AU633372B2 - Antimicrobial method and formulation employing type ii endoglycosidase and antimicrobial agent - Google Patents

Description

AUSTRALIA

Patents Act 7 COMPLETE S7FCIFICATIO-' 3 2

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Applicant(s): The Procter Gamble Company One Procter Gamble Plaza, Cincinnati,

AMERICA

Ohio 45202, UNITED STATES OF Genencor International, Inc.

4 Cambridge Place, 1870 South Winton Road, Rochester, New York, 14618, UNITED STATES OF AMERICA Address for Service is: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled; ANTIMICROBIAL METHOD AND FORMULATON EMPLOYING TYPE II ENDOGLYCOSIDASE AND ANTIMICROBIAL AGENT Our Ref 192975 POF Code: 44135/44135,121678 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 1 6006 I: a 4K) 4- #4 4 4 4444 4 4 4I 4 44 4 4 4 44 *44 4 4444 *44* e 4 44,4 4444 *4 44 4 444* 4 4 4444 4 444444 44 4~ 4 4 44 4 1 44 ANTIMICROBIAL METHOD AND FORMULATION EMPLOYING TYPE II ENDOGLYCOSIDASE AND ANTIMICROBIAL AGENT Field of the Invention The present invention relates to antimicrobial methods and antimicrobial compositions utilizing Type II endoglycosidase alone or in combination with an antimicrobial agent.

Background of the Invention The use of enzymes to remove stains comprising proteins and/or carbohydrates, in combination with various detergents, is well known in the art if detergent formulations. Such enzyme tormulations are designed to remove various types of stains from soft surfaces such as cloth and hard surfaces such as porcelain and metal. Thus, for example, proteases such as trypsin, pancreatin, papain and bromelain have reportedly been used in detergent formulations to remove proteinaceous stains with variable degrees of success. Specific glycosidases such as cellulase, lysozyme, amylase and glucanase, on the other hand, have been formulated with various detergents for removal of certain carbohydrate stains. Other detergent formulations have combinet proteases and glycosidases for stain treatment.

j -2- Some of the glycosidases used in detergent formulations, e.g. A-amylase, a-galactosidase and S-galactosidase, are exoglycosidases which cleave one or more terminal residues from an oligosaccharide or polysaccharide. Other glycosidases e.g. cellulase and a-amylase are endoglycosidases which are reactive with specific internal linkages within an oligo- or polysaccharide substrate. Such endoglycosidases are 0 'referred to herein as Type I endoglycosidases.

S. 10 Although formulations of detergent with one or more S, proteases and/or glycosidases (including Type I endoglycosidases) have greatly improved stain Alto removal, many stains, e.g. blood, fecal material and body soil stains, often leave a residual stain after treatment.

In the art of contact lens cleaning, similar a enzyme/detergent formulations have been used to clean and sterilize hard and soft contact lenses. In many cases, these formulations have been used to degrade the biofilm which forms on the surface of contact lenses and which is used by various ophthalmic pathogens such as Pseudomonas aeruinosa and Staohylococcus evidermidis to adhere to such lens.

See, e.g. Duran, et al. (1987), Arch. Ophthalmol, 15 106-109; Stern, et al.

(1987), Ophthalmolov, SA, 115-119 (which reports Sthe treatment of aucin coated conte~t lenses with various enzymes such as pancreatin, papain, trypsin and neuraminidase to inhibit Pseudoonas adherence); and Slucher, et al. (1987), Arch. OPhthalmol, 110-115.

The use of biofilms for microbial adhesion is not limited to contact lenses. Thus, StreDtoocus Sutans reporteciy uses extracellular polysaccharides -e -3to adhere to tooth enamel. EPO Publication No.

0195672 reports the use of a-1,3 glucanase or a-1,6 glucanase to cleave the extracellular polysaccharides used by Streptococcus mutans to adhere to tooth enamel.

The effect of certain enzymes on cells adhered to glass surfaces has also been reported by Danielsson, et al. (1977), Botanica Marina, 2Q, 13-17. As reported therein, Pseudomonas species isolated from 10 sea water was adhered to glass slides. Thereafter, the slides were treated with either pronase, trypsin, S0 a-amylase (a Type I endoglycosidase), or lysozyme (also a Type I endoglycosidase). In this report, treatment with the proteolytic enzymes pronase and trypsin resulted in the release of a portion of the O.o. population of adhered bacteria, whereas the cell oo degradative enzyme lysozyme showed diminished Sactivity compared to the proteolytic enzymes. The 000, a-amylase reportedly had no effect at all.

20 In addition to the attachment of microorganisms to contact lenses, tooth enamel and glass surfaces, many 00 other surfaces are subject to microbial attachment.

S° See, e.g. Marrie, et al. (1984), J. Clin.

S. Microbiology, 12, 991-914 (bacterial attachment to cardiac pacemaker leads and powerpacks); Freimer, et al. (1978), Acta. Path. Microbiol. Scand, SeCtb. g6, 53-57 (binding of microorganisms to macrophages); and Mirelman, at al. (1982), Tokai J.

Exp. Clin. Med., 1, 77-183 (microbial adherence to mammalian mucosal surfaces). Various mechanisms have been proposed to describe the adhesion of microorganisms, such as bacteria, to non-biological solid surfaces. See, e.g. Fletcher, M. (1987), Microbiological Sciences, 4, 133-136, and Duddridge, et al. (1983), Factors Affecting the Adhesion -4of Bacteria to-Surfaces--in Microbial Corrosion, Delco Printing Co., Ltd., pp. 28-35. Although these references discuss microbial adherence to various surfaces anrd the factors which may be involved in such attachment, they do not discuss the control of microorganism growth on such surfaces or their removal therefrom.

Type II endoglycosidases, as used herein, are a category of endoglycosidases which are capable of cleaving specific internal glycosidic linkages found in glycoproteins. These endoglycosidases cleave all or part of the carbohydrate moiety from a glycoprotein depending on the location of the reactive glycosidic linkage in the glycoprotein. Examples include endo- 1 5-N-acetylglucosaminidases (Endo-D, Endo-H, Endo-L, Endo-CI, Endo-CII, Endo-F-Gal type 12 and Endo-F), endo-a-N-acetylgalactosaminidase and endo-A-N-galactosidases. See, e.g. Tarentino, A.L., et al. (1985), Bioche 2A, 4665-4671; Arakawa, M., et al. (1974), J. 15ohm,1, 307-317; Plummer, Tarentino, et al. (1975), Biochem. and Biorhys.

Res. CoMm., 67, 455-462; and Triable, et al.

(19F4), Anal, Biochem., IA.I, 515-522; and "Glycoprotein and Proteoglycan Techniques" (1985) by J.G. Beeley, Chapter 6, pp. 153-300, Elsevier, Amsterdam, New York, Oxford. in addition to having a specificity for the internal glycosidic linkages of glycoproteins, at least one endoglycosidase (endo-,O- N-acetylglucosaminidase H) hes also deiuonstrated a specificity which produces the cleavage of lipidlinked oligosaccharides (Chalifour, et al.

(1983), Archives of Biochem. and-Biovhvs., 386-394) ecnd reportedly di-N-acetylchitobiose linkages in oligosaccharides and glycoproteins i,h (Tarention, et al. (1974), J. Biol. Chem., 249, 811-817).

Such Type II endoglycosidases, in general, have been used primarily for analytical purposes, e.g. the determination of protein or carbohydrate sequence and/or the structure and function of specific glycoproteins. See, e.g. Hsieh, et al. (1982), J. Biolchem., 258, 2555-2561, and Geyar, et al.

(1984), Eur. J. Biochem., 1A, 531-539. In a recent 10 report, a Type II endoglycosidase was reportedly used 5 to analyze a glycoprotein antigen from Leishmania mexicana amazonensis. Chin Shen Chang, et al.

(1986), Mol. Biochem. Parasitol 11, 197-210. This glycoprotein antigen was first immunologically bound to immunobeads. After reacting the immunologically bound glycoprotein with analytical amounts of Endo-H, the immunobeads were washed and boiled in buffer containing 1% SDS in preparation for polyacrylamide gel electrophoresis. This analysis revealed a 20 decrease in molecular weight attributed to the cleavage of carbohydrate from the immunologically bound glycoprotein antigen.

4 4 *o Type II endoglycosidases, however, have not been used to remove substances, including glycoproteins and glycolipids, from surfaces of substances such as fabric, contact lenses, metals, ceramics, cells, tissue and the like. Nor have they been used to control microorganism growth in suspension or on such surfaces.

Glycosidases have been used in combination with other enzymes for removal of various materials.

f-glycosidases are described as carbohydratemetabolizing enzymes in Anderson, et al. (1964), id -6- Biochem. 90, 30, Neuraminidase (N-acetylneuraminiate glycohydrolase) inhibitors are viewed as possible anti-viral, antibacterial agents in Khorlin, et al. (1979), FEBS Letters, 1, 17; and Haskell, et al. (1970), J. Med. Chem., 1, 48. Dextranase is described as catalyzing hydrolysis of bacterial polysaccharide, dextran (a-1,6-glucan), to isomaltose residues in Chalet, et al. (1970), ApD1. Microbiol., 421. LysozyTe (muramidase) is described as hydrolyzing glycosidic linkages in the mucopolysaccharide cell wall structure of a variety of microbes in Chipman, et al. (1969), Science, 165, 454 and Montague (1964), Biochem. Biophvs. Acta., 6, 588. Lastly, inhibition of lysozyme by D-glucosamine derivatives is described in Neuberger, et al. (1967), Nature, 215, 524.

ri@e Type II endoglycosidases such as endo-p-Nacetylglucosaminidase H, D, F and/or PNGase F have not, however, previously been combined with antimicrobial agents to form antimicrobial compositions.

4 t The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that such references are prior art or that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications.

Summary of the Invention The invention provides antimicrobial methods utilizing Type II endoglycosidases such as endo-p-Nacetylglucosaminidase ("Endo") H, D, F and/or PNGase F alone or in combination with a different 13 1 ig i: ji f I:i ::a 'i II t I 14 4 1r 1 0 414 4(1 I 1 *LIt 4' 4441 4414 4(( component comprising one or more antimicrobial agents. In one aspect of the invention, the antimicrobial methods provide for the killing of microorganisms or the inhibition of microbial growth.

In another aspect of the invention, such antimicrobial methods provide for enhanced removal of microorganisms attached to a surface and/or the prevention of microorganism attachment on surfaces.

The killing and/or inhibition method comprises 10 contacting microorganisms with an antimicrobialeffective concentration of Type II endoglycosidase or a combination of Type II endoglycosidase and a different component comprising an antimicrobial agent. The method for treating surfaces comprises 15 contacting a surface having microorganisms attached thereto with an effective amount of Type II endoglycosidase, e.g. Endo-H, D, F and/or PNGase F and, simultaneously or immediately thereafter, with an effective amount of a different component 20 comprising one or more antimicrobial agents. Also provided are antimicrobial compositions comprising at least two different components. The first component comprises a Type II endoglycosidase, e.g. Endo-H, D, F or PNGase. The second component comprises an antimicrobial agent. In one aspect of the invention, such antimicrobial compositions kill and/or inhibit growth of microorganisms. In another aspect, they facilitate the removal of microorganisms attached to a surface or prevent microorganism attachment to surfaces.

4444 4r 4 41 1 t 1 44 t -8- Brief Description of the Drawings Fig. 1 depicts the common core structure of N-linked and O-linked glycoproteins.

Fig. 2 depicts the substrates and known cleavage sites for various Type II endoglycosidases.

Fig. 3 is a generic presentation of the protein amino S, acids, carbohydrate residues and cleavage sites of S 'Fig. 2.

Fig. 4 depicts the core structure of an N-linked glycoprotein, the cleavage site of a Type II endoglycosidase and the relationship between the protein and carbohydrate units and the aglycon and carbohydrate portions produced upon cleavage with a Type II endoglycosidase.

49 Figs. 5A-5E depict various mechanisms wheret', a 'l glycoside-containing substance, microorganisms or substances reactive with Type II endoglycosidase may be released from a surface by treatment with Type II endoglycosidase alone or in combination with a second enzyme.

Figs. 6A and 6B are electron micrographs (8100X) of nylon swatches stained with fecal matter and treated either with or without Endo-H.

Figs. 7A through 7H are electron micrographs (5000X) showing the effect of Endo-H and other carbohydrase enzymes on cotton swatches stained with fecal matter.

Figs. 8, 9 10A and 10B demonstrate the effect of Svarious concentrations of Endo-H and chlorhexidine, alone or in combination, on the viability of E. coii Ii 1 -9- Figs. 11 and 12 demonstrate that a detergent composition containing Endo-H is more effective in the removal of S. aureaus from swine skin than a detergent composition not containing Endo-H.

Fig. 13 demonstrates that Endo-H is more effective in removing mold from a shower curtain than water or a detergent composition. The center photograph is of a portion of the shower curtain. The other four photographs are enlargements of the corresponding quadrants of the center photograph.

4 I 4 Fig. 14 demonstrates the antimicrobial effect of Endo-H in combination with different antimicrobial agents.

Figs. 15A-B and 16A-B demonstrate the effect of Endo- H on different species of yeast.

J Figs. 17 and 18 demonstrate the enhanced removal of fecal matter from diaper material by a deteigent composition containing Endo-H.

Detailed Description of the Invention Type II endoglycosidases and formulations employing such endoglycosidases are used in the methods of the present invention to release and/or remove substances reactive with Type II endoglycosidases from a surface. The mechanism of this reactivity is not known with certainty. In some cases, such substances are glycosides or glycoside-containing substances which are believed to have glycosidic linkages that are krawn cleavage sites for Type II endoglycosidases or l.nkages which are closely related to such cleavage sites.

F *%t i As used herein, "Type II endoglycosidases" are enzymes which are capable of cleaving linkages at or near the juncture of the protein and carbohydrate i units of a glycoprotein. Preferably, such Type II endoglycosidases are capable of cleaving at least one glycosidic linkage within about three glycosidic linkages of the protein-carbohydrate unit juncture (including the glycosidic linkage comprising the protein-carbohydrate junction). Most preferably, such glycosidic linkages are within about two v glycosidic linkages of the protein-carbohydrate unit juncture (see Figs. 1, 2 and 3).

#e Type II endoglycosidases are also defined by their specificities for the particular glycosidic linkages shown in Fig. 1 for the known core structures of N- 4, .4 and 0-linked glycoproteins. These correspond to the I 4 glycosidic linkages between the amino acids serine, threonine or asparagine and the first carbohydrate residue and the glycosidic linkages between at least 44***4 the first, second and third carbohydrate residues.

Although this core structure will be described in more detail hereinafter in terms of the specific glycosidic linkages which exist in known core structures, such specific linkages are not to be construed as limiting to this definition of Type II endoglycosidases. Accordingly, all possible glycosidic linkages between these amino acids and carbohydrate residues define the core structure of Nand 0- linked glycoprotein used to identify Type II endoglycosidases.

Typ- II endoglycosidases are not limited by the present knowledge of the glycoprotein core structure and the specificity of known endoglycosidases for such core structures. A comparison of the core j 41 4 4, 44 ,4 I 44 4.4t r 4- 9.

4r I 9.,l 44~ -11structures in Fig. 1 with the known substrates for Type II endoglycosidases in Fig. 2 indicates that Type T r endoglycosidases for each of the possible cleavage sites in the core structures in Fig. 1, if they exist, have not yet been identified. Moreover, other core structures may also exist which have not yet been identified. Endoglycosidases reactive with linkages in such, as yet, unknown core structures are also Type II endoglycosidases. Accordingly, the glycosidic linkages in glycoproteins which define Type II endoglycosidases are not limited to those located within the first three glycosidic linkages closest to the protein unit of the glycoprotein but may extend to more distant glycosidic linkages in the core structure, e.g. to the fourth or fifth glycosidic linkage from the protein unit depending on the core structure identified.

The specificity for the core structure of glycoptuteins provides a convenient definition of Type II endoglycosidases which distinguishes them from Type I endoglycosidases. Type I endoglycosidases cleave specific linkages in oligo- or polysaccharides but generally are not reactive with those core structure glycosidic linkages in glycoproteins which define Type II endoglycosidases. Examples of Type I endoglycosidases and the linkages with which they are reactive are shown in Table I.

-12-

TALEI

Type I Substrate Endo lvcosidaqp

I~~

oligo-gr lpolysaccharide a -aznylase al-4 (Gic-Gic) n

I

I

14 It

I

.1

I

I I 01-4 cellulase (Gi.-C-Gic) n hyaluronidase GlcA-GlcNAc-GlcA-GlcNAc *t lysozymes: pl-4 A11-4 p1-4 hen egg white GlcNAc-MurNAc-GlcNAc-MurNAc lysozyme T4 lysozyme mutanolysin al-4 al-4 al-6 al-4 Gic- (GIc-Glc-) GIC-Glc pullulanase *GlcA is D-Glucuronic Acid MurNAc is N-Acetylmuramic Acid t Indicates cleavage site.

S-13- Specific glycosidic linkages in glycoproteins which define Type II endoglycosidases and which identify 11 preferred Type II endoglycosideses are shown in Fig.

2. The cleavage sites are identified by a vertical arrow. A generic presentation of the protein amino Sacids, carbohydrate residues and cleavage sites of Fig.2 is shown in Fig.3. As can be seen, Type II endoglycosidases preferably cleave the first, second or third glycosidic linkages in N- or O-linked glycoproteins. These linkages comprise the glycosidic 'linkages between asparagine, serine or threonine in the protein unit and the first carbohydrate residue, between carbohydrate residues 1 and 2 and between carbohydrate residues 2 and 3, respectively. This specificity is defined primarily by the carbohydrate sequence of the glycoprotein with specificity and reactivity being influenced to some extent by the protein unit of the glycoprotein. Thus, with regard to glycosidic linkages 2 and 3 (comprising glycosidic linkages between carbohydrate residues only), Type II endoglyeosidases may be reactive with identical or similar glycosidic linkages located in other regions of a glycoprotein, perhaps quite distant from the juncture of the i 25 protein and carbohydrate units of the glycoprotein.

An application of the above definition to a particular glycoprotein is illustrative. Bovine I thyroglobulin has been analyzed using endo-9-Nt acetylglucosaminidase-H (Endo-H), a-mannosidase and p-mannosidase. Tarentino, A.L. at al. (1973) i.

Biol. Chem..218.5547. The Endo-H hydrolyzed the glycosidic linkage between the two N-acetyl Dglucosamines, one of which was N-linked to an asparagine in the protein unit of the thyroglobulin.

The oligosaccharide or carbohydraite portion of the Sor 3, they are not Type II endoglycosidases a nd can -14be characterized as either an exoglycosidase or Type iI endoglycosidase. The specificity of the Endo-H is the same as that shown for Endo-H in Fig. 2 and Endo- H is therefore a Type II endoglycosidase. This is of course a trivial application. But if a new endoglycosidase Endo-X) is discovered which also demonstrates this specificity or one or mere of ji the other specificities in Figs. 1, 2 or 3, that iEndo-X would also be a Type II endoglycosidase.

This definition of a Type II endoglycosidase based on its specificity for glycoproteins, however, should not be construed as a limitation on the mechanism utilized by Type II endoglycosidases to release Sand/or remove a substance from a surface. Although it will be assumed in some instances that Type II endoglycosidases cleave at least a part of a i glycoside from a surface by reacting with a glycosidic linkage in the glycoside, the invention is Snot limited to such cleavage. Rather, the action of Type II endoglycosidases is defined functionally by their ability to cleave from a *rirface at least a part of any substance reactive with a Type II endoglycosidase.

As used herein, the tern "endoglycosidase" comprises 'ype I and Type II endoglycosidases.

As used herein, "glycoside" refers to a polymer which has one or more "carbohydrate portions" covalently attached through a glycosidic linkage to an "aglycon

L-

-28- Of course, other Tvye II endoal~1 4 .enc r- I Firen~"~ ~31~ 1~~ portion.". This definition of glycoside is derived from the common definition of glycoside which refers to a compound that yields on hydrolysis a sugar and an aglycon, the aglycon being the non-sugar compound resulting from such hydrolysis, As used herein, a glycoside produces an aglycon and an oligo- or polysaccharide carbohydrate portion when cleaved by a Type II endoglycosidase. The aglycon unit, however, is not limited to a non-sugar compound since Type II endoglycosidases may hydrolyze a glycoside to produce an aglycon portion containing one or more sugar residues depending on the cleavage site of the Type II endoglycosidase. Further, the aglycon portion may be quite complex as might be the case with peptidoglyeans where crosslinked peptides can be found attached to a matrix of carbohydrate. Thus, o"o 0glycosides include glycoproteins, glycolipids, peptidoglycans and the like which upon treatment with a Type II endoglycosidase produce a carbohydrate 20 portion and aglycon portion wherein the carbohydrate portion and aglycon portion are defined by the cleavage site of the Type II endoglycosidase. This 0: definition of glycosida will be apparent from the discussion which follows.

As used herein, "glycoprotein" refers to a glycoside which has one or more oligo- or polysaccharides covalently attached to a peptide or protein. Oligoand polysaccharides are souetimes referred to herein as "carbohydrate units". Such carbohydrate units, however, may be diffarent from the "carbohydrate portion" of a glycoside. As shown in Fig. 4, a carbohydrate unit comprises the entire oligo- or polysaccharide attached to a ascond class of molecule, to a protein or peptide as in a glycoprotin or to a lipid as in a glycolipid. If -16the Type II endoglyeosidase cleaves the carbohydrate unit at its juncture with, for example, a protein then the carbohydrate unit is synonymous with the carbohydrate portion of a glycoside. If, however, the Type II endoglycosidase cleaves the carbohydrate unit at a glycosidic linkage within the carbohydrate unit, then the carbohydrate portion of the glycoside formed by such cleavage will be less than the entire carbohydrate unit. This difference is shown in Fig.

4 10 4 for a Type II endoglycosidase cleavage site indicated by the arrow.

4 I The carbohydrate units of a glycoprotein may be oligosaccharides containing 1 to 10 carbohydrate i (sugar) residues or short polysaccharides which usually contain between 10 to 25 carbohydrate residues. Many glycoproteins are produced by higher S Siammalian cells. The linkage bet'een the I carbohydrate unit and the peptide or protein unit of a glycoprotein is a glycosidic linkage which results from a condensation reaction between an amino acid side chain of the protein unit and the anomeric carbon on the first residue of the carbohydrate unit. Such glycosidic linkages in mammalian 25 glycoproteins are either N-glycosidic linkages (carbohydrate linked to the amido nitrogen of asparagine) or 0-glycosidic linkages (carbohydrate linked to the hydroxy oxygen of serine or threonine).

The carbohydrate residues (monosaccharides) of a carbohydrate unit (oligo or polysaccharide) may be joined together in many different ways. Thus, such carbohydrate units may be unbranched, linear |structures or may be complex branched structures. In -17general, however, each of the carbohydrate residues in the carbohydrate unit is linked by way of a glycosidic linkage wherein the anomeric carbon of one carbohydrate residue is condensed with the hydroxyl carbon in another carbohydrate residue. Such glycosidic bonds may be either alpha or beta depending on the configuration of the anomeric carbon. The anomeric carbon of one residue may be .combined with any of the hydroxyl carbons in another S 10 carbohydrate residue. Thus, the complexity of many .a 9 glycoproteins arises from the many different S'o glycosidio linkages which are found in the "carbohydrate units of such molecules.

40 f f t Many membrane glycoproteins carry asparagine-linked carbohydrate units (carbohydrate units linked to asparagine in a peptide via an N-glycosidic linkage).

The structure of such asparagine linked glycoproteins can be quite complex. See Schachterh (1984) Clinical Biochemistry 17, 3-14. The structure of ,oo 20 many of these asparagine linked membranous glycoproteins from a variet of sources eo erythrocyte plasma merAbrane glycoproteins, viral envelope glycoprotejis) as well as the structure of non-membranous able glycoproteins indicate that S 25 the two types of glycoproteins share many structural features. I4. at 3. The common core structure of such asparagine-linked glycoproteins is shown in Figs. 1 and 4, wherein GlcNAC is N-acetyl Dglucosamine and Man is mannose. The a1-6, al-3 and il-4 designations describe the type of glycosidic linkage between the various carbohydrate residues.

This core linkage forms the basis of numerous glycoproteins having any of a number of carbohydrate residues attached to the core. Id. at 0-linked glycoproteins contain a core structure wherein the protein unit of the glycoprotein is coupled to the carbohydrate unit through the hydroxyl .4 4 4 4 44 4 4 4 444 4 4 44 4 4 4 444, 44 44 4 4 4 '4 of this core structure is the presence of N-acetyl Dgalactosamine (GalNAc) linked to serine or threonine.

Other details of such glycoproteins are shown in Fig.

1 where Neu.Ac is N-acetylneuraminic acid, Gal is Galactose and L-Fuc is L-Fucose. When Gal is the second carbohydrate residue the glycosidic linkage .between GalNAc and Gal is usually pl-3. For review of the structure biosynthesis and function of glycoproteins including N-and 0- linked glycoprotein, see Berger E.G. et al. (1982) 15 Ecperimentia, 28, 1229-1258.

Lower organisms such as prokaryotes, the bacteria E. col, Psudmoa species, BaQili species and the like, produce peptidoglycans rather than glycoproteins. Peptidoglycans are found in bacterial cell walls and typically have a polysaccharide backbone of alternating N-acetylglucosamine and N-acetylmuramic acids. Peptide side chains are sometimes ssociated with the N-acetylmuramic acid residues with cross-linked peptide 25 bridges often being interposed between the peptide side chains. Tile call wall of Gram-positive bacteria typically comprises approximately 10% peptidoglycan whereas the cell vail of Gram-negative bacteria typically have a peptidoglycan content of about Peptidolyttns, however, are not glyJcoproteina, at least to the extent that specific glycosidic linkages in glycopror..eins are used to define the class of Type II endoglycosidase. Thus, Endo-H is a Type 11 endoglycosidase because it cleaves the glycosidic 1. -19- St I *5r I I Ir I I iI linkage between the two N-acetylglucosamine sugar residues found in some glycoproteins containing Nlinked oligosaccharides. See Fig. 4. Endo-H, however, may also have an as yet undefined reactivity with peptidoglycan since it is capable of facilitating the removal of fecal matter from a surface such as cloth swatches. Such fecal matter is known to contain peptidoglycans associated with intestinal bacteria. Lysozymes are enzymes which are 10 reactive with peptidoglycan. Lysozymes, such as hen egg white lysozyme, T4 lysozyme and mutanolysin (Goodman, et al. (1981), J. Bacteriol, 146, 755), however, are not Type II endoglycosidases. This is because they io not have a substantial reactivity with the unique glycosidic linkages found in N- and 0-linked glycoproteins used to define Type II endoglycosidases. They are, however, reactive with peptidoglycans to produce disaccharides of Nacetylglucosamine and N-acetylmuramic acid containing attached peptide side-chains. As such, lysozymes are more appropriately characterized as a Type I endoglycosidase. Thus, even though lysozymes and Endo-H may have an overlap in reactivity with peptidoglycans, they are mutually exclusive, for the 25 most part, with regard to Endo-H's specificity for, and lysozyme's substantial lack of reactivity with, the glycosidic linkages in glycoproteins which define Type II endoglycosidases.

As used herein, a "glycoside containing substance" or "glycoprotein containing substance" is a glycoside or glycoprotein alone or a glycoside or glycoprotein combined with another component. Thus, glycosidecontaining substances include glycosides such as glycoprotein enzymes, alkaline phosphatase, bromelain, carboxypeptidase-Y; glycoprotein hormones, chorionic gonadotropin, erythropoietin; lectins, those derived from potato and soybean; serum glycoproteins, IgG immunoglobulin, thyroglobulin, prothrombin and the like and I 5 miscellaneous glycoproteins such as hemoglobin and interferon; and complex carbohydrates. Examples of Sglycosides combined with another component include glycoprotains comprising membrane constituents, e.g., glycophorin contained by human erythrocytes, hemagglutinin contained by influenza virus, rhodopsin contained in bovine retina and collagen contained by fibroblasts. Further glycoside-containing substances include viral envelope glycoproteins and fecal matter which contains in part peptidoglycans associated with intestinal bacteria. Thus, viruses, fibroblasts, fecal matter etc. are considered glycoside-containing substances.

As used herein, a "microorganism" (sometimes referred ,to as a glycoside-containing microorganism) is one capable of being cleaved from the surface of a substance to which it is bound by a Type II endoglycosidase. Examples include the intestinal bacteria found in fecal matter and bacteria commonly contaminating contact lens. Other examples include fungi and algae which can be cleaved from a surface by Type II endoglycosidase.

As used herein, the term "in vitro" refers to the environment in which the processes and methods of the invention are practiced. It is used only to distinguish from the term "in vivo" which describes the environment in which Type II endoglycosidases are found naturally, e.g. within organisms whiJh naturally produce Type II endoglycosidase.

Accordingly, an in vito method employing a Type II -21-

I'

I I endoglycosidase is a method or process which does not occur in nature. The term in vitro, however, is not to be construed as a limitation of such methods to "in glass" or to exclude such methods from being practiced on or in a living organism. The methods of the invention may be practiced on a variety of surfaces other than glass including fabric, contact lenses, metallic surfaces, ceramic surfaces, cell surfaces, plastic surfaces, tissue and the like.

Further, such in vitro methods may be practiced for example in the human oral cavity as described in more detail hereinafter.

Some known Type II endoglycosidases are listed in Table II together with the natural biological source of such enzymes. The cleavage sites for some Type II endoglycosidases are shown in Fig. 2. See "Glycoprotein and Proteoglycan Techniques" (1985) by J.G. Beeley, Chapter 6, pp. 153-300, Elsevier, Amsterdam, New York, Oxford. A Type II -ndoglycosidase not listed in Table II is Glycopeptidase F also sometimes referred to as PNGase F. PNGase F may be obtained from Flavobacterium meninaosepticum. It is also commercially available 25 from Boehringer Mannheim Biochemical, Indianapolis, Indiana.

Ai I I t K I It Enzyme endo-P-N- Acetylglycosaminidases

D

L

C1

CII

F-Gal type

F

endo-ii-N- Acetylgalactosaminidase endo-p-N-Galactos idases TABLE II Source Diplococcus pneumoniae Streptomyces plicatus (Streptomyces griseus) Streptomyces plicatus Clostridium perfringens Clostridium. perfringens Sporotricum diinorphosphosphorum Flavobacterium meininogosepticum Diplococcus pneumoniae Diplococcus pneumoniae Escherichia freundli Flavobacterturn keratolyticus Typical Substrate N-linked complex type (peripheral sugars removed) N-linked hign-mannose and hybrid types N-linked low mol. wt. only N-linked complex type (peripheral sugars removed) N-linked high-mannose type N-linked complex type (biantennary only, requires terminal Gal) N-linked high-mannose and complex types 0-linked, only Gal-crl-3GalNAcl- Blood group A and B determinants Keratan sulphate and oligosaccharides containing sequence R GlcNAc-fil- 3Gal-131-4GlcNAc (or Glc) ii -23- I As can be seen, Endo-H, F, D, CI and Endo-F-Gal type all cleave the second glycosidic linkage in a glycoprotein. In the case of Endo-F-Gal type, this glycosidic linkage is between GlcNAc and Gal. For Endo-H, F, D, and CI, the cleavage is between two residues comprising GlcNAc, with specificity being defined by the substituents U, V, W, X, Y, and Z.

Endo-H cleaves N-linked glycoproteins having a high mannose content. Thus in Fig. 2, W comprises 2-150 'mannose residues, Y comprises 1-2 mannose residues and X,Z,V and U are H (hydrogen). Endo-H also I* cleaves hybrid structures wherein W comprises 1-2 Imannose residues and Y and/or Z comprise NeuNAc-Gal- GlcNAc or similar structures and V comprises H or GlcNAc. Endo-H is the preferred Type II endoglycosidase used in the formulations and methods of the invention.

Endo-D and Endo-C I have similar reactivities although these enzymes are derived from different sources.

Endo-D and Endo-C I are active on N-linked, oligosaccharides of glycoproteins and cleave a high mannose structure containing more than a carbohydrat residue in which case X comprises mannose linked by way of an al-3 glycosidic bond to the core structure, W comprises mannose linked by way of an al-6 glycosidic bond to the core structure and the remaining substituents are H in Fig. 2. Endo-D also cleaves core portion of a complex or hybrid structure after removal of most antennery residues with exoglycosidases, in which case Y comprises H or GlcNAc and U comprises H or fucose in Fig. 2.

The endoglycosidase Endo-F is active on N-linked glycoproteins having a high mannose content wherein -24in Fig. 2 X and Y are one or more mannose residues and the remaining substituents are H. Endo-F also cleaves biantennary hybrid structures wherein X and W comprise mannose linked to the core structure by way of a1-3 and al-6 glycosidic linkages and Y comprises NeuNAc-Gal-GlcNAc or similar structure and U comprises H or fucose. Biantennary complex structures are also cleaved by Endo-F. Such structures comprise the substrate core structure for Endo-F in Fig. 2 wherein X and Y comprise NeuNAc-Gal-GlcNAc or similar structures and U comprises H or fucose.

Endo-L has a similar reactivity in cleaving the f.

second glycosidic linkage in N-linked glycoproteins.

It is specific for low molecular weight substrates comprising Man-GlcNAc-GlcNAc-Asn. Endo-CII demonstrates a specificity similar to Endo H. Endoa-N-acetyl galactosaminidase hydrolyzes glycoprotein containing oligosaccharides O-linked to serine or threorine where GlcNAc and Gal are the first two carbohydrate residues. The specificity of endo-p-Ngalactosidase is also shown in Fig. 2 wherein Rl may be one of the man, ses from which antennas in the carbohydrate unit may be formed.

The Type II glycocidase glycopeptidasa F (PNGase F) cleaves the first glycosidic linkage in N-linked glycoproteins between asparagine and GlcNAc. It cleaves high mannose structures wherein W, X and Y comprise one or more mannose residues and V and Z comprise H with fucose being absent from the first carbohydrate residue GlcNAc. It also cleaves hybrid structures wherein W and X comprise mannose, Y and/or Z comprise NeuNAc-Gal-GlcNAc or similar structure,V comprises H or GlcNAc with fucose typically being absent from the first carbohydrate residue. Complex structures are also cleaved by glycopeptidase F. Such structures comprise the core structure shown in Fig.

2 wherein Y and W comprise NeuNAc-Gal-GlcNAc or similar structure, X and Z comprise H, NeuNAc-Gal- GlcNAc or similar structure, V comprises H or GlcNAc and fucose is sometimes present on the first carbohydrate residue GlcNAc.

Endo- -N-galactosidase is known to cleave glycosydic linkages within oligosaccharides on a glycoprotein or glycolipid. A typical glycoprotein substrate together with the cleavage site for Endo-p-Ngalactosidase is shown in Fig. 2 where R 2 is protein, lipid or carbohydrate, and R 1 is a sugar residue or hydrogen.

Of course, the invention is not limited by the present known specificity of endoglycosidases. Until recently, the endoglycosidases which hr-e been commercially available have been expensive due to their relatively low levels of expression in their o' 20 naturally occurring sources. Accordingly, the "reactivity of such enzymes has not been broadly investigated. However, with the advent of molecular cloning, greater amounts of endoglycosidase have been or will be made available. To the extent that alternate reactivity and specificity may be Sl discovered for these or other endoglycosidases, such reactivity is intended to be within the scope of the invention.

Accordingly, as used herein, a "Type II endoglycosidase-reactivei substance" (also referred to as a "Type II-reactive substance" or a substance containing a "Type II reactive linkage") is any substance which is reactive with a Type II -26endoglycosidase. Included within Type II reactive substances, of course, are glycoside-containing substances and glycoprotein. Also included, however, are other, as yet, unknown substrates reactive with Type II endoglycosidase at other than a glycosidic linkage, and multicomponent aggregates containing components having Type II reactive linkages.

For example, microorganisms, such as bacteria, can be removed from surfaces by treatment with Endo-H. It is presently not known how this result occurs.

a Bacteria are not known to contain linkages which are normally reactive with Endo-H and the details of *o 9 So their attachment to surfaces, other microorganisms o. 15 and other substances is not well understood. Yet, bacteria removal by Endo-H has been observed.

0 0a 0 *go Further, other stains may involve complex aggregates of substances some of which or all of which are reactive with Type II endoglycosidase. The term Type 20 II reactive substance covers all such situations.

Thus, uses of Type II endoglycosidase include (1) cleaning surfaces containing Type II-reactive substances, treating Type II-reactive substances to prevent attachment to a surface, and treating Type II-reactive substances such as microorganisms to produce an antimicrobial effect.

The Type II endoglycosidases used in the invention can be obtained from the organisms listed in Table II according to methods known to those skilled in the art. Some of the Type II endoglycosidases in Table II, Endo-H from Streptomvces plicatus (initially classified as Streptomvces Griseus) and produced in L.x licatus or S. lividans and Endo-D -27from Diplococgus Dnetj.,oniae, are commercially available from Boehringer Mannheim Biochemic'al, Indianapolis, IN. Besides the commercially available preparations, Endo-H may be derived from F,.

91i transformed with a plasmid encoding the Endo-H gene from Streptomyces plicatu and the prcimoter from alkaline phosphatase (Oka, et al, (1985) E~q NJatl. ca.S JA i 1 7212-7216) by methods similar to that reported for the cloning and expression of Endo-H from Streptomvy.es plca i n E. 1 Zil (Robbins, et al. (1981) J. Biol. Chem. 245; 640). See also Trumb.-ly R.J. et al. (1985) J Biol.em L 6_0, 5638. Endo-H may also be derived from otetoye cells engineered to express Endo-H derived from j reptomyce plicatu (EPO Publication 1 No. 0179449, April 30, 1986). Alternatively, Endo-H may be produced by any appropriate host cell such as Bacillus &uktiiij using techniques well known to2 those skilled in the art. The amino acid andDNA sequences of Endo-H for 5. pjjg~t ariseus) have been published. Robbins, at al. (1984) Bil Cb2M, 7577-7583.

The Endo-H used in the examples herein was obtained commercially or from L 2gj or IL subti1.is hosts trans~ormed to express Endo-H from i. Rlic±au. 2 One unit of Endo-H activity is the amount of enzyme required to release lpmole of 3 H)-dansyl-Asn-GlcNAc from 3 H)-dansyl-Asn-(GlcNAc) 4 (Xan) 6 at pH 5.5 at 379C in one minute. Tarantino, A. at al. (1978) I3 Hbthds in Enzymolga, U,574. The unit activity of other Type 11 endoglycosidames are similarly defined by an appropriate substrate.

-28- Of course, other Type II endoglycosidases may exist which have not yet been identified. Such Type II endoglycosidases as well as the ones described herein, including allelic variations and genetically engineered modifications of such endoglycosidases are within the scope of the present invention.

Glycosides and glycoside-containing substances often become bound to a wide variety of surfaces. Thus, for example, glycoproteins, such as those associated with blood glycosylated hemoglobin), can stain the surfaces of fabrics used for clothes, linen and the like. Such stains have heretofore been highly resistant to complete removal by treatment with detergents or detergents in combination with various enzymes not comprising the endoglycosidases utilized in the present invention. A further glycosidecontaining substance which stains surfaces such as fabric and which is also difficult to remove by known techniques comprises fecal matter. Such fecal stains include various glycosides and glycoside-containing substances associated with intestinal bacteria peptidoglycans), catabolic excretions, including glycoproteins, and non-absorbed nutrients and the like.

Other surfaces to which glycosidos or glycoside- S. containing substances may be bound include the surfaces of hard and soft contact lenses. Soft contact lenses are typically hydrophilic cross-linked polymers having a hydrogel structure or are made of silicon polymers. See, U.S. Patent Nos.

3,403,393 and 2,976,576. Hard contact lenses, on the other hand, are typically made of methacrylate or methylmethacrylate polymers. Other surfaces include naturally occurring biofilms, cardiac pacemaker leads -42- When used to treat contact lens, Type II rP -29and power packs, cellular and mucosal surfaces, tooth enamel, filters used to remove bacteria and particulate material in processing foods; chemicals and the like; air conditioning filters; the surfaces of various structural components exposed to an aqueous environment, boats, piers and the like; plastics and composites such as formica; and metals or metal alloys such as steel, aluminum, etc.

As will be shown in detail hereinafter, Type II endoglycosidases alone or in combination with a second enzyme such as subtilisin, either with or without detergent, effectively increases the removal •Q of blood and fecal stains from cloth swatches. It is not known precisely how such stains adhere to such 15 swatches. However, the enhanced removal of such substances from these swatches by Type II *o endoglycosidase, alone or in combination with other agents, suggests that at least one glycosidic linkage is interposed between the fabric and that part of the stain which is released upon treatment with Type II endoglycosidase. Based on these results, the following are proposed mechanisms of the binding of glycoside-containing substances to a surface and the orelease and/or removal of such substances by Type II 25 endoglycosidase. These proposed mechanisms, however, should not be considered as a limitation to a"g the scope of the invention.

0 Figures Thus, as shown in Fig. 5A, a glycoside-containing substance may be bound to a surface other than by an immunological bond. In this regard, an "immunological bond" is one which exists between an antigen and an antibody, specific for that antigen (polyclonal or monoclonal). As shown in Fig. 5A, the glycoside-containing substance has a proximal porzion bound to the surface and a distal portion extending outwardly from the proximal portion. The proximal and distal portions are joined by a glycosidic linkage with which Type II endoglycosidase is reactive. As further shown in Fig. 5A, when treated with Type II endoglycosidase, the distal portion of the glycoside-containing substance is "released" from the proximal portion of the glycoside-containing substance. To the extent that this distal portion is not bound by other means to the surface, it is also readily "removed" from the surface and may be washed away with a fluid.

9e° C. In Fig. 5B, a glycoside-containing substance, in this case a glycoprotein containing a carbohydrate unit and protein unit, is shown bound to a surface. This peso glycoside-containing substance further contains a carbohydrate portion and an aglycon portion joined by a glycosidic linkage which is reactive with Type II endoglycosidase. In this particular case, the glycoside-containing substance (glycoprotein) is Sbound to the surface through the carbohydrate portion of the glycoside-containing substance. When treated with Type II endoglycosidase, the aglycon portion is 25 released from the carbohydrate portion of the glycoside-containing substance. As in Fig. 5A, to the extent that the aglycon portion is not further bound to the surface by other means, the aglycon portion is also removed from the surface.

Fig. 5C depicts the situation where a glycosidecontaining substance is bound to a surface by way of at least two points of attachment. As indicated, a glycosidic first linkage exists between the surface and the glycoside-containing substance. In addition, r_ Ys r -31a second linkage reactive with a second enzyme is also present between the surface and the portion of the glycoside-containing substance to be removed. If treated only with Type II endoglycosidase, the portion of the glycoside containing substance distal from the first glycosidic linkage is released from the surface at least to the extent that it was bound through the first glycosidic linkage. If contacted with a second enzyme reactive with the second linkage shown, the portion of the glycoside-containing substance as distal from the first glycosidic linkage and the second linkage is released from the surface.

To the extent that this distal portion is not .otherwise bound to the surface, by other *o 15 contact points which may be reactive with other enzymes or susceptible to detergents and/or surfactants, this distal portion is effectively removed from the surface.

Fig. 5D shows a microorganism bound to a surface through at least part of the glycoside portion of said microorganism. The glycoside portion contains a glycosidic linkage reactive with Type II endoglycosidase. A cleaved portion of the microorganism distal from the glycosidic linkage is 25 released from the surface when treated with Type II endoglycosidase. To the extent that this cleaved portion is not otherwise bound to the surface it is A also removed from the surface. However, multiple points of contact may exist with the surface which may require further treatment with other enzymes and/or detergent or surfactant.

In Fig. 5E, a Type II endoglycosidase-reactive substance is shown bound to a surface. This Type II reactive substance has a proximal portion bound to -32the surface and a distal portion extending outwardly from the proximal portion. The proximal and distal portions are joined by a Type II reactive linkage which refers to a linkage reactive with a Type II endoglycosidase. When treated with Type II endoglycosidase, the distal portion of the Type II reactive substance is "released" from the proximal portion of the Type II reactive substance. It is to be understood that Type II reactive substances may comprise molecules, microorganisms or aggregates of various components which may become attached to a surface. To the extent that the distal portion of 0 o° the Type II reactive substance is not bound by other means to the surface, it is also readily "removed" .o 15 from the surface and may be washed away with a fluid.

0 go 0 The amount of Type II endoglycosidase used to produce the removal of the substances identified in the figures is defined functionally as an "amount 00* effective" for removal of the particular subutance 20 from a surface. This amount may vary depending on 0, the substance and surface to be treated. Typical amounts are disclosed in more detail herein with S* o regard to the specific embodiments disclosed.

c O 0 Second Enzymes 0 0 25 "Second enzymes" include proteases, lipases, glycosidases such as lysozyme and combinations thereof. Various protease which may be combined with Type II endoglycosidase include subtilisin, bromilain, papaine, trypsin, chymotrypsin, pancreatin, lysozyme and combinations thereof. Such enzymes may be derived from natural sources, e.g., subtilisin from Bacillius subtilis or from genetically engineered clones, subtilisin and -33mutant subtilisins as described in EPO Publication No. 0130756. See also, Wells, et al. (1983) Nucleic Acids Res., 11, 7911-7915; Yang, et al.

(1984) J. Bacteriology, 160, 15-21; Estell, et al. (1985) J. Biological Chemistry, 26., 6518-6521.

Many such enzymes, of course, are available from commercial sources.

In addition, Type II endoglycosidases may be combined with lipases such as bacterial, mammalian and fungal lipases and combinations thereof.

.Glycosidases which may be used as a second enzyme include exoglycosidases, a second Type II endoglycosidase and Type I endoglycosidases. Examples S. include a- and A-amylase, cellulase, pectinase, hemicellulase, dextranase, various glucanases, and the like and combinations thereof.

Moreover, Type II endoglycosidase may be combined with more than one of the above classes of second enzymes to facilitate the removal of a glycoside- 20 containing substance from a surface.

When a Type II endoglycosidase is combined with one or more second enzymes, the ratio of Type II endoglycosidase to second enzyme is preferably about 0.01 to 100 and most preferably 1 to 1.

Disulfide Cleavina Reaoents Type II endoglycosidases may also be used in combination with detergents, either alone or in combination with one or more second enzymes and/or disulfide-cleaving reagents to form a detergent formulation. Substances capable of cleaving disulfide bonds are varied, but fall generally into I

I

-34- *r U ae go 4 A U 4090 4 0 a~ a a.b 4 A ASr *4 9I three categories: oxidizing agents, reducing agents, and miscellaneous addition substrates such as those exemplified by fumaric acid and sodium sulfite.

Suitable oxidizing agents include hydrogen peroxide, performic acid, sodium perborate, and oxidizing bleaches. Effective reducing agents include dithiothreitol (DTT), p-mercaptoethanol (BME), sodium borohydride, and the like.

Alternate disulfide cleavage reagents which are not easily classified include mercuric chloride, nitroprusside, tributylphosphine, and phosphothiolate. A particularly useful cleavage reagent is sodium sulfite, which results in sulfitolysis of the disulfide according to the 15 reaction: R-S-S-R S0 3 2 R-S-S03 2 -SR. The equilibrium of this reaction may be shifted by removal of the thiol anion using heavy metal ions or oxidizing agents. The oxidizing power may be provided by aeration or an oxidizing agent, such as CuSO 4 or sodium perborate.

The foregoing list of substances capable of cleaving disulfides is not meant to be comprehensive, and conversely does include substances which are effective but not necessarily appropriate for a commercial product. In order to be successful commercially, the added substance must be relatively inexpensive and must not have undesirable properties for its intended use. Thus, for example, while the use of mercuric chloride would be workable in carrying out the process of the invention, it would not be suitable for ordinary detergent products intended for commercial use. P-mercaptoethanol and DTT are feasible commercially, except that they have mildly offensive odors. particularly preferred

-I

I

substances, therefore, for commercial formulation, are sodium sulfite (preferably in combination with an oxidizing agent) or hydrogen peroxide, which are inexpensive and are relatively safe. Reviews of materials which are useful in the cleavage of disulfide bonds are found, for example, in Chemical Modification of Proteins, Means, et al., eds (1971), Holden-Day, Inc. San Francisco, CA, Ch 8; and 'j Chemical Reacents for Protein Modification, Lundbald, i 10 R.L. et al., eds (1984), CRC Press, Inc., Boca Raton, FL, Ch. 7.

I t Typically, the Type II endoglycosidase alone or in S" combination with one or more second enzymes forms I 0.01-3% wt/wt of the detergent compositions of the S 15 invention, and may include disulfide-cleaving reagents, ranging from about 10-40% wt/wt thereof.

t" The amounts present depend, of course, on the nature of the endoglycosidase (and second enzyme, if used) and the disulfide cleavage reagent, the dilution of 20 the detergent in the wash solution, and the conditions of the wash. However, the ranges given t are generally typical.

In one embodiment of the invention, surfaces having 4 glycoprotein containing substances bound thereto are 25 treated with the combination (simultaneous or sequential) of a disulfide cleaving reagent, a Type II endoglycosidase and a second enzyme at suitable pH, temperature, for an appropriate period of time.

These conditions are, of course, variable according to convenience, and the selection of the Type II endoglycosidase, protease and the substance to cleave disulfides to some extent depends on this selection.

However, convenient conditions frequently encountered are pH values between 5 and 12. Temperatures of -36pmrticularly around 40*-55'C, and times of up to 20 minutes, usually around 10-15 minutes are typical and preferred. The preferred times and temperatures are those generally utilized in household washing machines, neighborhood laundromats, and professional laundry services, since in order to be commercially practical, the process needs to be conducted under conditions ordinarily available to the user.

In another embodiment of the invention, conventional washing procedures using commercial detergents are used and the Type II endoglycosidase, second enzyme .and disulfide-cleaving substance are provided, either separately or together, as an additive, much in the l 15 manner of the methods in which bleach is used. Thus, Sthese may be added along with the detergent at the beginning of the wash cycle or at some intermediate point, for example, after approximately half of the wash cycle is completed. If handled in this way, i 20 assuming an approximately 1:500 dilution of a solid i or liquid detergent composition (approximately 2 i mg/ml of the solid), arbitrary amounts of the Type II i endoglycosidase, second enzyme and disulfide cleaving reagents may be added without the upper limit imposed I 25 by this dilution. (If the Type II endoglycosidase, second enzyme and disulfide cleaving reagent had been added to the detergent composition originally, and if, for example, the disulfide cleaving reagent constituted 50% of the composition, only 1 mg/ml would result in the final wash solution. However, if these materials are added separately, amounts most effective for the particular Type II endoglycosidase, disulfide cleaving reagent and second enzyme may be added.) i -37- 4* 44 *o 4 4 44 94 @4 4 4~ i *41 4 4 0 44I 4 tr444 *444 4 4 4 4 @4,o ooc a a a 4 I o 4 4 4.

L

I 4 4 4 44 4 I 4 4 t 44 With respect to the Type II endoglycosidase and second enzyme, only very small quantities are usually required. Typically, the Type II endoglycosidase and second enzyme are added to a final concentration of approximately 1-500 pg/ml of wash solution for each enzyme. In the case of the disulfide-cleaving reagent, however, larger amounts than would be permitted by the dilution of the detergent may be desirable. For example, cleavage of disulfide bonds 10 using sodium borohydride may conveniently be carried out with concentrations as high as 0.2M reagent in the present of similar quantities of buffer (Lundbald, et al., Chemical Reagents for Protein Modification, supra).

Although such high amounts are conventional, they are not necessarily required, and lower concentrations are workable. Sulfitolysis is ordinarily carried out in sodium sulfite concentrations of the order of 0.1M, although concentrations as low as 0.01M and lower can also be used. DTT is effective when supplied at concentrations of the order of 0.02-0.1M.

In short, the disulfide-clea ing reagent concentration can be varied over a wide range for any of these reagents and effectiveness maintained. The optimum concentration for a particular application will, of course, depend on the nature of the stain and the nature of the reagent, as well as the conditions of the wash procedure, including time, temperature, and pH.

In an alternative and more convenient approach, the Type II endoglycosidase, second enzyme and disulfidecleaving substance are added to the original detergent composition, and the process is conducted as a standard wash procedure using these modified

I

*I-Y-lii li.-, -38detergents. Under these circumstances, the detergent composition will correspond to that described above, but the amount of the composition can also be varied over the range of approximately 0.5 mg/ml-10 mg/ml or greater of the wash solution, depending, again, on the conditions of the wash solution and procedure, and on the solubilities of the detergent components.

In any case, the inclusion of the Type II o endoglycosidase, disulfide-cleaving reagent and second enzyme in the detergent limits the concentrations of these components in accordance with the dilution of the detergent. Thus, even if a 1:100 dilution is used (10 mg/ml), and the disulfidecleaving reagent for example, is limited to 50% of the detergent composition, a maximum concentration of mg/ml disulfide-cleaving reagent in the resulting 0, wash solution is an upper limit. Typically, of course, the concentration of disulfide-cleaving "oo* reagent in the detergent will be less than 20 mandating even lower concentrations of the disulfidecleaving reagent.

The detergent compositions of the invention contain r mostly detergent active substances, relatively .e smaller amounts of disulfide-cleaving reagent, if used, and quite small amounts of Type II endoglycosidase and second enzyme, if used, which is especially desirable in view of the cost of enzymic components. Thus, in general, the preparation will contain 60-90% detergent active substances, including conventional comercial detergent additives such as surfactant builders and whiteners, 0.01-3% Type II endoglycosidase and second enzyme, and approximately 10-40% disulfide cleavage reagent.

-39- Of course, it is also possible to add only one of these three additives to the original detergent and to supply the other separately to the wash liquid.

In particular, the Type II endoglycosidase may be added to a prewash, followed by a detergent containing the second enzyme, or addition of the detergent containing endoglycosidase may be followed o. or preceded by treatment with the second enzyme.

0000 0 0 6 Cleaning Compositions GS 10 Endo D, F and H are preferred Type II endoglycosidases for use in cleaning compositions.

S'.0 Endo-H is most preferred.

*0a For removal of glycoside-containing substances, the compositions herein preferably comprise from about 15 0.1 ppm (parts per million) to 1200 ppm, more preferably from about 1 ppm to 1000 ppm, most preferably from about 20 ppm to about 200 ppm, of Type II endoglycosidase, depending on the type of composition. Cleaning compositions are preferred.

S 20 Laundry detergent compositions are most preferred for use herein, and preferably comprise from about 0.1 S ppm to 1200 ppm of Type II endoglycosidase, preferably from about 20 ppm to 200 ppm of Endo D, F or H, most preferably from about 50 ppm to 125 ppm Endo H.

When used to control or remove micr3organismas, the compositions preferably comprise from about 0.1 ppm to 1200ppm, more preferably from about 1 ppm to 1000 ppm, most preferably from about 20 ppm to 400 ppm, of Type II endoglycosidase, preferably Endo-H. Cleaning compositions are preferred and preferably comprise the same amounts of Type II endoglycosidase, preferably Endo-H.

Described below are suggested types of compositions which comprise Type II endoglycosidase for removal of glycoside-containing substances and/or Smicroorganisms. The compositions can be made and used in any way which does not destroy enzyme activity. They can be made up of any ingredients which do not unduly hinder the activity of the enzyme. The compositions can be laundry detergents, dishwashing detergents, hard surface cleaners, dental enamel cleaners, liquid and bar soaps, anti-acne compositions, antiperspirants, shampoos, face creams, fruit and vegetable surface preservatives, or S fabric softeners.

In addition to the cleaning of fabrics using common cycles in washing machines, the cleaning compositions herein may also be used for removing glycosidecontaining substances and/or microorganisms from other surfaces such as metals and metal alloys such as found in surgical instruments, pipelines, metal containers and the like, and plastics and composite materials such as Formica and the surfaces of boats, piers and the like. Depending upon the particular application, the composition may comprise Type II endoglycosidase alone or in combination with a disulfide cleaving reagent, second enzyme and/or detergent surfactant.

Type II endoglycosidase may also be formulated in a composition for removing glycoside-containing substances and/or microorganisms including yeast, fungi, algae and bacteria from "biological surfaces" such as surfaces of skin, skin pores, hair, hair follicles and tissue. Thus, those skilled in the art of shampoo formulations, conditioner formulations, soap formulations and the medicinal arts can readily -41- S adapt the above disclosure for detergent formulations to employ Type II endoglycosidase in such ;applications. When so formulated, such compositions Iare useful in removing glycoside-containing substances which may adhere to such surfaces.

Type II endoglycosidase may also be formulated in a composition for removing glycoside-containing substances and/or microorganisms, especially yeast and fungus, from the surfaces of plants such as fruits and vegetables. Such compositions preferably include nonionic .iurfactant.

In addition, Type II endoglycosidase may be formulated in deodorant compositions in a manner known to those skilled in the art to provide endoglycosidase activity to remove glycosidecontaining substances and/or microorganisms ;responsible for undesirable odors. Such deodorant formulations employing Type II endoglycosidase may include modifications of formulations for stick, creams and aerosol deodorants known to those skilled in the art.

Further, Type II endoglycosidase may be formulated for the treatment of acne which usually results from inflammation, at least to the extent that glycoside- 25 containing substances and/or microorgansims responsible for or involved in such inflamation are bound to a surface. As with the above formulations, those skilled in the art are capable of modifying known acne formulations to incorporate a Type II endoglycosidase alone or in combination with other enzymes, detergents and/or surfactants.

i UIU IIICI- iI 1ijj 1ii -42ii t r I When used to treat contact lens, Type II endoglycosidase suitably is supplied at a concentration of about 0.1-20 pg/ml in the cleaning compositions, ar the concentration of a second enzyme such as a protease is in the same range if such second enzymes are utilized. Treatment times can vary from about five minutes to about 15 hours, but a standard convenient cleaning time is overnight, so that the wearer can allow the lenses to soak while he sleeps. A variety of protocols are suitable, but ones that are particularly preferred are the use of a single solution containing Type II endoglycosidase and the second enzyme (if used) conducted from minutes to two hours or overnight at room temperature, or a 10-minute to two-hour presoak in the presence of Type II endoglycosidase solution, followed by a similar overnight treatment with a solution containing a second enzyme.

Preferred general purpose second enzymes for contact lens formulation include proteases such as papain, pancreatin and subtilisin. The preferred Type II endoglycosidase eiizyme is Endo-H from Streptomyces plicatus. A ningle second enzyme protease may be used, or the composition may contain a mixture of 25 second enzymes.

In addition, the contact lens compositions may include additiona components which aid in the overall enzymatic degradation. Particularly useful among these are disulfide cleavage reagents such as 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol, dithioerythritol, sodium bisulfate, sodium metabisulfite, thiourea, and the like, generally preferred in range of about 0.01-5% by weight preferably 0.05-1% by weight. In addition,

A

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_1I 'I 8 -56an antimicrobial effect tfea. nvn ^v-n -43detergents may be included in the composition to aid in the wetting of the lens with the enzymecontaining solution. Suitable detergents include sodium dodecyl sulfate, sodium monolaurate, nonionic surfactants such as alcohol ethoxylates polyethoxyethanol) anionic surfactants such as ether sulfonates, linear alkylbenzene sulfonates, sodium lauryl sulfate, and the like.

000 o. *Suitable buffers and stabilizers for contact lens cleaning may also be used and include sodium or potassium citrate, citric acid, boric acid, sodium '°oo EDTA, various mixed phosphate buffers and NaHCO 3 Generally buffers and stabilizers may be used in amounts ranging from about 0.001 to about 2.5% and preferably about 0.1 to 1% by weight. It should be understood that the foregoing description of the amounts of the various compounds which may be used in the present invention for cleaning contact lens are a S stated in percentage of ingredients in solution 20 (wt/vol). The formulation may also take the form of one or more conventional solid dosage forms such as Stablets suitable for use in measured quantity of a Ssuitable solvent such as water. The percentage composition of the solid dosage forms is such that when dissolved in a specified volume of water, the solution will have the percentage composition within the ranges set forth in the specification. If solid dosage forms are used, the formulation may include conventional lubricants, binders, and excipients which include glycerol, sorbitol, boric acid, propylene glycol, polyethylene glycols, dextran, methylcellulose, hydroxyethylcellulose, water soluble salts of carboxymethylcellulose, or naturally occurring hydrophilics such as gelatin, alginates, tragacanth, pectin, acacia and soluble starches.

-3 -44- STypical compositions and protocols useful in cleaning contact lens include the following: 1. The composition contains 1-100 pg/ml Type II endoglycosidase. The lenses are removed and placed in contact with the solution for a period of 12 hours at 22'C. The lenses are removed from the cleaning solution and rinsed.

2. Solution A contains 10 pg/ml of Type II endoglycosidase; solution B contains 5 pg/ml subtilisin. The lenses are soaked in solution A for 30 minutes at 25'C, removed, and immersed in solution B for 10 hours at 3. The cleaning solution contains 10 pg/ml of the protease pepsin and 10 pg/ml of Type II endoglycosidase. The lenses are soaked in this solution for 5 hours at 4. The cleaning solution contains 5 pg/ml subtilisin, 5 pg/ml Type II endoglycosidase, and mM 2-mercaptoethanol. The lenses are immersed in this solution for 5 hours at 5. The cleaning solution contains 7pg/ml subtilisin, 3pg/ml Type II endoglycosidase, 10mM 2mercaptoethanol, and 2% sodium dodecyl sulfate (SDS).

The lenses are soaked in this solution for 3 hours at 6. The cleaning solution contains 4pg/ml subtilisin, 2 pg/ml trypsin, 10 pg/al Type II endoglycosidase, and 2% SDS. The lenses are soaked in this solution for 7 hours at 7. Solution A contains 4gg/ml subtilisin and 2,ug/ml.trypsin in 2% SDS. Solution B contains Type II endoglycosidase plus 10mM 2mercaptoethanol. The lenses are immersed in solution B for 20 minutes at 30*C and then in solution A for 6 hours at 25 0

C.

In all the foregoing examples, the lenses are thoroughly rinsed in saline before being returned to the wearer's eyes.

S 10 The compositions herein can be formulated in a variety of physical forms, including liquids, gels, pastes and solid particles such as powders and granules. The compositions can be formulated as laundry detergents, such as disclosed in U.S. Patents 4,507,219, 4,318,818, 4,605,509 and 4,412,934; dishwashing detergents such as disclosed in U.S.

Patents 4,714,562, 3,630,923, 4,133,779, 4,316,824 S...and 4,555,360; hard surfaces cleaners such as S' disclosed in U.S. Patents 4,414,128, 3,679,608, 3,985,668 and 4,005,027; fabric softeners such as disclosed in U.S. Patents 3,944,694, 4,073,996, 4,424,134 and 4,661,269; bar soaps such as disclosed I in U.S. Patents 3,993,722 and 3,070,547; shampoos such as disclosed in U.S. Patents 4,345,080, 4,704,272 and 4,741,855; antiperspirants such as disclosed in U.S. Patent 4,725,432; anti-acne products such as disclosed in U.S. Patents 4,318,907 and 4,608,370; and oral compositions such as disclosed in U.S. Patent 4,684,518. The above patents are incorporated herein by reference.

The compositions preferably have a pH from about 4 to more preferably from about 5 to 8 for good enzyme performance.

S-46- SLaboratory work on microorganism removal has shown that, in order to obtain effective removal, the i bathing of the surface holding the microorganisms in Ssome instances requires a physical or chemical action to remove the microorganisms. Microorganisms tested include: Escherichia cli including Type 1 and 3 fimbriae Staphvlococcus Aureus 10 Staphylococcus epidermidis .Serratia marcescens Streptococcus mutans Streptococcus sanquis Bacillus so Candida Sz.

Asperaillus sD.

S' In the case of removal of bacteria such as oli, j I for example, the surface-bound microorganisms may be treated with Endo-H and then removed by chemical action, such as by treatment with an antimicrobial agent, or a physical action, such as by rinsing with water or hand wiping. It is preferred for liquid and I bar soaps, dental enamel cleaners, antiperspirants, anti-odor fabric softeners and anti-acne compositions that the composition include an anti-microbial agent, such as Irgasane (Cia-Geigy) or chlorhexidine, in addition to the Endo-H. An antimicrobial agent is not required in the composition (for example a hard i surface cleaner) when physical action such as water rinsing or wiping by hand will occur.

Preferred herein are detergent cleaning compositions, especially granular and liquid laundry detergent compositions. These detergent cleaning compositions preferably comprise from about 1% to 90%, more -47preferably from about 5% to 50%, by weight, of detergent surfactants, most preferably from about to 40% by weight Surfactants useful in the detergent compositions herein include well-known synthetic anionic, nonionic, amphoteric and zwitterionic surfactants.

o Typical of these are the alkyl benzene sulfonates, S' alkyl- and alkylether sulfates, paraffin sulfonates, S. olefin sulfonates, alkoxylated (especially 10 ethoxylated) alcohols and alkyl phenols, amine oxides, alpha-sulfonates of fatty acids and of fatty S, acid esters, alkyl betaines, and the like, which are well known from the detergency art. In general, such detersive surfactants contain an alkyl group in the C9-C 18 range. The anionic detersive surfactants can used in the form of their sodium, potassium or triethanolammonium salts; and the nonionic surfactants generally contain from about 5 to about 17 ethylene oxide groups. C 11

-C

16 alkyl benzene sulfonates, C 12

-C

18 paraffin-sulfonates and alkyl sulfates are especially preferred in the compositions of the present type.

A detailed listing of suitable surfactants for the compositions herein can be found in U.S. Patent 3,936,537, Baskerville, issued February 3, 1976, incorporated by reference herein. Commercial sources J of such surfactants can be found in McCutcheon's SEmulsifiers and Deteralts. North American Edition, 1984, McCutcheon Division, MC Publishing Company, also incorporated herein by reference.

Useful detergency builders for the detergent compositions herein include any of the conventional inorganic and organic water-soluble builder salts, as -48well as various water-insoluble and so-called "seeded" builders. The instant laundry detergent compositions preferably comprise from about 1% to more preferably from about 5% to 40%, most preferably from about 10% to 20%, by weight of detergent builders. These compositions preferably have a pH of from about 6 to 9* 99 0 0 Nonlimiting examples of suitable water-soluble, .inorganic alkaline detergent builder salts include the alkali metal carbonates, borates, phosphates, Spolyphosphates, tripolyphosphates, bicarbonates, O4 «silicates and sulfates. Specific examples of such 0060 salts include the sodium and potassium tetraborates, bicarbonates, carbonates, tripolyphosphates, pyrophosphates, and hexametaphosphates.

9 4 Examples of suitable organic alkaline detergency builder salts are: water-soluble amino polyacetates, sodium and potassium ethylenediaminetetraacetates, nitrilotriacetates, and N-(2-hydroxyethyl)nitrilodiacetates; watersoluble salts of phytic acid, sodium and Spotassium phytates; water-soluble polyphosphonates, including sodium, potassium and lithium salts of ethane-l-hydroxy-l,l-diphophonic acid, sodium, potassium, and lithium sAlts of methylenediphosphonic acid and the like.

Seeded builders include such materials as sodium carbonate or sodium silicate, seeded with calcium carbonate or barium sulfate. Hydrated stWium zeolite A having a particle siz less than about 5 microns is particularly desirable.

-49- A detailed listing of suitable detergency builders can be found in U.S. Patent 3,936,537, incorporated herein by reference. Preferred builders are fatty acids, polycarboxylates, polyphosphates and mixtures thereof.

Optional detergent composition components include enzymes proteases and amylases), peroxygen bleaches and bleach activators, halogen bleaches sodium and potassium dichloroisocyanurates), S" 10 soil release agents methylcellulose), soil .1 suspending agents sodium carboxymethyl- SoBo cellulose), fabric brighteners, enzyme stabilizing agents, color speckles, suds boosters or suds suppressors, anticorrosion agents, dyes, fillers, germicides, pH adjusting agents, nonbuilder e alkalinity sources, and the l~ks.

EndoQlycosidase Plus Antimicrobial Agents Of the Type II endoglycosidases, endo-p-Nt.oo acetylglucosaminidase H, D, F and/or PNGase F are preferred for formulating antimicrobial compositions and for use in the antimicrobial methods herein.

Endo-H is most preferred.

When the Type II endoglycosidase is used alone, it is formulated such that its concentration produces an antimicrobial effect. When the antimicrobial composition comprises at least two different components, i.e. a Type II endoglycosidase and one or more antimicrobial agents, each of the components are present at a concentration sufficient to produce an antimicrobial effect. The amount of at least one component in said compositions is generally less than the amount required for that component to produce the same antimicrobial effect if used alone in a similar composition.

As used herein, an "antimicrobial effect" includes the ramoval, killing, inhibition of growth, change in gross morphology, protoplast formation and/or degradation of the cell wall of a microorganism when contacted with a Type II endoglycosidase alone or in combination with a second component comprising an antimicrobial agent.

As used herein, an "antimicrobial method" refers to a method which produced an antimicrobial effect. In fit# one aspect of the invention, the antimicrobial method causes the killing of microorganisms, the inhibition of microorganism growth, and/or changes in -the gross morphology of the microorganism. In another aspect of the invention, the antimicrobial method causes the removal of a microorganism from a surface. In the antimicrobial methods to remove microorganisms from surfaces, it is preferred that the surface be treated with the antimicrobial agent and the Type II endoglycosidase simultaneously, rather than treating with the additional antimicrobial agent immediately after treating with Type II endoglycosidase. In some applications of the antimicrobial methods, a combined antimicrobial effect may be produced, e.g. killing and/or growth inhibition may occur in combination with microorganism removal from a surface.

As used herein, an wantimicrobial composition" refers to a composition containing at least two different components: a Type II endoglycosidase and a different component comprising an antimicrobial agent. Such antimicrobial compositions have variable antimicrobial effects depending upon the amount and -51choice of Type II endoglycosidase and antimicrobial agent. Observed antimicrobial effects include the killing of microorganisms and/or inhibiting microorganism growth, the removal of microorganisms from a surface and the prevention of microorganism attachment to surfaces.

As used herein, an "antimicrobial-effective concentration" of Type II endoglycosidase generally oo refers to the final concentration of Type II S 1 0 endoglycosidase used alone to contact a microorganism So to produce an antimicrobial effect.

00 u*0 As used herein, an "antimicrobial agent" is a second different component of an antimicrobial composition.

Such antimicrobial agents in general are antibiotics 15 and include agents which kill microorganisms and those which inhibit microorganism growth. Examples of such antimicrobial agents include bacteriocides, fungicides and algicides each of which are capable of killing or inhibiting the growth of bacteria, fungi o 20 or algae, respectively. Bacteriocides include compounds such as chlorhexidine, 2,4,4'-trichloro-2'- 00 0 fg 0 hydroxydiphenyl ether, Triclocarbane, penicillins, tetracycline and Bacitracin*. Fungicides include Nystatin, Amphotericin Be, Benomyle, Captane and Dichlorar.4. Other examples of antimicrobial agents include surfactant-stable antimicrobial enzymes such as surfactant-stable p-1,3-glucanases, lysozymes, proteases and chitinpses, and detergent surfactants such as anionic, nonionic, zwitterionic, ampholytic and cationic surfactants known to those skilled in the art. The latter should be employed in an amount sufficient to produce an antimicrobial effect. The above antimicrobial agents identified by generic name or trademark are compositions as identified in the -52- Merck Index, 10th Ed. (198. 'Wck Co., Inc., Rahway, N.J.

Type II endoglycosidase diffEi m the first component of the antimit 'saXial ipositions may also be used as an antimicrobi. avt'c. Thus, to the extent Type II endoglycosic ,ie are themselves antimicrobial agents are capable of producing an antimicrobial effect, such as morphological changes or protoplast formation), they may be combined with a different Type It endoglycosidase to Sform an antimicrobial composition. Antimicrobial I compositions may therefore comprise one or more different Type II endoglycosidase with or without one or more antimicrobial agents not comprising Type II endoglycosidase.

Preferred antimicrobial agents for use herein are chlorhexidine, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, Triclocarbane, Nystatine Amphotericin Be antibiotic, anionic and nonionic detergent surfactants. A surfactant-stable antimicrobial lysozyme is disclosed in the copending U.S.

Application Serial No. entitled Methods and Compositions Employing Certain Lysozymes and Endoglycosidases in the names of Richard S. Carpenter and Ann M. Wolff, filed on even date as this application. Other lysozymes, e.g. hen egg white lysozyme, have been used in combination with Endo-H to produce antimicrobial effects albeit to a lesser extent and with variability in the results obtained.

The antimicrobial compositions and methods of the invention can produce an antimicrobial effect on a wide range of microorganisms including Gram-positive and negative bacteria, fungi, and algae. Such -53bacteria include Escherichia coli, Streptococcus mutans, Staphylococcus eDidermidis, and Staphvlococcus aureus. Such fungi include yeasts such as Candida and Saccharovces, and species and filamentous fungi such as Aspergillus, Sporobolomyces, Basidiobolus and Entomoththora.

A specific advantage of combining a Type II endoglycosidase Endo-H, D, F and/or PNGase F) with an antimicrobial agent is that less of the antimicrobial agent can be used to produce an antimicrobial effect. In some aspects of the Sinvention, the antimicrobial agent when used with a Type II endoglycosidase produces an antimicrobial effect comprising the removal of microorganisms attached to surfaces or the prevention of their attachment to such ,',rfaces. In other aspects, there is a negative effect on microorganism viability or S' microorganism morphology.

Sturface treatment(s) with Type II endoglycosidase and antimicrobial agent car be performed periodically so as to prevent further growth or attachment or adhesion of microorganisms to the surfaces exposed to the treatment.

Of the Type II endoglycosidases, Endo-H, D, F and/or PNGase F are preferred. Of these, Endo-H is most preferred. In general, an antimicrobial-effective amount of Type II endoglycosidases for use in combination with antimicrobial agents is from about 1 Sto 1200 ppm Endo-H, D, F, and/or PNGase F, preferably from about 1 to 1200 ppm Endo-H, more preferably from about 20 to 1000 ppm Endo-H, most preferably from about 50 to 400 ppm Endo-H. The amount used depends upon the type of treatment and amount of exposure to I: 5 It *i -54the surface or microorganism to be treated. In general, an effective amount of antimicrobial agent, which depends upon which agent is usea, is from about to 1200 ppm, preferably 2 to 1200 ppm, most preferably from about 5 to 350 ppm chlorhexidine or 2,4,4'-trichlco-2'-hydroxydiphenyl ether, or 0.5 to 100 ppm Nystatin®.

4 t4 0r When Type II endoglycosidase is used alone to kill and/or inhibit microorganisms, the use of substantially more Type II endoglycosidase is generally required. For example, about 100 ppm to 1000 ppm of Endo-H has been shown to substantially decrease the viability of yeast cells exposed to such concentrations. When yeast is exposed to less than 100 ppm of Endo-H, however, a significant decrease in viability has not been observed. Although the lower limit of Endo-H necessary to adversely affect yeast viability has not yet been determined, the lower limit of its antimicrobial-effective concentration is believed to be between 10 and 100 ppm. Similar amounts of Endo-H are believed to be useful to kill and/or inhibit other microorganisms such as algae and fungi. The xa.ct effect of Endo-H and other Type II endoglycosidases on these organisms and others, e.g., bacteria, when not used in combination with antimicrobial agents has not yet been ieterzined.

The range of antimicrobial-effective concentrations of Type II endoglycosidase for use against such organisms, houever, can be routinely determined.

Thq. antimicrobial methods and compositions of uji invention have a wide applicability and include antimicrobial methods and compositions for personal care, health care and household and industrial cleaning. Thus, such methods and compositions may be used to formiulate and use antimicrobial mouthwash, dentifrice or denture cleaner, as well as antimicrobial liquid or solid. hand or body soaps, aniti-acne medication, deodorant, shampoo and face creams and compositions for cleansing woundls or suppressing infections. Typical, householei applications include antimicrobial cleaning products such as liquid soap, hard surface cleaners, and liquid and granular laundry detergents, Heavy duty antimicrobial deterqpnt compositions may also be formulated for industrial use.

Chlorhexidine is an effective oral antibacterial agent and i rt-.l~.erred for use in dental applications. 2,4, 4'-trichloro-2'-hydroxy,!iphenyl ether is available as IrgasanOD DP 300 from ciba-Geigy and is a broad-spectrum antimicrobial effectiv%,t in personal care and laundry applications.

Triclocarbane from Monsanto is a bacteriostat usetul In bar soaps. Traditional antibiotics can also e employed as the additional ant"~ icrobial agent herein, Lastly, surf actant -stab le arntimicrobial enzymes can be used in dental applications and for preservation of shampoos and other surfactantcontaining formulations. A preferred surfactantstable antimicrobial enzyme is the lysozyno disclosed in the previously Idanntified copending Application in the names of Carpenter and Wolff. Surfactanitstabilit.y of antimicrobial enzymes can be qg4,uged hervin by retained activity in the presence of representative amounts of alkyl. ether sulfate or linear alkylbenzene sulfate, for example.

The anf,4crobial composition may be formulated as an antimicrobial mouthwash, d.ntirc o Id~r cleaner. The treatment of yaitrooraims to produce lpP i;'

I

-56an antimicrobial effect to remove or prevent microorganism attachment to natural or synthetic s 't and/or hard surfaces in the oral cavity or to kill microorganisms or inhibit their growth in the oral cavity), then, essentially comprises rinsing with an antimicrobial mouthwash, cleaning the teeth with an antimicrobial dentifrice, and/or cleaning dentures with an antimicrobial denture cleaner. The antimicrobial mouthwash, dentifrice and denture cleaners herein preferably comprise Endo-H, and chlorhexidine and/or surfactant stable antimicrobial enzyme as the antimicrobial agent. Where t #e chlorhexidine is used, the antimicrobial mouthwash, dentifrice, or denture cleaner preferably comprises from about 50 to 1200 ppm Endo-H and from about 50 to lt 350 ppm chlorhexidine. Where surfactant-stable antimicrobial enzyme is used, the antimicrobial mouthwash, dentifrice or denture cleaner preferably o comprises from about 50 :to 150 ppm Endo-H and from C1o 0 20 about 50 to 1,000 ppm surfactant-stable a antimicrobial enzyme.

t The antimicrobial composition may also be formulated Sas antimicrobial personal care or household cleaning products. In such products, Endo-H is preferably used at a concentration of from about 1 to 1200 ppm.

The antimicrobial agent for use in these products is preferably chlorhexidine, most preferably at a concentration of from about 150 to 1200 ppm, or 2,4,4 -trichloro-2'-hydroxydiphenyl ether, most preferably at a earnAntration of from about 2 to 500 ppm. Preferred pirsonal care or household cleaning products are liquid hand soaps, hard surface cleaners, laundry detergents and shampoo (described below).

-57- A preferred antimicrobial liquid hand soap comprises from about 50 to 400 ppm Erdo-H, from about 5 to 100 Sppm 2,4,4'-trichloro-2'--hydroxydiphenyl ether, and preferably from about 1 to 40 weight to detergent surfactant. Preferably from about 2 to 20 weight most preferably from about 3 to 10 weight detergent surfactant is employed, preferably selected from the group consisting of anionic, nonionic, zwitterionic, ampholytic and cationic surfactants.

The liquid hand soap can further comprise emollient (up to about 30 weight and minor amounts of perfume, colorant, solvent, and opacifier.

it 0 The antimicrobial hard surface cleaners herein can be glass cleaners, abrasive hard surface cleaners, scouring cleansers, or toilet bowel cleaners. These S' should be substantially free of hypochloritegenerating bleaches, and other endoglycosidaseincompatible ingredients. A preferred hard surface cleaner comprises from about 100 to 1000 ppm Endo-H, 20 and antimicrobial agent, and from about 0.1 to t weight detergent surfactant. From about 2 to weight detergent surfactant is most preferred, preferably selected from the group consisting of anionic, nonionic, zwitterionic, ampholytic and 25 cationic surftants. The antimicrobial hard surface cleaners herein optionally further comprise abrasive, j builder, diluent, solvent, suspending agent (such as clay, carboxymethylcellulose, and polyacrylate), perfume, and/or colorant.

Th antimicrobial laundry detergent herein, in addition to Type II endoglycosidase and antimicrobial agent, preferably comprises from about 1 to 99 weight more preferably from about 5 to 60 weight most preferably from about 10 to 40 weight detergent cI I I I I- -58surfactant, preferably selected from the group consisting of anionic, nonionic, zwitterionic, ampholytic and cationic surfactants. A preferred liquid or granular antimicrobial laundry detergent comprises from about 2 to 250 ppm Endo-H, from about to 40 ppm 2,4,4'-trichloro-2'-hydroxydiphenyl ether, and from about 1 to 99 weight t, preferably from about 5 to 60 weight detergent surfactant.

The antimicrobial laundry detergents herein optionally further comprise builder, perfume, bleach, diluent, suds suppressor, colorant, brightener, soil suspending agent, antiredeposition aids, softeners, ae and/or soil release agents.

a.* a The antimicrobial shampoo for use herein preferably comprises Endo-H, an antimicrobial agent, and from about 5 to 60 weight detergent surfactant, preferably selected from the group consisting of lauryl sulfate, isoethionate, acyl amidobetaine, a alkyl glyceryl ether sulfonate, and alkyl ether 20 sulfate. Optional ingredients are suds booster, a conditioner, dye, colorant, perfume and/or antidandruff agent.

The present antimicrobial compositions may also be in the form of a preservative or microorganism control 25 agent for treatment of plant surfaces. Preferred are a preservative for the surfaces of fruits or vegetables or an antimicrobial product to be applied on crops for microorganism control. The latter is preferably in the form of a solution to be sprayed on crops such as corn, citrus, wheat, tobacco, uoybeans, tomatoes and strawberries for control and prevention of microorganism growth.

-59- The following is presented by way of example only and Sis not to be construed as limiting the scope of the invention.

Example 1 Removal of blood and fecal matter from fabric jSeparate blood and fecal matter stained (cotton ifabric) swatches were washed with commercial detergents in an automatic washing machine using a warm (approximately 37*C) wash cycle. The swatches were then rinsed and air dried. They were then .Ot incubated with various amounts and tyj'.a of endoglycosidase [(0.005 U of Endo-D (Boehringer Mannheim Biochemical), or Endo-H (Boehringer Mannheim Biochemical from S, riseus, Catalog No. 752 967), i 15 and 0.25U N-glycanase (PNGase F or peptide endoglycosidase F) Genzyme, Boston, MA] in 0.75 ml of mM Tris-HCl, pH 7.0 at 37'C for 30 minutes in a test tube. The control contained buffer but no endoglycosidase. At the end of the incubation period, 0.25 ml of detergent solution (1:125 dilution of a commercial liquid detergent composition which did not contain dyes, perfumes, enzymes or brighteners in 1M Tris-HCl, pH 7.5) containing of subtilisin BPN'/ml was added to the control and S 25 entyme containing samples and incubated for an i additional 20 minutes. At the end of this treatment, the tubes were centrifuged and the protein content in the supernatants were determined by measuring absorbance at 280nm. For each treatment, a reaction blank was prepared which contained no swatch during the assay. The blank values were subtracted from the absorbance of treated samples to determine the release of 280nm absorbing material during incubation. Higher absorbance represents increased release of protein from fibers. The results are shown in Table III.

jTABLE 11 Treatment Absorbance at 280nm Blood Stain Fecal Matter Stain Control 0.79 2.07 Endo-D 0.84 2.14 *Endo-H 0.83 2.12 N-Glycanase 0.78 2.10 t II r f t t These results suggested that the zndoglycosidases, r Endo-D and Endo-H, in combination with the second enzyme subtilisin increased the release of 280nm absorbing material from the blood stained swatches as compared to the control. In addition, Endo-D, Endo-H and N-glycanase all showed an increase in the release of 280nm absorbing material from the fecal stained swatches.

Example 2 Effect of Endo-H on removal of fecal matter stain This example is similar to Example 1 but was performed by using fecal matter stained swatches made of nylon fabric. The swatches were washed in detergent solution, rinsed and dried. The detergent consisted of liquid commercial detergent which did not contain enzymes, brighteners, dyes or perfumes.

One set of swatches was kept aside and referred as "untreated control". These swatches were treated the same as the sample swatches except that they were not treated with Endo-H. The sample swatches were I incubo ted with 0.01U Endo-H (Boehringer Mannheim -61- Biochemical Catalog No. 752 967) in buffer (10 mM Naacetate, pH 6.0) at 37*C for 15 minutes. Then 0.25 ml of detergent solution (1:125 dilution in 1.0 M Tris-HCl, pH 7.5) was added and incubated for an additional 15 minutes. At the end, tubes were centrifuged and the supernatants removed by suction.

The swatches were air dried. Fibers from the swatches were examined by scanning electron microscopy following critical point drying. An electron micrograph of a detergent-washed swatch stained with fecal matter is shown in Fig. 6A. As i can be seen. rod like bacteria and particulate matter t *so are found on the surface of the fabric. Fig. 6B S* shows a swatch treated with Endo-H and detergent.

t 4 15 This figure shows a smooth clean fabric which demonstrates that Endo-H and detergents facilitates the removal of particulate material and bactCrial debris.

Example 3 20 Effect of Endo-F on :ecal matter stain iSwatches stained with fecal matter (1 inch diameter) were washed in detergent solution, rinsed and dried.

Swatches were cut into quarters and used in the I following experiments.

25 A. Swatches were incubated in 1 ml 10 mM 4 sodium-acetate buffer, pH 5.5 with or without Endo-F (Boehringer Mannheim Biochemical) (0.15 units) for minutes at 37*C. The tubes were then centrifuged for eight minutes. Supernatants were removed and the absorbance of each was measured at 280 mm. Change in A280 was determined by subtracting appropriate blanks (see Example Higher absorbance includes the increase in the amount of protein or material absorbing at 280 mm released from the swatches. For I -Y -62the controls, the average change in A280 was 0.93.

For swatches treated with Endo-F the average change K in A280 was 1.05. This indicated that Endo-F I increases the efficiincy of fecal stain removal.

B. Swatches were incubated in 0.75 10 mM sodium-acetate buffer pH 5.5 with or without Endo-F (0.15 units) for 15 minutes at 37'C. At the end of this treatment, 0.25 ml of detergent solution (in 0.1 M Tris-HCl, pH 7.5) containing 10 pg of the protease subtilisin BPN' was added and the tubes were incubated at 37'C for another 15 minutes. At the i endtubes were centrifuged, supernatants were removed and absorbance at 280 nm was measured. In the case S" of the control (no Endo-F), the average change in 15 A280 was 1.08 whereas the sample treated with Endo-F showed a change in A280 of 1.36. This indicated that the effect of Endo-F was enhanced by the presence of the detergent.

C. An experiment similar to was performed except the detergent solution contained 10 mM 2-mercaptoethanol instead of subtilisin. The average change in A280 for the control was 1.05 whereas the sample treated with Endo-F produced a change in A280 of 1.24. These results demonstrated the ability of 25 Endo-F in the presence of disulfide cleaving reagents to remove fecal stains.

4 D. An experiment similar to was performed except that the detergent solution contained 10 mM 2-mercaptoethanol and 10 pg subtilisin BPN'. The average change in A280 for the control was 1.14 whereas the Endo-F treated sample had a change in A280 of 1.29. These results indicate that Endo-F is capable of removing fecal matter in the presence of i -I -3 C- -~U ~t -63detergent, a protease and a disulfide cleaving i reagent (2-mercaptoethanol).

Example 4 Comparison of Endo-H with other enzymes Experiments similar to those described in part B of Exa le 3 were repeated with Endo-H (Boehringer Mannheim Biochemical Catalog No. 100 119) and other carbohydrase enzymes except that no protease such as subtilisin was used. Changes in A280 were monitored and fibers were examined by scanning electron microscopy. Removal of particulate and bacterial S* debris from fabric was seen with Endo-H and "Lysing Enzymes" (a mix of proteases and glyconases obtained from Sigma Chemical Company). However, the enzymes, lysozyme, a-glycosidase, 9-glucosidase and p-glucorinadase, showed little or no benefit, (Results not shown.) The results of electron microscopy for this experiment for treatment with or without the above enzymes are shown in Figs. 7A through 7H. Fig. 7A is a control which was not treated with endoglycosidase. Fig. 7B is an electron micrograph of a swatch treated with lysozyme; Fig. 7C is a swatch treated with Endo-H; Fig. 7D is a swatch treated with a-glucosidase; Fig. 7E is a swatch 25 treated with p-glucosidase; Fig. 7F is an electron micrograph of a fiber treated with "Lysing Enzymes"; jFig. 7G is an electron micrograph of a swatch treated with p-glucorinadase; and Fig. 7H is an electron micrograph of a swatch treated with chitinase. As can be seen, the swatch treated with Endo-H (Fig. 7C) has been thoroughly cleansed of the fecal matter stain. Similar results were obtained for the swatches treated with "Lysing Enzymes" as shown in Fig. 7F.

I~t t I I It I 14 I a k 4 I -64- Example Removal of bacteria from a solid surface To test the effect of Endo-H on removal of bacteria from solid surfaces (glass),the following protocol was used. Trypticase soy broth (TSB) (10 ml) was inoculated with a microbial species (Staphylococcus aureus ATCC culture #6538 or Escherichia coli ATCC culture #10536) from a stock culture slant and incubated overnight at 37'C. A suspension of about 108 cells/ml TSB was prepared and 100 pl of this suspension was placed within the etched ring on a glass slide. Each slide was incubated for 5 minutes at 37 0 C in a dry incubator oven after which excess microbial solution was tapped off. The slides were 15 then rinsed with 100 pl of sterile distilled water.

The excess solution and loose organisms were then tapped off.

After the bacteria were adhered to the glass slides (2 or more hours at 37*C), 100 pl of the following solutions were applied to separate slides: 10 mM acetate buffer, pH 5.5, 10 mM acetate buffer, pH 5.5 1 ppm Endo-H (Boehringer Mannheim Biochemical Catalog No. 100 119), detergent solution, detergent solution 1 ppm Endo-H. A set of slides were kept aside as untreated controls and were not treated with any solutions. The noncontrol slides were then incubated for 15 minutes at 37'C. At the end of the incubation, the solutions wure tapped off. The slides were then rinsed with 100 pl of sterile distilled water and air dried at room temperature. The bacteria which remained after this treatment were heat fixed and stained by a standard Gram staining method. The slides were then examined by a light microscope (bright field illumination, 125x magnification) and the number of l~nr~ 6 1 organi I count organi organi isms/field was determined. Twenty fields were id for each slide from which the average .sms/field was calculated.

The following results were obtained: A. For hvlococcus aureus i) No treatment ii) Buffer iii) Buffer Endo-H iv) Detergent solution .0 v) Detergent Endo-H >100 >100 10 >100 10 organisms/field organisms/field organisms/field organisms/field organisms/field *1 4 4 44 These results indicate that Endo-H buffer alone or in combination with detergent reduced the number of S.

aureus bacteria retained on the glass slides 10 fold as compared to treatment with detergent alone.

61*4 I #4 #4 #4 0r 4 0 4* B. For Escherichia coli i) No treatment ii) Buffer iii) Buffer Endo H iv) Detergent v) Detergent Endo H >100 organisms/field >100 organisms/field >100 organisms/field >100 organisms/field 10 organisms/field These results indicate that Endo-H in combination with a detergent reduced the number of E. coli retained on the glass slide 10-fold as compared to treatment with detergent alone.

Example6 Removal of bacteria from solid surface An experiment similar to Example 5 was performed with two slime-producing Staphylococcus aureus cultures (determined by their abilities to bind to polystyrene tubes). Microscope slides were modified by forming -66two rings cm diameter) with nail polish.

Overnight culture of the organisms were diluted 1:10 with 1% peptone solution. Diluted culture (100 pl) was put in rings. Slides were put in 150 cm petri dishes and incubated at 370C. After two hours incubation, slides were rinsed with distilled water and treated wth three different conditions Naacetate buffer, B. detergent, and C. detergent plus 1 pg Endo-H/ml) as in Example 5. The Endo-H was obtained from E. co 9 l transformed to produce Endo-H from plicatus. At end of 15 minutes, incubation slides were rinsed with distilled water and Gram stained. The number of bacteria was counted under microscope per 100X field for 20 fields. The results are expressed as the average number of cells per field.

Condition Culture I Culture II A. Control 23 202 B. Detergent 9 58 C. Detergent Endo-H 2 33 Exampe. 7 Removal of bacteria frolpa cloth surface To test the effect of Endo-H on the removal of bacteria from a cloth surface, the following protocol S 25 was used. Staphylocpcus aureus (ATCC S538) and I StaDhvlococcus eidermidis (ATCC 155) were separately cultured in 5 ml of Luria's broth and allowed to grow at 37*C for 12 hours. The cultures were then added to 30 ml of 0.2M NaCitrate, pH 5.5 buffer at about 103 cells/ml, in two 100 ml shake flasks. Twelve cloth swatches (0.5 x 0.5 inch cotton swatches) were also added to the flasks after inoculation. After incubation at 37*C for two hours with gentle rotation (150 rpm), the swatches wmre transferred to sterile tubes and washed 3x with buffer comprising 200 mM I I If -67- I, O

D~

1' 44~ 44 9 81 8 8 4P 0 4 91 8484 NaCitrate, pH 5.5 which had been previously sterilized by 0.22 micron filtration. Six swatches were then added to a shake flask containing mg/ml ErEdo-H in 30 ml citrate buffer, and six swatches were added to a shake flask containing only citrate buffer as the control. The Endo-H was obtained from E. gli producing L, 2licatus Endo-H.

After incubation at 37* for 1.5 hours with gentle rotation (100 rpm), the swatches were transferred to sterile tubes and washed as previously described.

Swatches were then plated carefully on trypticase soy agar plates and overlaid with enough liquid trypticase soy agar to cover the swatches. After the plates were dry, they were incubated at 37"C for 18 15 hours, and colonies of Staphylococcus aureus and Staphvlococcus epidermidis on the cloth surface were counted using a dissecting scope.

The following results were obtained: A. For Staphvlococcus aureus Control 103 24 colonies per swatch Endo-H 53 18 colonies per swatch 49% decrease in bacterial colonies by Endo-H treatment B. For Staphylococcus cidermidis t 64 Control Endo-H 57 11 colonies per swatch 16 10 colonies per swatch 72t decrease in bacterial colonies by Endo-H treatment.

*rurn~ :*gCs ;q 1 -68- These results indicate that Endo-H treatment Ssignificantly reduces the number of bacteria adhered to a cloth surface.

Example 8 Binding of Endo-H to bacteria The following experiment was conducted to determine if the Type II endoglycosidase, Endo-H, interacts with a surface component on the bacteria Staphvlococcus aureus and Streptococcus mutans. Such an interaction was detected. Although not completely characterized herein, this interaction was not f previously known and may form the basis of the above described ability of Endo-H to remove such bacteria from a surface.

Endo-H from transformed E. c£o1 and purified by modifying the methods described by Trimble R.J. et al. (1985), J. Biol. Chem., 2.Q, 5638-5690, was labelled with biotin according to the procedure described by Updyke, T.V. and Nicolson, G, T. (1986): Methods in Enzymoloy, 121, 717-725. Aftlr such labelling, the Endo-H retained most of its reactivity |with the glycoprotein ovalbumin.

|I Overnight cultures of Staphylococcus aureus (ATCC i 6538) grown in Luria's broth, and Streptococcus 1 25 mutans (ATCC 27607) grown in Difco Brain Heart Infusion media, were centrifuged and washed three times with 200 mM NaCitrate pH 5.5 buffer and suspended in the same buffer to a concentration of about 109 cells/ml. Aliquots of 0.5 al were placed in 31.5 ml Eppendorf tubes and incubated under various conditions and times.

-69- SIncu- BJotiny- 0.2 M bation lated NaCitrate Time Tube Cells 2% BSA Endo-H 2H 5.5 (min.) 1 0.5 ml 5 pi 0.5 ml 2 0.5 ml 5 il 0.5 ml 2 3 0.5 ml -5 .1 0.5 ml Incubation was done at room temperature using a slow S speed rocker for either two or 30 minutes. BSA (bovine serum albumin), diluted in tris-buffered saline was used as a control solution in order to S' 5 prevent any non-specific protein binding to the cells. After incubation, the tubes were centrifuged and the supernatants were discarded. Two cell washes with 2% BSA solution were done by adding 1.0 ml BSA to the cells, vortexing well, centrifuging and discarding the supernatant. To the washed cells, ml of streptavidin-HRP (streptavidin-labeled horse radish peroxidase, Kirkegaard and Perry Laboratories, Inc.) was used and incubated for 30 minutes at room temperature. The tubes were again centrifuged and washed as previously described. Detection of Endo-H jbinding to the bacterial cells was determined by the detection of HRP-streptavidin, which will bind very tightly to the biotinylated Endo-H bound to the cells. HRP detection was determined by adding 0.5 ml of the HRP substrate OPD (O-phenylenediamine) diluted 4 in citrate phosphate buffer solution containing i hydrogen peroxide. The chromogen generation was Iquenched with 2 M H 2 S0 4 one minute after adding OPD.

The cells were centrifuged and the supernatant was read at 490 run.

ii The following results were obtained: For Staphylococcus Aureus Control 0.13 Endo-H, 2 minutes 1.89 Endo-H, 30 minutes 1L.90 For Streptococcus mutans OD 490 nm ifControl 0.18 Endo-H, 2 minutes 3.76 3. i 'fEndo-H, 30 minutes 3.80 These results indicate that there is binding of Endo- H to the bacteria Stahy1Qspoccus aureus and Streptococcus mutans. The data show that the majority of Endo-H that binds occurs in the first two minutes or less after contact with the cells. The higher absorbance obtained with Streptococcus-mutans may indicate a higher level of Endo-H binding.

A heavy duty liquid laundry detergent composition of :the present invention is as follows: Active Qgponnt eih

C

13 linear alkylbenzene sulfonic acid 8.00

C

14 15 alkyl polyethoxylate (2.25) sulfonic acid 12.00 1,2 propanediol. 3.50 Sodium diethylenetriamine pentaacetate 0.30 Monoethanolamine 2.00

C

12 13 alcohol polyethoxylate 5.00 Ethanol 8.50 Potassium hydroxide L wTf i'T- .11 nmlri i i~iii~ij.n -71- Sodium hydroxide

C

12 14 fatty acid Citric acid Calcium formate Sodium formate

C

12 alkyltrimethylammonium chloride Tetraethylene pentamine ethoxylate (15-18) Water Dye 10 Perfume Protease** Endoglycosidase H 3.85 10.00 4.00 0.12 0.86 0.50 2.00 35.12 0.08 0.25 0.125 2000 ppm o O00 o o r0 0 0 0 0 o00 0 a 0900 0 9 o a .9 00 0 0 u 0000 0 000 00 9 00 06 00 0 3 0 0r 0 60 *0 Notes Alcohol and monoethoxylated alcohol removed.

mg active enzyme/g (@34 mg active enzyme/g stock) 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 20 perfume are added, the pH of the mix is adjusted so that a 10% by weight solution in water at 20*C has a pH of about This composition provides superior cleaning of carbohydrate-containing stains, even compared to protease-containing and/or anylase-containing detergents.

Example A heavy duty liquid laundry detergent composition of the present invention is as follows: lpr;anv ~S~m ii L~ I- -72- Active Component Weight

C

13 linear alkylbenzene sulfonic acid 8.00

C

14 15 alkyl polyethoxylate (2.25) sulfonic acid 12.00 1,2 Propanediol 3.50 Sodium diethylenetriamine pentaacetate 0.30 Monoethanolamine 2.00

C

12 13 alcohol polyethoxylate 5.00 Ethanol 8.50 Potassium hydroxide 1.80 Sodium hydroxide 3.85 C12-1 4 fatty acid 10.00 SCitric acid 4.00 Calcium formate 0.12 Sodium formate 0.86

C

12 alkyltrimethylammonium chloride 0.50 Tetraethylene pentamine ethoxylate (15-18) 2.00 Water 37.12 Dye 0.08 Perfume 0.25 Protease** 0.125 Endoglycosidase H 125 ppm Notes S 25 Alcohol and monoethoxylated alcohol removed.

S' mg active enzyme/g (@34mg active enzyme/g stock) 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 are added, the pH of the mix is adjusted so that a 10o by weight solution in water at 20*C has a pH of about i 1 ii -73- This composition provides superior cleaning of carbohydrate-containing stains, particularly fecal stains.

Other compositions of the present invention are obtained when the Endo H level Is reduced to 0.40 mg/ml, water is decreased to 35.72, and 1% Irgasan (a Ciba-Geigy antibacterial) is added.

Example 11 A liquid soap composition of the present invention is 10 as follows: Active Component Weight t f t t f V 0 ft t t 0 V t 0 0 II 0 t0 o N )fItI* Ammonium lauryl sulfate Sodium lauryl sarcosinate Cocamidopropyl betaine Coconut fatty acid Quaternary amine Ethylenediamine tetraacetic acid Ammonium sulfate Perfume Kathon Water Endoglycosidase H Triclocarban 5.7 6.3 0.3 0.2 0.4 0.25 5 ppm 72.0 1000 ppm 1.50 The ingredients listed above are added to a mixing tank with a single agitator in the order in which they appear below.

This composition provides antibacterial action for removal of common skin flora, even when compared to non-glycosidase containing, antibacterial soaps.

-74- Example 12 A hard surface scouring cleanser of the present invention is as follows: Component False Body Fluid Phase (Specific Gravity 1.1) Barasum NAS-100 (Sodium saponite clay) Tetrapotassium pyrophosphate Tripotassium phosphate Sodium hypochlorite bleach Sodium lauryl alkyl sulfate Surfactant Dye and Perfume Endoglycosidase H Soft Water Abrasive (Expanded Perlite-Specific Gravity Average Particle Diameter microns) Hercoflat 135 Filler (powdered polypro-pylene, Specific Gravity 0.9 Average Particle Diameter 35 microns) Ratio Average Particle Diameter Abrasive/Filler 1.43:1 Weight 93.5 4.25 6.00 2.00 0.90 0.25 0.26 1000 ppm -1.50 4 4 II 4 4. 9 44 The composition is prepared by mixing tetrapotassium pyrophosphate, tripotassium phosphate, sodium 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 surfactant is then blended into this mixture followed by the polypropylene filler material. A separate aqueous slurry of sodium

_C

i hypochlorite and perlite abrasive is prepared and then blended into the false body fluid phase while it is being liquified under moderate shear agitation.

The resulting scouring composition is false bodied, gel-like in its quiescent state but easily fluidized by application of shear stress. Such a composition is especially effective for removal or stains and soil from hard surfaces.

I

Example 13 A shampoo composition of the present invention follows: is as Component Ammonium alkyl sulfate (29% Aqueous solution) Zinc pyridinethione crystals of Ex. I of USP 4,345,080 Coconut monoethanolamide Ethylene glycol distearate Sodium citrate Citric acid Color solution Perfume Endoglycosidase H Water 55.25% 0.2 0.1 1000 ppm q.s. 100.00% Example_ 14 An antiperspirant stick of the przsent invention is made utilizing the following components: Component Cyclomethicone Fluid AP Stearyl alcohol Castor wax Level 42.55 4.99 11.49 4.99 -76- Talc Zirconiuin/aluminum/glycine complex Fragrance masking agent

C

20 alcohol Pyridoxal Pjhosphate Endoglycosidase H 6.99 26.67 0.80 0.12 1.00 500 ppm 0o oa 0 a 00 o 0 ooj a o 04 0 0 000 0 O 00 0 0 4000 0000 4 0 0004 hoot 00 00 0 0404 0 0004 4 0 ~04 044 V04~ 0 00 44 0 A liquid soap composition of the present invention is as follows: Component Amrr~nium lauryl sulfate Sodium alkyl sarcosinate 15 Cocamidopropyl betaine Coconut fatty acid Ethylenediamine tetraacetic acid Ammonium sulfate Per fume 20 Dye Water Endo-H 2, 4,4' -trichloro-2' -hydroxydiphenyl ether Active 5.7 6.3 140O 0.2 0.4 0.25 5 ?pm 80.15 50 ppm 100 ppm The ingredients listed above are added to a mixing tank with a single agitator in the order in which they appear above. Before the dye and perfume are added, the pH of the six is adjusted so that a 10% by weight solution in water at 200C has a pH of about This cc%po~ition provides antibacterial action for the rc.Aoval of common skin flora.

mm F

M

OVVIF -77- Eample 16 A hard surface cleanser of the present invention is as follows: Component Sodium lauryl alkyl sulfate Sodium alkyl sulfate Butyl carbitol Sodium bicarbonate Citric acid Formaldehyde Perfume Tartrate mono/disuccinate Endo-H Water Active Weight 0.04 0.03 0.05 1000 ppm 88.4 The ingredients listed above are added to a mixing tank with a single agitator in the order in which they appear above. Before the perfume is added, the pH of the mix is adjusted so that a 10% by weight solution in water at 20'C hau a pH of about 7.

This composition is effective for the removal of soap scum and mold from hard surfaces, and is more efficacious than a cleanser without the endoglycosidase.

Example 17 A composition used for the cleaning and/or preservation of whole fruit, vegetables or other plant surfaces is as follows: Active Comonent Weight *1 I

II

4 41 Water

C

12 -1 3 alcohol polyethoxylate (6.5) Endo-H 96.4 0.1 3500 ppm -78- This composition is prepared by mixing the alcohol polyethoxylate and Endo-H in water at their respective levels and adjusting the final pH to between 6-7. The final composition, when sprayed on plant surfaces such as whole fruit or vegetables, is useful in preventing microbial growth on said surfaces.

Example 18 Potentiation of bacteriocidal effect of antimicrobial by Endo-H.

An overnight culture of Escherichia coli was diluted into fresh nutrient broth and grown for four hours at 37°C. Cells were obtained by centrifugation and washed in 0.2 M Na-citrate buffer (SCB) pH After centrifuging, cells were resuspended in SCB, The following tubes (in duplicate) were prepared: 5000 ppm 1000 ppm Chlor- Condition Endo-H hexidine Water Control 0 pl 0 pl 200 pl 10 p1 Chlorhexidine 0 pl 10 pl 200 pl 10 p1 Endo-H 200 pj 0 pI 0 pl 10 pi Endo-H Chlorhexidine 200 pl 10 pl 0 pl 0 p1 S The Endo-H was from c2li producing licats Endo-H. To each tube, 790 pl of cell suspension added (final volume now 1 al) and 10 p1 samples were Staken out as a 0 min control. Tubes were incubated at 37C, on a rotary shaker and 10 pI samples were removed at 1 and 3 hours. The 10 pi aliquots were mixed with 990 pl of PBS (Phosphate buffered saline) 2 dilution) and diluted further sequentially (1:10) in PBS (100 pl in 900 pl of PBS). 10 pl of each diluted solution was plated on Luria-Bertani agar plates. The plates were incubated at 37'C -79overnight and colonies were counted. Number of colony forming bacteria in tubes were calculated according to dilutions made and the logarithm of this number used for further arAphs and calculations.

0 minute 1 hour 3 hours Condition Control (log kill) (log kill) Control 8.62 8.57 8.53 (.09) 200 ppm Endo-H 8.64 8.55 8.55 (.09) 50 ppm Chlorhexidine 8.60 4.42 (4.15) 2.44 (6.59) 200 ppm Endo-H Chlorhexidine 8.61 2.40 (6.17) 2.00 (>6.53) These results are plotted in Fig. 8. As can be seen, 200 ppm Endo-H enhances the bacteriocidal effect of ppm chlorhexidine.

Similar results were obtained for slightly different concentrations of chlorhexidine and Endo-H as measured over a one hour time period. These results are depicted in Fig- 9. As can be seen, 140 ppm of Endo-H enhances the efficacy of 40 ppm chlorhexidine.

L i To further investigate this effect, a §il,,ar experiment was conducted using 20 ppm chlorhexidine (final concentration) with varying concentrations of Endo-H. The results are shown in Figs. 10A and These plots represent the change in the log of colony forming units (CFU). As can be seen, a relatively linear relationship exists between the amount of Endo-H added through about 280 ppm Endo-H. Further increases in Endo-H concentration enhance the adverse tetfec effe I

PPM

ct on bacterial viability through at least 1000 Endo-H in combination with 20 ppm chlorhexidine.

4 Example 19 Effect of Endo-H alone and in combination with antimicrobial on viability of fungi A log phase culture of Cajda albicans was grown, diluted into fresh growth medium, and treated with 0, 1, 10, 100 and 1000 ppm Endo-H (final concentration) for 4 hours while incubating with agitation at 37'C.

The Endo-H was from Bacillus subtilis transformed to produce Endo-H from g. plicatus. One, ten and one hundred fold dilutions were made and plated to give viable cell counts. Zero through 10 ppm Endo-H did ncit significantly reduce cell viability, although in one case 10 ppm Endo-H reduced viability by about 36% after 18 hours of incubation. However, IJ0 ppm to 1000 ppm Endo-H reduced the number of viable cells recovered by about 50% to 88%, respectively, compared to the control not treated with Endo-H when treated for four hours.

In a separate experiment, a culture of Candida allicans was grown, diluted into fresh medium, and treated with 2.5 pg/ml Nystatine in addition to either 0, 1, 10, 100 or 1000 ppm Endo-H (final 2$ concentration) for 18 hours, while incubating with agitation at 37*C. One, ten, one hundred and one thousand fold dilutions were made and plated to give viable cell counts. Endo-H reduced viable cells recovered as follows as compared to that obtained with Nystatine alone:

I

-81- 0 1 ppm ppm 100 ppm 0% 69% 93% 99% As can be seen, as little as 1 ppm Endo-H sig~nificantll enhances t~he mycocidal effect of NystatinO whereas 10 ppm and 100 ppm Endo-H kill almost all of the fungi surviving NystatinO treatment alone.

A similar experiment was conducted using Amphotericin BO at a concentration of 0.5 micrograms per ml for three hours. The results were as follows:

I

pp Endo-H reduction in viability 17% 96% 94% 100 1000 ti ti~ ~1

I

As can be seen, 100 ppm of Endo-H enhances the mycocidal af fect of Amphotericin Be.

E~amle 2 Antimicrobial effect of Endo-H alone or in combination igith a lysozyMe A 48-hour subculture of Lj S21 (ATCC 31617) was used to test the effect of the lysozyme autanolysin (sigma Chernical Co.) alone or in combination with detergent and/or Endo-H. The Endo-H was from ZL, g1i transformed to produce Endo-H from L R1.ignau. The following protocol and results were obtained after treatment for two hours at 370C: Ai -82citrate MRtano- pDH Tide Endo-H Ivsin 5.5 7.0 200QBS 200 Results 1 control 2 200 pp 3 200 ppn 4 200 ppm 200 n 6 200 ppn 7 200 ppn 8 200 ppn 9 200 ppr, Fimbriae, tight cell wall Fiitriae, tight cell wall Fimbriae, tight cell wall Fimbriae, tight cell wall Fitbriae, cell cdensaticr Ioss of fimbriae Few cells, scoe ghosts, cell wall disintegration Sane fimbriae oells in bad shape (oonensed) ht still present I I I Ii It I I I tat.

S4 4 9 9 4 As can be seen, the gross morphology of the bacteria exposed to Endo-H and mutanolysin either with or without detergent at various pH, was significantly modified. The most dramatic effects occurred at pH 7 when Endo-H was used alone or in combination with detergent. Cell viability, however, was apparently hot effected. Endo-H and mutanolysin did not reduce the number of colonies obtained in a plating experiment as compared to a buffer control.

EPsle 21 Bacterial removal from class surfaces by Endo-H and PNGase F Escherichia gqji (ATCC 31617) and Staphylococcus epidermidis (ATCC 155) were used to inoculate glass slides. Each slide contained two etched circles and each was inoculated with EL g1.i or EL PDiderridis.

-83- The bacteria were allowed to incubate at 37*C for two hours.

After rinsing with distilled water, the slides were treated with either 1) PBS buffer, 2) Endo-H (100 ppm) in PBS buffer, or 3) PNGase F (100 ppm) in PBS buffer. The Endo-H was derived from oli producing S plicatus Endo-H. After 30 minutes at 37"C the slides were rinsed in distilled water.

After Gram staining, the slides were read with bright a 10 field optics on a light microscope.

o o In the case of the buffer control, the number of bacteria remaining on the slide was greater than 100 per field. The slides treated with Endo-H contained So. far fewer teria. In the case of S. epidermidis, only about i to 3 bacteria were observed per field.

In the case of L_ coli. about 5 to 10 wer( observed per field. For those slides treated with PNGase F, moderate numbers of bacteria were observed for both S. epidermidis and E. coli (approximately 20 per 20 field).

0 0 Mo 6a" These results indicated that PNGase F is capable of removing bacteria from glass surfaces albeit not as efficiently as Endo-H.

Example 22 Tablet denture cleaner with Endo-H Sodium bicarbonate, sodium perborate monohydrate, tartaric acid, sodium tripolyphosphate,. ulphamic acid, polyethylene glycol (20,000 m.wt.) and ethylene diamine tetraacetate are separately granulated by fluidizing in a hot air bed at 60-65'C for minutes. Such granulates are then tumble mixed with the other ingredients to produce a "first layer" r -84mixture and a "second layer" mixture, wherein the "first layer" mixture has the following composit: .on: Soditun bicarbonate Tartaric acid Potassium monopersuiphate Suiphamic acid Oisodium pyrophosphate Sodium carbonate Polyethylene glycol Sodium sulphate Peppermint powder Silicon dioxide Sodium dodecyl benzene sulphonate %by Weight 30.00 23.00 16. 00 11. 00 8.20 3.*90 12. 2.00 2.50 1.30 0.*50 if I I S S I I I 4111 15 and t',Ie "'second layer" mixture has the following composition: 5114 I I ''It

III'

4 I I till 4444 4 4* 44 4 $4 4 9 4 44 Sodium perborate monohydrate Potassium monopersulphate 20 Sodium bicarbonate Sodium tripolyphosphte Sodium bicarbo 4 /colour Trilon B Sodium carbonate 25 Polyethylene glycol Silicone dioxide Peppermint powder Wasag ester 7 Wasag ester 15 Hardened triglycerides Sodium dodecyl benzene sulphonate Succinate detergent Blue Lake No. 1 Endo-H %by Weight 30.00 28 .00 13.34 10.00 4.00 3 .00 3.*00 2.50 2.00 1.50 0.70 0.70 0.50 0.40 0.30 0.06 100 ppm 1 ul A tablet is produced by compressing in a HORN rotary tableting press of 39 stations. Compressing is in two stages: Initially the "second layer", blue mixture is compressed to very low pressure (10 kN per tablet) by way of tamping. The "first layer", white mixture is then instilled and pressed to 70 kN per tablet. In this way a tablet of 4 grams is produced being 2.7 grams blue and 1.3 grams white.

Tablets are dissolved in water by the consumer 10 to clean dentures placed in the water.

o ,o Example 23 Light cream with Endo-H 9 o An oil-in-water sunscreen emulsion base is made from o the following ingredients, which are indicated by their chemical or Cosmetic, Toiletry and Fragrance Association (CTFA) name: Ingredient Weight Water Phase: Methylparaben (preservative) 0.20 2antethine (moisturizer) 0.10 o Carbomer 934 (thickener) 0.08 AL Sodium hydroxide, 10% (neutralizer) 1.00 Endo-H 100 ppm oO Purified water, q.a. to 100% Oil 9hase: Heavy mineral oil 4.00 Stearic acid, double pressed (anionic ieulsifier) 3.00 Cholesterol (auxiliary emulsifier) 1.00 Cetyl alcohol (auxiliary emulsifier) 1.80 Castor oil (emollient) 1.00 Cetyl palmitate (emollient) 1.20 Octyl dimethyl PABA (U.V.-absorber) 1.40 Propylparaben (preservative) 0.10

I

'I;

-86- In a mixing vessel equipped with a mechanical stirrer, water and the water phase ingredients other than the sodium hydroxide and Endo-H aqueous solution are added and mixed with heating to about 75-80"C to form a uniform aqueous dispersion. The sodium hydroxide solution is then added and mixed into the aqueous phase to neutralize the acidic Carbomer thickener.

In a separate mixing vessel, the mineral oil and oil 1, 0 phase ingredients are added and mixed with heating to about 80-82*C to form a uniform oil phase. The heated oil phase 4s slowly added to the heated water phase using high speed mechanical dispersing means.

Mixing is continued until a homogeneous oil/water 0 15 emulsion is obtained. The emulsion is cooled to room temperature. If desired, optional colorants such as water-soluble dyes are preferably mixed into the emulsion at about 45-50'C and fragrant oils are preferably added at about 35-40'C. Endo-H is mixed 20 into the emulsion at about 35-40'C.

Example 24 Removal of S. aureus from pig skin Pig skin was inoculated with 5. aureus (1.2 x 10 7 colonies/al) by spreading 0.1 cc of the culture on 25 the skin surface. The organisms were allowed to set on the skin for two hours at room temperature.

Duplicate pieces of skin were then treated for seconds with: 1) untreated control 2) water alone 3) 10% soap solution 4) #3 Endo-H (20 ppm) 20 ppm Endo-H in buffer i L -87- The Endo-H was obtained from E coli transformed to produce. Endo-H from plicatus. After treatment the samples were rinsed in distilled water and placed in 2% osmin, tetroxide followed by fixation in Ryter- Kellenberger fixative. The samples were then processed alternatively in osmium and thiosemicarbizone. After critical point drying, all samples were examined on the SEM. Photomicrographs were taken.

S. aureus colonies were found in abundance on the untreated, water treated, or plain soap treated samples. See, e.g. Fig. 11 which demonstrates the effect of treatment with liquid hand soap. The Endo- H-treated samples demonstrated a significant loss of S. 15 organisms. See, e.g. Fig. 12 which demonstrates the removal of S. aureus from swine skin wheii treated with liquid hand soap plus Endo-H.

~Example Mold removal from shower curtain A plastic shower curtain was moistened with tap water 'and placed in the dark for 3 weeks. At the end of that time, a small sample of the curtain that was covered with molz was treated with: 1) distilled water 25 2) 2000 ppm Joy detergent 3) 1000 ppm Endo-H 4) untreated The Endo-H was obtained from Ej. coli transformed to produce Endo-H from pipicatus. The treatments lasted 10-15 seconds at room temperature. The shWwer curtain was wiped off after treatment with a cotton swab.

S88- Fig. 13 depicts the results obtained. The nontreated control (lower right photograph lower right quadrant of center photograph) showed abundant mold and mildew particles both macro and microscopically.

The distilled water control (upper right photograph upper right quadrant of center photograph) showed less organisms, although particles still remained and discoloration was evident.

The Joy-treated control (lower left photograph lower left quadrant of center photograph) showed less organisms than the water treated sample, but discoloration was still evident.

The Endo-H treated sample (upper left photograph upper left quadrant of center photograph) was free of both organisms and any discolorations.

Example 26 SBacterial removal from fabric Fabric swatches were cut to the size of a petri dish. Additional fabric was added to reach a fabric load (which was not inoculated). The swatches were sterilized in an autoclave for 15 minutes at 15 lbs. 121*C. One fabric load is needed for each treatment. Glass beads (40 g) and 100 mils 0.2M pH 7.0 citrate buffer was placed into 250 ml K 25 Erlenmeyer flasks. The flasks were plugged with 1 rubber stoppers and aluminum foil and sterilized in an autoclave. E. coli ubcultured into fresh nutrient broth and allowed to incubate for 48 hours at 37*C. Half strength trypticase soy agar plates (10 g/500 mls) were prepared and sterilized. After cooling, tetrazolium (1 ml/liter) was added.

-89- The agar plates were inoculated as follows: 1) serial dilutions from the 48-hour culture were prepared (1:10, and 10 fold dilutions through three more tubes in peptone water); 2) Thereafter, each swatch was inoculated with 2 mls of the last dilution (104).

3) The swatches were then incubated at 37*C for two hours (two swatches/treatment).

After incubation, the swatches were laundered as follows: Wash 100 mls sterile 0.2 M pH 7.0 citrate buffer 40 q glass beads the treatment described in Fig. 14 (where AWA is Endo-H from E. coli transformed to produce Endo-H from S. plicatus) in a 250 ml Erlenmeyer flask (sterile). Two inoculated swatches sterile fabric to make 5% fabric load were washed at 95°F for 12 minutes with shaking.

Rinse After washing, the swatches were rinsed by adding 100 mls sterile doubly distilled/deionized water g glass beads in a 250 ml Erlenmeyer flask (sterile) at room temperature for two minutes with *t shaking.

The fabric swatches were than placed in petri dishes and overlaid with 3 als of one-half strength trypticase soy agar with tetrazolium. After incubation for 48 hours, the coloniev were counted.

The results are shown in Fig. 15. These results indicate that 2% Irgasan plus Liquid Tide provide a two log decrease in bacterial growth as compared to Tide alone. The addition of 40 ppm Endo-H, however, reduces bacterial growth another log unit.

Example 27 Effect of Endo-H on yeast Broth cultures (18 hour) of Candida albicans and Sacchromyces cerevisiae were treated with: 1) 0.2 M Na citrate buffer, pH 2) #1 plus 200 ppm Endo-H (from co1i producing S. plicatus Endo-H) 'The treatments lasted 2 hours at 370C.

After treatment, an aliquot of each was placed on a formvar-coated 200 mesh copper grid, and examined by TEM. Photomicrographs of the examinations were taken and are presented in Figs. 15 and 16.

As can be seen in Fig. 15A, Candida treated with buffer alone was in good morphological condition. As indicated in Fig. 15B, Candida treated with Endo-H leaked material at a rapid rate and lost structural integrity.

Saccjrhmycgs treated with buffer alone was in good morphological condition as can be seen in Fig. 16A.

When treated with Endo-H, however, all that remained were very limited pieces of membranous material. See Fig. 163.

Example 28 Effect of Endo-H and Lysozyme on viability of E. coli A culture of EL coll K12 grown overnight in Laurie Broth was diluted 1:1000 in LB and regrown for 4 hours at 37'C. Cells were centrifuged, washed and resuspended in 0-1M NA-acetate pH 5.5 (NA) buffer.

Eight tubes were set up as follows: -91- Tube Number 1,2 3,4 5,6 7,8 p 1 cells 800 800 800 800 p 1 NA buffer 200 200 p 1 Endo-H (1 mg/ml) 200 200 The Endo-H was from B subtil transformed to produce Endo-H from plicatus. Tubes were incubated for one hour at 37*C. Tubes were centrifuged, washed and resuspended in 8.00 pl of 0.1 M Na-phosphate, pH 7.2 (NP) buffer containing 10 0.1 M EDTA. Buffer or hen egg white lysozyme solution was added to tubes as follows: Tube Number 1,2 34, 56, 7,8 p 1 NP buffer 200 200 p 1 lysozyme (1 mg/ml) 200 200 Aliquots were taken at this time to determine colony forming units (CFU) (Column After incubation for one hour at 37°C aliquots were used to determine CFUs (Column The log of colony forming units were calculated. Th, Jecrease in log CFUs was determined by subtracting B from A. The results are shown below: Lcci CFUs Change Condition A B in log CFUs Control 7.89 7.90 +0.01 Endo-H (200 ppm) 8.21 7.92 -0.29 Lysozyme (200 ppm) 7.87 7.68 -0.19 Endo-H lysozyme 8.17 7.53 -0.64 These results indicate that the combination of Endo-H and lysozyme decreases the viability of E coliJ as compared to Endo-H or lysozyme alone.

I

-92- Example 29 Comparison of Endo-H with T-4 or hen egg white Lysozyme on viability of E. coli coli cells were washed and suspended in 0.1 M Naacetate pH 5.5 buffer, Cells were aliquoted (10 ml) j in two tubes. To one tube, only buffer was added 1 (control) and to another Endo-H was added (treated).

The Endo-H was from B. subtilis transformed to produce Endo-H from j plicatus. Cells were incubated for one hour at 37'C. Cells were i centrifuged, washed and resuspended in 0.1 M Na- S phosphate (pH 7.2) buffer. Cells were aliquoted I equally and incubated either with buffer or lysozyme.

Hen egg white (HL) and T4 (TL) lysozymes were compared in this experiment. Tubes were incubated S for 1.5 hours. Samples were diluted and plated for CFU determination before and after (B) i incubation. The log of CFUs were determined. The jfollowing results were obtained.

Incubation Condition Log CFUs Change J First Second A B in loc CFUs Endo-H (300 ppm) 7.60 7.23 -0.37

HEWL

S(445 ppm) 6.69 6.73 -0.23 Endo-H HEWL S(300 ppm) (445 ppm) 7.41 6.81 -0.60

I

TL

(445 ppm) 4.98 4.53 -0.45 Endo-H TL (300 ppm) (445 ppm) 5.27 4.30 -0.93 These results indicate that T-4 lysozyme is also effective in reducing the viability of Ej. Sl in combination with Endo-H.

y! -93- Example Treatment of soiled diaper material with Endo-H Samples were obtained from a soiled diaper. Each sample was divided. The left side of the sample was washed in 2000 ppm T'de and 1 ppm BPN' (subtilisin protease from Bacillus amvloliquifaciens). The right side was washed in 2000 ppm Tide, 1 ppm BPN' and ppm Endo-H (Boehringer Mannheim Biochemical Catalog No. 100 119). Each sample was washed for 12 minutes at 95'F. The results of two experiments are shown in Figs. 17 and 18. As can be seen, the diaper j H material on the right side of Figs. 17 and 18 j contains substantially less fecal stain as compared S, to the Tide-protease treated diaper shown on the left a 15 of Figs. 17 and 18.

j Having described the preferred embodiments of the present invention, it will appear to those of ordinary skill in the art that various modifications may be made and that such modifications are intended 20 to be within the scope of the present invention.

~e0 Other compositions of the preseAt invention are obtained when Endo-D or F or PNGase F are substituted a for Endo-H in the Examples.

All references cited herein are expressly 25 incorporated by reference.

d*

Claims (19)

1. An antimicrobial composition comprisn. at least two different components, said first component comprising a Type 1I endoglycosidase and said second component comprising an antimicrobial agent.

2. The antimicrobial composition of Claim I wherein said Type II endoglycosidase is selected from the group consisting of Endo-O, Endo-H, Endo-F and PNGase F. 0m S 3. Tih antimicrobial composition of Claim 2 wherein said Type II endoglycosidase is Endo-H, S 0

4. The antimicrobial composition of Claim 1 wherein said Type II S o endoglycosidase comprises about 1 ppm to 1200 ppm and said antimicrobial agent comprises about 2 ppm to about 1200 ppm of said composition.

5. The antimicrobial composition of Claim I wherein said Type II .0 endoglycosidase is Endo-H and comprises about 1 ppm to 1200 ppm of said composition, 0 6, The antimicrobial composition of Claim 1 wherein said O antimicrobial agent is an antibiotic selected from the group o- consisting of bacteriocides, fungicides and algicides.

7. The antimicrobial composition of Claim 6 wherein said antimicrobial agent is a bacteriocide selected from the group consisting of chlorhexidine, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, Triclocarban, penicillins, tetracycline and Bacitracin.

8. The antimicrobial composition of Claim 7 wherein said bacteriocide is chlorhexidine, SP&G Case 4040& Claims 95

9. The antimicrobial composition of Claim 8 in the form of a mouthwash, dentifrice or denture cleaner. The antimicrobial composition of Claim 9 wherein said chlorhexidine comprises from about 50 to 350 ppm of said composition.

11. The antimicrobial composition of Claim 7 wherein said bacteriocide is 2,4,4'-trichloro-2'-hydroxydiphenyl ether icomprising about 5 to 350 ppm of said composition. o I ft

12. The antimicrobial composition of Claim 6 wherein said ,r antimicrobial agent is a fungicide selected from the group consisting of Nystatin, Amphotericin B, Benonyl, Captan and lot Dichloran.

13. The antimicrobial composition of Claim 12 wherein said .tt Ifungicide is Nystatin.

14. The antimicrobial composition of Claim 13 wherein said Nystatin comprises from about 0.5 to 100 ppm of said composition. The antimicrobial composition of Claim 12 wherein said fungicide is Amphotericin B. 4 t1 4

16. The antimicrobial composition of Claim 14 in the form of a liquid hand soap comprising from about 50 to 400 ppm Endo-H,

17. The antimicrobial composition of Claim 1 wherein said antimicrobial agent is surfactant-stable antimicrobial enzyme.

18. The antimicrobial composition of Claim 17 wherein said surfactant-stable antimicrobial enzyme is lysozyme. P&G Case 4040& Claims 96

19. The antimicrobial composition of Claim 1 in the form of personal care or household cleaning products selected from the group consisting of liquid soap, hard surface cleaner, laundry detergent, anti-acne medication, deodorant, shampoo, face cream, mouthwash, dentifrice, and denture cleaner. The antimicrobial composition of Claim 19 in the form of a hard surface cleaner comprising from about 100 to 1000 ppm endo-B-N-acetylglucosaminidase H and from abut 0.1 to 20 weight detergent surfactant of said composition. 0 8i 4 40 90 0 9 .o a

21. The antimicrobial composition of Claim 19 in the liquid or granular laundry detergent comprising from 250 ppm Endo-H and from about 5 to 60 weight surfactant of said composition. form of a about 2 to detergent 08' #006 8099 0 9 9i 8 980* 9r 22, The antimicrobial method comprising contacting a microorganism or a surface containing a microorganism with an antimicrobial composition comprising at least two different components, said first component comprising a Type II endoglycosidase and said second component comprising an antimicrobial agent.

23. The antimicrobial method of Claim 22 wherein said Type II endoglycosidase is selected from the group consisting of Endo-D, Endo-H, Endo-F and PNGaseF.

24. The antimicrobial method of Claim 23 wherein said Type II endoglycosidase is Endo-H. The antimicrobial method of Claim 22 wherein said Type II endoglycosidase comprises about 1 ppm to 1200 ppm and said antimicrobial agent comprises about 2 ppm to about 1200 ppm of said composition. P&G Case 4040& Claims

97- 26. The antimicrobial method of Claim 22 wherein said Type II endoglycosidase is Endo-H and comprises about 1 ppm to 1200 ppm. Sn an a o a o s S a 4 anna S r d i Vv t a es 4 5 27. The antimicrobial method of Claim 22 wherein said antimicrobial agent is an antibiotic selected from the group consisting of bacteriocides, fungicides and algicides. 28. The antimicrobial method of Claim 27 wherein said antimicrobial agent is a bacteriocide selected from the group consisting of chlorhexidine, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, Triclocarban, penicillins, tetracycline and Bacitracin. 29. The antimicrobial method of Claim 28 wherein said bacteriocide is chlorhexidine. The antimicrobial method of Claim 29 wherein said chlorhexidine comprises from about 5 to 350 ppm. 31. The antimicrobial method of Claim 28 wherein said bacteriocide is 2,4,4'-trichloro-2'-hydroxydiphenyl ether comprising about to 350 ppm. 32. The antimicrobial method of Claim 27 wherein said antimicrobial agent is a fungicide selected from the group consisting of Nystatin, Amphotcricin 8, Benonyl, Captan and Dichloran. 33. The antimicrobial method of Claim 32 wherein said fungicide is Nystatin. 34. The antimicrobial method of Claim 33 wherein said Nystatin comprises from about 0.5 to 100 ppm. P&G Case 4040& Claims 98 The antimicrobial method of Claim 32 wherein said antimicrobial agent is Amphotericin B. 36. The antimicrobial method of Claim 22 wherein said antimicrobial agent is surfactant-stable antimicrobial enzyme. 37. The antimicrobial method of Claim 35 wherein said surfactant-stable antimicrobial enzyme is lysozyme. 38. The antimicrobial method of Claim 24 wherein said Endo-H and said antimicrobial agent are contained in mouthwash, S' dentifrice, or denture cleaner. o A e ,o 39. The antimicrobial method of Claim 38 wherein said antimicrobial agent is chlorhexidine or surfactant-stable antimicrobial enzyme. s40. The antimicrobial method of Claim 38 wherein said rnouthwash, Soo o dentifrice, or denture cleaner comprises from about 1 to 1200 ppm Endo-H. 4 0444 A ,E 41. The antimicrobial method of Claim 22 wherein said antimicrobial composition comprises a liquid medium containing from about 1 to about 1200 ppm of said Type II endoglycosidase and from oa about 2 ppm to about 1200 ppm of said antimicrobial agent. 42. The antimicrobial method of Claim 22 wherein said microorganism is selected from the group consisting of bacteria, fungus, and JIgae. 43. The antimicrobial method of Claim 42 wherein said microorganism is bacteria. r P&G Case 4040& Claims 99 44. The antimicrobial method of Claim 42 wherein said bacteria are selected from the group consisting of Escherichia coli, Streptococcus mutans, Staphylococcus epidermidis, and Staphylococcus aureus. The antimicrobial method of Claim 42 where said microorganism is fungus. 46. The method of Claim 45 wherein said fungus is Candida albicans. t 47. The antimicrobial method of Claim 22 wherein said method kills said microorganism or inhibits the growth of said microorganism. 48. The antimicrobial method of Claim 22 wherein said microorganism is attached to a surface and said method removes said microorganism from said surface. 49. The antimicrobial method of Claim 22 wherein said antimicrobial composition is contained in personal care or household cleaning products. The antimicrobial method of Claim 47 wherein said Type II endoglycosidase is Endo-H and is present at a concentration of from about 1 to 1200 ppm in said antimicrobial composition. 51. The antimicrobial method of Claim 49 wherein said antimicrobial agent is chlorhexidine and is present at a concentration of from about 150 to 1200 ppm in said antimicrobial composition. 52. The antimicrobial method of Claim 49 wherein said antimicrobial agent is 2,3,4'-trichloro-2'-hydroxydiphenyl ether and is present at a concentration of from about 2 to 500 ppm in said antimicrobial composition. ;arb yl i I U~ P&G Case 4040& Claims 100 53. The antimicrobial method of Claim 49 wherein said personal care or household cleaning products are selected from the group consisting of liquid soap, hard surface cleaner, laundry detergent, anti-acne medication, deodorant, shampoo, and face cream. 54. The antimicrobial method of Claim 53 wherein said personal care product is liquid hand soap containing an antimicrobial composition comprising from about 50 to 400 ppm Endo-H and about 5 to 100 ppm 2,4,4'-trichloro-2'-hydroxydiphenyl ether. The antimicrobial method cleaning product is a antimicrobial composition Endo-H and from about 0.1 of Claim 53 wherein said household hard surface cleaner containing an comprising from about 100 to 1000 ppm to 20 weight detergent surfactant. Ir At A &r 11 *1 56. The antimicrobial method of Claim 53 wherein said household cleaning product is a liquid or granular laundry detergent containing an antimicrobial composition comprising from about 2 to 250 ppm Endo-H and from about 5 to 60 weight detergent surfactant. 57. The antimicrobial method of Claim 22 wherein said antimicrobial composition is applied periodically so as to prevent further adhesion of said microorganisms to said surface. 58. The antimicrobial method of Claim 22 wherein said contacting is of plant, vegetable or fruit surfaces. 59. The antimicrobial method of the surface of crops corn, citrus, wheat, strawberries. of Claim 22 wherein said contacting is selected from the group consisting of tobacco, soybeans, tomatoes and -~laarsrrrrBaxcrslu~I-~--~ P&G Cas e 4040& Claims 101 An antimicrobial method for killing microorganisms, said method comprising microorganisms with a first component antimicrobial-effective concentration endoglycosidase. or inhibiting contacting said comprising an of Type II 61. The antimicrobial method of Claim 60 contacting said microorganism with a comprising an antimicrobial agent. 62. The antimicrobial method of Claim 60 wh endoglycosidase is selected from the g Endo-0, Endo-H, Endo-F and PNGase F. further second comprising component erein said Type II roup consisting of I 63. The antimicrobial method of Claim 62 wherein said Type II endoglycosidase is Endo-H. 64. The antimicrobial method of Claim 63 wherein said antimicrobial-effective concentration is about 1000 ppm Endo-H. The antimicrobial method of Claim 60 wherein said microorganism is selected from the group consisting of bacteria, fungi and algae. 66. The antimicrobial method of Claim 65 wherein said microorganism is fungi. 67. The antimicrobial method of Claim 66 wherein said fungi is yeast. DATED: 9 October 1990 lltr I 4 rI I "c 4 r S 4'* PHILLIPS ORMONDE FITZPATRICK Attorneys for: THE PROCTER GAMBLE COMPANY AND GENENOR INTERNATIONAL, INC ~au~P~ 9 Y I' A;