No. 428,362, Oct. 27, 1989, aban- "",(List continuedNY) on next page.) 51) Int, Cl...... C12N 9/24; C12N 9/40; C12N 9/78; D06M 16/00 Primary Examiner-David M. Naff 52 U.S. Cl...... 435/200; 435/208; Assistant Examiner-Michael V. Meller 435/264; 435/206,435/227; 252/DIG. 12; Attorney, Agent, or Firm-Margaret A. Horn 252/17412 58) Field of Search ...... 435/264, 208, 200, 206, 57 ABSTRACT 435/227; 252/DIG. 12, 174.12 An antimicrobial composition consisting essentially of (56) References Cited from about 1 ppm to about 1200 ppm of a Type II endo glycosidase and from about 0.5 ppm to about 1200 ppm U.S. PATENT DOCUMENTS of an antimicrobial agent is disclosed. The preferred 4,062,941 12/1977 Davies ...... 424/94 Type II endoglycosidases to be used in the invention are 4,144,327 3/1979 Davies et al. . 424/94.61 Endo-Ds Endo-H, Endo-F and PNGaseF. The pre : 3. y ------: ferred antimicrobial agents are bactericides, fungicides 4,619,8259. 9. 10/1986 EigeneSO et al...... 424/49 and algicides. The composition can be used in the form 4,639,375 l/1987 Tsai ...... 26/49 of personal care or household cleaning products such as 4,710,313 12/1987 Miyajima et al...... 252/105 liquid soap, hard surface cleaner, laundry detergent, 4,749,511 6/1988 Lad et al...... 252/174.12 anti-acne medication, deodorant, shampoo, face cream, 4,801,451 1/1989 Hellgren et al...... 424/94.63 mouthwash, dentifrice and denture cleaner. 4,812,404 3/1989 Kuboki ...... 435/175 4,939,123 7/1990 Neeser et al...... 514/8 5,041,236 8/1991 Carpenter et al...... 252/17412 15 Claims, 28 Drawing Sheets 5,356,803 Page 2

FOREIGN PATENT DOCUMENTS Glycoprotein of Friend Murine Leukemia Virus”, Eur. 63-002911 1/1988 Japan. J. Biochem., vol. 143, pp. 531-539. 1942236 3/1971 Netherlands . Hsieh et al., 1983, “Selective Cleavage by En 693380 6/1970 South Africa. do-g-N-acetylglucosaminidase H at Individual Glyco 86O7738 4/1987 South Africa . sylation Sites of Sindbis Virion Envelope Glycopro 1272135 4/1972 United Kingdom. teins', The Journal of Biological Chemistry, vol. 258, 1311375 3/1973 United Kingdom . No. 4, issue of Feb. 25, pp. 2555-2561. 2120240 11/1983 United Kingdom . Chipman et al., 1969, “Mechanism of Lysozyme Ac tion', Science, vol. 165, pp. 454-465. OTHER PUBLICATIONS Haskell et al., 1970, "Neuraminidase Inhibition and Thotakura et al., 1987, “Enzymatic Deglycosylation of Viral Chemotherapy”, Journal of Medicinal Chemistry, Glycoproteins', Methods in Enzymology, vol. 138, pp. vol. 13, No. 4, pp. 697-704. 350-359. Tute, M. S., 1970, "The Inhibition of Viral Neuramini Boehringer Mannheim, Biochemicals Division, Indian dase by 1-Phenoxymethyl-3,4-dihydroisoquinolines', apolis, Ind., "Glycohydrolases'. Part II, vol. 13, pp. 48-51. Cohen, 1986, "B-N-Acetylglucosaminidase from Chang et al., 1986, "Expression and Size Heterogeneity of a 63 Kilodalton Membrane Glycoprotein During Phycomyces Blakesleeanus', Plant Science, vol.43, pp. Growth and Transformation of Leishmania Mexicana 93-101. Amazonensis’, Molecular and Biochemical Parasitol Abeles et al., 1970, "Preparation and Purification of ogy, vol. 18, pp. 197-210. Glucanase and Chitinase from Bean Leaves', Plant Tarentino, A. L. and Maley, F., "Purification and Prop Physiology, vol. 47, pp. 129-134. erties of an Endo-g-N-acetyl glucosaminidase from Chaiet et al., 1970, "Isolation of a Pure Dextranase from Streptomyces griseus', The Journal of Biological Chem Penicillium funiculosun', Applied Microbiology, vol. istry vol. 249, No. 3, Issue of Feb. 10, pp. 811-817, 1974. 20, No. 3, pp. 421-426. Neuberger et al., 1967, "Inhibition of Lypozyme by Montague, M.D., 1964, “The Enzymic Degradation of N-Acyl-D-Glucosamine Derivatives', Nature, vol. Cell Walls of Streptococcus Faecalis', Biochim. Bio 215, Jul. 29 Issue, pp. 524-525. phys. Acta, vol. 86, pp. 588-595. Thotakura et al., Methods in Enzymology (138), pp. Anderson et al., 1964, “Studies on Carbohydrate-Meta 350-359, 1987. bolizing Enzymes', Biochem. J., vol. 90, pp. 30-35. Merck Index, 10th ed. (1983), Merck & Co., Inc., Rah Geyer et al., 1984, “Structure of the Oligosaccharides way, N.J. (pp. 135-136, 293, 1018-1020, 1315-1316, Sensitive to Endo-3-N-acetylglucosaminidase Hin the 1380-1381). U.S. Patent Oct. 18, 1994 Sheet 1 of 28 5,356,803

N-LINKED CORE STRUCTURE

Asn-GlcNAc-glCno C-Man Z

D-LINKED CORE STRUCTURE

-GONAc-Neuac - GolNAc-Got

Ser- DR, -GONAC-Go-Neu Act Thr NeuNAC -GolNAc-Got L-FuC

Figure-1 U.S. Patent Oct. 18, 1994 Sheet 2 of 28 5,356,803

TYPE II ENDDGLYCSIDASE SUBSTRATE X Glycopeptidase-F Asn-GlcNAc-GlcNAc-Mon-vMan-w (PNGOSeF) A N CFuC) Mary Z

Mor X M. N. Endo-H, F, D, CI Asn-GNAGlcNAc-Marvt A Mon-Y W N Z

Endo-F-got type Asn-GlcNAC-Go-Mor Man A N Mon

Ser Enco-O-N- Or golNAc-Gol Acetylgalactosorinidase Thr

Endo-R-N- V Golactosidose R1-GlcNAc-Gol-GlcNAc-R2

Figure-2 U.S. Patent 5,356,803

ç-ºun61-I

I

U.S. Patent 5,356,803

3.LIS350k}/\VETTO #-eun61-I U.S. Patent 5,356,803

3.SVGIISTJOATEJDJOINE

LNDO-EUIISDJOÅT19ONINI? LS8ITÈSEONº

wg-eun612 gg-ean513 OG-ºun614

U.S. Patent Oct. 18, 1994 Sheet 7 of 28 5,356,803

FIGURE-6A. U.S. Patent Oct. 18, 1994 Sheet 8 of 28 5,356,803

FIGURE-6B U.S. Patent Oct. 18, 1994 Sheet 9 of 28 5,356,803

FIGURE-7A U.S. Patent Oct. 18, 1994 Sheet 10 of 28 5,356,803

FIGURE-7B U.S. Patent Oct. 18, 1994 Sheet 11 of 28 5,356,803

FIGURE-7C U.S. Patent Oct. 18, 1994 Sheet 12 of 28 5,356,803

FIGURE-7D U.S. Patent Oct. 18, 1994 Sheet 13 of 28 5,356,803

FIGURE-7E U.S. Patent Oct. 18, 1994 Sheet 14 of 28 5,356,803

FIGURE-7F U.S. Patent Oct. 18, 1994 Sheet 15 of 28 5,356,803

FIGURE -7G U.S. Patent Oct. 18, 1994 Sheet 16 of 28 5,356,803

FIGURE -7H U.S. Patent Oct. 18, 1994 Sheet 17 of 28 5,356,803

(INV0+IuiddH–opu? 0+uidd3NICIIXEHNDTHO 0+uidd3NICIIXBHNDTHO 09 g–Qun614

02

O 9 U.S. Patent Oct. 18, 1994 Sheet 18 of 28 5,356,803

OGudd3NICIIXEHNDTHO

LINDJOH-OKOUETID]>| 002viddH–opu3CINº OGudd3NICIIXEHNDTHO an5136–3

O O. U.S. Patent Oct. 18, 1994 Sheet 19 of 28 5,356,803

3.4 A LG CFU E.Coll VS, Enco-H 3.3 3.2 3.1 3.0 2.9 2,8 2.7 2,6 2.5 2,4 2,3 2.2 2.1. 2.0 1.9 18 1.7 16 15 14 1.3 O 40 80 120 160 200 240 280 Figure-10A(Endo-H) ppm

3 A LOG CFU E.coli VS, Endo-Hi 6 200, 500, 1000 ppm Endo-H

O 200 400 600 800 1000 (Endo-H) ppm Figure-10B U.S. Patent Oct. 18, 1994 Sheet 20 of 28 5,356,803

U.S. Patent Oct. 18, 1994 Sheet 21 of 28 5,356,803

£1–HHn?IH

U.S. Patent Oct. 18, 1994 Sheet 22 of 28 5,356,803

ANTMICROBIAL EFFECTS THROUGH THE WASH

LDGS GROWTH

2L/D5 1/D5 1/05/A 2TM D5 D5

D5 = DETERGENT L = IRGASAN T = TRICLDCARBAN A = E 2 D R 1 =2A DR A RESPECTIVELY Figure-14 U.S. Patent Oct. 18, 1994 Sheet 23 of 28 5,356,803

FIGURE - 15A U.S. Patent Oct. 18, 1994 Sheet 24 of 28 5,356,803

U.S. Patent Oct. 18, 1994 Sheet 25 of 28 5,356,803

FIGURE-16A U.S. Patent Oct. 18, 1994 Sheet 26 of 28 5,356,803

FIGURE-6B U.S. Patent Oct. 18, 1994 Sheet 27 of 28 5,356,803

FIGURE-17B U.S. Patent Oct. 18, 1994 Sheet 28 of 28 5,356,803

FIGURE - 18B 5,356,803 1. 2 et al. (1977), Botanica Marina, 20, 13-17. As reported ANTMCROBAL COMPOSITION CONTAINING therein, Pseudomonas species isolated from sea water TYPE II ENDOGLYCOSIDASE AND was adhered to glass slides. Thereafter, the slides were ANTIMICROBAL AGENT treated with either pronase, trypsin, a-amylase (a Type I endoglycosidase), or lysozyme (also a Type I endo This is a continuation of application Ser. No. 428,362 glycosidase). In this report, treatment with the proteo filed Oct. 27, 1989, now abandoned. lytic enzymes pronase and trypsin resulted in the release of a portion of the population of adhered bacteria, FIELD OF THE INVENTION whereas the cell degradative enzyme lysozyme showed The present invention relates to antimicrobial meth 10 diminished activity compared to the proteolytic en ods and antimicrobial compositions utilizing Type II zymes. The a-amylase reportedly had no effect at all. In endoglycosidase alone or in combination with an anti addition to the attachment of microorganisms to microbial agent. contact lenses, tooth enamel and glass surfaces, many BACKGROUND OF THE INVENTION other surfaces are subject to microbial attachment. See, 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., 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. (3-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-g-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-g-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, New York, Oxford. In addition to to adhere to such lens. See, e.g. Duran, J. A., et al. having a specificity for the internal glycosidic linkages (1987), Arch. Ophthalmol, 105 106-109; Stern, G. A., et of glycoproteins, at least one endoglycosidase (endo-g- al. (1987), Ophthalmology, 94, 115-119 (which reports N-acetylglucosaminidase H) has also demonstrated a the treatment of mucin coated contact lenses with vari 55 specificity which produces the cleavage of lipid-linked ous enzymes such as pancreatin, papain, trypsin and oligosaccharides (Chalifour, R. J., et al. (1983), Archives neuraminidase to inhibit Pseudomonas adherence); and of 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

5,356,803 5 6 surface. The mechanism of this reactivity is not known with certainty. In some cases, such substances are TABLE I-continued glycosides or glycoside-containing substances which Type I Substrate are believed to have glycosidic linkages that are known Endoglycosidase oligo- or polysaccharide cleavage sites for Type II endoglycosidases or linkages f cellulase g1-4 which are closely related to such cleavage sites. (Glc-Glc) As used herein, "Type II endoglycosidases' are en f zymes which are capable of cleaving linkages at or near hyaluronidase £31-3 (31-4 & 1-3 the juncture of the protein and carbohydrate units of a GlcA-GlcNAc-GlcA-GlcNAc 10 st glycoprotein. Preferably, such Type II endoglycosi lysozymes: £31-4 (31-4 (31-4 dases are capable of cleaving at least one glycosidic hen egg white GlcNAc-Murnac-GlcNAc-Murnac linkage within about three glycosidic linkages of the lysozyme protein-carbohydrate unit juncture (including the gly T4 lysozyme cosidic linkage comprising the protein-carbohydrate mutanolysin junction). Most preferably, such glycosidic linkages are 15 pullulanase o1-4 al-4 oil-6 oil-4 Glc-(Glc-Glc-)Glc-Glc within about two glycosidic linkages of the protein-car t bohydrate unit juncture (see FIGS. 1, 2 and 3). 'GlcA is D-Glucuronic Acid Type II endoglycosidases are also defined by their Murnac is N-Acetylmuramic Acid specificities for the particular glycosidic linkages shown f Indicates cleavage site. in FIG. 1 for the known core structures of N-and O 20 linked glycoproteins. These correspond to the glyco Specific glycosidic linkages in glycoproteins which sidic linkages between the amino acids serine, threonine define Type II endoglycosidases and which identify or asparagine and the first carbohydrate residue and the preferred Type II endoglycosidases are shown in FIG. glycosidic linkages between at least the first, second and 2. The cleavage sites are identified by a vertical arrow. third carbohydrate residues. Although this core struc 25 A generic presentation of the protein amino acids, car ture will be described in more detail hereinafter in terms bohydrate residues and cleavage sites of FIG. 2 is of the specific glycosidic linkages which exist in known shown in FIG. 3. As can be seen, Type II endoglycosi core structures, such specific linkages are not to be dases preferably cleave the first, second or third glyco construed as limiting to this definition of Type II endo sidic linkages in N- or O-linked glycoproteins. These glycosidases. Accordingly, all possible glycosidic link 30 linkages comprise the glycosidic linkages (1) between ages between these amino acids and carbohydrate resi asparagine, serine or threonine in the protein unit and dues define the core structure of N-and O-linked glyco the first carbohydrate residue, (2) between carbohy protein used to identify Type II endoglycosidases. drate residues 1 and 2 and (3) between carbohydrate Type II endoglycosidases are not limited by the pres residues 2 and 3, respectively. This specificity is defined ent knowledge of the glycoprotein core structure and 35 primarily by the carbohydrate sequence of the glyco the specificity of known endoglycosidases for such core protein with specificity and reactivity being influenced structures. A comparison of the core structures in FIG. to some extent by the protein unit of the glycoprotein. 1 with the known substrates for Type II endoglycosi Thus, with regard to glycosidic linkages 2 and 3 (com dases in FIG. 2 indicates that Type II endoglycosidases prising glycosidic linkages between carbohydrate resi for each of the possible cleavage sites in the core struc dues only), Type II endoglycosidases may be reactive tures in FIG. 1, if they exist, have not yet been identi with identical or similar glycosidic linkages located in fied. Moreover, other core structures may also exist other regions of a glycoprotein, perhaps quite distant which have not yet been identified. Endoglycosidases from the juncture of the protein and carbohydrate units reactive with linkages in such, as yet, unknown core of the glycoprotein. structures are also Type II endoglycosidases. Accord 45 An application of the above definition to a particular ingly, the glycosidic linkages in glycoproteins which glycoprotein is illustrative. Bovine thyroglobulin has define Type II endoglycosidases are not limited to those been analyzed using endo-g-N-acetylglucosaminidase located within the first three glycosidic linkages closest H (Endo-H), a-mannosidase and 3-mannosidase. Taren to the protein unit of the glycoprotein but may extend 50 tino, A. L. et al. (1973) J. Biol. Chem., 218, 5547. The to more distant glycosidic linkages in the core structure, Endo-H hydrolyzed the glycosidic linkage between the e.g. to the fourth or fifth glycosidic linkage from the two N-acetyl D-glucosamines, one of which was N protein unit depending on the core structure identified. linked to an asparagine in the protein unit of the thyro The specificity for the corestructure of glycoproteins globulin. The oligosaccharide or carbohydrate portion provides a convenient definition of Type II endo 55 of the thyroglobulin released upon treatment with En glycosidases which distinguishes them from Type I do-Hwas also treated with a- and (3-mannosidase. Since endoglycosidases. Type I endoglycosidases cleave spe neither of these enzymes has a specificity for the sub cific linkages in oligo- or polysaccharides but generally strates corresponding to those shown in FIGS. 1, 2 or 3, are not reactive with those core structure glycosidic they are not Type II endoglycosidases and can be char linkages in glycoproteins which define Type II endo acterized as either an exoglycosidase or Type I endo glycosidases. Examples of Type I endoglycosidases and glycosidase. The specificity of the Endo-H is the same the linkages with which they are reactive are shown in as that shown for Endo-H in FIG. 2 and Endo-H is Table I. therefore a Type II endoglycosidase. This is of course a TABLE I trivial application. But if a new endoglycosidase (e.g. Type I Substrate 65 Endo-X) is discovered which also demonstrates this Endoglycosidase oligo- or polysaccharide specificity or one or more of the other specificities in a-amylase ai-4 FIGS. 1, 2 or 3, that Endo-X would also be a Type II (Glc-Glc) endoglycosidase. 5,356,803 7 8 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 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 The carbohydrate residues (monosaccharides) of a Type II endoglycosidases is defined functionally by O 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. eral, however, each of the carbohydrate residues in the As used herein, “glycoside' refers to a polymer 15 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 20 of the anomeric carbon. The anomeric carbon of one and an aglycon, the aglycon being the non-sugar con 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 different glycosidic linkages which are found in the II endoglycosidase. The aglycon unit, however, is not 25 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 aglycon portion containing one or more sugar residues asparagine in a peptide via an N-glycosidic linkage). depending on the cleavage site of the Type II endo The structure of such asparagine linked glycoproteins glycosidase. Further, the aglycon portion may be quite 30 can be quite complex. See e.g., Schachterh (1984) Clini complex as might be the case with peptidoglycans cal Biochemistry 17, 3-14. The structure of many of where crosslinked peptides can be found attached to a these asparagine linked membranous glycoproteins matrix of carbohydrate. Thus, glycosides include glyco from a variety of sources (e.g., erythrocyte plasma proteins, glycolipids, peptidoglycans and the like which membrane glycoproteins, viral envelope glycoproteins) upon treatment with a Type II endoglycosidase pro 35 as well as the structure of non-membranous soluble duce a carbohydrate portion and aglycon portion glycoproteins indicate that the two types of glycopro wherein the carbohydrate portion and aglycon portion teins share many structural features. Id. at 3. The com are defined by the cleavage site of the Type II endo mon core structure of such asparagine-linked glycopro glycosidase. This definition of glycoside will be appar teins is shown in FIGS. 1 and 4, wherein GlcNAC is ent from the discussion which follows. N-acetyl D-glucosamine and Man is mannose. The As used herein, “glycoprotein' refers to a glycoside a 1-6, a 1-3 and 61-4 designations describe the type of which has one or more oligo- or polysaccharides cova glycosidic linkage between the various carbohydrate lently attached to a peptide or protein. Oligo- and poly residues. This core linkage forms the basis of numerous saccharides are sometimes referred to herein as "carbo glycoproteins having any of a number of carbohydrate hydrate units'. Such carbohydrate units, however, may 45 residues attached to the core. Id. at 5. be different from the "carbohydrate portion' of a gly O-linked glycoproteins contain a core structure coside. As shown in FIG. 4, a carbohydrate unit com wherein the protein unit of the glycoprotein is coupled prises the entire oligo- or polysaccharide attached to a to the carbohydrate unit through the hydroxyl group of second class of molecule, e.g., to a protein or peptide as either serine or threonine. A common feature of this in a glycoprotein or to a lipid as in a glycolipid. If the 50 core structure is the presence of N-acetyl D-galactosa Type II endoglycosidase cleaves the carbohydrate unit mine (GalNAc) linked to serine or threonine. Other at its juncture with, for example, a protein then the details of such glycoproteins are shown in FIG. 1 where carbohydrate unit is synonymous with the carbohydrate NeuAc is N-acetylneuraminic acid, Gal is Galactose portion of a glycoside. If, however, the Type II endo and L-Fuc is L-Fucose. When Gal is the second carbo glycosidase cleaves the carbohydrate unit at a glyco 55 hydrate residue the glycosidic linkage between Gal sidic linkage within the carbohydrate unit, then the NAc and Gal is usually 61-3. For review of the struc carbohydrate portion of the glycoside formed by such ture biosynthesis and function of glycoproteins includ cleavage will be less than the entire carbohydrate unit. ing N-and O-linked glycoprotein, see Berger E.G. et al. This difference is shown in FIG. 4 for a Type II endo (1982) Experimentia, 38, 1229-1258. glycosidase cleavage site indicated by the arrow. 60 Lower organisms such as prokaryotes, e.g., the bac The carbohydrate units of a glycoprotein may be teria E. coli, Pseudomonas species, Bacillis species and oligosaccharides containing 1 to 10 carbohydrate the like, produce peptidoglycans rather than glycopro (sugar) residues or short polysaccharides which usually teins. Peptidoglycans are found in bacterial cell walls contain between 10 to 25 carbohydrate residues. Many and typically have a polysaccharide backbone of alter glycoproteins are produced by higher organisms such 65 nating N-acetylglucosamine and N-acetylmuramic as eukaryotes including yeast and mammalian cells. The acids. Peptide side chains are sometimes associated with linkage between the carbohydrate unit and the peptide the N-acetylmuramic acid residues with cross-linked or protein unit of a glycoprotein is a glycosidic linkage peptide bridges often being interposed between the 5,356,803 10 peptide side chains. The cell wall of Gram-positive brane constituents, e.g., glycophorin contained by bacteria typically comprises approximately 10% pepti human erythrocytes, hemagglutinin contained by influ doglycan whereas the cell wall of Gram-negative bac enza virus, rhodopsin contained in bovine retina and teria typically have a peptidoglycan content of about collagen contained by fibroblasts. Further glycoside 50%. containing substances include viral envelope glycopro Peptidoglycans, however, are not glycoproteins, at teins and fecal matter which contains in part peptido least to the extent that specific glycosidic linkages in glycans associated with intestinal bacteria. Thus, vi glycoproteins are used to define the class of Type II ruses, fibroblasts, fecal matter etc. are considered glyco endoglycosidase. Thus, Endo-H is a Type II endo side-containing substances. glycosidase because it cleaves the glycosidic linkage 10 As used herein, a "microorganism' (sometimes re between the two N-acetylglucosamine sugar residues ferred to as a glycoside-containing microorganism) is found in some glycoproteins containing N-linked oligo one capable of being cleaved from the surface of a sub saccharides. See FIG. 4. Endo-H, however, may also stance to which it is bound by a Type II endoglycosi have an as yet undefined reactivity with peptidoglycan dase. Examples include the intestinal bacteria found in since it is capable of facilitating the removal of fecal 15 fecal matter and bacteria commonly contaminating matter from a surface such as cloth swatches. Such fecal contact lens. Other examples include fungi and algae matter is known to contain peptidoglycans associated which can be cleaved from a surface by Type II endo with intestinal bacteria. Lysozymes are enzymes which glycosidase. are reactive with peptidoglycan. Lysozymes, such as As used herein, the term "in vitro' refers to the envi hen egg white lysozyme, T4 lysozyme and mutanolysin 20 ronment in which the processes and methods of the (Goodman, et al. (1981), J. Bacteriol. 146, 755), how invention are practiced. It is used only to distinguish ever, are not Type II endoglycosidases. This is because from the term "in vivo' which describes the environ they do not have a substantial reactivity with the unique ment in which Type II endoglycosidases are found glycosidic linkages found in N- and O-linked glycopro naturally, e.g. within organisms which naturally pro teins used to define Type II endoglycosidases. They are, 25 duce Type II endoglycosidase. Accordingly, an in vitro however, reactive with peptidoglycans to produce di method employing a Type II endoglycosidase is a saccharides of N-acetylglucosamine and N-acetyl method or process which does not occur in nature. The muramic acid containing attached peptide side-chains. term in vitro, however, is not to be construed as a limi As such, lysozymes are more appropriately character tation of such methods to "in glass' or to exclude such ized as a Type I endoglycosidase. Thus, even though 30 methods from being practiced on or in a living organ lysozymes and Endo-H may have an overlap in reactiv ism. The methods of the invention may be practiced on ity with peptidoglycans, they are mutually exclusive, a variety of surfaces other than glass including fabric, for the most part, with regard to Endo-H's specificity contact lenses, metallic surfaces, ceramic surfaces, cell for, and lysozyme’s substantial lack of reactivity with, surfaces, plastic surfaces, tissue and the like. Further, the glycosidic linkages in glycoproteins which define 35 such in vitro methods may be practiced for example in Type II endoglycosidases. the human oral cavity as described in more detail here As used herein, a 'glycoside containing substance' or inafter. 'glycoprotein containing substance' is a glycoside or Some known Type II endoglycosidases are listed in glycoprotein alone or a glycoside or glycoprotein com Table II together with the natural biological source of bined with another component. Thus, glycoside-con such enzymes. The cleavage sites for some Type II taining Substances include glycosides such as glycopro endoglycosidases are shown in FIG. 2. See "Glycopro tein enzymes, e.g., alkaline phosphatase, bromelain, tein and Proteoglycan Techniques' (1985) by J. G. carboxypeptidase-Y; glycoprotein hormones, e.g., cho Beeley, Chapter 6, pp. 153-300, Elsevier, Amsterdam, . rionic gonadotropin, erythropoietin; lectins, e.g., those New York, Oxford. A Type II endoglycosidase not derived from potato and soybean; serum glycoproteins, 45 listed in Table II is Glycopeptidase F also sometimes e.g., IgG immunoglobulin, thyroglobulin, prothrombin referred to as PNGase F. PNGase F may be obtained and the like and miscellaneous glycoproteins such as from Flavobacterium meningosepticum. It is also com hemoglobin and interferon; and complex carbohy mercially available from Boehringer Mannheim Bio drates. Examples of glycosides combined with another chemical, Indianapolis, Ind. component include glycoproteins comprising mem 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-a-N- Diplococcus pneumoniae O-linked, only Acetylgalactosaminidase Gal-ol-3GalNAc endo-3-N-Galactosidases Diplococcus pneumoniae Blood group A and B determinants 5,356,803 11 12 TABLE II-continued Enzyme Source Typical Substrate Escherichia freundi Keratan sulphate and oligo Flavobacterium saccharides containing keratolyticus sequence R GlcNAc-6-3Gal g1-4GlcNAc (or Glc) As can be seen, Endo-H, F, D, CI and Endo-F-Gal type all cleave the second glycosidic linkage in a glyco protein. In the case of Endo-F-Gal type, this glycosidic 10 with fucose being absent from the first carbohydrate linkage is between GlcNAc and Gal. For Endo-H, F, residue GlcNAc. It also cleaves hybrid structures D, and CI, the cleavage is between two residues com wherein W and X comprise mannose, Y and/or Z com prising GlcNAc, with specificity being defined by the prise NeuNAc-Gal-GlcNAc or similar structure, V substituents U, V, W, X, Y, and Z. comprises H or GlcNAc with fucose typically being Endo-H cleaves N-linked glycoproteins having a 5 absent from the first carbohydrate residue. Complex high mannose content. Thus in FIG. 2, W comprises structures are also cleaved by glycopeptidase F. Such 2-150 mannose residues, Y comprises 1-2 mannose resi structures comprise the core structure shown in FIG. 2 dues and X,Z.V and U are H (hydrogen). Endo-H also wherein Y and W comprise NeuNAc-Gal-GlcNAc or cleaves hybrid structures wherein W comprises 1-2 similar structure, X and Z comprise H, NeuNAc-Gal mannose residues and Y and/or Z comprise NeuNAc 20 GlcNAc or similar structure, V comprises H or Gal-GlcNAc or similar structures and V comprises H GlcNAc and fucose is sometimes present on the first or GlcNAc. Endo-H is the preferred Type II endo carbohydrate residue GlcNAc. glycosidase used in the formulations and methods of the Endo-3-N-galactosidase is known to cleave glycosy invention. dic linkages within oligosaccharides on a glycoprotein Endo-D and Endo-CI have similar reactivities al 25 or glycolipid. A typical glycoprotein substrate together though these enzymes are derived from different with the cleavage site for Endo-3-N-galactosidase is sources. Endo-D and Endo-CI are active on N-linked shown in FIG. 2 where R2 is protein, lipid or carbohy oligosaccharides of glycoproteins and cleave a high drate, and R1 is a sugar residue or hydrogen. mannose structure containing more than a 5-mannose Of course, the invention is not limited by the present carbohydrate residue in which case X comprises man 30 known specificity of endoglycosidases. Until recently, nose linked by way of an a 1-3 glycosidic bond to the the endoglycosidases which have been commercially core structure, W comprises mannose linked by way of available have been expensive due to their relatively an a 1-6 glycosidic bond to the core structure and the low levels of expression in their naturally occurring remaining substituents are H in FIG. 2. Endo-D also sources. Accordingly, the reactivity of such enzymes cleaves a core portion of a complex or hybrid structure 35 has not been broadly investigated. However, with the after removal of most antennary residues with exo advent of molecular cloning, greater amounts of endo glycosidases, in which case Y comprises H or GlcNAc glycosidase have been or will be made available. To the and U comprises H or fucose in FIG. 2. extent that alternate reactivity and specificity may be The endoglycosidase Endo-F is active on N-linked discovered for these or other endoglycosidases, such glycoproteins having a high mannose content wherein reactivity is intended to be within the scope of the in in FIG. 2 X and Y are one or more mannose residues vention. and the remaining substituents are H. Endo-F also Accordingly, as used herein, a "Type II endoglycosi cleavesbiantennary hybrid structures wherein XandW dase-reactive substance' (also referred to as a "Type comprise mannose linked to the core structure by way II-reactive substance' or a substance containing a of a 1-3 and a 1-6 glycosidic linkages and Y comprises 45 "Type II reactive linkage') is any substance which is NeuNAc-Gal-GlcNAc or similar structure and U com reactive with a Type II endoglycosidase. Included prises H or fucose. Biantennary complex structures are within Type II reactive substances, of course, are glyco also cleaved by Endo-F. Such structures comprise the side-containing substances and glycoprotein. Also in substrate core structure for Endo-Fin FIG. 2 wherein cluded, however, are (1) other, as yet, unknown sub X and Y comprise NeuNAc-Gal-GlcNAc or similar 50 strates reactive with Type II endoglycosidase at other structures and U comprises H or fucose. than a glycosidic linkage, and (2) multicomponent ag Endo-L has a similar reactivity in cleaving the second gregates containing components having Type II reac glycosidic linkage in N-linked glycoproteins. It is spe tive linkages. cific for low molecular weight substrates comprising For example, microorganisms, such as bacteria, can Man-GlcNAc-GlcNAc-Asn. Endo-CII demonstrates a 55 be removed from surfaces by treatment with Endo-H. It specificity similar to Endo H. Endo-a-N-acetylgalac is presently not known how this result occurs. Bacteria tosaminidase hydrolyzes glycoprotein containing oligo are not known to contain linkages which are normally saccharides O-linked to serine or threorine where reactive with Endo-H and the details of their attach GlcNAc and Gal are the first two carbohydrate resi ment to surfaces, other microorganisms and other sub dues. The specificity of endo-g-N-galactosidase is also stances is not well understood. Yet, bacteria removal by shown in FIG. 2 wherein R1 may be one of the man Endo-H has been observed. noses from which antennas in the carbohydrate unit Further, other stains may involve complex aggre may be formed. gates of substances some of which or all of which are The Type II glycosidase glycopeptidase F (PNGase reactive with Type II endoglycosidase. The term Type F) cleaves the first glycosidic linkage in N-linked glyco 65 II reactive substance covers all such situations. Thus, proteins between asparagine and GlcNAc. It cleaves uses of Type II endoglycosidase include (1) cleaning high mannose structures wherein W, X and Y comprise surfaces containing Type II-reactive substances, (2) one or more mannose residues and V and Z comprise H treating Type II-reactive substances to prevent attach 5,356,803 13 14 ment to a surface, and (3) treating Type II-reactive e.g., U.S. Pat. Nos. 3,403,393 and 2,976,576. Hard substances such as microorganisms to produce an anti contact lenses, on the other hand, are typically made of microbial effect. methacrylate or methylmethacrylate polymers. Other The Type II endoglycosidases used in the invention surfaces include naturally occurring biofilms, cardiac can be obtained from the organisms listed in Table II pacemaker leads and power packs, cellular and mucosal according to methods known to those skilled in the art. surfaces, tooth enamel, filters used to remove bacteria Some of the Type II endoglycosidases in Table II, e.g., and particulate material in processing foods; chemicals Endo-H from Streptomyces plicatus (initially classified as and the like; air conditioning filters; the surfaces of Streptomyces griseus) and produced in S. plicatus or S. various structural components exposed to an aqueous lividans and Endo-D from Diplococcus pneumoniae, are 10 environment, e.g., boats, piers and the like; plastics and commercially available from Boehringer Mannheim composites such as formica; and metals or metal alloys Biochemical, Indianapolis, Ind. Besides the commer such as steel, aluminum, etc. cially available preparations, Endo-H may be derived As will be shown in detail hereinafter, Type II endo from E. coli transformed with a plasmid encoding the glycosidases alone or in combination with a second Endo-H gene from Streptomyces plicatus and the pro 15 enzyme such as subtilisin, either with or without deter moter from alkaline phosphatase (Oka, T., et al. (1985) gent, effectively increases the removal of blood and Proc. Natl. Acad. Sci USA, 82, 7212-7216) by methods fecal stains from cloth swatches. It is not known pre similar to that reported for the cloning and expression cisely how such stains adhere to such swatches. How of Endo-H from Streptomyces plicatus in E. coli (Rob ever, the enhanced removal of such substances from bins, et al. (1981).J. Biol. Chem. 256; 10 640). See also 20 these swatches by Type II endoglycosidase, alone or in Trumbly R. J. et al. (1985) J Biol. Chem., 260, 5638. combination with other agents, suggests that at least one Endo-H may also be derived from Streptomyces cells glycosidic linkage is interposed between the fabric and engineered to express Endo-H derived from Streptomy that part of the stain which is released upon treatment ces plicatus (EPO Publication No. 0179449, Apr. 30, with Type II endoglycosidase. Based on these results, 1986). Alternatively, Endo-H may be produced by any 25 the following are proposed mechanisms of the binding appropriate host cell such as Bacillus subtilis using tech of glycoside-containing substances to a surface and the niques well known to those skilled in the art. The amino release and/or removal of such substances by Type II acid and DNA sequences of Endo-H for S. plicatus (S. endoglycosidase. These proposed mechanisms, how griseus) have been published. Robbins, P. W., et al. ever, should not be considered as a limitation to the (1984) J. Biol. Chem, 259, 7577-7583. 30 scope of the invention. The Endo-H used in the examples herein was ob tained commercially or from E. coli or B. subtilis hosts Figures transformed to express Endo-H from S. plicatus. Thus, as shown in FIG. 5A, a glycoside-containing One unit of Endo-H activity is the amount of enzyme substance may be bound to a surface other than by an required to release 1 umole of (H)-dansyl-Asn 35 immunological bond. In this regard, an 'immunological GlcNAc from (3H)-dansyl-Asn-(GlcNAc)4(Man)6 at bond' is one which exists between an antigen and an pH 5.5 at 37 C. in one minute. Tarentino, A. et al. antibody, specific for that antigen (polyclonal or mono (1978) Methods in Enzymology, 50, 574. The unit activity clonal). As shown in FIG. 5A, the glycoside-containing of other Type II endoglycosidases are similarly defined substance has a proximal portion bound to the surface by an appropriate substrate. and a distal portion extending outwardly from the prox Of course, other Type II endoglycosidases may exist imal portion. The proximal and distal portions are which have not yet been identified. Such Type II endo joined by a glycosidic linkage with which Type II en glycosidases as well as the ones described herein, in doglycosidase is reactive. As further shown in FIG. 5A, cluding allelic variations and genetically engineered when treated with Type II endoglycosidase, the distal modifications of such endoglycosidases are within the 45 portion of the glycoside-containing substance is "re scope of the present invention. leased' from the proximal portion of the glycoside-con Glycosides and glycoside-containing substances often taining substance. To the extent that this distal portion is become bound to a wide variety of surfaces. Thus, for not bound by other means to the surface, it is also example, glycoproteins, such as those associated with readily “removed' from the surface and may be washed blood (e.g., glycosylated hemoglobin), can stain the 50 away with a fluid. surfaces of fabrics used for clothes, linen and the like. In FIG. 5B, a glycoside-containing substance, in this Such stains have heretofore been highly resistant to case a glycoprotein containing a carbohydrate unit and complete removal by treatment with detergents or de protein unit, is shown bound to a surface. This glyco tergents in combination with various enzymes not com side-containing substance further contains a carbohy prising the endoglycosidases utilized in the present in 55 drate portion and an aglycon portion joined by a glyco vention. A further glycoside-containing substance sidic linkage which is reactive with Type II endo which stains surfaces such as fabric and which is also glycosidase. In this particular case, the glycoside-con difficult to remove by known techniques comprises taining substance (glycoprotein) is bound to the surface fecal matter. Such fecal stains include various glyco through the carbohydrate portion of the glycoside-con sides and glycoside-containing substances associated taining substance. When treated with Type II endo with intestinal bacteria (e.g., peptidoglycans), catabolic glycosidase, the aglycon portion is released from the excretions, including glycoproteins, and non-absorbed carbohydrate portion of the glycoside-containing sub nutrients and the like. stance. As in FIG. 5A, to the extent that the aglycon Other surfaces to which glycosides or glycoside-con portion is not further bound to the surface by other taining substances may be bound include the surfaces of 65 means, the aglycon portion is also removed from the hard and soft contact lenses. Soft contact lenses are surface. typically hydrophilic cross-linked polymers having a FIG. 5C depicts the situation where a glycoside-con hydrogel structure or are made of silicon polymers. See, taining substance is bound to a surface by way of at least 5,356,803 15 16 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 5 SOCCS. substance to be removed. If treated only with Type II 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 10 include exoglycosidases, a second Type II endoglycosi tacted with a second enzyme reactive with the second dase and Type I endoglycosidases. Examples include O linkage shown, the portion of the glycoside-containing and 3-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 15 Moreover, Type II endoglycosidase may be com the surface, i.e., by other contact points which may be 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 20 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 Disulfide Cleaving Reagents glycosidic linkage is released from the surface when 25 Type II endoglycosidases may also be used in combi treated with Type II endoglycosidase. To the extent 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, which may require further treatment with other en 30 but fall generally into three categories: oxidizing agents, zymes and/or detergent or surfactant. reducing agents, and miscellaneous addition substrates In FIG. 5E, a Type II endoglycosidase-reactive sub such as those exemplified by fumaric acid and sodium stance is shown bound to a surface. This Type II reac sulfite, Suitable oxidizing agents include hydrogen per tive substance has a proximal portion bound to the sur oxide, performic acid, sodium perborate, and oxidizing face and a distal portion extending outwardly from the 35 bleaches. Effective reducing agents include dithio proximal portion. The proximal and distal portions are threitol (DTT), (3-mercaptoethanol (BME), sodium joined by a Type II reactive linkage which refers to a borohydride, and the like. linkage reactive with a Type II endoglycosidase. When Alternate disulfide cleavage reagents which are not treated with Type II endoglycosidase, the distal portion easily classified include mercuric chloride, nitroprus of the Type II reactive substance is "released' from the 40 side, tributylphosphine, and phosphothiolate. A particu proximal portion of the Type II reactive substance. It is larly useful cleavage reagent is sodium sulfite, which to be understood that Type II reactive substances may results in sulfitolysis of the disulfide according to the comprise molecules, microorganisms or aggregates of reaction: R-S-S-R--SO3-2R-S-SO32- SR. The various components which may become attached to a equilibrium of this reaction may be shifted by removal surface. To the extent that the distal portion of the Type 45 of the thiol anion using heavy metal ions or oxidizing II reactive substance is not bound by other means to the agents. The oxidizing power may be provided by aera surface, it is also readily "removed' from the surface tion or an oxidizing agent, such as CuSO4 or sodium and may be washed away with a fluid. perborate. The amount of Type II endoglycosidase used to pro The foregoing list of substances capable of cleaving duce the removal of the substances identified in the 50 disulfides is not meant to be comprehensive, and con figures is defined functionally as an "amount effective' versely does include substances which are effective but for removal of the particular substance from a surface. not necessarily appropriate for a commercial product. This amount may vary depending on the substance and In order to be successful commercially, the added sub surface to be treated. Typical amounts are disclosed in stance must be relatively inexpensive and must not have more detail herein with regard to the specific embodi 55 undesirable properties for its intended use. Thus, for ments disclosed. example, while the use of mercuric chloride would be workable in carrying out the process of the invention, it Second Enzymes would not be suitable for ordinary detergent products "Second enzymes' include proteases, lipases, glycosi intended for commercial use. S-mercaptoethanol and dases such as lysozyme and combinations thereof. Vari 60 DTT are feasible commercially, except that they have ous proteases which may be combined with Type II mildly offensive odors. Particularly preferred sub endoglycosidase include subtilisin, bromilain, papaine, stances, therefore, for commercial formulation, are so trypsin, chymotrypsin, pancreatin, lysozyme and con dium sulfite (preferably in combination with an oxidiz binations thereof. Such enzymes may be derived from ing agent) or hydrogen peroxide, which are inexpensive natural sources, e.g., subtilisin from Bacilius subtilis or 65 and are relatively safe. Reviews of materials which are from genetically engineered clones, e.g., subtilisin and useful in the cleavage of disulfide bonds are found, for mutant subtilisins as described in EPO Publication No. example, in Chemical Modification of Proteins, Means, 0130756. See also, Wells, J. A., et al. (1983) Nucleic G. E., et al., eds (1971), Holden-Day, Inc. San Fran 5,356,803 17 18 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 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% wit/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 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 15 tions of the wash procedure, including time, tempera treated with the combination (simultaneous or sequen 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 20 composition, and the process is conducted as a standard nience, and the selection of the Type II endoglycosi 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 25 approximately 0.5 mg/ml-10 mg/ml or greater of the larly around 40-55 C., and times of up to 20 minutes, 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 hood laundromats, and professional laundry services, 30 gent and second enzyme in the detergent limits the since in order to be commercially practical, the process concentrations of these components in accordance with needs to be conducted under conditions ordinarily the dilution of the detergent. Thus, even if a 1:100 dilu available to the user. tion is used (10 mg/ml), and the disulfide-cleaving rea In another embodiment of the invention, conven gent for example, is limited to 50% of the detergent tional washing procedures using commercial detergents 35 composition, a maximum concentration of 5 mg/ml are used and the Type II endoglycosidase, second en disulfide-cleaving reagent in the resulting wash solution zyme and disulfide-cleaving substance are provided, is an upper limit. Typically, of course, the concentration either separately or together, as an additive, much in the of disulfide-cleaving reagent in the detergent will be less manner of the methods in which bleach is used. Thus, than 50%, mandating even lower concentrations of the these may be added along with the detergent at the disulfide-cleaving reagent. beginning of the wash cycle or at some intermediate The detergent compositions of the invention contain point, for example, after approximately half of the wash mostly detergent active substances, relatively smaller cycle is completed. If handled in this way, assuming an amounts of disulfide-cleaving reagent, if used, and quite approximately 1:500 dilution of a solid or liquid deter small amounts of Type II endoglycosidase and second gent composition (approximately 2 mg/ml of the solid), 45 enzyme, if used, which is especially desirable in view of arbitrary amounts of the Type II endoglycosidase, sec the cost of enzymic components. Thus, in general, the ond enzyme and disulfide cleaving reagents may be preparation will contain 60-90% detergent active sub added without the upper limit imposed by this dilution. stances, including conventional commercial detergent (If the Type II endoglycosidase, second enzyme and additives such as surfactant builders and whiteners, disulfide cleaving reagent had been added to the deter 0.01-3% Type II endoglycosidase and second enzyme, gent composition originally, and if, for example, the and approximately 10-40% disulfide cleavage reagent. disulfide cleaving reagent constituted 50% of the com Of course, it is also possible to add only one of these position, only 1 mg/ml would result in the final wash three additives to the original detergent and to supply solution. However, if these materials are added sepa the other separately to the wash liquid. In particular, rately, amounts most effective for the particular Type II 55 the Type II endoglycosidase may be added to a pre endoglycosidase, disulfide cleaving reagent and second wash, followed by a detergent containing the second enzyme may be added.) enzyme, or addition of the detergent containing endo With respect to the Type II endoglycosidase and glycosidase may be followed or preceded by treatment second enzyme, only very small quantities are usually with the second enzyme. required. Typically, the Type II endoglycosidase and 60 second enzyme are added to a final concentration of Cleaning Compositions approximately 1-500 ug/ml of wash solution for each Endo D, F and Hare preferred Type II endoglycosi enzyme. In the case of the disulfide-cleaving reagent, dases for use in cleaning compositions. Endo-H is most however, larger amounts than would be permitted by preferred. the dilution of the detergent may be desirable. For ex 65 For removal of glycoside-containing substances, the ample, cleavage of disulfide bonds using sodium boro compositions herein preferably comprise from about 0.1 hydride may conveniently be carried out with concen ppm (parts per million) to 1200 ppm, more preferably trations as high as 0.2M reagent in the present of similar from about 1 ppm to 1000 ppm, most preferably from 5,356,803 19 20 about 20 ppm to about 200 ppm, of Type II endo Further, Type II endoglycosidase may be formulated glycosidase, depending on the type of composition. for the treatment of acne which usually results from Cleaning compositions are preferred. Laundry deter inflammation, at least to the extent that glycoside-con gent compositions are most preferred for use herein, taining substances and/or microorgansims responsible and preferably comprise from about 0.1 ppm to 1200 5 for or involved in such inflamation are bound to a sur ppm of Type II endoglycosidase, preferably from about face. As with the above formulations, those skilled in 20 ppm to 200 ppm of Endo D, F or H, most preferably the art are capable of modifying known acne formula from about 50 ppm to 125 ppm Endo H. tions to incorporate a Type II endoglycosidase alone or When used to control or remove microorganisms, the in combination with other enzymes, detergents and/or compositions preferably comprise from about 0.1 ppm 10 surfactants. to 1200ppm, more preferably from about 1 ppm to 1000 When used to treat contact lens, Type II endoglycosi ppm, most preferably from about 20 ppm to 400 ppm, of dase suitably is supplied at a concentration of about Type II endoglycosidase, preferably Endo-H. Cleaning 0.1-20 ug/ml in the cleaning compositions, and the compositions are preferred and preferably comprise the concentration of a second enzyme such as a protease is same amounts of Type II endoglycosidase, preferably 15 in the same range if such second enzymes are utilized. Endo-H. Treatment times can vary from about five minutes to Described below are suggested types of compositions about 15 hours, but a standard convenient cleaning time which comprise Type II endoglycosidase for removal is overnight, so that the wearer can allow the lenses to of glycoside-containing substances and/or microorgan soak while he sleeps. A variety of protocols are suitable, isms. The compositions can be made and used in any 20 but ones that are particularly preferred are the use of a way which does not destroy enzyme activity. They can single solution containing Type II endoglycosidase and be made up of any ingredients which do not unduly the second enzyme (if used) conducted from 10 minutes hinder the activity of the enzyme. The compositions can to two hours or overnight at room temperature, or a be laundry detergents, dishwashing detergents, hard 10-minute to two-hour presoak in the presence of Type surface cleaners, dental enamel cleaners, liquid and bar 25 II endoglycosidase solution, followed by a similar over soaps, anti-acne compositions, antiperspirants, sham night treatment with a solution containing a second poos, face creams, fruit and vegetable surface preserva enzyme. tives, or fabric softeners. Preferred general purpose second enzymes for In addition to the cleaning of fabrics using common contact lens formulation include proteases such as pa cycles in washing machines, the cleaning compositions 30 pain, pancreatin and subtilisin. The preferred Type II herein may also be used for removing glycoside-con endoglycosidase enzyme is Endo-H from Streptomyces taining substances and/or microorganisms from other plicatus. A single second enzyme protease may be used, surfaces such as metals and metal alloys such as found in or the composition may contain a mixture of second surgical instruments, pipelines, metal containers and the enzymes. like, and plastics and composite materials such as For 35 In addition, the contact lens compositions may in mica and the surfaces of boats, piers and the like. De clude additional components which aid in the overall pending upon the particular application, the composi enzymatic degradation. Particularly useful among these tion may comprise Type II endoglycosidase alone or in are disulfide cleavage reagents such as 2-mercaptoe combination with a disulfide cleaving reagent, second thanol, cysteine hydrochloride, dithiothreitol, dithioe enzyme and/or detergent surfactant. 40 rythritol, sodium bisulfate, sodium metabisulfite, thio Type II endoglycosidase may also be formulated in a urea, and the like, generally preferred in a range of composition for removing glycoside-containing sub about 0.01-5% by weight preferably 0.05-1% by stances and/or microorganisms including yeast, fungi, weight. In addition, detergents may be included in the algae and bacteria from “biological surfaces' such as composition to aid in the wetting of the lens with the surfaces of skin, skin pores, hair, hair follicles and tissue. 45 enzyme-containing solution. Suitable detergents include Thus, those skilled in the art of shampoo formulations, sodium dodecyl sulfate, sodium monolaurate, nonionic conditioner formulations, soap formulations and the surfactants such as alcohol ethoxylates (e.g., polyethox medicinal arts can readily adapt the above disclosure yethanol) anionic surfactants such as ether sulfonates, for detergent formulations to employ Type II endo linear alkylbenzene sulfonates, sodium lauryl sulfate, glycosidase in such applications. When so formulated, 50 and the like. such compositions are useful in removing glycoside Suitable buffers and stabilizers for contact lens clean containing substances which may adhere to such sur ing may also be used and include sodium or potassium faces. citrate, citric acid, boric acid, sodium EDTA, various Type II endoglycosidase may also be formulated in a mixed phosphate buffers and NaHCO3. Generally buff. composition for removing glycoside-containing sub 55 ers and stabilizers may be used in amounts ranging from stances and/or microorganisms, especially yeast and about 0.001 to about 2.5% and preferably about 0.1 to fungus, from the surfaces of plants such as fruits and 1% by weight. It should be understood that the forego vegetables. Such compositions preferably include non ing description of the amounts of the various com ionic surfactant. pounds which may be used in the present invention for In addition, Type II endoglycosidase may be formu cleaning contact lens are stated in percentage of ingredi lated in deodorant compositions in a manner known to ents in solution (wt/vol). The formulation may also take those skilled in the art to provide endoglycosidase ac the form of one or more conventional solid dosage tivity to remove glycoside-containing substances and forms such as tablets suitable for use in measured quan /or microorganisms responsible for undesirable odors. tity of a suitable solvent such as water. The percentage Such deodorant formulations employing Type II endo 65 composition of the solid dosage forms is such that when glycosidase may include modifications of formulations dissolved in a specified volume of water, the solution for stick, creams and aerosol deodorants known to those will have the percentage composition within the ranges skilled in the art. set forth in the specification. If solid dosage forms are 5,356,803 21 22 used, the formulation may include conventional lubri Laboratory work on microorganism removal has cants, binders, and excipients which include glycerol, shown that, in order to obtain effective removal, the sorbitol, boric acid, propylene glycol, polyethylene bathing of the surface holding the microorganisms in glycols, dextran, methylcellulose, hydroxyethylcellu some instances requires a physical or chemical action to lose, water soluble salts of carboxymethylcellulose, or 5 remove the microorganisms. Microorganisms tested naturally occurring hydrophilics such as gelatin, algi include: nates, tragacanth, pectin, acacia and soluble starches. Escherichia coli including Type 1 and 3 fimbriae Typical compositions and protocols useful in cleaning Staphylococcus aureus contact lens include the following: Staphylococcus epidermidis 1. The composition contains 1-100 ug/ml Type II O Serratia marcescens endoglycosidase. The lenses are removed and Streptococcus mutans placed in contact with the solution for a period of Streptococcus Sanguis 12 hours at 22 C. The lenses are removed from the Bacillus sp. cleaning solution and rinsed. Candida sp. 2. Solution. A contains 10 pg/ml of Type II endo 15 Aspergillus sp. glycosidase; solution B contains 5 g/ml subtilisin. In the case of removal of bacteria such as E. coli, for The lenses are soaked in solution. A for 30 minutes example, the surface-bound microorganisms may be at 25 C., removed, and immersed in solution B for treated with Endo-H and then removed by chemical 10 hours at 25 C. action, such as by treatment with an antimicrobial 3. The cleaning solution contains 10 ug/ml of the 20 agent, or a physical action, such as by rinsing with protease pepsin and 10 g/ml of Type II endo water or hand wiping. It is preferred for liquid and bar glycosidase. The lenses are soaked in this solution soaps, dental enamel cleaners, antiperspirants, anti-odor for 5 hours at 20 C. fabric softeners and anti-acne compositions that the 4. The cleaning solution contains 5 g/ml subtilisin, 5 composition include an anti-microbial agent, such as ug/ml. Type II endoglycosidase, and 10 mM2-mer 25 Irgasane (Ciba-Geigy) or chlorhexidine, in addition to captoethanol. The lenses are immersed in this solu the Endo-H. An antimicrobial agent is not required in tion for 5 hours at 30 C. the composition (for example a hard surface cleaner) 5. The cleaning solution contains 7 g/ml subtilisin, 3 when physical action such as water rinsing or wiping by ug/ml Type II endoglycosidase, 10 mM 2-mercap hand will occur. toethanol, and 2% sodium dodecyl sulfate (SDS). 30 Preferred herein are detergent cleaning compositions, The lenses are soaked in this solution for 3 hours at especially granular and liquid laundry detergent com 20 C. positions. These detergent cleaning compositions pref 6. The cleaning solution contains 4 g/ml subtilisin, 2 erably comprise from about 1% to 90%, more prefera ug/ml trypsin, 10 pug/ml Type II endoglycosidase, bly from about 5% to 50%, by weight, of detergent 35 surfactants, most preferably from about 10% to 40% by and 2% SDS. The lenses are soaked in this solution weight. for 7 hours at 20 C. Surfactants useful in the detergent compositions 7. Solution. A contains 4 ug/ml subtilisin and 2 g/ml herein include well-known synthetic anionic, nonionic, trypsin in 2% SDS. Solution B contains 10 pg/ml amphoteric and zwitterionic surfactants. Typical of Type II endoglycosidase plus 10 mM 2-mercaptoe these are the alkyl benzene sulfonates, alkyl- and al thanol. The lenses are immersed in solution B for kylether sulfates, paraffin sulfonates, olefin sulfonates, 20 minutes at 30° C. and then in solution. A for 6 alkoxylated (especially ethoxylated) alcohols and alkyl hours at 25 C. phenols, amine oxides, alpha-sulfonates of fatty acids In all the foregoing examples, the lenses are thor and of fatty acid esters, alkyl betaines, and the like, oughly rinsed in saline before being returned to the 45 which are well known from the detergency art. In gen wearer’s eyes. eral, such detersive surfactants contain an alkyl group in The compositions herein can be formulated in a vari the C9-C18 range. The anionic detersive surfactants can ety of physical forms, including liquids, gels, pastes and be used in the form of their sodium, potassium or trie solid particles such as powders and granules. The com thanolammonium salts; and the nonionic surfactants positions can be formulated as laundry detergents, such 50 generally contain from about 5 to about 17 ethylene as disclosed in U.S. Pat. Nos. 4,507,219, 4,318,818, oxide groups. C11-C16 alkyl benzene sulfonates, 4,605,509 and 4,412,934; dishwashing detergents such as C12-C18 paraffin-sulfonates and alkyl sulfates are espe disclosed in U.S. Pat. Nos. 4,714,562, 3,630,923, cially preferred in the compositions of the present type. 4,133,779, 4,316,824 and 4,555,360; hard surfaces clean A detailed listing of suitable surfactants for the com ers such as disclosed in U.S. Pat. Nos. 4414,128, 55 positions herein can be found in U.S. Pat. No. 3,936,537, 3,679,608, 3,985,668 and 4,005,027; fabric softeners such Baskerville, issued Feb. 3, 1976, incorporated by refer as disclosed in U.S. Pat. Nos. 3,944,694, 4,073,996, ence herein. Commercial sources of such surfactants 4,424,134 and 4,661,269; bar soaps such as disclosed in can be found in McCutcheon's Emulsifiers and Deter U.S. Pat. Nos. 3,993,722 and 3,070,547; shampoos such gents, North American Edition, 1984, McCutcheon as disclosed in U.S. Pat. Nos. 4,345,080, 4,704,272 and Division, MC Publishing Company, also incorporated 4,741,855; antiperspirants such as disclosed in U.S. Pat. herein by reference. No. 4,725,432; anti-acne products such as disclosed in Useful detergency builders for the detergent compo U.S. Pat. Nos. 4,318,907 and 4,608,370; and oral compo sitions herein include any of the conventional inorganic sitions such as disclosed in U.S. Pat. No. 4,684,518. The and organic water-soluble builder salts, as well as vari above patents are incorporated herein by reference. 65 ous water-insoluble and so-called 'seeded' builders. The compositions preferably have a pH from about 4 The instant laundry detergent compositions preferably to 10, more preferably from about 5 to 8 for good en comprise from about 1% to 75%, more preferably from zyme performance. about 5% to 40%, most preferably from about 10% to 5,356,803 23 24 20%, by weight of detergent builders. These composi ganism growth, and/or changes in the gross morphol tions preferably have a pH of from about 6 to 10. ogy of the microorganism. In another aspect of the Nonlimiting examples of suitable water-soluble, inor invention, the antimicrobial method causes the removal ganic alkaline detergent builder salts include the alkali of a microorganism from a surface. In the antimicrobial metal carbonates, borates, phosphates, polyphosphates, methods to remove microorganisms from surfaces, it is tripolyphosphates, bicarbonates, silicates and sulfates. preferred that the surface be treated with the antimicro Specific examples of such salts include the sodium and bial agent and the Type II endoglycosidase simulta potassium tetraborates, bicarbonates, carbonates, tri neously, rather than treating with the additional antimi polyphosphates, pyrophosphates, and hexametaphos crobial agent immediately after treating with Type II phates. 10 endoglycosidase. In some applications of the antimicro Examples of suitable organic alkaline detergency bial methods, a combined antimicrobial effect may be builder salts are: (1) water-soluble amino polyacetates, produced, e.g. killing and/or growth inhibition may e.g., sodium and potassium ethylenediaminetetraace occur in combination with microorganism removal tates, nitrilotriacetates, and N-(2-hydroxyethyl)nitrilod from a surface. iacetates; (2) water-soluble salts of phytic acid, e.g., 15 sodium and potassium phytates; (3) water-soluble poly 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 com dium, potassium, and lithium salts of methylenediphos ponent comprising an antimicrobial agent. Such antimi phonic acid and the like. 20 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. 25 moval of microorganisms from a surface and the pre A detailed listing of suitable detergency builders can 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 Optional detergent composition components include 30 alone to contact a microorganism to produce an antimi enzymes (e.g., proteases and amylases), peroxygen crobial effect. bleaches and bleach activators, halogen bleaches (e.g., As used herein, an "antimicrobial agent' is a second sodium and potassium dichloroisocyanurates), soil re different component of an antimicrobial composition. lease agents (e.g., methylcellulose), soil suspending Such antimicrobial agents in general are antibiotics and agents (e.g., sodium carboxymethyl-cellulose), fabric 35 include agents which kill microorganisms and those brighteners, enzyme stabilizing agents, color speckles, which inhibit microorganism growth. Examples of such Suds boosters or suds suppressors, anticorrosion agents, antimicrobial agents include bacteriocides, fungicides dyes, fillers, germicides, pH adjusting agents, non and algicides each of which are capable of killing or builder alkalinity sources, and the like. inhibiting the growth of bacteria, fungi or algae, respec tively. Bacteriocides include compounds such as chlor Endoglycosidase Plus Antimicrobial Agents hexidine, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, Of the Type II endoglycosidases, endo-3-N-acetyl triclocarban, penicillins, tetracycline and bacitracin. glucosaminidase H, D, F and/or PNGase F are pre Fungicides include nystatin (Fungicidin (R), amphoteri ferred for formulating antimicrobial compositions and cin B (Fungizone (R)), benomyl (Benlate (R)), captan for use in the antimicrobial methods herein. Endo-H is 45 (Merpan (R)), dichlorobenzalkonium chloride (Di most preferred. chlorane (R)). Other examples of antimicrobial agents When the Type II endoglycosidase is used alone, it is include surfactant-stable antimicrobial enzymes such as formulated such that its concentration produces an surfactant-stable (3-1,3-glucanases, lysozymes, proteases antimicrobial effect. When the antimicrobial composi and chitinases, and detergent surfactants such as ani tion comprises at least two different components, i.e. a 50 onic, nonionic, zwitterionic, ampholytic and cationic Type II endoglycosidase and one or more antimicrobial surfactants known to those skilled in the art. The latter agents, each of the components are present at a concen should be employed in an amount sufficient to produce tration sufficient to produce an antimicrobial effect. The an antimicrobial effect. The above antimicrobial agents amount of at least one component in said compositions identified by generic name or trademark are composi is generally less than the amount required for that com 55 tions as identified in the Merck Index, 10th Ed. (1983), ponent to produce the same antimicrobial effect if used Merck & Co., Inc., Rahway, N. J. alone in a similar composition. Type II endoglycosidases different from the first As used herein, an “antimicrobial effect' includes the component of the antimicrobial compositions may also removal, killing, inhibition of growth, change in gross be used as an antimicrobial agent. Thus, to the extent morphology, protoplast formation and/or degradation Type II endoglycosidases are themselves antimicrobial of the cell wall of a microorganism when contacted agents (e.g. are capable of producing an antimicrobial with a Type II endoglycosidase alone or in combination effect, such as morphological changes or protoplast with a second component comprising an antimicrobial formation), they may be combined with a different agent. Type II endoglycosidase to form an antimicrobial com As used herein, an “antimicrobial method' refers to a 65 position. Antimicrobial compositions may therefore method which produced an antimicrobial effect. In one comprise one or more different Type II endoglycosi aspect of the invention, the antimicrobial method causes dase with or without one or more antimicrobial agents the killing of microorganisms, the inhibition of microor not comprising Type II endoglycosidase. 5,356,803 25 26 Preferred antimicrobial agents for use herein are concentration is believed to be between 10 and 100 chlorhexidine, 2,4,4'-trichloro-2'-hydroxydiphenyl ppm. Similar amounts of Endo-H are believed to be ether, Triclocarban (R), Nystatin (R) Amphotericin B (R) useful to kill and/or inhibit other microorganisms such antibiotic, anionic and nonionic detergent surfactants. A as algae and fungi. The exact effect of Endo-H and surfactant-stable antimicrobial lysozyme is disclosed in other Type II endoglycosidases on these organisms and the copending U.S. application Ser. No. 428,273 now others, e.g., bacteria, when not used in combination issued as U.S. Pat. No. 5,041,236 entitled Methods and with antimicrobial agents has not yet been determined. Compositions Employing Certain Lysozymes and En The range of antimicrobial-effective concentrations of doglycosidases in the names of Richard S. Carpenter Type II endoglycosidase for use against such organ and Ann M. Wolff, filed on even date as this applica O isms, however, can be routinely determined. tion. Other lysozymes, e.g. hen egg white lysozyme, The antimicrobial methods and compositions of the have been used in combination with Endo-H to produce invention have a wide applicability and include antimi antimicrobial effects albeit to a lesser extent and with crobial methods and compositions for personal care, variability in the results obtained. health care and household and industrial cleaning. The antimicrobial compositions and methods of the 15 Thus, such methods and compositions may be used to invention can produce an antimicrobial effect on a wide formulate and use antimicrobial mouthwash, dentifrice range of microorganisms including Gram-positive and or denture cleaner, as well as antimicrobial liquid or negative bacteria, fungi, and algae. Such bacteria in solid hand or body soaps, anti-acne medication, deodor clude Escherichia coli, Streptococcus mutans, Staphylo ant, shampoo and face creams and compositions for coccus epidermidis, and Staphylococcus aureus. Such 20 cleansing wounds or suppressing infections. Typical fungi include yeasts such as Candida and Saccharomy household applications include antimicrobial cleaning ces, and species and filamentous fungi such as Aspergil products such as liquid soap, hard surface cleaners, and lus, Sporobolomyces, Basidiobolus and Entomoph liquid and granular laundry detergents. Heavy duty thora. antimicrobial detergent compositions may also be for A specific advantage of combining a Type II endo 25 Inulated 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 an(R) DP300 from Ciba-Geigy and is a broad-spectrum when used with a Type II endoglycosidase produces an 30 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 attachment to such surfaces. In other aspects, there is a also be employed as the additional antimicrobial agent negative effect on microorganism viability or microor herein. Lastly, surfactant-stable antimicrobial enzymes ganism morphology. 35 can be used in dental applications and for preservation Surface treatment(s) with Type II endoglycosidase of shampoos and other surfactant-containing formula and antimicrobial agent can be performed periodically tions. A preferred surfactant-stable antimicrobial en so as to prevent further growth or attachment or adhe zyme is the lysozyme disclosed in the previously identi sion of microorganisms to the surfaces exposed to the fied copending application in the names of Carpenter treatment. and Wolff. Surfactant-stability of antimicrobial enzymes Of the Type II endoglycosidases, Endo-H, D, F and can be gauged herein by retained activity in the pres /or PNGase F are preferred. Of these, Endo-His most ence of representative amounts of alkyl ether sulfate or preferred. In general, an antimicrobial-effective amount linear alkylbenzene sulfate, for example. of Type II endoglycosidases for use in combination The antimicrobial composition may be formulated as with antimicrobial agents is from about 1 to 1200 ppm 45 an antimicrobial mouthwash, dentifrice, or denture Endo-H, D, F, and/or PNGase F, preferably from cleaner. The treatment of microorganisms to produce about 1 to 1200 ppm Endo-H, more preferably from an antimicrobial effect (e.g. to remove or prevent mi about 20 to 1000 ppm Endo-H, most preferably from croorganism attachment to natural or synthetic soft about 50 to 400 ppm Endo-H. The amount used depends and/or hard surfaces in the oral cavity or to kill micro upon the type of treatment and amount of exposure to 50 organisms or inhibit their growth in the oral cavity), the surface or microorganism to be treated. In general, then, essentially comprises rinsing with an antimicrobial an effective amount of antimicrobial agent, which de mouthwash, cleaning the teeth with an antimicrobial pends upon which agent is used, is from about 0.5 to dentifrice, and/or cleaning dentures with an antimicro 1200 ppm, preferably 2 to 1200 ppm, most preferably bial denture cleaner. The antimicrobial mouthwash, from about 5 to 350 ppm chlorhexidine or 2,4,4'-tri 55 dentifrice and denture cleaners herein preferably com chloro-2'-hydroxydiphenyl ether, or 0.5 to 100 ppm prise Endo-H, and chlorhexidine and/or surfactant sta Nystatin (R). ble antimicrobial enzyme as the antimicrobial agent. When Type II endoglycosidase is used alone to kill Where chlorhexidine is used, the antimicrobial mouth and/or inhibit microorganisms, the use of substantially wash, dentifrice, or denture cleaner preferably com more Type II endoglycosidase is generally required. 60 prises from about 50 to 1200 ppm Endo-H and from For example, about 100 ppm to 1000 ppm of Endo-H about 50 to 350 ppm chlorhexidine. Where surfactant has been shown to substantially decrease the viability of stable antimicrobial enzyme is used, the antimicrobial yeast cells exposed to such concentrations. When yeast mouthwash, dentifrice or denture cleaner preferably is exposed to less than 100 ppm of Endo-H, however, a comprises from about 50 to 150 ppm Endo-H and from significant decrease in viability has not been observed. 65 about 50 to 1,000 ppm surfactant-stable antimicrobial Although the lower limit of Endo-H necessary to ad enzyme. versely affect yeast viability has not yet been deter The antimicrobial composition may also be formu mined, the lower limit of its antimicrobial-effective lated as antimicrobial personal care or household clean 5,356,803 27 28 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 5 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 and shampoo (described below). 10 EXAMPLE 1. A preferred antimicrobial liquid hand soap comprises Removal of Blood and Fecal Matter from Fabric from about 50 to 400 ppm Endo-H, from about 5 to 100 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 ric) swatches were washed with commercial detergents surfactant. Preferably from about 2 to 20 weight %, 15 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, am with various amounts and types of endoglycosidase pholytic and cationic surfactants. The liquid hand soap (0.005 U of Endo-D (Boehringer Mannheim Biochemi can further comprise emolient (up to about 30 weight 20 cal), or Endo-H (Boehringer Mannheim Biochemical %) and minor amounts of perfume, colorant, solvent, from S. griseus, Catalog No. 752 967), and 0.25 UN 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-HCl glass cleaners, abrasive hard surface cleaners, scouring , pH 7.0 at 37° C. for 30 minutes in a test tube. The cleansers, or toilet bowel cleaners. These should be 25 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 enzymes or brighteners in 1M Tris-HCl, pH 7.5) con from about 0.1 to 20 weight % detergent surfactant. 30 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 consisting of anionic, nonionic, zwitterionic, am ment, the tubes were centrifuged and the protein con pholytic and cationic surfactants. The antimicrobial tent in the supernatants were determined by measuring hard surface cleaners herein optionally further comprise 35 absorbance at 280 nm. For each treatment, a reaction abrasive, builder, diluent, solvent, suspending agent blank was prepared which contained no swatch during (such as clay, carboxymethylcellulose, and polyacryl the assay. The blank values were subtracted from the ate), perfume, and/or colorant. absorbance of treated samples to determine the release The antimicrobial laundry detergent herein, in addi of 280 nm absorbing material during incubation. Higher tion to Type II endoglycosidase and antimicrobial absorbance represents increased release of protein from agent, preferably comprises from about 1 to 99 weight fibers. The results are shown in Table III. %, more preferably from about 5 to 60 weight %, most preferably from about 10 to 40 weight % detergent TABLE III surfactant, preferably selected from the group consist Absorbance at 280 nm. ing of anionic, nonionic, zwitterionic, ampholytic and 45 Treatment Blood Stain Fecal Matter Stain cationic surfactants. A preferred liquid or granular anti Control 0.79 2.07 Endo-D 0.84 2.14 microbial laundry detergent comprises from about 2 to Endo-H 0.83 2.12 250 ppm Endo-H, from about 2.5 to 40 ppm 2,4,4'-tri N-Glycanase 0.78 2.10 chloro-2'-hydroxydiphenyl ether, and from about 1 to 99 weight %, preferably from about 5 to 60 weight %, 50 detergent surfactant. The antimicrobial laundry deter These results suggested that the endoglycosidases, gents herein optionally further comprise builder, per Endo-D and Endo-H, in combination with the second fume, bleach, diluent, suds suppressor, colorant, bright enzyme subtilisin increased the release of 280 nm ab ener, soil suspending agent, antiredeposition aids, soft sorbing material from the blood stained swatches as eners, and/or soil release agents. 55 compared to the control. In addition, Endo-D, Endo-H The antimicrobial shampoo for use herein preferably and N-glycanase all showed an increase in the release of comprises Endo-H, an antimicrobial agent, and from 280 nm absorbing material from the fecal stained about 5 to 60 weight% detergent surfactant, preferably swatches. selected from the group consisting of lauryl sulfate, isoethionate, acyl amidobetaine, alkyl glyceryl ether EXAMPLE 2 sulfonate, and alkyl ether sulfate. Optional ingredients Effect of Endo-H on Removal of Fetal Matter Stain are suds booster, conditioner, dye, colorant, perfume This example is similar to Example 1 but was per and/or anti-dandruff agent. formed by using fecal matter stained swatches made of The present antimicrobial compositions may also be nylon fabric. The swatches were washed in detergent in the form of a preservative or microorganism control 65 solution, rinsed and dried. The detergent consisted of agent for treatment of plant surfaces. Preferred are a liquid commercial detergent which did not contain preservative for the surfaces of fruits or vegetables or enzymes, brighteners, dyes or perfumes. One set of an antimicrobial product to be applied on crops for swatches was kept aside and referred as “untreated 5,356,803 29 30 control'. These swatches were treated the same as the removing fecal matter in the presence of detergent, a sample swatches except that they were not treated with protease and a disulfide cleaving reagent (2-mercaptoe Endo-H. The sample swatches were incubated with thanol). 0.01 U Endo-H (Boehringer Mannheim Biochemical Catalog No. 752967) in buffer (10 mM Naacetate, pH EXAMPLE 4 6.0) at 37 C. for 15 minutes. Then 0.25 ml of detergent Comparison of Endo-H with Other Enzymes solution (1:125 dilution in 1.0M Tris-HCl, pH 7.5) was 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 Example 3 were repeated with Endo-H (Boehringer removed by suction. The swatches were air dried. Fi 10 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 copy. Removal of particulate and bacterial debris from be seen, rod like bacteria and particulate matter are 15 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 dase, showed little or no benefit. (Results not shown.) ulate material and bacterial debris. 20 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. 25 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 mM sodium tron micrograph of a fiber treated with "Lysing En acetate buffer, pH 5.5 with or without Endo-F (Boehr zymes'; FIG. 7G is an electron micrograph of a swatch inger Mannheim Biochemical) (0.15 units) for 30 min 30 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) bance of each was measured at 280 mm. Change in has been thoroughly cleansed of the fecal matter stain. A280 was determined by subtracting appropriate blanks Similar results were obtained for the swatches treated (see Example 1). Higher absorbance includes the in 35 with “Lysing Enzymes' as shown in FIG. 7F. crease in the amount of protein or material absorbing at 280 mm released from the swatches. For the controls, EXAMPLE 5 the average change in A280 was 0.93. For swatches Removal of Bacteria from a Solid Surface treated with Endo-F the average change in A280 was 1.05. This indicated that Endo-F increases the effi To test the effect of Endo-H on removal of bacteria ciency of fecal stain removal. from solid surfaces (glass), the following protocol was B. Swatches were incubated in 0.75 10 mM sodium used. Trypticase soy broth (TSB) (10 ml) was inocu acetate buffer pH 5.5 with or without Endo-F (0.15 lated with a microbial species (Staphylococcus aureus units) for 15 minutes at 37° C. At the end of this treat ATCC culture if6538 or Escherichia coli ATCC culture ment, 0.25 ml of detergent solution (in 0.1M Tris-HCl, 45 #10536) from a stock culture slant and incubated over pH 7.5) containing 10 ug of the protease subtilisin BPN' night at 37° C. A suspension of about 108 cells/ml TSB was added and the tubes were incubated at 37 C. for was prepared and 100 ul of this suspension was placed another 15 minutes. At the end, tubes were centrifuged, within the etched ring on a glass slide. Each slide was supernatants were removed and absorbance at 280 nm incubated for 5 minutes at 37 C. in a dry incubator oven was measured. In the case of the control (no Endo-F), 50 after which excess microbial solution was tapped off. the average change in A280 was 1.08 whereas the sam The slides were then rinsed with 100 pil of sterile dis ple treated with Endo-F showed a change in A280 of tilled water. The excess solution and loose organisms 1.36. This indicated that the effect of Endo-F was en were then tapped off. hanced by the presence of the detergent. After the bacteria were adhered to the glass slides (2 C. An experiment similar to “B” was performed ex 55 or more hours at 37° C), 100 ul of the following solu cept the detergent solution contained 10 mM 2-mercap tions were applied to separate slides: (a) 10 mM acetate toethanol instead of subtilisin. The average change in buffer, pH 5.5, (b) 10 mMacetate buffer, pH 5.5-1 ppm A280 for the control was 1.05 whereas the sample Endo-H (Boehringer Mannheim Biochemical Catalog treated with Endo-F produced a change in A280 of No. 100 119), (c) detergent solution, (d) detergent solu 1.24. These results demonstrated the ability of Endo-F 60 tion--1 ppm Endo-H. A set of slides were kept aside as in the presence of disulfide cleaving reagents to remove untreated controls and were not treated with any solu fecal stains. tions. The non-control slides were then incubated for 15 D. An experiment similar to “B” was performed minutes at 37 C. At the end of the incubation, the solu except that the detergent solution contained 10 mM tions were tapped off. The slides were then rinsed with 2-mercaptoethanol and 10 pig subtilisin BPN'. The aver 65 100 ul of sterile distilled water and air dried at room age change in A280 for the control was 1.14 whereas temperature. The bacteria which remained after this the Endo-F treated sample had a change in A280 of treatment were heat fixed and stained by a standard 1.29. These results indicate that Endo-F is capable of Gram staining method. The slides were then examined 5,356,803 31 32 by a light microscope (bright field illumination, 37° C. for 12 hours. The cultures were then added to 30 125Xmagnification) and the number of organisms/field ml of 0.2M NaCitrate, pH 5.5 buffer at about 103 was determined. Twenty fields were counted for each cells/ml, in two 100 ml shake flasks. Twelve cloth slide from which the average organisms/field was cal swatches (0.5x0.5 inch cotton swatches) were also culated. 5 added to the flasks after inoculation. After incubation at The following results were obtained: 37° C. for two hours with gentle rotation (150 rpm), the swatches were transferred to sterile tubes and washed A. For Staphylococcus aureus 3X with buffer comprising 200 mM. NaCitrate, pH 5.5 i) No treatment > 100 organisms/field which had been previously sterilized by 0.22 micron ii) Buffer > 100 organisms/field 10 filtration. Six swatches were then added to a shake flask iii) Buffer -- Endo-H C 10 organisms/field containing 0.5 mg/ml Endo-H in 30 ml citrate buffer, iv) Detergent solution > 100 organisms/field and six swatches were added to a shake flask containing v) Detergent -- Endo-H < 10 organisms/field only citrate buffer as the control. The Endo-H was obtained from E. coli producing S. plicatus Endo-H. These results indicate that Endo-H buffer alone or in 15 After incubation at 37 C. for 1.5 hours with gentle combination with detergent reduced the number of S. rotation (100 rpm), the swatches were transferred to aureus bacteria retained on the glass slides 10 fold as sterile tubes and washed as previously described. compared to treatment with detergent alone. Swatches were then plated carefully on trypticase soy agar plates and overlaid with enough liquid trypticase B. For Escherichia coli soy agar to cover the swatches. After the plates were i) No treatment > 100 organisms/field dry, they were incubated at 37 C. for 18 hours, and ii) Buffer > 100 organisms/field colonies of Staphylococcus aureus and Staphylococcus iii) Buffer + Endo H > 100 organisms/field epidermidis on the cloth surface were counted using a iv) Detergent > 100 organisms/field dissecting scope. v) Detergent -- Endo H < 10 organisms/field 25 The following results were obtained:

These results indicate that Endo-H in combination A. For Staphylococcus aureus with a detergent reduced the number of E. coli retained Control 103 +/- 24 colonies per swatch on the glass slide 10-fold as compared to treatment with Endo-H 53 +/- 18 colonies per swatch detergent alone. 30 49% decrease in bacterial colonies by Endo-H treatment EXAMPLE 6 B. For Staphylococcus epidermidis Control 57 --/- 11 colonies per swatch Removal of Bacteria from a Solid Surface Endo-H 16 +/- 10 colonies per swatch An experiment similar to Example 5 was performed 35 72% decrease in bacterial colonies by Endo-H with two slime-producing Staphylococcus aureus cul treatment. tures (determined by their abilities to bind to polysty rene tubes). Microscope slides were modified by form These results indicate that Endo-H treatment signifi ing two rings (st 1.7 cm diameter) with nail polish. cantly reduces the number of bacteria adhered to a Overnight culture of the organisms were diluted 1:10 cloth surface. with 1% peptone solution. Diluted culture (100 ul) was put in rings. Slides were put in 150 cm petri dishes and EXAMPLE 8 incubated at 37 C. After two hours incubation, slides Binding of Endo-H to Bacteria were rinsed with distilled water and treated with three The following experiment was conducted to deter different conditions (A. Naacetate buffer, B. detergent, 45 mine if the Type II endoglycosidase, Endo-H, interacts and C. detergent plus 1 ug Endo-H/ml) as in Example with a surface component on the bacteria Staphylococ 5. The Endo-H was obtained from E. coli transformed cus aureus and Streptococcus mutans. Such an interaction to produce Endo-H from S. plicatus. At end of 15 min was detected. Although not completely characterized utes, incubation slides were rinsed with distilled water herein, this interaction was not previously known and and Gram stained. The number of bacteria was counted 50 may form the basis of the above described ability of under microscope per 100X field for 20 fields. The Endo-H to remove such bacteria from a surface. results are expressed as the average number of cells per Endo-H from transformed E. coli and purified by field. modifying the methods described by Trimble R.J. et al. (1985), J. Biol. Chem, 260,5638-5690, was labelled with Condition Culture I Culture II biotin according to the procedure described by Updyke, A. Control 23 202 T. V. and Nicolson, G. L. (1986), Methods in Enzymol B. Detergent 9 58 ogy, 21, 717-725. After such labelling, the Endo-H re C. Detergent -- Endo-H 2 33 tained most of its reactivity with the glycoprotein oval bumin. 60 Overnight cultures of Staphylococcus aureus (ATCC EXAMPLE 7 6538) grown in Luria's broth, and Streptococcus mutans (ATCC 27607) grown in Difco Brain Heart Infusion Removal of Bacteria from a Cloth Surface media, were centrifuged and washed three times with To test the effect of Endo-H on the removal of bac 200 mM NaCitrate pH 5.5 buffer and suspended in the teria from a cloth surface, the following protocol was 65 same buffer to a concentration of about 109 cells/mi. used. Staphylococcus aureus (ATCC 6538) and Staphylo Aliquots of 0.5 ml were placed in 31.5 ml Eppendorf coccus epidermidis (ATCC 155) were separately cul tubes and incubated under various conditions and times. tured in 5 ml of Luria's broth and allowed to grow at 5,356,803 33 34 -continued Incu Active Biotiny- 0.2M bation Component Weight % lated NaCitrate Time Tube Cells 2% BSA Endo-H pH 5.5 (min.) Sodium hydroxide 3.85 C12-4 fatty acid 10.00 0.5 5 Jul 0.5 m 30 Citric acid 4.00 2 0.5 m -- 5 ul 0.5 ml 2 Calcium formate 0.12 3 0.5 ml - 5 pull 0.5 ml 30 Sodium formate 0.86 C12 alkyltrimethylammonium chloride 0.50 10 Tetraethylene pentamine ethoxylate (15-18) 2.00 Incubation was done at room temperature using a Water 35,12 slow speed rocker for either two or 30 minutes. BSA Dye 0.08 (bovine serum albumin), diluted in tris-buffered saline Perfume 0.25 was used as a control solution in order to prevent any Protease 0.125 non-specific protein binding to the cells. After incuba Endoglycosidase H 2000 ppm 15 Notes tion, the tubes were centrifuged and the supernatants Alcohol and monoethoxylated alcohol removed. were discarded. Two cell washes with 2% BSA solu "mg active enzyme/g (G34 mg active enzyme/gstock) tion were done by adding 1.0 ml BSA to the cells, vor texing well, centrifuging and discarding the superna The ingredients listed above are added to a mixing tant. To the washed cells, 0.5 ml of streptavidin-HRP tank with a single agitator in the order in which they (streptavidin-labeled horse radish peroxidase, Kirkeg 20 appear. Before the protease enzyme, dye and perfume aard and Perry Laboratories, Inc.) was used and incu are added, the pH of the mix is adjusted so that a 10% bated for 30 minutes at room temperature. The tubes by weight solution in water at 20° C. has a pH of about were again centrifuged and washed as previously de 8.5. scribed. Detection of Endo-H binding to the bacterial cells was determined by the detection of HRP-strep 25 This composition provides superior cleaning of car tavidin, which will bind very tightly to the biotinylated bohydrate-containing stains, even compared to pro Endo-H bound to the cells. HRP detection was deter tease-containing and/or amylase-containing detergents. mined by adding 0.5 ml of the HRP substrate OPD EXAMPLE 10 (O-phenylenediamine) diluted in citrate phosphate buffer solution containing hydrogen peroxide. The 30 A heavy duty liquid laundry detergent composition chromogen generation was quenched with 2M H2SO4 of the present invention is as follows: one minute after adding OPD. The cells were centri fuged and the supernatant was read at 490 nm. Active Component Weight % The following results were obtained: 35 C13 linear alkylbenzene sulfonic acid 8.00 C14-15 alkyl polyethoxylate (2.25) 12.00 OD 490 mm sulfonic acid For Staphylococcusaureus 1,2 Propanediol 3.50 Control 0.13 Sodium diethylenetriamine pentaacetate 0.30 Endo-H, 2 minutes 1.89 Monoethanolamine 2.00 Endo-H, 30 minutes 1.90 C12-13 alcohol polyethoxylate (6.5)" 5.00 Ethanol 8.50 For Streptococcus mutans - Potassium hydroxide 1.80 Control 0.8 Sodium hydroxide 3.85 Endo-H, 2 minutes 3.76 Endo-H, 30 minutes 3.80 C12-14 fatty acid 10.00 45 Citric acid 400 Calcium formate 0.12 Sodium formate 0.86 These results indicate that there is binding of Endo-H C12 alkyltrimethylammonium chloride 0.50 to the bacteria Staphylococcus aureus and Streptococcus Tetraethylene pentamine ethoxylate (15-18) 2.00 mutans. The data show that the majority of Endo-H Water 37.12. that binds occurs in the first two minutes or less after 50 Dye 0.08 Perfume 0.25 contact with the cells. The higher absorbance obtained Protease 0.25 with Streptococcus mutans may indicate a higher level of Endoglycosidase H 125 ppm Endo-H binding. Notes Alcohol and monoethoxylated alcohol removed. EXAMPLE 9 "mg active enzyme/g (G34 mg active enzyme/g stock) A heavy duty liquid laundry detergent composition 55 of the present invention is as follows: The ingredients listed above are added to a mixing tank with a single agitator in the order in which they appear. Before the protease enzyme, dye and perfume Active Component Weight % are added, the pH of the mix is adjusted so that a 10% C13 linear alkylbenzene sulfonic acid 8.00 by weight solution in water at 20° C. has a pH of about C14-15 alkyl polyethoxylate (2.25) 12.00 8.5. sulfonic acid This composition provides superior cleaning of car 1,2 propanediol 3.50 bohydrate-containing stains, particularly fecal stains. Sodium diethylenetriamine pentaacetate 0.30 65 Monoethanolamine 2.00 Other compositions of the present invention are ob C12-13 alcohol polyethoxylate (6.5) 5.00 tained when the Endo Hlevel is reduced to 0.40 mg/ml, Ethanol 8.50 water is decreased to 35.72, and 1% Irgasan (a Ciba Potassium hydroxide 1.80 Geigy antibacterial) is added. 5,356,803 35 36 EXAMPLE 11 EXAMPLE 13 A liquid soap composition of the present invention is Ashampoo composition of the present invention is as as follows: follows: 5

Active Component Weight % Component Level Ammonium lauryl sulfate 6.0 Ammonium alkyl sulfate 55.25% Sodium lauryl sarcosinate 5.7 (29% Aqueous solution) Cocamidopropyl betaine 6.3 10 Zinc pyridinethione crystals of 2.0 Coconut fatty acid 1.0 Ex. I of USP 4,345,080 Quaternary amine 0.3 Coconut monoethanolamide 3.0 Ethylenediamine tetraacetic acid 0.2 Ethylene glycol distearate 5.0 Ammonium sulfate 0.4 Sodium citrate 0.5 Perfume 0.25 Citric acid 0.2 Kathon 5 ppm 15 Color solution 0. Water 720 Endoglycosidase H 1000 ppm Perfume 0.5 Triclocarban 150 Endoglycosidase H 1000 ppm Water qs. 100.00% The ingredients listed above are added to a mixing 20 tank with a single agitator in the order in which they EXAMPLE 14 appear below. This composition provides antibacterial action for An antiperspirant stick of the present invention is removal of common skin flora, even when compared to made utilizing the following components: non-glycosidase containing, antibacterial soaps. 25 EXAMPLE 12 Component Level Cyclomethicone 42.55 A hard surface scouring cleanser of the present inven Fluid AP 4.99 tion is as follows: Stearyl alcohol 1.49 30 Castor wax 4.99 Talc 6.99 Component Weight % Zirconium/aluminum/glycine complex 26.67 False Body Fluid Phase 93.5 Fragrance masking agent 0.80 (Specific Gravity 1.1) C20 alcohol 0.12 Barasum NAS-00 4.25 35 Pyridoxal phosphate 1.00 (Sodium saponite clay) Tetrapotassium pyrophosphate 6.00 Endoglycosidase H 500 ppm Tripotassium phosphate 2.00 Sodium hypochlorite bleach 0.90 Sodium lauryl alkyl sulfate 0.25 EXAMPLE 15 Surfactant Dye and Perfume 0.26 40 A liquid soap composition of the present invention is Endoglycosidase H 1000 ppm as follows: Soft Water 78.86 Abrasive 5.0 (Expanded Perlite-specific Gravity 2.0 Active Average Particle Diameter 45 Component Weight % 50 microns) Hercoflat 135 Filler 1.50 Annonium lauryl sulfate 6.0 (powdered polypro-pylene, Sodium alkyl sarcosinate 5.7 Specific Gravity 0.9 Cocamidopropyl betaine 6.3 Average Particle Diameter Coconut fatty acid 1.0 35 microns) 50 Ethylenediamine tetraacetic acid 0.2 Ratio Average Particle Ammonium sulfate 0.4 diameter Abrasive/Filler = 1.43:1 Perfume 0.25 Dye 5 ppm Water 80.15 The composition is prepared by mixing tetrapotas Endo-H 50 ppm sium pyrophosphate, tripotassium phosphate, sodium 55 2,4,4'-trichloro-2'-hydroxydiphenyl ether 100 ppm saponite clay, dye, perfume and deionized water using relatively high shear agitation to the extent necessary to form a false body fluid phase. The alkyl sulfate surfac The ingredients listed above are added to a mixing tant is then blended into this mixture followed by the tank with a single agitator in the order in which they polypropylene filler material. A separate aqueous slurry 60 appear above. Before the dye and perfume are added, of sodium hypochlorite and perlite abrasive is prepared the pH of the mix is adjusted so that a 10% by weight and then blended into the false body fluid phase while it solution in water at 20° C. has a pH of about 6.5. is being liquified under moderate shear agitation. The This composition provides antibacterial action for the resulting scouring composition is false bodied, i.e., gel removal of common skin flora. like in its quiescent state but easily fluidized by applica 65 tion of shear stress. Such a composition is especially EXAMPLE 16 effective for removal of stains and soil from hard sur A hard surface cleanser of the present invention is as faces. follows: 5,356,803 37 38 900 ul of PBS). 10 ul of each diluted solution was plated Active on Luria-Bertaniagar plates. The plates were incubated Component Weight % at 37 C. overnight and colonies were counted. Number Sodium lauryl alkyl sulfate 0.5 5 of colony forming bacteria in tubes were calculated Sodium alkyl sulfate 0.5 according to dilutions made and the logarithm of this Butyl carbitol 4.0 Sodium bicarbonate 0.5 number used for further graphs and calculations. Citric acid 0.04 Formaldehyde 0.03 Perfume 0.05 0 minute 1 hour 3 hours Tartrate mono/disuccinate 5.0 10 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 (0.9) 8.55 (09) Endo-H 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 15 200 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 Chlorhexidine water at 20° C. has a pH of about 7. This composition is effective for the removal of soap scum and mold from hard surfaces, and is more effica- 20 These results are plotted in FIG. 8. As can be seen, cious than a cleanser without the endoglycosidase. 200 ppm Endo-Henhances the bacteriocidal effect of 50 ppm chlorhexidine. EXAMPLE 1.7 Similar results were obtained for slightly different A composition used for the cleaning and/or preserva concentrations of chlorhexidine and Endo-H as mea tion of whole fruit, vegetables or other plant surfaces is 25 sured over a one hour time period. These results are as follows: depicted in FIG. 9. As can be seen, 140 ppm of Endo-H enhances the efficacy of 40 ppm chlorhexidine. Active To further investigate this effect, a similar experiment Component Weight % 30 was conducted using 20 ppm chlorhexidine (final con Water 96.4 centration) with varying concentrations of Endo-H. C12-13 alcohol polyethoxylate (6.5) 0. The results are shown in FIGS. 10A and 10B. These Endo-H 3500 ppm plots represent the change in the log of colony forming units (CFU). As can be seen, a relatively linear relation This composition is prepared by mixing the alcohol 35 ship exists between the amount of Endo-H added polyethoxylate and Endo-Hinwater at their respective through about 280 ppm Endo-H. Further increases in levels and adjusting the final pH to between 6-7. The Endo-H concentration enhance the adverse effect on final composition, when sprayed on plant surfaces such bacterial viability through at least 1000 ppm Endo-H in as whole fruit or vegetables, is useful in preventing combination with 20 ppm chlorhexidine. microbial growth on said surfaces. EXAMPLE 19 EXAMPLE 1.8 Effect of Endo-H alone and in Combination with Potentiation of Bacteriocidal Effect of Antimicrobial by Antimicrobial on Viability of Fungi Endo-H. A log phase culture of Candida albicans was grown, An overnight culture of Escherichia coli was diluted 45 diluted into fresh growth medium, and treated with 0, into fresh nutrient broth and grown for four hours at 37 1, 10, 100 and 1000 ppm Endo-H (final concentration) C. Cells were obtained by centrifugation and washed in for 4 hours while incubating with agitation at 37 C. 0.2M Na-citrate buffer (SCB) pH 5.5. After centrifug The Endo-H was from Bacillus subtilis transformed to ing, cells were resuspended in SCB. The following produce Endo-H from S. plicatus. One, ten and one tubes (in duplicate) were prepared: 50 hundred fold dilutions were made and plated to give viable cell counts. Zero through 10 ppm Endo-H did 5000 ppm not significantly reduce cell viability, although in one 1000 ppm Chlor case 10 ppm Endo-H reduced viability by about 36% Condition Endo-H hexidine SCB Water after 18 hours of incubation. However, 100 ppm to 1000 Control 0 ul 0 ul 200 ul 10 ul 55 Chlorhexidine 0 ful 10 ul 200 ul 10 ul ppm Endo-H reduced the number of viable cells recov Endo-H 200 ul 0 ul 0 pil 10 ul ered by about 50% to 88%, respectively, compared to Endo-H -- 200 ul 10 ul 0 ul 0 ul the control not treated with Endo-H when treated for Chlorhexidine four hours. 60 In a separate experiment, a culture of Candida albi The Endo-H was from E. coli producing S. plicatus cans was grown, diluted into fresh medium, and treated Endo-H. To each tube, 790 ul of cell suspension added with 2.5 g/ml Nystatin (R) in addition to either 0, 1, 10, (final volume now 1 ml) and 10 ul samples were taken 100 or 1000 ppm Endo-H (final concentration) for 18 out as a 0 min control. Tubes were incubated at 37 C., hours, while incubating with agitation at 37° C. One, on a rotary shaker and 10 ul samples were removed at 65 ten, one hundred and one thousand fold dilutions were 1 and 3 hours. The 10 ul aliquots were mixed with 990 made and plated to give viable cell counts. Endo-H ul of PBS (Phosphate buffered saline) (10-2 dilution) reduced viable cells recovered as follows as compared and diluted further sequentially (1:10) in PBS (100 ul in to that obtained with Nystatin (R) alone: 5,356,803 39 40 EXAMPLE 21 ppm Endo-H % Reduction Bacterial Removal from Glass Surfaces by Endo-Hand O 0% PNGase F 1 ppm 69% 5 10 ppm 93% Escherichia coli (ATCC 31617) and Staphylococcus 100 ppm 99% epidermidis (ATCC 155) were used to inoculate glass slides. Each slide contained two etched circles and each As can be seen, as little as 1 ppm Endo-H significantly WaS inoculated with E. coli or S. epidermidis. O enhances the mycocidal effect of Nystatin (R) whereas 10 The bacteria were allowed to incubate at 37 C. for 10 ppm and 100 ppm Endo-H kill almost all of the fungi W. hours. surviving Nystatin (R) treatment alone. After rinsing with distilled water, the slides were A similar experiment was conducted using Ampho- treated with either FBS buff2) Endo-H(00 ppm) tericin B(eat a concentration of 0.5 micrograms per ml in PBS bufi or 3)PNGasef (100ppm) in PBS buffer. for three hours. The results were as follows: 15 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 Gram staining, the ppm Endo-H % reduction in viability slides were read with bright field optics on a light mi O O CIOScope. 1. 17% In the case of the buffer control, the number of bac 5% 2O teria remaining on the slide was greater than 100 per 18 field. The slides treated with Endo-H contained far fewer bacteria. In the case of S. epidermidis, only about 1 to 3 bacteria were observed per field. In the case of E. As can be seen, 100 ppm of Endo-H enhances the is 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 EXAMPLE 20 bacteria were observed for both S. epidermidis and E. coli (approximately 20 per field). Antimicrobial Effect of Endo-H Alone or in These results indicated that PNGase F is capable of Combination with a Lysozyme 3o removing bacteria from glass surfaces albeit not as effi A 48-hour subculture of E. coli (ATCC 31617) was ciently as Endo-H. used to test the effect of the lysozyme mutanolysin EXAMPLE 22 (Sigma Chemical Co.) alone or in combination with detergent and/or Endo-H. The Endo-H was from E. Tablet Denture Cleaner with Endo-H coli transformed to produce Endo-H from S. plicatus. 35 Sodium bicarbonate, sodium perborate monohydrate, The following protocol and results were obtained after tartaric acid, sodium tripolyphosphate, sulphamic acid, treatment for two hours at 37 C.: polyethylene glycol (20,000 m.wt.) and ethylene di amine tetraacetate are separately granulated by fluidiz Na. ing in a hot air bed at 60-65 C. for 30 minutes. Such Citrate 40 granulates are then tumble mixed with the other ingre Mutano- pH Tide Endo-H dients to produce a "first layer” mixture and a "second lysin 5.5 7.0 200 ppm 200 ppm Results layer” mixture, wherein the "first layer” mixture has the 1 Control Fimbriae, tight following composition: cell wall 2 200 ppm -- Fimbriae, tight 45 cell wall % by Weight 3 200 ppm -- Fimbriae, tight Sodium bicarbonate 30.00 cell wall Tartaric acid 23.00 4 200 ppm -- -- Fimbriae, tight Potassium monopersulphate 16.00 cell wall Sulphamic acid 1100 5 200 ppm -- -- Fimbriae, cell 50 Oisodium pyrophosphate 8.20 condensation Sodium carbonate 3.90 6 200 ppm + -- Loss of fimbriae Polyethylene glycol 12.60 7 200 ppm -- -- Few cells, some Sodium sulphate 2.00 ghosts, cell wall Peppermint powder 2.50 disintegration Silicon dioxide 1.30 8 200 ppm - -- -- Some fimbriae 55 Sodium dodecyl benzene sulphonate 0.50 9 200 ppm ------Cells in bad shape (condensed) but still present and the "second layer” mixture has the following com position: As can be seen, the gross morphology of the bacteria 60 exposed to Endo-H and mutanolysin either with or % by Weight without detergent at various pH, was significantly mod- Sodium perborate monohydrate 30.00 ified. The most dramatic effects occurred at pH7 when E. notic 2800 Endo-H was used alone or in combination with deter- Sodium bicarbonate 3.34 gent. Cell viability, however, was apparently not ef- 65 Sodium tripolyphosphate 10.00 fected. Endo-H and mutanolysin did not reduce the icarbonate/colour : number of colonies obtained in a plating experiment as Sodium carbonate 300 compared to a buffer control. Polyethylene glycol 2.50 5,356,803 41 42 -continued EXAMPLE 24 % by Weight Removal of S. aureus from Pig Skin Silicone dioxide 2.00 Peppermint powder 1.50 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 Hardened triglycerides 0.50 surface. The organisms were allowed to set on the skin Sodium dodecyl benzene sulphonate 0.40 for two hours at room temperature. Duplicate pieces of Succinate detergent 0.30 skin were then treated for 30 seconds with: Blue Lake No. 1 0.06 10 1) untreated control Endo-H 100 ppm 2) water alone 3) 10% soap solution A tablet is produced by compressing in a HORN 4) #3--Endo-H (20 ppm) rotary tableting press of 39 stations. Compressing is in 5) 20 ppm Endo-H in buffer two stages: Initially the "second layer', blue mixture is 15 The Endo-H was obtained from E. coli transformed compressed to very low pressure (10 kN per tablet) by to produce Endo-H from S. plicatus. After treatment the way of tamping. The "first layer', white mixture is then samples were rinsed in distilled water and placed in 2% instilled and pressed to 70 kN per tablet. In this way a osmium tetroxide followed by fixation in Ryter-Kellen tablet of 4 grams is produced being 2.7 grams blue and berger fixative. The samples were then processed alter 1.3 grams white. 20 natively in osmium and thiosemicarbizone. After criti Tablets are dissolved in water by the consumer to cal point drying, all samples were examined on the clean dentures placed in the water. SEM. Photomicrographs were taken. S. aureus colonies were found in abundance on the EXAMPLE 23 untreated, water treated, or plain soap treated samples. 25 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 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: 30 soap plus Endo-H. EXAMPLE 25 Ingredient Weight % Mold Removal from Shower Curtain Water Phase: - Methylparaben (preservative) 0.20 35 A plastic shower curtain was moistened with tap Pantethine (moisturizer) 0.10 water and placed in the dark for 3 weeks. At the end of Carbomer 934 (thickener) 0.08 that time, a small sample of the curtain that was covered Sodium hydroxide, 10% (neutralizer) 1.00 with mold was treated with: Endo-H 100 ppm Purified water, q.s. to 100% 1) distilled water Oil Phase: 2) --2000 ppm Joy detergent Heavy mineral oil 4.00 3) +1000 ppm Endo-H Stearic acid, double pressed 3.00 4) untreated (anionic emulsifier) The Endo-H was obtained from E. coli transformed Cholesterol (auxiliary emulsifier) 1.00 Cetyl alcohol (auxiliary emulsifier) 1.80 to produce Endo-H from S. plicatus. The treatments Castor oil (emollient) 1.00 45 lasted 10-15 seconds at room temperature. The shower Cetyl palmitate (emollient) 1.20 curtain was wiped off after treatment with a cotton Octyl dimethyl PABA (U.V.-absorber) 140 Swab. Propylparaben (preservative) 0.10 FIG. 13 depicts the results obtained. The non-treated control (lower right photograph - lower right quadrant In a mixing vessel equipped with a mechanical stirrer, of center photograph) showed abundant mold and mil water and the water phase ingredients other than the 50 dew particles both macro and microscopically. sodium hydroxide and Endo-H aqueous solution are The distilled water control (upper right photograph added and mixed with heating to about 75-80 C. to upper right quadrant of center photograph) showed less form a uniform aqueous dispersion. The sodium hydrox organisms, although particles still remained and discol ide solution is then added and mixed into the aqueous 55 oration was evident. phase to neutralize the acidic Carbomer thickener. The Joy-treated control (lower left photograph In a separate mixing vessel, the mineral oil and oil lower left quadrant of center photograph) showed less phase ingredients are added and mixed with heating to organisms than the water treated sample, but discolor about 80-82 C. to form a uniform oil phase. The ation was still evident. heated oil phase is slowly added to the heated water The Endo-H treated sample (upper left photograph phase using high speed mechanical dispersing means. upper left quadrant of center photograph) was free of Mixing is continued until a homogeneous oil/water both organisms and any discolorations. emulsion is obtained. The emulsion is cooled to room EXAMPLE 26 temperature. If desired, optional colorants such as water-soluble dyes are preferably mixed into the emul 65 Bacterial Removal from Fabric sion at about 45-50° C. and fragrant oils are preferably Fabric swatches were cut to the size of a petri dish. added at about 35-40 C. Endo-H is mixed into the Additional fabric was added to reach a 5% fabric load emulsion at about 35-40 C. (which was not inoculated). The swatches were steril 5,356,803 43 44 ized in an autoclave for 15 minutes at 15 lbs. 121 C. One fabric load is needed for each treatment. Glass beads (40 EXAMPLE 28 g) and 100 mls 0.2M pH 7.0 citrate buffer was placed Effect of Endo-H and Lysozyme on Viability of E. coli into 250 ml Erlenmeyer flasks. The flasks were plugged with rubber stoppers and aluminum foil and sterilized in A culture of E. coli K12 grown overnight in Laurie an autoclave. E. coli subcultured into fresh nutrient Broth (LB), was diluted 1:1000 in LB and regrown for broth and allowed to incubate for 48 hours at 37 C. 4 hours at 37 C. Cells were centrifuged, washed and Half strength trypticase soy agar plates (10 g/500 mls) resuspended in 0-1M NA-acetate pH 5.5 (NA) buffer. were prepared and sterilized. After cooling, tetrazolium Eight tubes were set up as follows: (1 ml/liter) was added. 10 The agar plates were inoculated as follows: Tube Number 1) serial dilutions from the 48-hour culture were pre 1,2 3,4 5,6 7,8 pared (1:10, and 10 fold dilutions through three pu 1 cells 800 800 800 800 more tubes in peptone water); pu 1 NA buffer 200 - 200 2) Thereafter, each swatch was inoculated with 2 mls 5 1 Endo-H (1 mg/ml) u- 200 200 - of the last dilution (10). 3) The swatches were then incubated at 37 C. for two hours (two swatches/treatment). The Endo-H was from B. subtilis transformed to pro After incubation, the swatches were laundered as duce Endo-H from S. plicatus. Tubes were incubated for follows: 20 one hour at 37 C. Tubes were centrifuged, washed and resuspended in 8.00 pull of 0.1M Na-phosphate, pH 7.2 Wash (NP) buffer containing 0.1M EDTA. Buffer or hen egg 100 mls sterile 0.2M pH 7.0 citrate buffer + 40g glass white lysozyme solution was added to tubes as follows: beads--the treatment described in FIG. 14 (where AWA is Endo-H from E. coli transformed to produce 25 Endo-H from S. plicatus) in a 250 ml Erlenmeyer flask (sterile). Two inoculated swatches--sterile fabric to Tube Number make 5% fabric load were washed at 95 F. for 12 min 1,2 34, 56, 7,8 utes with shaking. pl. 1 NP buffer 200 200 30 pu 1 lysozyme (1 mg/ml) - 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 glass beads in a 250 ml Erlenmeyer flask (sterile) at one hour at 37 C. aliquots were used to determine room temperature for two minutes with shaking. 35 CFUs (Column B). The log of colony forming units The fabric swatches were then placed in petri dishes were calculated. The decrease in log CFUs was deter and overlaid with 3 mls of one-half strength trypticase mined by subtracting B from A. The results are shown soy agar with tetrazolium. After incubation for 48 below: hours, the colonies were counted. The results are shown in FIG. 15. These results indi cate that 2% Irgasan plus Liquid Tide provide a two log Log CFUs Change decrease in bacterial growth as compared to Tide alone. Condition A. B in log CFUs The addition of 40 ppm Endo-H, however, reduces Control 7.89 7.90 --0.01 bacterial growth another log unit. Endo-H (200 ppm) 8.21 7.92 --0,29 45 Lysozyme (200 ppm) 7.87 7.68 - 0.19 EXAMPLE 27 Endo-H -- lysozyme 8.17 7.53 -0.64 Effect of Endo-H on Yeast These results indicate that the combination of Endo Broth cultures (18 hour) of Candida albicans and H and lysozyme decreases the viability of E. coli as Sacchromyces cerevisiae were treated with: 50 compared to Endo-H or lysozyme alone. 1) 0.2M Na citrate buffer, pH 5.5 2) #1 plus 200 ppm Endo-H (from E. coli producing EXAMPLE 29 S. plicatus Endo-H) Comparison of Endo-H with T-4 or Hen Egg White The treatments lasted 2 hours at 37 C. Lysozyme on Viability of E. coli After treatment, an aliquot of each was placed on a 55 formvar-coated 200 mesh copper grid, and examined by E. coli cells were washed and suspended in 0.1M TEM. Photomicrographs of the examinations were Naacetate pH 5.5 buffer. Cells were aliquoted (10 ml) in taken and are presented in FIGS. 15 and 16. two tubes. To one tube, only buffer was added (control) As can be seen in FIG. 15A, Candida treated with and to another Endo-H was added (treated). The Endo buffer alone was in good morphological condition. As 60 H was from B. subtilis transformed to produce Endo-H indicated in FIG. 15B, Candida treated with Endo-H from S. plicatus. Cells were incubated for one hour at leaked material at a rapid rate and lost structural integ 37 C. Cells were centrifuged, washed and resuspended rity. in 0.1M Na-phosphate (pH 7.2) buffer. Cells were aliq Sacchromyces treated with buffer alone was in good uoted equally and incubated either with buffer or lyso morphological condition as can be seen in FIG. 16A. 65 zyme. Hen egg white (HL) and T4 (TL) lysozymes When treated with Endo-H, however, all that remained were compared in this experiment. Tubes were incu were very limited pieces of membranous material. See bated for 1.5 hours. Samples were diluted and plated for FIG. 16B. CFU determination before (A) and after (B) incubation. 5,356,803 45 46 The log of CFUs were determined. The following re agent; provided that the antimicrobial agent is not a sults were obtained. lysozyme. 2. An antimicrobial composition of claim 1 wherein said Type II endoglycosidase in Endo-H. Incubation Condition Log CFUs Change 5 3. An antimicrobial composition of claim 1 wherein First Second A. B in log CFUs said antimicrobial agent is an antibiotic selected from Endo-H --- 7.60 7.23 -0.37 (300 ppm) the group consisting of bactericides, fungicides and HEWL 6.69 6.73 -0.23 algicides. (445 ppm) 4. An antimicrobial composition of claim 3 wherein Endo-H HEWL 7.41 6.81 -0.60 10 said antimicrobial agent is a bactericide selected from (300 ppm) (445 ppm) the group consisting of chlorhexidine, 2,4,4'-trichloro m TL 4.98 4.53 -0.45 (445 ppm) 2'-hydroxydiphenyl ether, triclocarban, penicillins, tet Endo-H TL 5.27 4.30 -0.93 racycline and bacitracin. (300 ppm) (445 ppm) 5. An antimicrobial composition of claim 4 wherein 15 said bactericide is chlorhexidine. 6. An antimicrobial composition of claim 5 in the These results indicate that T-4 lysozyme is also effec form of a mouthwash, dentifrice or denture cleaner. tive in reducing the viability of E. coli in combination 7. An antimicrobial composition of claim 6 wherein with Endo-H. said chlorhexidine comprises from about 50 to 350 ppm EXAMPLE 30 20 of said composition. 8. An antimicrobial composition of claim 4 wherein Treatment of Soiled Diaper Material with Endo-H said bactericide is 2,4,4'-trichloro-2'-hydroxydiphenyl Samples were obtained from a soiled diaper. Each ether comprising from about 5 to 350 ppm of said com sample was divided. The left side of the sample was position. washed in 2000 ppm Tide and 1 ppm BPN' (subtilisin 25 9. An antimicrobial composition of claim 3 wherein protease from Bacillus amyloliquifaciens). The right side said antimicrobial agent is a fungicide selected from the was washed in 2000 ppm Tide, 1 ppm. BPN' and 40 ppm group consisting of nystatin, amphotericin B, benomyl, Endo-H (Boehringer Mannheim Biochemical Catalog captan and dichlorbenzalkonium chloride. No. 100 119). Each sample was washed for 12 minutes 10. An antimicrobial composition of claim 9 wherein at 95 F. The results of two experiments are shown in 30 said fungicide is nystatin. FIGS. 17 and 18. As can be seen, the diaper material on 11. An antimicrobial composition of claim 10 wherein the right side of FIGS. 17 and 18 contains substantially said nystatin comprises from about 0.5 to 100 ppm of less fecal stain as compared to the Tide-protease treated said composition. diaper shown on the left of FIGS. 17 and 18. 12. An antimicrobial composition of claim 9 wherein Having described the preferred embodiments of the 35 said fungicide is amphotericin B. present invention, it will appear to those of ordinary 13. An antimicrobial composition of claim 11 in the skill in the art that various modifications may be made form of a liquid hand soap comprising from about 50 to and that such modifications are intended to be within 400 ppm Endo-H. the scope of the present invention. Other compositions 14. An antimicrobial composition of claim 1 in the of the present invention are obtained when Endo-D or 40 form of personal care or household cleaning products For PNGase F are substituted for Endo-H in the Exam selected from the following: liquid soap, hard surface ples. cleaner, laundry detergent, anti-acne medication, de All references cited herein are expressly incorporated odorant, shampoo, face cream, mouthwash, dentifrice, by reference. and denture cleaner. What is claimed is: 45 15. An antimicrobial composition of claim 14 in the 1. An antimicrobial composition consisting essentially form of a hard surface cleaner comprising from about of from about 1 ppm to about 1200 ppm of a Type II 100 to 1000 ppm endo-3-N-acetylglucosaminidase H endoglycosidase selected from the group consisting of and from about 0.1 to 20 weight % detergent surfactant Endo-D, Endo-H, Endo-F and PNGaseF and from of said composition. about 0.5 ppm to about 1200 ppm of an antimicrobial 50 k k k k

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