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Researchers Submit Patent Application, "Preparation and Use of Biofilm-Degrading, Multiple-Specificity, Hydrolytic Enzyme Mixtures", for Approval

May 13, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Manyak, David M. (Ellicot City, MD); Weiner, Ronald M. (Potomac, MD); Carlson, Peter S. (Chevy Chase, MD); Quintero, Ernesto J. (Coco del Mar, PA), filed on October 18, 2012, was made available online on May 1, 2014 (see also Patents).

No assignee for this patent application has been made.

News editors obtained the following quote from the background information supplied by the inventors: "Naturally occurring biofilms are continuously produced and often accumulate on numerous industrial surfaces and on biological surfaces. In an industrial setting, the presence of these biofilms causes a decrease in the efficiency of industrial machinery, requires increased maintenance, and presents potential health hazards. For example, the surfaces of water cooling towers become increasingly coated with microbially produced biofilm slime which both constricts water flow and reduces heat exchange capacity. Water cooling tower biofilms may also harbor pathogenic microorganisms such as Legionella pneumophila. Food preparation lines are routinely plagued by biofilm build-up both on the machinery and on the food product where biofilms often include potential pathogens. Industrial biofilms are complex assemblages of insoluble polysaccharide-rich biopolymers which are produced and elaborated by surface dwelling microorganisms. The chemical composition of industrial biofilms are diverse and are specific to each species of surface dwelling microorganism. Because of this complexity and diversity, non-specific hydrolytic enzymes are ineffective in degrading these biofilms and consequently ineffective in reducing or eliminating the undesirable biofilm.

"On a biological surface, the presence of these biofilms results in the growth of, and subsequent colonization by, pathogenic microorganisms on an internal or external surface of a host animal or on the surface of objects introduced into the animal (e.g surgical implants). Animal pathogens which colonize surfaces are often maintained and protected by unique polysaccharide rich biofilms produced by the pathogen. Such biofilms coat the infected or colonized surface of the animal or implanted object and continue to be produced during the disease process. For many diseases, biofilms are required for the disease process to become established and to progress. The chemical compositions of pathogen-associated surface biofilms, which consist of complex mixtures of biopolymers, are specific to each species of pathogen. Because of this complexity, non-specific hydrolytic enzymes or hydrolytic enzymes with a single specificity are ineffective in degrading these biofilms and consequently ineffective in reducing or eliminating the disease condition. At the present time, there are no therapeutic products which are commercially employed to degrade and remove these disease related, pathogen-produced biofilms.

"Currently, biofilms are most commonly removed using physical abrasion, a process which is both inefficient and incomplete. Antimicrobials (biocides and antibiotics) are employed to slow biofilm build-up by killing the microbes that produce biofilms; however, once established, the biofilms protect the embedded, biofilm producing bacteria from the action of these agents. Furthermore, many antimicrobial agents are toxic and damaging to the environment. Consequently, there is a need for a method to readily remove and control biofilms that does not depend solely on physical abrasion or on the action of antimicrobial agents. This need could be met by a mixture of multiple specificity, hydrolytic enzymes which have been tailored to degrade the specific complex biopolymer composition of a target biofilm. A tailored mixture of multiple hydrolytic enzymes could be employed to degrade biofilms resulting in their more complete removal and in enhanced antimicrobial activity.

"It has recently become apparent that insoluble complex polysaccharides (ICP) in the environment are most efficiently degraded by a cascade of enzymes acting in concert. The degradation of these insoluble complex polysaccharides require more than 'simple' exoenzymes. Normally, an array of enzymes, part of a complex system, is required to fully hydrolyze the polysaccharide into its final monosaccharide end product (Betas et al., 1988; Bassler et al., 1991 b; Bayer & Lamed 1992; Salyers et al., 1996; Svitil et al., 1997). Most of the carbohydrate-degrading enzymes are highly specific for glycosidic sugar and the anomeric configuration of the glycosidic bond.

"They can act endolytically, hydrolyzing internal carbohydrate bonds, generating oligosaccharide intermediates resulting in relatively rapid viscosity decreases of the polymer; others act exolytically, degrading the polymer from the non-reducing termini generating a single monosaccharide end product.

"These enzymes tend to show a higher specificity with high molecular weight substrates than lower molecular weight substrates.

"For the degradation of the insoluble complex polysaccharides, enzyme localization relative to other enzymes and biomolecules is often important for enzyme efficiency, as is the chemistry of its active site. Many reports have been published describing the properties of numerous isolated polysaccharide-degrading bacteria; however, relatively little is understood concerning how intact bacteria degrade insoluble complex polysaccharides or how the multiple enzymes produced by the organism interact (Salyers et al., 1996). It should be noted that degradation of the insoluble complex polysaccharides into its monosaccharide requires multiple enzymes and possibly other proteins (e.g. substrate-binding).

"The present invention teaches general methods for preparing biofilm-degrading, multiple-specificity hydrolytic enzyme mixtures which are specifically tailored to remove targeted industrial and/or disease-related biofilms. These biofilm degrading hydrolytic enzyme mixtures can be employed to remove or degrade biofilms from the target surface causing a reduction of the biofilm and resulting in increased efficiency and improved hygiene in industrial settings and in improved treatment in therapeutic settings. The present invention will find application in numerous settings where biofilms currently present efficiency and health problems.

"Hydrolytic enzyme mixtures can be employed, via direct application to the biofilm, to remove or degrade disease-associated and/or industrial biofilms from the surfaces colonized by the pathogen. The present invention will find application in industrial settings, such as water cooling towers, waste water piping, heat exchangers, and food preparation lines. The present invention will also find application as a therapeutic agent for the treatment of numerous currently uncontrolled animal, and particularly human, diseases. For example: i) Oral plaque-forming bacterial species, the causal agents of dental caries, are maintained by complex biofilms required for their continued colonization of the tooth surface and their disease causing action. Animal species, particularly humans, exposed to these oral plaque-forming bacteria are at risk of developing caries. These pathogen-related biofilms are currently removed by physical abrasion. ii) Porphyromonas gingivalis, the causal agent of periodontal disease, requires a glycocalyx biofilm for its disease action. Human periodontal disease is currently the major cause of tooth loss world-wide. iii) Cystic fibrosis, which has a frequency of 1 in every 2,000 live births, frequently is associated with infection by Pseudomonas aeruginosa in the lungs, P. aeruginosa produces a complex, alginate-based biofilm which directly results in the hyperviscous mucus characteristic of cystic fibrosis patients. This biofilm is also the substrate for pulmonary infections by opportunistic pathogens characteristic of the disease. iv) Implantable medical devices, such as artificial valves, stents, and catheters, can become colonized by pathogens such as Streptococcus sp., leading to premature failure of the devices and/or life-threatening secondary infections. v) contact lenses can become coated with biofilms and colonized by pathogens. The enzyme mixtures of the present invention will conveniently remove these biofilms.

"Microorganisms which degrade complex polysaccharides are known in the art. Some marine microorganisms faced with oligotrophic conditions in the pelagic zone, have evolved powerful enzyme systems to take advantage of the ubiquitous marine snow, which are potential oases in the nutritionally poor open waters. As a consequence, selected marine species have developed very efficient mechanisms to utilize complex polysaccharides. Marine bacterium Microbulbifer [e.g. 2-40 (deposited at the American Type Culture Collection as ATCC 43961) and IRE-31 (deposited at the American Type Culture Collection as ATCC 700072)] and Marinobacterium [e.g. KW-40 (deposited at the American Type Culture Collection as ATCC 700074)] have been identified as a potentially important bioremediation species, since they synthesize an unusually large number of degradative enzymes. Marine bacterium Microbulbifer 2-40 is described in U.S. Pat. No. 5,418,156 (described as Alteromonas 2-40 in U.S. Pat. No. 5,418,156 but subsequently determined through nucleic acid sequencing to be a Microbulbifer) which is hereby incorporated by reference into the present document. The marine/estuarine bacterium, 2-40, is a periphytic organism isolated from a salt marsh growing on Spartina alterniflora. It is Gram negative, pleomorphic, rod-shaped and motile. This aerobe requires sea salts and carbohydrates for growth. It produces numerous proteases, lipases, and carbohydrases that allow Microbulbifer to degrade a variety of complex, insoluble polysaccharides of plant, fungi, and animal origin. These polysaccharides include alginate, araban, carrageenan, carboxymethylcellulose, chitin, glycogen, .beta.-glucan, pectin, laminarin, pullulan, starch, xylan, and agar.

"Relatively recently, a novel structure relating to insoluble substrate degradation was discovered in a Gram positive bacterium. It was a cellulose-binding and multicellulase-containing cell-surface protuberance produced by Clostridium thermocellum and C. cellulovorans. Coined 'cellulosomes' they were found to attach directly to the insoluble substrates, via special cellulose binding proteins. Thus, they bring cellulases into contact with cellulose, targeting the enzyme substrate complex. Cellulosomes are comprised of at least 14 different proteins. Cip A refers to the largest of the cellulosome proteins, approximately 250 kDa. It serves as the scaffolding protein, binding and anchoring the enzymatic components and securing the entire cellulosome on the cell surface. This protein has a 166 amino acid sequence that is repeated 9 times and is a receptor for the enzymatic cellulosome components, such as CeID. CeID is a 68 kDa endoglucanase isolated from the cellulosome whose carboxy-terminus has a docking sequence that binds to the CipA receptors.

"Such structures (hereinafter 'degradosomes') may be involved in the depolymerization of other insoluble polymers in addition to cellulose. Degradosome components could also consist of spatially arrayed enzymes, adhesions and scaffold protein. Not only do degradosomes maintain the released monomer product close to the cell for metabolic utilization, but the degradosome may place a cascade of hydrolytic enzymes in proper juxtaposition for optimal enzyme activity. It is also an attachment organelle, incorporating specific polymer binding proteins. Because of whole cell/degradosome efficiency and the potential for continued enzyme synthesis, the use of living bacteria as bioreactors in the degradation of not only cellulose, but also potential chitin (aquaculture), algae slimes (algal culture) and biofouled surfaces may be quite advantageous.

"It has been found that Microbulbifer expresses cell surface protruberances on its outer membrane and that they are expressed coincidentally with insoluble biopolymer degradation. Furthermore, results suggest that insoluble carbohydrate degradation is indeed most efficient in Microbulbifer when the carbohydrase systems are introduced and degradosome structures are expressed on the outer membrane of this Gram negative rod. Microbulbifer has been shown to a) synthesize greater quantities, and a greater variety, of degradable carbohydrase systems when grown in media containing several complex carbohydrate carbon sources than when grown in a single complex carbohydrate minimal media, b) package agarases and chitinases in different degradosome structures from one another, and c) undergo morphogenesis and synthesize interesting tubular is structures under conditions of carbon limitation. In Microbulbifer a system of enzymes in the degradosome, acting in concert, degrade a portion of the carbohydrate to monomers, thus converting waste into usable nutrients. Living Microbulbifer may be used for bioremediation since it not a pathogen of animals or invertebrates.

"Other genera shown to synthesize polysaccharide degrading enzymes (e.g. agarases) include Vibrio, Alteromonas, Flavobacterium, Streptomyces, and Pseudomonas. Microbulbifer produces three agarases with activities which are analogous to those of P. atlantica. However, the Microbulbifer agarases have different molecular weights, higher specific activity and are generally more resistant to denaturation than those of other species.

"Alginate is commonly produced by both algae, such as Macrocystis pyrifera, and prokaryotes, such as Azotobacter vinelandii, and is consequently a major component of many biofilms. The alginic acid of mucoid Pseudomonas aerugnosa is of medical importance in the exacerbation of cystic fibrosis where it acts as a virulence factor, inhibiting host phagocytosis. Bacterial alginates differ from algal alginates in the degree of O-acetylation of the mannuronic acid residues. Chronic pulmonary infection with Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis patients. Pseudomonas aeruginosa produces a number of virulence factors including extracellular toxins, proteases, hemolysins and exopolysaccharides. The exopolysaccharide alginate shields the bacterium from the host defense mechanisms and anti-microbial agents. The exopolysaccharides may also promote adherence of mucoid strains to the epithelial cells of the respiratory tract. The use of an alginate lyase obtained from Flavobactedum OTC-6 as a therapeutic medicine for cystic fibrosis is described in U.S. Pat. No. 5,582,825. The alginate enzyme system produced by Microbulbifer differs from alginases purified from other organisms in that Microbulbifer produces an enzyme system made up of several enzymes which act together to more effectively degrade polysaccharides.

"Pseudomonas aeruginosa infections also occur in burn victims, individuals with cancer and patients requiring extensive stays in intensive care units. Therefore, these patients would also benefit from an improved method for treating Pseudomonas aeruginosa infections.

"In addition, many strains of Streptococcus mutans have been shown to be cariogenic in experimental animals and are directly associated with human dental caries (Hardie, J, M., 1981. The microbiology of dental canes. In: Silverstone, Johson, Hardie and Williams (ed.), Dental Caries: Aetiology, Pathology, and Prevention, The Macmillian Press Ltd, London, pp 48-69.; Tenzer, J. M. (ed), 1981, Animal Models in Cariology, Special Supplement, Microbiology Abstracts, Information Retrieval, Washington D.C. and London.) and can be isolated from cases of infective endocarditis. The primary habitat of S. mutans is the tooth surface of humans, and its colonization of this surface is favored by high levels of dietary sucrose. S. mutans produces biofilms which are composed of several types of extracellular polysaccharides which are manufactured from sucrose and which are important in the colonization of hard tissue surfaces in the mouth (Gibbons, R. J. and J. van Houte, 1973. On the formation of dental plaques. J. Periodontal. 44-347-360.; Gibbons, R. J. and J. van Houte, 1975. Bacterial adherence in oral microbial ecology. Ann. Rev. Microbiol. 29:19-44.; Hamada, S, and H. D. Slade, 1980. Mechanisms of adherence of Streptococcus mutans to smooth surfaces in vitro. In: Beachey (ed.), Bacterial Adherence, Chapman and Hall, London, pp. 105-135.) These glucans include a water soluble .alpha.-(1-6)-linked linear glucose polymer with .alpha.(1-3) glucosidic branch linkages (Long, L. and J. Edwards, 1972. Detailed structure of a dextran from a cariogenic bacterium. Carbohydr. Res. 24:216-217.), and other essentially water-insoluble, cell-associated polymers. These water-insoluble polymers contain a high proportion of .alpha.(1-3) glucosidic linkages and are generally resistant to degradation by enzymes commonly present in the oral cavity. Because these S. mutans produced biofilms are resistant to enzymatic degradation they build up on the tooth surface, are a major component of dental plaque, and provide an additional habitat for dental cary causing microbes and microbes which contribute to 'bad breath.' Currently, dental plaque is removed by physical scraping of the tooth surface which is most often performed by dental technicians. The S. mutans biofilm is only partially removed from the tooth surface by brushing with a dentifrice or by mouthwash. Consequently, an enzymatic preparation able to degrade the S. mutans produced polysaccharide biofilm and aid in the removal of dental plaque could be incorporated into a toothpaste, into a mouth rinse, or into other vehicles which contact the tooth surface. Enzymatically degraded S. mutans biofilm and the biofilm-associated microorganisms can be more easily and readily removed from the oral cavity resulting in fewer dental caries and objectionable mouth odors.

"In view of the above discussion, one object of the present invention is to develop nontoxic, environmentally friendly methods for removing industrial biofilms.

"Another object of the present invention is to develop a method for removing various disease related biofilms on an internal or external surface of an animal or on an implant prior to or after implantation in an animal by applying to the affected surface or administering to the animal an effective amount of a) an organism which produces a hydrolytic enzyme mixture, b) a hydrolytic enzyme mixture and/or c) a component of a hydrolytic enzyme mixture."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "In the present invention, multiple specificity, hydrolytic enzyme mixtures are produced using certain bacterial species (e.g. marine saprophytic bacteria such as Microbulbifer 2-40 also known as Alteromonas 2-40). The bacteria are selected for their ability to grow on and catabolize or degrade a wide range of complex polysaccharide sources such as those that are present in biofilms. The bacteria are cultured in the presence of one or more polysaccharides which are present in the targeted biofilm. The polysaccharides are used as the primary carbon source to support the growth and metabolism of the bacteria. During the growth of the bacteria in the presence of the polysaccharide, a mixture of hydrolytic enzymes with multiple specificities capable of degrading a complex biofilm material containing the polysaccharide is produced. By altering the composition of the polysaccharides in the culture medium, a custom tailored mixture of enzymes can be produced. The hydrolytic enzyme mixture can then be isolated and applied to the affected industrial or biological surface.

"The industrial application of a multiple specificity, hydrolytic enzyme mixture will remove or degrade biofilms from the target industrial surface causing a reduction of the biofilm thereby resulting in increased efficiency and improved hygiene.

"The therapeutic administration of a hydrolytic enzyme mixture or a component thereof will reduce the biofilm and thereby enable antibiotics and/or the animal recipient's immune system to fight an infection with a bacterial pathogen. The therapeutic, multiple specificity, hydrolytic enzyme mixture of the present invention will therefore be useful as an adjunct to standard anti-infective therapies when a biofilm producing pathogen is involved.

"When used therapeutically, the hydrolytic enzyme mixture of the present invention can be administered by any route, including but not limited to oral, pulmonary (by aerosol or by other respiratory device for respiratory tract infections), nasal, IV, IP and intra-ocularly.


"FIG. 1. Growth in monosaccharides or ICP as sole carbon sources. 2-40 was grown in MM+0.2% of the indicated sole carbon source. Batch culture growth (OD.sub.600nm) was plotted vs. time: Growth in monosaccharides (A.) and Growth in ICP (B. & C.). Growth in other sole carbon sources is shown in subsequent figures: glucose, FIG. 2; agarose, FIG. 3; and chitin, FIG. 4. The cultures were sampled in time course for enzyme activity (see Table 4).

"FIG. 2. Batch growth in 0.2% glucose minimal medium. A. Batch culture growth (Abs 600 nm) (- -) and cell number (cfu/ml) ( were plotted vs time. Inset graph shows the relationship between OD(600 nm) (y-axis) and cell number (cfu/ml) (x-axis) in glucose MM vs a glucose MM blank (- -). Solid black line on inset graph is the linear regression.

"FIG. 3. Batch growth in 0.2% agarose minimal medium. A. Batch culture growth (Abs 600 nm) (-.tangle-solidup.-) and cell number (cfu/ml) (- -) were plotted vs time. Inset graph shows the relationship between OD (600 nm) (y-axis) and cell number (cfu/ml) (x-axis) in agarose MM (- -). Solid line on the inset graph is the linear regression.

"FIG. 4. Batch growth in 0.2% chitin minimal medium. A. Batch culture growth (Abs 600 nm) (- -) and relative extracellular chitinase activity (-.quadrature.-) (.mu.g/ml reducing sugar) were plotted vs time. B. Relative carbohydrase activity (.mu.g/ml reducing sugar) of cellular chitinases (-.quadrature.-) and cellular agarases (-.DELTA.-) produced by 2-40 grown in chitin MM (graphed in A.) were plotted vs time. There was no detectable extracellular agarase activity in 2-40 grown in chitin MM.

"FIG. 5. Fine structure of 2-40 grown in 0.2% glucose (A), agarose (B), or chitin (C). Cells grown in the respective substrate to mid-log phase were observed by SEM. Note the rough surface (degradosome covered) of cells grown in ICP (B & C) and their absence in glucose (A). The production of these structures correlated to the respective, homologous ICP degradase activity. Scale bar=300 nm.

"FIG. 6. Immunolabeling of agarase and chitinase synthesized simultaneously by 2-40. Whole cells of 2-40, grown to mid-log phase in MM containing 0.2% agarose and chitin, were labeled with anti-agarase antibody (primary) and 10 nm gold labeled goat anti-rabbit IgG antibody (secondary). They were additionally labeled with 20 mm gold labeled anti-chitinase antibody. Cells were stained with 1% uranyl acetate and observed by TEM. Arrows indicate agarase labeling. Note the segregation of agarase (small gold particles) and chitinase (larger gold is particles) labeling in the micrograph.

"FIG. 7. Zymogram of 8% native-PA gel overlay with molecular weights added for reference. For both A and B Lane 1, 60 .mu.g total protein of 0.2% glucose grown 2-40; Lane 2, 40 .mu.g total protein of partially purified concentrated alginase preparations. Duplicate lanes of glucose 2-40 cell prep and alginase prep from the gel were (A) silver stained and (B) used in the zymogram overlay, then stained with toluidine blue O. There are eight bands with alginase activity with approximate molecular weight of 87, 66, 43, 39, 35, 27, 25 and 23 kD.

"FIG. 8. SEQ ID NO: 1 from position 300 to 1500 of the 16S gene of Microbulbifer 240."

For additional information on this patent application, see: Manyak, David M.; Weiner, Ronald M.; Carlson, Peter S.; Quintero, Ernesto J. Preparation and Use of Biofilm-Degrading, Multiple-Specificity, Hydrolytic Enzyme Mixtures. Filed October 18, 2012 and posted May 1, 2014. Patent URL:

Keywords for this news article include: Antibodies, Patents, Bacteria, Peptides, Immunology, Amino Acids, Cellulosomes, Blood Proteins, Immunoglobulins, Enzymes and Coenzymes, Cell Surface Extensions.

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Source: Life Science Weekly

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