WO1999014312A1 - Preparation and use of biofilm-degrading, multiple-specificity, hydrolytic enzyme mixtures - Google Patents
Preparation and use of biofilm-degrading, multiple-specificity, hydrolytic enzyme mixtures Download PDFInfo
- Publication number
- WO1999014312A1 WO1999014312A1 PCT/US1998/018167 US9818167W WO9914312A1 WO 1999014312 A1 WO1999014312 A1 WO 1999014312A1 US 9818167 W US9818167 W US 9818167W WO 9914312 A1 WO9914312 A1 WO 9914312A1
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- WO
- WIPO (PCT)
- Prior art keywords
- microbulbifer
- specificity
- hydrolytic enzyme
- bacterial strain
- enzyme mixture
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01081—Beta-agarase (3.2.1.81)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L12/00—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor
- A61L12/08—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor using chemical substances
- A61L12/082—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor using chemical substances in combination with specific enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
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- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C12N9/14—Hydrolases (3)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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- A—HUMAN NECESSITIES
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- C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
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- C11D2111/14—Hard surfaces
- C11D2111/20—Industrial or commercial equipment, e.g. reactors, tubes or engines
Definitions
- the present invention relates to a method for preparing biofilm degrading, multiple specificity, hydrolytic enzyme mixtures which are specifically tailored to remove targeted biofilms.
- the present invention also is directed to methods for using hydrolytic enzyme mixtures in both industrial and therapeutic applications.
- the industrial applications include but are not limited to the use of biofilm- degrading, multiple specificity, hydrolytic enzyme mixtures for removing or preventing the formation of biofilms in water cooling towers, industrial process piping, heat exchangers, in food processing or food preparation, in potable water systems, reservoirs, swimming pools, or related sanitary water systems, and on membranes such as those used for desalinization, industrial processes, or related applications.
- biofilms are continuously produced and often accumulate on numerous industrial surfaces and on biological surfaces.
- the presence of these biofilms causes a decrease in the efficiency of industrial machinery, requires increased maintenance, and presents potential health hazards.
- 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 poiysaccharide-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.
- biofilms 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.
- biofilms coat the infected or colonized surface of the animal or implanted object and continue to be produced during the disease process.
- 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.
- biofilms are most commonly removed using physical abrasion, a process which is both inefficient and incomplete.
- Antimicrobials biocides and antibiotics
- biofilms protect the embedded, biofilm-producing bacteria from the action of these agents.
- 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.
- ICP insoluble complex polysaccharides
- cascade of enzymes acting in concert The degradation of these insoluble complex polysaccharides require more than "simple" exoenzymes.
- an array of enzymes, part of a complex system is required to fully hydrolyze the polysaccharide into its final monosaccharide end product (Belas et al., 1988; Bassler ef al., 1991 b; Bayer & Lamed 1992; Salyers et al., 1996; Svitil et al. A 997).
- 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.
- 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.
- 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.
- 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 worldwide, 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.
- 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,
- 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
- Marine bacterium Microbulbifer 2-40 is described in U.S. Patent No. 5,418,156 (described as Alteromonas 2-40 in U.S. Patent 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, ⁇ -glucan, pectin, laminarin, pullulan, starch, xylan, and agar.
- This protein has a 166 amino acid sequence that is repeated 9 times and is a receptor for the enzymatic cellulosome components, such as CelD.
- CelD is a 68 kDa endoglucanase isolated from the cellulosome whose carboxy-terminus has a docking sequence that binds to the CipA receptors.
- 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.
- 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 structures under conditions of carbon limitation.
- 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.
- agarases 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 Flavobacterium OTC-6 as a therapeutic medicine for cystic fibrosis is described in U.S. Patent 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.
- 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.),
- glucans include a water soluble ⁇ -(1 -6 ) - linked linear glucose polymer with ⁇ (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 ⁇ (A3) 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.”
- 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.
- 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.
- 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.
- hydrolytic enzymes with multiple specificities capable of degrading a complex biofilm material containing the polysaccharide is produced.
- 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.
- 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. 6 Immunolabeling of agarase and chitinase synthesized simultaneously by 2-40.
- 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 particles) labeling in the micrograph.
- Figure 7 Zymogram of 8% native-PA gel overlay with molecular weights added for reference.
- Lane 1 60 ⁇ g total protein of 0.2% glucose grown 2-40; Lane 2, 40 ⁇ 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 23kD.
- Figure 8 Sequence from position 300 to 1500 of the 16S gene of Microbulbifer 2-40.
- biofilm- degrading, multiple-specificity, hydrolytic enzyme mixtures are as follows. First, certain bacterial species (e.g. marine saprophytic bacteria as exemplified by Microbulbifer ) are selected for their ability to grow on and cataboiize or degrade a wide range of complex polysaccharide sources including those that comprise biofilms. These selected bacterial species are then cultured in a medium or series of media containing one or more of the specific polysaccharides comprising the targeted biofilm, or derivatives thereof. The polysaccharides are used as the primary carbon source to support the growth and metabolism of the bacterial species.
- certain bacterial species e.g. marine saprophytic bacteria as exemplified by Microbulbifer
- These selected bacterial species are then cultured in a medium or series of media containing one or more of the specific polysaccharides comprising the targeted biofilm, or derivatives thereof.
- the polysaccharides are used as the primary carbon source to support the growth and metabolism of the bacterial species.
- a mixture of hydrolytic enzymes with multiple specificities capable of degrading the complex biofilm material is produced on the surface of the organism in enzyme containing protuberances [for example through the formation of enzyme- containing appendages (degradosomes) from the bacterial cell surface] and elaborated from the cells in tubules or vesicles or otherwise released into the medium by the bacteria in increasing quantities as the insoluble complex polysaccharides are depleted, as exemplified by certain marine saprophytic bacteria such as Microbulbifer 2-40.
- a custom-tailored consortia of hydrolytic enzymes can be produced.
- the biofilm-degrading, multiple-specificity, hydrolytic enzyme mixtures are separated from the culture, preferably from the culture supernatant and more preferably from supernatant having hydrolytic enzyme-containing appendages or vesicles.
- the enriched hydrolytic enzyme mixture is appropriately formulated and applied to the biofilm which results in the degradation and removal of the biofilm targeted for the application.
- the living organism itself, the degradosomes, tubules, vesicles or purified enzymes can be directly applied to the biofilm. It should be clear that since each biofilm forming microbial species produces a unique biofilm material, each biofilm will require a different, custom tailored, multiple-specificity, hydrolytic enzyme mixture to achieve biofilm control.
- the enzyme mixture can then be purified and applied to the targeted biofilm.
- the enzyme mixture can be purified as a mixture or the various enzyme systems present in the mixture can be purified individually. If the enzymes are purified individually an enzyme mixture can be reformulated after purification or the enzymes can be used individually (e.g. in some specific therapeutic applications). It is not necessary to completely purify the enzyme systems prior to use.
- the enzymes may be present in and purified from degradosomes, vesicles or tubules or the degradosomes, vesicles or tubules can be applied directly to the targeted biofilm.
- the amount of the enzyme mixture to be applied to the targeted biofilm is not critical.
- the amount to be applied can easily be determined by routine experimentation and will be related to the composition of the biofilm.
- the enzyme mixture is applied by contacting the targeted biofilm with the appropriate enzyme mixture.
- the composition can be administered according to a treatment protocol which depends on the patient's age, sex and other factors, the severity of disease, etc.
- a spray or an infusion can be directly applied to the affected site.
- a tablet, solution, emulsion, powder or capsule can be administered orally.
- An injection can be administered in admixture with an ordinary infusion fluid such as glucose solution, amino acid infusion, etc.
- the routes of administration include but are not limited to: oral, aerosol or other device for delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, and topical.
- Excipients which can be used as a vehicle for the delivery of the enzyme mixture will be apparent to those skilled in the art.
- the enzyme mixture could be in lyophilized form and be dissolved just prior to administration or the enzyme mixture could be present in liposomes.
- the enzyme mixture could be applied in the form of a toothpaste or mouth rinse.
- the dosage of administration of the enzyme mixture for reducing biofilms on an oral surface is between 0.1 mg-1g per ml of delivery excipient.
- the dosage of administration of the enzyme mixture for treating P. aeruginosa infections is contemplated to be in the range of about 0.1- 100 mg/per kg body weight, and preferably about 1-10 mg/per kg body weight.
- the hydrolytic enzyme mixture is incorporated into an aerosol formulation specifically designed for administration to the lungs by inhalation.
- aerosols are known in the art, and the present invention is not limited to any particular formulation.
- An example of such an aerosol is the Proventil tm inhaler manufactured by Schering-Plough, the propellant of which contains trichloromonofluoromethane, dichiorodifluoromethane and oleic acid.
- the concentrations of the propellant ingredients and emulsifiers are adjusted if necessary based on the enzyme mixture being used in the treatment.
- the enzyme mixture can also be administered using a nebulizer.
- a bronchodilator such as aminophylline, an antibiotic drug such as a ⁇ -lactam (e.g. penicillin, cephalosporin) or quinolone, a DNase, a protease inhibitor and/or an amiloride, can be combined with the enzyme mixture for enhanced therapeutic efficacy.
- an antibiotic drug such as a ⁇ -lactam (e.g. penicillin, cephalosporin) or quinolone
- a DNase e.g. penicillin, cephalosporin
- protease inhibitor e.g. amiloride
- the enzyme mixture can be administered in combination with antibiotics or other antimicrobial substances, other therapeutic proteins and/or mild abrasives.
- Suitable antibiotics include but are not limited to tobramycin and duramycin. Other suitable antibiotics will be apparent to those in the art once the organism producing the biofilm is determined.
- Therapeutic proteins useful in combination with the enzyme mixtures of the present invention include but are not limited to Dnases.
- MM minimal medium 23 g/L sea salts 1.0 g/L yeast extract 2.0 g/L polysaccharide 3 50 ml/L Tris-HCL, pH 7.6 0.5 g/L NH 4 CI
- LM Lia Marine 10 g/L bacto-tryptone 5 g/L yeast extract 20 g/L NaCI 0.1 g/L chitin adjust pH to 7.5
- MA plates (Marine agar) 37.4 g/L Marine broth 1.5g/L agar
- a Insoluble polysaccharides were added to media prior to autoclaving.
- Other carbon sources were filter sterilized in 20mM Pipes buffer, pH 6.8 and added to cooled media to produce a final concentration of 0.2%.
- b Vibrio harveyi BB7-1 was cultured in this medium
- c chitin paste was produced as outlined by Lingappa and Lockwood (1962).
- Silver Stain Developer 2.5% sodium carbonate 0.02% formaldehyde
- Periodic Acid Oxidizing Solution 0.833% periodic acid
- Triton Solution 2.5% Triton X-100 in 20mM Pipes buffer, pH 6.8
- SDS-PAGE Treatment buffer Native PAGE treatment buffer with 2% sodium dodecyl sulfate (SDS)
- Viable plate counts For each time point viable plate counts and optical density (OD 600 ) were made in triplicate. The culture was vortexed thoroughly, to disrupt aggregated or substrate-bound cells, and plated on
- MM containing the sole carbon source. Plates, depending on the type of MM, were incubated for 24 to 48 hours.
- Carbohydrase activity was determined in crude enzyme preparations. These preparations consisted of whole cells or supernatant. At each time point, 100ml of culture was centrifuged (10,000 x g, 15 min., 4°C) and the supernatant and cell pellet were separated. The supernatant was stored at -20°C until used. The whole cells were washed twice in 50ml of Pipes buffer and then resuspended in 2ml of buffer. The concentrated whole cells were also stored at -20°C until enzyme activity was assayed.
- Dinitrosalicyclic acid (DNSA) reducing sugar assay uses dinitrosalicyclic acid reagent, developed by Sumner and coworkers (Sumner & Sisler, 1944), to quantitate the amount of carbohydrase activity ( ⁇ g/ ml) by measuring the resulting reducing sugars present in the sample.
- the 3,5-dinitrosalicyclic acid is reduced to 3-amino-5-nitrosalicyciic acid and the aldehyde groups are oxidized to o carboxyl groups (Hostettler et al. , 1951 ).
- Color change in DNSA reagent is detected spectrophotometrically as it becomes reduced by any reducing sugar present in a reaction mixture.
- the enzyme preparation (spent media, whole cells, or concentrated enzyme preparations) (0.3ml) was incubated with 0.7ml of substrate (the various carbohydrates listed above). Substrates were prepared as 0.5% stocks except for agarose, which was 0.2%, in buffer of either pH 5.0 (0.025M sodium citrate buffer) or pH 7.0 (0.01 M potassium phosphate buffer) depending on the polysaccharide. Carboxymethyl cellulose (Pettersson & Porath, 1966), chitin (Jeuniaux, 1966), laminarin (Ruse & Mandels, 1966) and pectin (Albersheim, 1966) were prepared in pH 5.0 buffer.
- Agarose, alginic acid (Preiss, 1966), carrageenan, pectin, pullulan, starch, and xylan were prepared in buffer of pH 7.0.
- Agarose alginic acid, chitin and carrageenan were boiled for 5 min. to dissolve them in the respective buffer prior to their addition to the reaction mixture. The reaction incubation time and temperature were also dependent upon substrate.
- DNSA assay values are recorded as ⁇ g of reducing sugar equivalents generated per ml of sample. Triplicate samples were prepared for each reaction and their average was taken to determine the ⁇ g/ml reducing sugar produced.
- Carbohydrase activity in sole carbon sources To assess the regulation of the carbohydrases by sole growth substrate, 2-40 was grown in minimal medium containing a final concentration of 0.2% of one of 16 sole carbon sources. Monosaccharides included: glucose, D-galactose, glucosamine, N-acetyl-D-glucosamine (NAG), and xylose.
- Insoluble complex polysaccharides included: agarose, alginic acid, carrageenan, carboxymethyl cellulose (CMC), chitin, glucan, laminarin, pectin, pullulan, starch, and xylan.
- Batch culture growth (OD 600nm ) was monitored (Fig. 1 , 2, 3 & 4) and carbohydrase activity was assayed in both cellular and supernatant culture fractions (Tables 4 & 5).
- Enzymatic activity was reported either as total relative ⁇ g/ ml of carbohydrase activity to report 2-40 carbohydrase activity, or as units ( ⁇ g/ ml carbohydrase activity per ⁇ g/ ml total sample protein per DNSA assay reaction time.
- Example 2 Provide and Purification of Enzyme Systems Chemicals, media and bacterial growth conditions.
- Chitin paste was purified from commercial chitin as outlined by Liggappa and Lockwood (1962). Practical grade chitin was soaked in 1 M NaOH for 24 hours. After the chitin was washed with dH 2 O, it was soaked in 1 M HCI for 24 hours, washed again with dH 2 O, and transferred to 1 M NaOH. The alternate base/ acid soaking step was repeated four times as described. Following the final washing, the chitin was washed 4 times with 95% EtOH and dissolved in 2 vol.
- the resulting 15ml was referred to the crude chitinase preparation and was stored at -70°C until used. Total protein concentration and enzyme activity was determined for the crude enzyme preparation.
- Vibrio harveyi chitinase harvest V. harveyi BB7-1 was obtained and chitinase produced by it was harvested to use as a control in developing chitinase enzyme assays and zymograms.
- the bacterium was grown in 1 L of LM supplemented with chitin (Table 1 ), to induce the clone's overproduction of chitinase, at 25 °C with shaking (100rpm) for 3 days. The sample was centrifuged (10,000 x g, 15 min., 4°C) and the supernatant collected. The extracellular chitinase was concentrated in
- Polyacrylamide gel electrophoresis of crude enzyme preparations were analyzed on a discontinuous SDS- or native- PAGE as described by Laemmli (1970) using non-denaturing conditions (excluding boiling of the sample prior to electrophoresis and the addition of ⁇ -mercaptoethanol to the 2X PAGE treatment buffer), unless otherwise indicated.
- Non-denaturing conditions allowed for the enzymatic activity of separated protein bands to be determined directly in the separating gel or an overlay gel following electrophoresis. Gels used as agarase zymograms, to detect agarase activity, had 0.1 % agarose incorporated in the separating gel.
- glycol chitin prepared as described below, was included in a duplicate overlay gel.
- Protein samples were diluted in the appropriate 2X PAGE treatment buffer (Table 3), either native- or SDS-, and allowed to incubate for 20 min. prior to gel loading of the samples.
- SDS-PAGE molecular weight standards were included on SDS-PAGES to allow for calculation of separated protein molecular weights (Bio-Rad, Richmond, CA).
- the separating gel was stained by one of the following methods or processed as a zymogram. Zymograms (activity gels).
- Chitinase zymograms containing 0.05% glycol chitin in the separating gel, were developed as described by Pan et al. (1991 ). Following electrophoresis, the native-PAGE was incubated for 5 min. In 150mM sodium acetate buffer, pH 5.0. The buffer was replaced with fresh buffer and the gel was incubated for 2 hrs. at 37°C. After the buffer was removed, the gel was stained with 0.01 % (w/v) Calcoflour White M2R in 500mM Tris-HCI, pH 8.9 for 5 min. at room temperature. Calcoflour is light sensitive, so the gel was covered for staining and the remaining steps. The stain was removed and the gel destained overnight in dH 2 O at room temperature. Chitinase bands were detected by observation of the gel on a transilluminator and photographic documentation was made with a thermal printer.
- Silver staining of proteins allows for the most sensitive detection of them, detecting as little as 0.1 - 1.0ng of protein in a single band, and is approximately 100- to 1000- times more sensitive than Coomassie Blue staining.
- the silver ions following staining with silver nitrate, are bound to the side chains of the proteins' amino acids and are differentially reduced upon development (Merril et al., 1984). Free amines and sulfur groups are the principal reactive groups of the protein (Freeman, 1973; Heukeshoven & Dernick, 1985; Neilsen & Brown, 1984).
- Silver staining was performed as outlined by Sambrook et al. (1989), a modification of the original staining procedure described by Sammons et al. (1981 ). All procedures were performed at 25°C, the gel was gently shaken during incubation periods, and HPLC grade water was used. Proteins were fixed in the gel following separation by electrophoresis by overnight incubation in 300ml silver stain fixing solution (Table 2). The fixing solution was discarded and the gel incubated for 30 min. in 30% EtOH. The gel was washed with 4 changes of HPLC water, for 10min. each, and stained with 0.1 % AgNO 3 for 30 min. The AgNO 3 solution was then discarded and the gel was washed under a stream of HPLC water for 40 sec.
- the protein bands were developed by 300ml of silver stain developing solution (Table 2) for 10 - 30 min., until the desired visual development of the bands was achieved. Development was ceased by incubation of the gel for 10min. in 1 % acetic acid. All gels were washed and stored at 4°C in dH 2 O.
- Coomassie blue protein staining The Coomassie Blue R-250 protein stain described by Diezel et al. (1972) was used to detect predominant proteins in preparative gel when purity and trace protein detection was not of significant importance. Coomassie blue stain (Table 2) was heated to 45°C and incubated with the gel at 25°C for 2 hours with gentle shaking.
- the gel was destained with Coomassie blue destaining solution (Table 2) at 25°C with constant shaking and several changes until protein bands were distinguishable in the gel. If stained gel sections were used to identify enzyme bands to be excised from a preparative gel, the gel fragments were shrunken (in methanol) or swollen (in dH 2 O) until the gel fragments matched the size of the initial separating gel, just prior to excising of the protein band. The stained outer gel fragments were matched up with the mid-section of the separating gel to estimate the position of the enzymatic band to be excised from the gel. The excised gel band was washed with PBS and crushed as described below. This staining method was only used for native PAGE, since imidazole-zinc staining is only compatible with SDS-PAGEs.
- Imidazole-zinc protein staining The imidazole-zinc protein stain allowed for visual detection of the protein band of interest on preparative SDS-PAGE. The protein could then be excised and destained. This method, described by Fernandez-Patron et al. (1992), allows for detection of proteins by negatively staining them and is only slightly less sensitive that silver staining. Following electrophoretic separation of the proteins, the SDS-PAGE was soaked in dH 2 O for 10 seconds. The gel was incubated in 200ml of 0.2M imidazole for 10 min. at 25°C while gently shaking. The imidazole solution was removed and the gel was negatively stained for 2 min. in 200ml of 0.3M ZnSO 4 at room temperature while rocking.
- the protein band of interest a clear band against an opaque background, was excised form the gel and destained for 10 min. in 2% citric acid.
- the gel fragment was washed with several changes of Pipes buffer + 2.5% Triton X-100.
- the gel fragment, containing the enzyme of interest was washed with PBS and finely sliced.
- the gel fragments were loaded into a syringe with PBS and crushed by passing the mixture back and forth between two glass syringes connected by an 18 gauge hub. This mixture of crushed acrylamide and PBS was frozen at -70°C until use.
- Topographical protuberances (Degradosomes). 2-40, grown in MM containing 0.2% agarose or chitin, attach to the insoluble substrate while growing on it.
- Double labeling immunoelectron microscopy using both anti-agarase and -chitinase to label the respective enzyme, was done to: 1 ) determine if both enzyme systems are induced in a single cell; 2) when both were produced; 3) see whether both enzyme systems were localized in the same degradosome. 2-40, grown in MM plus 0.2% agarose and chitin to mid-log phase, was labeled with anti-agarase antibody and anti-chitinase antibody. Both the agarase and chitinase systems were active in cells sampled during growth. The agarase was labeled with smaller colloidal gold particles, 10nm, and chitinase with larger particles, 20 - 30nm.
- Double labeled whole cells and ultrathin sections showed that both enzyme systems were synthesized in a single cell and that each segregated into a different degradosome.
- Microbulbifer 2-40 synthesizes a ⁇ - agarase system comprised of numerous extracellular agarases, with predominant agarases of 98, 90, 60, and 42 kDa. Many of these degradative agarases are packaged in tubules, vesicles or other elaborated structures. Other species, P. atlantica, Vibrio sp. strain JT0107, and a Pseudomonas-Wke bacteria, synthesize multiple agarases, which arguably work cooperatively to degrade the substrate (Sugano et al., 1994; Bibb et ai, 1987; Belas et al., 1988; Malmqvist, 1978).
- the multiple agarases of Microbulbifer 2-40 appear to be discrete enzymes, not dissociated into lower molecular weight agarases under fully reducing conditions, determined by comparison of silver stained native-PAGE to fully reducing SDS-PAGE and Western blots of both of gels probed with anti- agarases antiserum.
- Such attachment is a common mechanism used by numerous microorganisms for ICP hydrolysis (Svitil et al., 1997; Montgomery & Kirchman, 1993; Miron & Ben-Ghedaiia, 1993; Haack & Breznak, 1993). This is an efficient degradative mechanism, especially for marine bacteria, maintaining contact between the organism, its enzymes and the substrate, and the end products. Also, the carbohydrases would not be so vulnerable to proteolysis, "poisoning", or dilution (Montgomery & Kirchman, 1993; Svitil er a/., 1997).
- chitinases 200, 98, 66, & 52.5 kDa, are synthesized by Microbulbifer 2-40 when cultured in chitin MM.
- Microbulbifer 2-40 agarases and other insoluble complex polysaccharides (ICP) degradative systems, microorganisms commonly synthesize several enzymes with like activity to degrade ICP substrates.
- ICP insoluble complex polysaccharides
- These individual enzymes may be the result of bacterial processing of a single chitinase, smaller proteolytic degradative products of a single genetically encoded enzyme, or they may actually be unique enzymes, each encoded by an individual gene (Techkarnijanaruk et al., 1997; Wantabe et al., 1992 & 1990; Keyhani ef al., 1996; Harman et al., 1993).
- Microbulbifer 2-40 chitinases appear to be individual enzymes, not concatamers of one another. This was determined by comparison of silver stained native-PAGE to fully-reducing SDS-PAGE and Western blots of both gels probed with anti-chitinase antibody.
- the 98kDa chitinase was selected as the antigen for polyclonal antibody production.
- the homologous chitinase, as well as 3 immunogically related chitinases, were identified by the antibody. Serological cross reactivity may result from these chitinases sharing common domains for substrate binding or hydrolysis. This has been shown for other bacterial chitinases by sequence homology and immunological cross reactivity (Robbins et al., 1992; S. Roseman, personal communication). Additionally, the antiserum inhibited 64% of Microbulbifer 2-40 chitinase activity, under experimental conditions used. Microbulbifer 2-40 chitinase does not appear to share antigenically-related domains with
- V. harveyi chitinase since antiserum raised against V. harveyi chitinase is not reactive against any Microbulbifer 2-40 chitinases.
- Microbulbifer 2-40 cells grown in glucose MM have smooth surfaces during early logarithmic phase growth. Bleb-like vesicles were formed during mid-log through stationary culture phases. Vesicles were formed due to separation of the inner and outer membrane of the cell. (These vesicles eventually partition from the cell body being released in late culture stages).
- late culture phases in glucose MM late stationary to death phase, an abundance of long, filamentous tubules, coated with small nodules, were synthesized. The tubules were -20 - 50nm in diameter and their length extended up to several micrometers. The nodules have an approximate diameter of 20 - 40 nm.
- filamentous tubules and bleb-like vesicles were produced during logarithmic phase growth in MM containing agar or chitin.
- the appearance and abundance of tubules and blebs during early growth stages in ICP were similar to those produced during late culture phase in glucose MM.
- Microbulbifer 2-40 during batch growth in neoagarohexose.
- Neoagarohexose induced agarase 575 ⁇ g/ ml reducing sugar equivalents , during log phase growth at levels consistent with those induced by agarose (Table 4).
- Cells used to inoculate neoagarohexose MM were carbohydrase-uninduced, typical in appearance (rods approximately 2.0 x 0.5 ⁇ m on average), and their surfaces were smooth, lacking degradosomes. However, some tubules were transfered from washed, glucose-grown inoculum.
- cells grown to mid-log phase, in either agarose or chitin MM were labeled with antiserum raised against the homologous carbohydrase.
- Tubules produced during growth in agarose were labeled with anti-agarase antibody.
- Cells and tubules produced during growth in chitin MM were not labeled by the anti-agarase antibody.
- tubules produced during growth in chitin were labeled with anti-chitinase antibody.
- Cells and tubules produced during growth in agarose MM were not labeled by anti-chitinase antibody.
- the circular nodules, attached to the tubules or released into the cultures were labeled by anti-LPS, - agarase, and -chitinase in the respective cultures.
- tubules are membraneous and contain carbohydrases, specifically agarase or chitinase.
- Microbulbifer 2-40 produces tubular extensions from the outer membrane.
- the filamentous tubules are -30 - 60nm in diameter and reach lengths up to several micrometers. They are produced during late culture phases in glucose MM, during logarithmic growth in MM containing ICP or neoagarohexose as carbon source(s), and in mid- to late-log phase growth in MM containing both glucose and ICP(s).
- These tubules are membranous and localize agarase(s) or chitinase(s), as determined by immunoelectron microscopy.
- Nodules are random on the surface of these tubules and also free in the culture. They also package agarases or chitinases. 35
- Pseudomonas aeruginosa FRD462 polymannuronicacid producing mutant
- Microbulbifer 2-40 was grown to late log phase (10 9 cells ml "1 ), harvested, brought up in 2% instant ocean (IO) and seeded onto moist biofilms of Pseudomonas aeruginosa FRD1 & 462. (Grown on nutrient broth + 0.5% yeast extract with the spent medium decanted.) Microbulbifer 2-40 was incubated at 30 °C with biofilms over a time course of seven days.
- Biofilm degradation This was monitored visually by examination of the film and by the elaboration of reducing sugars (glucuronic and mannuronic acids). Following the procedures of the above examples.
- Example 6 Production of S. mutans Biofilm Degrading Enzyme Mixtures Strains a) Microbulbifer 2-40 b)Streptococcus mutans ATTC 25175 (type strain) Biomass. Microbulbifer 2-40 cells are grown in broth to late log phase (10 9 cells ml "1 ), harvested, brought up in 2% instant ocean (IO) and seeded onto moist biofilms of Streptococcus mutans, (S. mutans is grown on trypticase soy agar with 5% defibrinated sheep blood at 37° C.) Microbulbifer 2-4O is incubated at 30° C with the biofilms in time course. Biofilm degradation.
- Biofilm degradation is monitored visually by examination of the film and by the elaboration of reducing sugars. Following the procedures of the above examples, biofilms of Streptococcus mutans that are not exposed to Microbulibifer 2-40 and the enzymes it produces are utilized as control materials.
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Cited By (14)
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US6251674B1 (en) | 1997-01-17 | 2001-06-26 | Maxygen, Inc. | Evolution of whole cells and organisms by recursive sequence recombination |
WO2001049255A2 (en) * | 1999-12-30 | 2001-07-12 | University Of Louisville Research Foundation, Inc. | Methods and compositions for inhibiting adhesion by microorganisms |
US6576226B1 (en) * | 2000-11-17 | 2003-06-10 | Gary R. Jernberg | Local delivery of agents for disruption and inhibition of bacterial biofilm for treatment of periodontal disease |
US6726898B2 (en) | 2000-11-17 | 2004-04-27 | Gary R. Jernberg | Local delivery of agents for disruption and inhibition of bacterial biofilm for treatment of periodontal disease |
US7439034B2 (en) | 2004-06-01 | 2008-10-21 | University Of Maryland | Alginases, systems containing alginases and methods of cloning, purifying and/or utilizing alginases |
EP2027259A2 (en) * | 2007-04-30 | 2009-02-25 | University Of Maryland | Carbohydrase expression during degradation of whole plant material by saccharophagus degradans |
WO2009085743A1 (en) * | 2007-12-20 | 2009-07-09 | Danisco Us Inc., Genencor Division | Enzymatic prevention and control of biofilm |
US8173787B2 (en) | 2004-05-04 | 2012-05-08 | University Of Maryland | Plant wall degradative compounds and systems |
US8377681B2 (en) | 1998-01-16 | 2013-02-19 | Codexis Mayflower Holdings, Llc | Evolution of whole cells and organisms by recursive sequence recombination |
US8835139B2 (en) | 2004-05-04 | 2014-09-16 | University Of Maryland | Methods of producing ethanol using hydrolytic enzyme mixtures for saccharification of lignocellulosic polysaccharides |
US9877983B2 (en) | 2007-11-27 | 2018-01-30 | Algipharma As | Use of alginate oligomers in combating biofilms |
CN110438035A (en) * | 2019-07-08 | 2019-11-12 | 威海银河生物技术有限公司 | It is a kind of produce protease Georgia sea bacillus and its application |
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CN104388411B (en) * | 2014-12-03 | 2017-02-22 | 福州大学 | Agarase as well as gene and application thereof |
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US8377681B2 (en) | 1998-01-16 | 2013-02-19 | Codexis Mayflower Holdings, Llc | Evolution of whole cells and organisms by recursive sequence recombination |
WO2001049255A2 (en) * | 1999-12-30 | 2001-07-12 | University Of Louisville Research Foundation, Inc. | Methods and compositions for inhibiting adhesion by microorganisms |
WO2001049255A3 (en) * | 1999-12-30 | 2002-02-21 | Univ Louisville Res Found | Methods and compositions for inhibiting adhesion by microorganisms |
US6576226B1 (en) * | 2000-11-17 | 2003-06-10 | Gary R. Jernberg | Local delivery of agents for disruption and inhibition of bacterial biofilm for treatment of periodontal disease |
US6726898B2 (en) | 2000-11-17 | 2004-04-27 | Gary R. Jernberg | Local delivery of agents for disruption and inhibition of bacterial biofilm for treatment of periodontal disease |
US8173787B2 (en) | 2004-05-04 | 2012-05-08 | University Of Maryland | Plant wall degradative compounds and systems |
US8835139B2 (en) | 2004-05-04 | 2014-09-16 | University Of Maryland | Methods of producing ethanol using hydrolytic enzyme mixtures for saccharification of lignocellulosic polysaccharides |
US8541563B2 (en) | 2004-05-04 | 2013-09-24 | University Of Maryland | Plant wall degradative compounds and systems |
US7439034B2 (en) | 2004-06-01 | 2008-10-21 | University Of Maryland | Alginases, systems containing alginases and methods of cloning, purifying and/or utilizing alginases |
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EP2027259A4 (en) * | 2007-04-30 | 2009-06-10 | Univ Maryland | Carbohydrase expression during degradation of whole plant material by saccharophagus degradans |
EP2027259A2 (en) * | 2007-04-30 | 2009-02-25 | University Of Maryland | Carbohydrase expression during degradation of whole plant material by saccharophagus degradans |
US9057081B2 (en) | 2007-04-30 | 2015-06-16 | Aemetis Technologies, Inc. | Carbohydrase expression during degradation of whole plant material by Saccharophagus degradans |
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US10624920B2 (en) | 2007-11-27 | 2020-04-21 | Algipharma As | Use of alginate oligomers in combating biofilms |
WO2009085743A1 (en) * | 2007-12-20 | 2009-07-09 | Danisco Us Inc., Genencor Division | Enzymatic prevention and control of biofilm |
US8597927B2 (en) | 2007-12-20 | 2013-12-03 | Danisco Us Inc. | Enzymatic prevention and control of biofilm |
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