Compositions and methods for inhibiting biofilm deposition and production - Patents.com

Abstract

To provide a composition and a method for inhibiting deposition and production of a biofilm.SOLUTION: Provided is a method for decomposing and / or removing biofilm includes bringing a biofilm into contact with a composition including an effective amount of a combination of anti-microorganism peptide preferably in the form of fusion protein and biofilm enzyme. The biofilm can be present on an animate surface or on an inanimate surface, and both medical and non-medical applications and methods are provided. In one aspect, provided is a composition to be used in treatment or prevention of biofilm infection in a subject (particularly oral cavity).SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 335,650, filed May 12, 2016, the entire disclosure of which is incorporated by reference herein as if fully set forth.

[0002] This invention was made with Government support under Grant Nos. R01 HL107904 and R01 HL109442 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present invention relates to the field of biofilm deposition and disease treatment. More specifically, the present invention provides compositions and methods useful for treating dental caries and other oral diseases. The present invention also provides a method of coating a biomedical device to inhibit unwanted biofilm deposition on the biomedical device. [Background technology]

[0004] Several publications and patent documents are cited throughout this specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as if fully set forth.

[0005] Biologics produced by the current system are prohibitively expensive and unaffordable for the majority of the world's population. The cost of protein drugs ($140 billion in 2013) exceeds the GDP of over 75% of countries worldwide [Walsh 2014], making them unaffordable. One-third of the world's population earns less than $2 per day and cannot afford any protein drugs (e.g., disadvantaged, elderly and lower socioeconomic groups in the United States). Such high costs are associated with prohibitively expensive protein production in fermenters, purification, transportation / storage at low temperatures, short shelf life and sterile delivery methods [Daniell et al 2015,2016].

[0006] Biofilms are formed by a complex population of microbial cells that adhere to an exopolysaccharide matrix present on the surface of a medical device. Biofilm-associated infections associated with the implantation of a medical device cause serious problems and negatively affect the function of the device. Medical implants used in oral and orthopedic surgery are manufactured using alloys such as stainless steel and titanium. Since bacterial adhesion is often associated with damage to the surrounding tissues and leads to the failure of the medical implant, surface treatment of medical implants by various physical and chemical techniques has been attempted to improve the surface properties, promote biointegration, and inhibit bacterial adhesion.

[0007] Many infectious diseases in humans are caused by biofilms, e.g., those occurring in the oral cavity [Hall-Stoodley et al., 2004; Marsh, et al 2011]. For example, dental caries (or dental caries) remains the single most prevalent biofilm-associated oral disease, primarily affecting underprivileged children and adults in the United States and worldwide, resulting in over $81 billion in expenditures annually [Beiker and Flemmig, 2011; Dye et al., 2015; Kassebaum et al, 2015]. Cavity-causing (cariogenic) biofilms develop when bacteria accumulate on tooth surfaces, forming organized communities of bacterial cells that eventually become sticky and entangled in an extracellular matrix composed of polymeric substances such as exopolysaccharides (EPS) [Bowen and Koo, 2011]. Current topical antimicrobial therapies to control cariogenic biofilms are limited. Chlorhexidine (CHX) is considered the "gold standard" of oral antimicrobial therapy, but has adverse side effects such as tooth staining and calculus formation and is not recommended for daily therapeutic use [Jones, 1997; Autio-Gold, 2008]. Alternatively, several antimicrobial peptides (AMPs) have emerged with potential anti-biofilm effects against cariogenic oral pathogens (e.g., Streptococcus mutans) [da Silva et al., 2012; Guo et al., 2015].

[0008] Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune response and are found naturally in various organisms, including humans. Compared to conventional antibiotics, resistance to AMPs is less likely to develop. AMPs are highly active against bacteria, fungi, and viruses and can be adapted to target specific pathogens by fusion with their surface antigens (Lee et al., 2011; DeGray et al., 2001; Gupta et al., 2015). Linear AMPs have lower stability and antimicrobial activity compared to AMPs with complex secondary structures. For example, retrocyclins or protegrins have high antimicrobial activity or stability when cyclized (Wang et al., 2003) or when they form hairpin structures through disulfide bond formation (Chen et al., 2000). RC101 is highly stable at pH 3, 4, 7 and temperatures 25°C-37°C as well as in human vaginal fluid over 48 hours (Sassi et al., 2011a), and at the same time, the antimicrobial activity of this RC101 was maintained for up to 6 months (Sassi et al., 2011b). Similarly, protegrins are highly stable in salt or in human body fluids (Lai et al., 2002; Ma et al., 2015), but lose potency when linearized. These interesting features of antimicrobial peptides with complex secondary structures may facilitate the development of novel therapeutic agents. However, the high cost of producing sufficient amounts of antimicrobial peptides is the main barrier to their clinical development and commercialization. Summary of the Invention [Means for solving the problem]

[0009] According to the present invention, there is provided a multi-component composition comprising at least one antimicrobial peptide (AMP) and at least one biofilm degrading enzyme that act synergistically to degrade biofilm structures and inhibit biofilm deposition. In certain embodiments, the AMP is selected from protegrin 1, RC-101, and the AMPs listed in Table 1. The biofilm degrading enzymes include, for example, mutanase, dextranase, glucoamylase, deoxyribonuclease I, DNAase, dispersin B, glycoside hydrolase, and the enzymes listed in Table 2. In certain embodiments, the coding sequences of these enzymes are codon-optimized for expression in plant chloroplasts. In particularly preferred embodiments, the at least one AMP and the at least one biofilm degrading enzyme are recombinantly produced. In particularly preferred embodiments, the AMP and the biofilm degrading enzyme are expressed as a fusion protein. When the composition is for treating oral diseases, the composition optionally further comprises an antibiotic, a fluoride, CHX, or all of the above. The composition may be included in chewing gum, hard candy or oral rinse. Preferred fusion proteins of the present invention include, but are not limited to, PG-1-Mut, PG-1-Dex, PG-1-Mut-Dex, RC-101-Mut, RC-101-Dex, RC-101-Mut-Dex for use alone or in combination to degrade biofilms. In particular, any of the AMPs listed in Table 1 may replace either PG-1 or RC-101 in the above-mentioned fusion protein to modify or improve the bactericidal action of the fusion protein. To modify the degradative activity of the fusion protein, the enzymes listed above and herein below may replace Mut, Dex or both in the fusion protein of the present invention. In another embodiment, when two different EPS enzymes are used in the composition, such enzymes may be delivered in various ratios (e.g., 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, etc.).When Mut and Dex are delivered together, for example in a gum or mouth rinse, the preferred ratio is 5:1 Dex:Mut.

[0010] In another aspect, the present invention provides a method for disintegrating and / or removing biofilm, comprising contacting a surface containing the biofilm with the composition described above, which has a bactericidal effect and reduces or removes the biofilm containing one or more undesirable microorganisms, and the biofilm is present in or on an animal subject in need of the reduction or removal. In certain embodiments, the biofilm is present in the oral cavity. In other embodiments, the biofilm is present on an implanted medical device. This method can also be used to remove biofilms present on internal or external surfaces of the body selected from the group consisting of surfaces in the urinary tract, the middle ear, the prostate, the lining of blood vessels, heart valves, skin, scalp, nails, teeth, and inside wounds.

[0011] In yet another embodiment, the composition of the present invention, comprising said at least one AMP and said at least one biofilm degrading enzyme, is produced in plant plastid.The plant may be a tobacco plant, and said AMP and enzyme coding sequences are codon-optimized for expression in plant plastid.In a preferred embodiment, said AMP and biofilm degrading enzyme are expressed in lettuce plant as fusion protein under the control of endogenous regulatory elements present in lettuce plastid. [Brief description of the drawings]

[0012] [Figure 1](Figure 1A-1D) Purification of GFP-fused retrocyclin (RC101) and GFP-fused protegrin (PG1) expressed in tobacco chloroplasts. Figure 1A. Western blot analysis of purified GFP-RC101 fusion using anti-GFP antibody. Figure 1B. Natural fluorescence gel of purified GFP-RC101 fusion. Figure 1C. Western blot of purified GFP-PG1 fusion using anti-GFP antibody. Figure 1D. Natural fluorescence gel of purified GFP-PG1. Note - All samples in Figure 1A-1D were loaded based on the total protein value obtained from the Bradford method. Densitometry using Image J software was performed to determine GFP concentration expression level, purity and yield. Expression level and yield were calculated from GFP concentration relative to total protein value. Yield was determined by multiplying GFP concentration by recovery value after purification. Individual peptide yield was determined by dividing GFP yield by molar concentration factor 14 (ratio of GFP MW to peptide MW). Fold enrichment was calculated by dividing the % purity by the % expression in the crude plant extract. [Figure 2-1] (Fig. 2A-2E) Graphs showing the antimicrobial activity of AMPs (GFP-PG1 and GFP-RC101) against Streptococcus mutans and other oral microorganisms. Cell viability was determined by absorbance (A600nm) and colony forming unit (CFU) counting over time. (Fig. 2A) Time-death curves (A600nm) of S. mutans treated with various concentrations of GFP-PG1 and synthetic PG1. (Fig. 2B) Viable cells (CFU / ml) of S. mutans treated with GFP-PG1 and synthetic PG1 at each time point. (Fig. 2C) Time-death curves (A600nm) of S. mutans treated with various concentrations of GFP-RC101. (Fig. 2D) Viable cells (CFU / ml) of S. mutans treated with GFP-RC101 at various time points. (Fig. 2E) Viable cells (CFU / ml) of S. gordonii, A. naeslundii, and C. albicans treated with GFP-PG1 at 10 μg / ml for 1 and 2 hours. [Figure 2-2] (Fig. 2A-2E) Graphs showing the antimicrobial activity of AMPs (GFP-PG1 and GFP-RC101) against Streptococcus mutans and other oral microorganisms. Cell viability was determined by absorbance (A600nm) and colony forming unit (CFU) counting over time. (Fig. 2A) Time-death curves (A600nm) of S. mutans treated with various concentrations of GFP-PG1 and synthetic PG1. (Fig. 2B) Viable cells (CFU / ml) of S. mutans treated with GFP-PG1 and synthetic PG1 at each time point. (Fig. 2C) Time-death curves (A600nm) of S. mutans treated with various concentrations of GFP-RC101. (Fig. 2D) Viable cells (CFU / ml) of S. mutans treated with GFP-RC101 at various time points. (Fig. 2E) Viable cells (CFU / ml) of S. gordonii, A. naeslundii, and C. albicans treated with GFP-PG1 at 10 μg / ml for 1 and 2 hours. [Diagram 3] (Figures 3A-3C) Bacterial killing by GFP-PG1 as determined by confocal fluorescence and SEM imaging. (Figure 3A) Time-lapse killing of S. mutans treated with GFP-PG1 at 10 μg / ml. (Figure 3B) Control group consisted of S. mutans cells treated with buffer only. Propidium iodide (PI) (red) was used in confocal microscopy to measure bacterial viability over time at the single cell level. PI is cell impermeant and enters only cells with compromised membranes, and dying and dead cells show bright red fluorescence upon binding of PI to DNA. GFP-PG1 is shown in green. (Figure 3C) Morphological observation of S. mutans exposed to GFP-PG1 at a concentration of 10 μg / ml for 1 hour using scanning electron microscopy. Red arrows indicate the recessed membrane and release of intracellular components. [Figure 4](Figure 4A-4C) Inhibition of biofilm formation by a single topical treatment of GFP-PG1. This figure shows representative images of three-dimensional (3D) rendering of S. mutans biofilms. Bacterial cells were stained with SYTO 9 (green) and EPS was labeled with Alexa Fluor 647 (red). Saliva-coated hydroxyapatite (sHA) disk surfaces were treated with a single topical treatment of GFP-PG1 by a short exposure of 30 min (Figure 4B). The control group (Figure 4A) was treated with buffer only. The treated sHA disks were then transferred to culture medium containing 1% (wt / vol) sucrose and actively growing S. mutans cells (105 cfu / ml) and incubated at 37 °C, 5% CO2 for 19 h. After biofilm growth, the biofilms were analyzed by two-photon confocal microscopy. (Figure 4C) Quantitative analysis of the percentage of viable and dead S. mutans cells by quantitative PCR (qPCR) with or without propidium monoazide (PAM) treatment (Klein et al., 2012). Viable cells with intact membranes are quantified by a combination of PMA and qPCR (PMA-qPCR). Prior to genomic DNA isolation and qPCR quantification, PMA is added to selectively crosslink to the DNA of dead cells, thereby preventing PCR amplification (Klein et al., 2012). Asterisks indicate that values ​​from GFP-PG1 treatment are significantly different from the control (P<0.05). [Diagram 5] Figure 1. EPS-degrading enzymes digesting biofilm matrix. Representative time-lapse images of EPS degradation in S. mutans biofilms treated with a combination of dextranase and mutanase. Bacterial cells were stained with SYTO 9 (green) and EPS was labeled with Alexa Fluor 647 (red). White arrows indicate the "opening" of the space between the bacterial cell population and the "uncoated" cells after enzymatic degradation of EPS. [Figure 6](Figures 6A-6C) Graphs showing biofilm destruction by synthetic PG1 alone or in combination with EPS-degrading enzymes. (Figure 6A) Time-lapse quantification of EPS degradation in intact biofilms using COMSTAT. (Figure 6B) Viability of S. mutans biofilms treated with synthetic PG1 and EPS-degrading enzymes (Dex / Mut), either alone or in combination, by ImageJ. (Figure 6C) Anti-biofilm activity of synthetic PG1 was enhanced by EPS-degrading enzymes (Dex / Mut). Asterisks indicate that values ​​of various experimental groups are significantly different from each other (P<0.05). [Figure 7]Confocal microscopy shows in vitro uptake of purified fusion proteins CTB-GFP, PTD-GFP, protegrin-1-GFP (PG1-GFP) and retrocyclin 101-GFP (RC101-GFP) in different human periodontal cell lines: human periodontal ligament stem cells (HPDLS), maxillary mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (SCC), gingival-derived mesenchymal stromal cells (GMSC), adult gingival keratinocytes (AGK) and osteoblasts (OBC). 2x104 cells of human cell lines HPDLS, MMS, SCC, GMSC, AGK and OBC were cultured overnight at 37°C in 8-well chamber slides (Nunc) and incubated with the following purified GFP fusion proteins: CTB-GFP (8.8μg), PTD-GFP (13μg), PG1-GFP (1.2μg), RC101-GFP (17.3μg) in 100μl PBS supplemented with 1% FBS for 1h at 37°C. After fixation with 2% paraformaldehyde for 10min at RT and washing three times with PBS, cells were stained with antifade mounting medium containing DAPI. For negative control, cells were incubated with commercial GFP (2μg) in PBS containing 1% FBS and then treated under the same conditions. All fixed cells were imaged using a confocal microscope. Green fluorescence indicates GFP expression, and blue fluorescence indicates DAPI-labeled cell nuclei. Images were observed with a 100x objective lens, and at least 10-15 GFP-positive cells or images were observed for each cell lineage. Scale bars represent 10 µm. All image studies were analyzed in triplicate. [Figure 8] FIG. 1D: Downstream processing of GFP fusions from transplastomic tobacco: Flow diagram showing the various steps involved in the production of purified GFP fusions from greenhouse-grown transplastomic tobacco plants. [Figure 9-1](FIGS. 9A-9B) Vector and codon-optimized sequences for mutanase (FIG. 9A) and dextranase (FIG. 9B). Codon-optimized mutanase: SEQ ID NO: 1. Codon-optimized dextranase: SEQ ID NO: 2. [Figure 9-2] (FIGS. 9A-9B) Vector and codon-optimized sequences for mutanase (FIG. 9A) and dextranase (FIG. 9B). Codon-optimized mutanase: SEQ ID NO: 1. Codon-optimized dextranase: SEQ ID NO: 2. [Figure 10] FIG. 10 is a schematic diagram showing chloroplast vectors expressing tandem repeats of AMP fused to GVGVP for use alone or to express fusion proteins with the EPS protein of FIG. [Figure 11] FIG. 1 shows a novel purification strategy: the reverse temperature cycling purification process shows high yields. [Figure 12-1] (FIGS. 12A-12B) Expression of functional codon-optimized mutanase in E. coli. FIG. 12 shows a Western blot showing mutanase expression in E. coli. FIG. 12B shows E. coli spread on a 0.5% blue dextran plate. The transformed clones can produce recombinant dextranase normally made in S. mutans, and can eliminate the blue halo around the colonies. FIG. 12C shows a gel diffusion assay comparing the degradative activity of recombinant dextranase present in crude lysate (total protein loading) from transformed E. coli against blue dextrin with commercially purified enzyme from penicillin. [Figure 12-2](FIGS. 12A-12B) Expression of functional codon-optimized mutanase in E. coli. FIG. 12 shows a Western blot showing mutanase expression in E. coli. FIG. 12B shows E. coli spread on a 0.5% blue dextran plate. The transformed clones can produce recombinant dextranase normally made in S. mutans, and can eliminate the blue halo around the colonies. FIG. 12C shows a gel diffusion assay comparing the degradative activity of recombinant dextranase present in crude lysate (total protein loading) from transformed E. coli against blue dextrin with commercially purified enzyme from penicillin. [Figure 13] FIG. 1 is a flow diagram showing the process of engineering lettuce plants for the production of AMP / biofilm degrading enzymes. [Figure 14] Figure 1 shows a chewing gum tablet preparation, where GFP is exemplified, but chewing gum containing AMP-enzyme fusion proteins (such as those shown in Figures 9 and 10) are also within the scope of the invention. [Figure 15] Figure 1: Gum tablets were assessed by fluorescence and confirmed the concentration of GFP by Western Blot. (i) Western Blotting (ii) Quantification of GFP release from chewing gum based on fluorometer (Fluoroskan Ascent™ Microplate Fluorometer-Thermo; λex 485 nm; λem 538 nm). Commercially available GFP (Vector Laboratories, Cat. No. MB-0752) was used as standard. Chewing gum was ground in protein extraction buffer. [Figure 16] FIG. 1 shows a chewing simulator that uses artificial saliva to evaluate the release kinetics of a biofilm disintegrating agent from a gum tablet of the present invention. [Figure 17]Quantification of GFP released from chewing gum. Gum tablets containing increasing concentrations of GFP expressed in lettuce leaves were evaluated in a chewing simulator in the presence of artificial saliva to determine GFP release kinetics. [Figure 18] Figure 1 shows that crude extracts containing enzymes expressed from chloroplast vectors are as effective in inhibiting CFU formation as commercial enzymes when mixed with Listerine. Enzymatic degradation of S. mutans biofilms in vitro using mutanase and dextranase from E. coli (ratio 1:5) supplemented with Listerine. Commercial mutanase (from Baciollus species, Amano) and dextranase (from Penicillium species, Sigma) were used as positive controls, and crude E. coli extract served as negative control. CFU / ml expressed as mean ± standard deviation (n=2). ***, P<0.001 vs. E. coli extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Many infectious diseases in humans, such as those occurring in the oral cavity (e.g., dental caries and periodontal disease), are caused by pathogenic biofilms. Dental caries (or tooth decay) remains the single most costly and widespread biofilm-associated oral disease throughout the United States and worldwide. Dental caries afflicts children and adults alike and is the leading reason for emergency room visits leading to absenteeism from work and school. Unfortunately, the prevalence of dental caries remains high (>90% of the U.S. adult population) and dental caries remains the most common chronic disease afflicting children and adolescents, especially those from poor socio-economic backgrounds. Furthermore, poor oral health often leads to systemic consequences that affect overall health. Importantly, the cost of treating the destruction caused by this disease (e.g., caries pathogens and dental pulp infections) exceeds $40 billion / year in the United States alone. Fluoride is central to caries prevention. However, this widespread use of fluoride results in an incomplete defense against this disease. Although fluoride is effective in reducing demineralization and enhancing demineralization of early caries lesions, it has limited effect on biofilms. Conversely, current antimicrobial therapies are largely ineffective in controlling caries-causing biofilms.

[0014] There is an urgent need to develop effective therapeutics to control pathogenic oral biofilms. The present invention provides methods for low-cost production and delivery of therapeutically effective plant-expressed biologics that are superior to current anti-biofilm / anti-caries therapeutics.

[0015] Definition: As used herein, antimicrobial peptides are small peptides with any bacterial activity. "RC-101" is a retrocyclin analog (cyclic octadecapeptide) that can protect human CD4+ cells from infection by T-tropic and M-tropic strains of HIV-1 in vitro and can prevent HIV-1 infection in human cervicovaginal tissue. RC-101's ability to prevent HIV-1 infection and retain full activity in the presence of vaginal fluid makes it a good candidate for other topical microbicidal applications, especially in oral biofilms. The sequence of RC-101 is described in Plant Biotechnol J. 2011 Jan;9(1):100-115 (incorporated herein by reference).

[0016] "C16G2" is a novel synthetic antimicrobial peptide with specificity against S. mutans.

[0017] "Protegrin-1 (PG)" is a cysteine-rich 18-residue β-sheet peptide. Protegrin-1 has strong antimicrobial activity against a wide range of microorganisms, including bacteria, fungi, viruses, and especially some clinically relevant antibiotic-resistant bacteria. For example, the bacterial pathogen E. coli and the opportunistic fungus C. albicans are effectively killed by PG in laboratory tests. The sequence of PG-1 is described in Plant Biotechnol J. 2011 Jan;9(1):100-115 (incorporated herein by reference).

[0018] Further antimicrobial peptides include those listed in Table 1 below.

[0019] [Table 1]

[0020] "Biofilms" are complex structures that adhere to surfaces that regularly come into contact with water and consist of colonies of bacteria and usually other microorganisms (e.g. yeasts, fungi and protozoa) that secrete a slimy protective coating that encases them. Biofilms can occur on solid or liquid surfaces as well as on soft tissues within living organisms and are typically resistant to conventional disinfection methods. Dental plaque, the slimy coatings that clog pipes and tanks, and algal mats on bodies of water are examples of biofilms. Biofilms are generally pathogenic in the body, causing diseases such as dental caries, cystic fibrosis and otitis media.

[0021] "Biofilm degrading enzymes" include, but are not limited to, exopolysaccharide degrading enzymes such as dextranase, mutanase, DNAse, endonuclease, deoxyribonuclease I, disperdin B, and glycoside hydrolases such as 1→3)-α-D-glucan hydrolase, although the use of chloroplast codon-optimized sequences encoding dextranase and mutanase is preferred, the skilled artisan will be well aware of the many different biofilm degrading enzymes in the art. Additional enzyme sequences for use in the fusion proteins of the present invention are provided below.

[0022] As used herein, the term "administering" or "administration" of an agent, drug or peptide to a subject includes any route of introduction or delivery of a compound to a subject that performs the intended function of the compound. This administering or administration can be performed by any suitable route, such as oral, topical, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), or rectal. Administering or administration includes administration by oneself and administration by another.

[0023] As used herein, the term "disease," "disorder," or "complication" refers to any deviation from a normal state in a subject.

[0024] As used herein, the terms "effective amount," "amount effective," or the like, mean an amount effective, at dosages and for periods of time necessary to achieve the desired result.

[0025] As used herein, the terms "inhibit" or "prevent" refer to not worsening or developing clinical symptoms of a disease state (e.g., inhibiting the onset of the disease) in a subject who may be exposed to or afflicted by the disease state but who has not yet experienced or exhibited symptoms of the disease state.

[0026] As used herein, the term "expression" in the context of a gene or polynucleotide includes transcription of the gene or polynucleotide into RNA. The term may also, but does not necessarily, include the subsequent translation of the RNA into a polypeptide chain and assembly of the polypeptide chain into a protein.

[0027] Plant residues may include one or more molecules (e.g., but not limited to, proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant components, etc.) derived from the plant in which the protein of interest was expressed. Thus, compositions of whole plant material (e.g., whole or parts of plant leaves, stems, fruits, etc.) or crude plant extracts should certainly contain high concentrations of plant residues, as should compositions that include purified proteins of interest having one or more detectable plant residues. In a specific embodiment, the plant residue is rubisco.

[0028] In another embodiment, the present invention relates to an administrable composition for the treatment or prevention of biofilm formation in situ (e.g., in the oral cavity) and on biomedical devices useful for surgical implantation (e.g., stents, artificial joints, and the like). In this embodiment, the device is coated with a composition for inhibiting unwanted biofilm deposition on the device. The composition comprises a therapeutically effective amount of one or more antimicrobial peptides (AMPs) in combination with one or more enzymes having biofilm degrading activity, each of which is expressed by plants and plant residues and acts synergistically to degrade the biofilm. In certain embodiments, the AMPs and enzymes are expressed from separate plastid transformation vectors. In other embodiments, a plasmid transformation vector containing a polycistronic coding sequence in which both the AMPs and enzymes are expressed from a single vector.

[0029] Proteins expressed according to certain embodiments taught herein can be used in vivo by administration to a subject (human or animal) in a variety of ways: The pharmaceutical compositions can be administered orally, topically, subcutaneously, intramuscularly or intravenously, although oral topical administration is preferred.

[0030] Oral compositions made according to embodiments of the present invention can be administered by ingestion of food produced from transgenic plants producing therapeutic proteins from plastids. The edible parts of the plants or parts thereof are used as dietary components. The therapeutic compositions can be formulated in a classical manner using solid or liquid vehicles, diluents and additives suitable for the desired mode of administration. Orally, the compositions can be administered in the form of tablets, capsules, granules, powders, gums and the like, with at least one vehicle (e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc.). The therapeutic protein of interest can be optionally purified from plant homogenates. The preparation can be emulsified. The active ingredient is often mixed with additives that are pharma- ceutically acceptable and compatible with the active ingredient. Suitable additives are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if necessary, the compositions can contain minor amounts of auxiliary substances (e.g., wetting agents, emulsifiers, pH buffers, or adjuvants). In a preferred embodiment, the edible plants, juice, grains, leaves, tubers, stems, seeds, roots or other plant parts of the transgenic plants for drug production are ingested by humans or animals, thereby providing a very inexpensive means of treating disease.

[0031] In a particular embodiment, plant material (e.g., lettuce material) containing AMP-expressing chloroplasts, biofilm-degrading enzymes, and combinations thereof, is homogenized and encapsulated. In a particular embodiment, an extract of the lettuce material is encapsulated. In an alternative embodiment, the lettuce material is powdered prior to encapsulation. As previously mentioned, the biofilm-degrading protein may be purified from the plant after expression.

[0032] In an alternative embodiment, for convenience of administration, the composition may be provided together with the juice of a transgenic plant. For said purpose, the plant to be transformed is preferably selected from edible plants consisting in particular of tomatoes, carrots and apples, which are usually consumed in the form of juice.

[0033] According to another embodiment, the present invention relates to a transformed chloroplast genome that has been transformed with a vector containing a heterologous gene expressing a combination of peptides disclosed herein.

[0034] Of particular interest are transformed chloroplast genomes that have been transformed with vectors that contain heterologous genes expressing one or more AMPs and biofilm degrading enzymes or a combination thereof. In a related embodiment, the present invention relates to a plant comprising at least one cell that has been transformed to express the peptides disclosed herein.

[0035] Reference to gene sequence herein refers to single-stranded or double-stranded nucleic acid sequences, including the coding sequence of a polypeptide of interest or the complement of this coding sequence. Degenerate nucleic acid sequences encoding polypeptides, as well as homologous nucleotide sequences that are at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96 or 98% identical to cDNA, may be used according to the polynucleotide teachings herein. The percent sequence identity between two polynucleotide sequences is determined using a computer program such as ALIGN using the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs and variants of nucleic acid sequences encoding biologically active polypeptides are also useful polynucleotides.

[0036] Variants and homologs of the nucleic acid sequences described above are also useful nucleic acid sequences. Typically, homologous polynucleotide sequences can be identified by hybridization of a candidate polynucleotide with a known polynucleotide under stringent conditions, as known in the art. For example, the following washing conditions can be used to determine homologous sequences containing up to about 25-30% base pair mismatches: 2xSSC (0.3M NaCl, 0.03M sodium citrate, pH 7.0), 0.1% SDS, room temperature, twice for 30 minutes each; then 2xSSC, 0.1% SDS, 50°C, once for 30 minutes; then 2xSSC, room temperature, twice for 10 minutes each. More preferably, the homologous nucleic acid strand contains 15-25% base pair mismatches, and even more preferably contains 5-15% base pair mismatches.

[0037] Species homologs of the polynucleotides referred to herein may also be identified by making appropriate probes or primers to screen cDNA expression libraries. It is known that the Tm of double-stranded DNA decreases by 1-1.5°C for every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973)). Nucleotide sequences or their complements that hybridize to a polynucleotide of interest after stringent hybridization and / or washing conditions are also useful polynucleotides. Stringent washing conditions are known and understood in the art and are described, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2001, pp. 211-215 (1973) and in the literature. nd ed., 1989, at pages 9.50-9.51.

[0038] In order to facilitate the practice of the invention, the following materials and methods are provided.

[0039] Microorganisms and growth conditions In this study, Streptococcus mutans UA159 serotype c (ATCC 700610), Actinomyces naeslundii ATCC 12104, Streptococcus gordonii DL-1 and Candida albicans SC5314 were used. These strains were chosen because S. mutans is a well-established pathogenic cariogenic bacterium [Ajdic D et al, 2002]. S. gordonii is a pioneer colonizer of dental biofilms, A. naeslundii is also detected in the early stages of dental biofilm formation, and A. naeslundii may be associated with the development of root caries [Dige I et al, 2009]. C. albicans is a fungal organism that colonizes human mucosal surfaces and is also detected in dental plaque from children with early childhood caries [Hajeshengallis E et al, 2015]. All strains were stored at -80°C in tryptic soy broth containing 20% ​​glycerol. Blood agar plates were used for the culture of S. mutans, S. gordonii, and A. naeslundii. Sabouraud agar plates were used for C. albicans. All these strains were grown to mid-exponential phase in ultrafiltered (10 kDa molecular weight cut-off membrane; Prep / Scale, Millipore, MA) buffered tryptone-yeast extract broth (UFTYE; 2.5% tryptone and 1.5% yeast extract, pH 7.0) containing 1% glucose before use (37°C, 5% CO2).

[0040] Generation of transplastomic lines expressing various tagged GFP fusion proteins Transplastomic plants expressing GFP fused with CTB, PTD, retrocyclin and protegrin were generated as described in previous studies [Limaye et al., 2006; Kwon et al., 2013; Xiao et al., 2016; Lee et al., 2011]. Transplastomic lines expressing GFP fusion proteins were confirmed using Southern blot assay as previously described [Verma et al., 2008]. Similarly, expression of GFP-tagged proteins was confirmed by visualizing green fluorescence from leaves of each construct under UV illumination.

[0041] Purification of GFP-tagged proteins Purification of GFP-fused protegrin-1 (PG1) and retrocyclin (RC101) from transplastomic tobacco was achieved by organic extraction and subsequent hydrophobic chromatography as previously described (Lee et al., 2011). Approximately 0.2-1 gm of freeze-dried leaf material was harvested and reconstituted in 10-20 ml of plant extraction buffer (0.2 M TrisHCl pH 8.0, 0.1 M NaCl, 10 mM EDTA, 0.4 M sucrose, 0.2% TritonX supplemented with 2% phenylmethylsulfonyl fluoride and 1 protease inhibitor cocktail). The resuspension was incubated on ice for 1 hour with homogenization by vortexing every 15 minutes. The homogenate was then sedimented at 75000 g (Beckman LE-80K Optimal Ultracentrifuge) at 4°C for 1 hour to obtain a clear lysate. The lysate was pretreated with 70% saturated ammonium sulfate and 1 / 4 volume of 100% ethanol followed by vigorous shaking for 2 min (Yakhnin et al, 1998). The treated solution was sedimented at 2100 g for 3 min. The upper ethanol phase was collected and the process was repeated with 1 / 16 volume of 100% ethanol. The pooled ethanol phase was further treated with 1 / 3 volume of 5M NaCl and 1 / 4 volume of 1-butanol, homogenized vigorously for 2 min, and sedimented at 2100 g for 3 min. The bottom phase was collected and loaded onto a 7 kDa MWCO Zevaspin desalting column (Thermo scientific) and desalted according to the manufacturer's recommendations.

[0042] The desalted extract was then subjected to hydrophobic interaction chromatography during the capture phase for further purification. The desalted extract was injected into a Toyopearl Butyl-650S hydrophobic interaction column (Tosoh bioscience) running on an FPLC unit (Pharmacia LKB-FPLC system). The column was equilibrated with 2.3 column volumes of salted buffer (10 mM Tris-HCl, 10 mM EDTA and 50% saturated ammonium sulfate) to a final 20% salt saturation to promote the binding of GFP to the resin. Following this, the column was washed with a mixture of salted and unsalted buffer for 5.8 column volumes and then eluted with unsalted buffer (10 mM Tris-HCl, 10 mM EDTA). The GFP fraction was identified and collected based on the peaks observed in the chromatogram. The collected fractions were subjected to a final polishing step by overnight dialysis. After dialysis, the purified protein was lyophilized (labconco freeze dryer) to concentrate the final product, which was then stored at -20°C.

[0043] Quantification of purified GFP fusions Quantification of GFP-RC101 and GFP-PG1 was performed by both Western blot-based and fluorescence-based methods. Lyophilized purified proteins were resuspended in sterile 1x PBS and total protein was determined by Bradford method. Purified proteins were then quantified by SDS-PAGE method by loading denatured protein samples on a 12% SDS gel together with commercial GFP standards (Vector labs), then Western blotting was performed using mouse anti-GFP antibody (Millipore) at 1:3000 dilution, followed by probing with secondary HRP-conjugated goat anti-mouse antibody (Southern biotech) at 1:4000 dilution.

[0044] The purified protein was also quantified using GFP fluorescence. The protein samples were run on a 12% SDS gel under native conditions. After the run, the gel was placed under a UV lamp and then imaged. GFP concentrations in both Western and native fluorescence were determined by densitometric analysis using Image J software with a commercial GFP standard to obtain a standard curve. Purity was determined based on GFP quantification relative to total protein values ​​determined by Bradford method.

[0045] Uptake of purified tagged GFP proteins by human periodontal cell lines Briefly, cells (2 × 10 ) of each human cell line were cultured in various human periodontal cell lines (e.g., human periodontal ligament stem cells (HPDLS), maxillary mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (SCC-1), gingival-derived mesenchymal stromal cells (GMSC), adult gingival keratinocytes (AGK) and osteoblasts (OBC)) to measure the uptake of four tags: CTB, PTD, PG1 and RC101, as previously described (Xiao, et al 2016). 4 ) were cultured overnight at 37°C in 8-well chamber slides (Nunc) and subsequently incubated with the following purified GFP fusion tags: CTB-GFP (8.8 μg), PTD-GFP (13 μg), GFP-PG1 (1.2 μg) and GFP-RC101 (17.3 μg) in 100 μl of PBS supplemented with 1% FBS for 1 h at 37°C. After fixation with 2% paraformaldehyde for 10 min at RT and washing three times with PBS, all cells were stained with antifade mounting medium containing DAPI (Vector laboratories, Inc). For negative controls, cells were incubated with commercial GFP (2 μg) in PBS containing 1% FBS for 1 h at 37°C. All fixed cells were imaged using a confocal microscope. Images were observed with a 100x objective and for each cell line in three independent analyses. At least 10–15 GFP-positive cells were scored.

[0046] Evaluation of antibacterial activity The killing kinetics of AMPs (Gfp-PG1 and Gfp-RC101) against S. mutans was analyzed by time-lapse killing assay. S. mutans were grown to logarithmic phase and incubated for 10 min in growth medium. 5 The concentrations of GFP-PG1 and GFP-RC101 were 0-10μg / ml and 0-80μg / ml, respectively, added to S.mutans suspensions. Samples were taken at 0, 1, 2, 4, 8 and 24 hours, serially diluted in 0.89% NaCl, then spread on agar plates and colonies were counted after 48 hours. The absorbance at 600nm was also checked at each time point. S.gordonii suspension, A.naeslundii suspension and C.albicans suspension were mixed with Gfp-PG1 at a concentration of 10μg / ml and at 0, 1 and 2 hours, aliquots were taken for counting of CFU.

[0047] The effect of AMPs on the viability of S. mutans cells was also evaluated by time-lapse measurements. S. mutans were grown to logarithmic phase and harvested by centrifugation (5500 g, 10 min), the pellet was washed once with sodium phosphate-buffered saline (PBS) (pH 7.2), resuspended in PBS, and diluted with 1 × 10 5The final concentration of CFU / ml was adjusted. GFP-PG1 was added to the S. mutans suspension at a concentration of 10 μg / ml, and 2.5 μM propidium iodide-PI (Molecular Probe Inc., Eugene, OR, USA) was added to label dead cells. 5 μl of the mixture was loaded onto an agarose pad for confocal imaging. Confocal images were acquired using a Leica SP5-FLIM inverted single photon laser scanning microscope equipped with a 100× (numerical aperture, 1.4) oil immersion objective. The excitation wavelengths were 488 nm and 543 nm for GFP and PI, respectively. The emission filters for GFP were 495 / 540 OlyMPFC1 filter and PI was 598 / 628 OlyMPFC2 filter. For time-lapse series, images of the same field were acquired at 0, 10, 30 and 60 min and generated using ImageJ 1.44 (http: / / rsbweb.nih.gov / ij / download.html).

[0048] The morphology of AMP-treated S. mutans was also examined by scanning electron microscopy (SEM). S. mutans was grown to logarithmic phase and diluted with PBS for 10 min. 5 The bacteria suspension was mixed with GFP-PG1 (final concentration of 10 μg / ml) for 1 h at 37°C. After treatment, the bacteria were harvested by filtration using a 0.4 μm Millipore filter. The deposits were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature and processed for SEM (Quanta FEG 250, FEI, Hillsboro, OR) observation. Untreated or buffer-only treated bacteria served as controls.

[0049] Assessment of anti-biofilm activity The well-characterized EPS-matrix-producing oral pathogen S. mutans UA159 was used to form biofilms on saliva-coated hydroxyapatite disk surfaces. Briefly, hydroxyapatite disks (diameter 1.25 cm, surface area 2.7 ± 0.2 cm) were used to form biofilms on saliva-coated hydroxyapatite disks. 2 Filter-sterilized, clarified human whole saliva (sHA) was applied to a 10-well plate (Clarkson, Chromatography Products, Inc., South Williamsport, PA) [Xiao J et al., 2012]. S. mutans was grown to mid-exponential phase in UFTYE medium containing 1% (wt / vol) glucose (37°C, 5% CO2). Each sHA disk was coated with 10 5CFU of actively growing S. mutans cells were seeded and incubated at 37°C and 5% CO2 for 19 hours. Prior to seeding, the sHA disks were locally treated with GFP-PG1 solution (10ug) for 30 minutes. The inhibitory effect of GFP-PG1 treatment on 3D biofilm structure was observed by confocal imaging. Briefly, EPS was labeled using 2.5μM Alexa Fluor 647-labeled dextran conjugate (10kDa; 647 / 668nm; Molecular Probes Inc.), and bacterial cells were stained with 2.5μM SYTO9 (485 / 498nm; Molecular Probes Inc.). Imaging was performed using a Leica SP5 microscope equipped with a 20× (numerical aperture, 1.00) water immersion objective. The excitation wavelength was 780 nm, the emission wavelength filter for SYTO 9 was 495 / 540 OlyMPFEC1 filter, and the filter for Alexa Fluor 647 was HQ655 / 40M-2P filter. Confocal image series were generated by optical sectioning at each selected position, and the step size of z-series scanning was 2 μm. Amira 5.4.1 software (Visage Imaging, San Diego, CA, USA) was used to generate 3D renderings of the biofilm structure [Xiao J et al. 2012, Koo H et al. 2010].

[0050] To investigate the effect of PG1 on biofilms formed by S. mutans on sHA disks for 19 h, we used time-lapse confocal microscopy to examine the 3D structure of the EPS-matrix and in situ cell viability after incubation of biofilms with 1) control, 2) EPS-degrading enzymes only, 3) PG1 only, or 4) PG1 and EPS-degrading enzymes for up to 60 min. The EPS-degrading enzymes used here were dextranase and mutanase, which could digest EPS from S. mutans by hydrolyzing α-1,6 and α-1,3 glucosidic linkages [Hayacibara et al. 2004]. Dextranase produced from Penicillium sp. was purchased commercially from Sigma (St. Louis, MO), and mutanase produced from Trichoderma harzianum was kindly provided by Dr. William H. Bowen (Center for Oral Biology, University of Rochester Medical Center). Dextranase and mutanase were mixed in a 5:1 ratio before application to the biofilms [Mitsue F. Hayacibara et al. 2004]. Alexa Fluor 647-labeled dextran conjugate was used to label the EPS-matrix, and SYTO 9 and PI were used to label live and dead cells. Biofilms were examined using confocal fluorescence imaging at 0, 10, 30, and 60 min and subjected to AMIRA / COMSTAT / ImageJ analysis. The total biomass of EPS matrix, live and dead cells in each series of confocal images was quantified using COMSTAT and ImageJ. The ratio of live to total bacteria at each time point was calculated and the viability of live cells (relative to live cells at 0 min) was plotted. The initial number of live cells at time point 0 min was considered to be 100%. Percent viability was determined by comparison with time point 0 min.

[0051] Microbial assays At selected time points (19 h), biofilms were removed, homogenized by sonication, and subjected to microbial analysis as previously described [Xiao J et al. 2012, Koo H et al. 2010], where our sonication procedure does not kill bacterial cells, and furthermore results in optimal dispersion and maximum recoverable counts. Aliquots of biofilm suspensions were serially diluted and plated on blood agar plates using an automated Eddy Jet Spiral Plater (IUL, SA, Barcelona, ​​Spain). Meanwhile, propidium monoazide (PAM) coupled with quantitative PCR (PMA-qPCR) was used to analyze S. mutans cell viability as described by Klein MI et al. [Klein MI et al. 2012]. The combination of PMA and qCPR quantifies only cells with intact membranes (i.e., viable cells) because PMA crosslinks to dead cell DNA and extracellular DNA, modifying this DNA and inhibiting PCR amplification of the extracted DNA. Briefly, biofilm pellets were resuspended in 500 μl of TE (50 mM Tris, 10 mM EDTA, pH 8.0). Using a pipette, the biofilm suspension was transferred to a 1.5 ml microcentrifuge tube and then mixed with PMA. 1.5 μl of PMA (20 mM in 20% dimethyl sulfoxide; Biotium, Hayward, CA) was added to the biofilm suspension. The tube was incubated in the dark at room temperature for 5 min with occasional mixing. The samples were then exposed to light (600-W halogen light source) for 3 min. After light-induced crosslinking, the biofilm suspension was centrifuged (13,000 g / 10 min / 4° C.) and the supernatant was discarded. The pellet was resuspended in 100 μl of TE and subsequently incubated with 10.9 μl of lysozyme (100 mg / ml stock) and 5 μl of mutanillysin (5 U / μl stock) (37° C. / 30 min). Genomic DNA was then isolated using a MasterPure DNA purification kit (Epicentre Technologies, Madison, WI).Ten pictograms of genomic DNA per sample and a negative control (no DNA) were amplified using the MyiQ real-time PCR detection system with iQ SYBR Green supermix (Bio-Rad Laboratories Inc., CA) and S. mutans specific primers (16S rRNA) [Klein MI et al 2010].

[0052] statistical analysis Data are expressed as mean ± standard deviation (SD). All assays were performed in duplicate in at least two separate experiments. Pairwise comparisons between test and control were performed using Student's t-test. The significance level chosen for all statistical tests in the current study was P<0.05.

[0053] The following examples are provided to illustrate certain embodiments of the invention and are not intended to limit the invention in any way. EXAMPLES

[0054] Example I Generation and characterization of transplastomic lines All fusion tags (CTB, PTD, protegrin, retrocyclin) were fused to green fluorescent protein (smGFP) at the N-terminus to evaluate the efficiency and specificity of these fusion tags. Fusion constructs encoding these fusion proteins were cloned into a chloroplast transformation vector, as described in U.S. Patent Application No. 13 / 101,389, incorporated herein by reference, and then the vector was used to transform plants of interest. To generate plants expressing GFP fusion proteins, tobacco chloroplasts were transformed using a biolistic particle delivery system. As seen in FIG. 1B, each tag fusion GFP is driven by the same regulatory sequence (light-regulated psbA promoter and 5'UTR), and the transcribed mRNA is stabilized by the 3'psbA UTR. The psbA gene is the most highly expressed chloroplast gene, and therefore, our laboratory uses the psbA regulatory sequence for transgene expression [7, 34]. To facilitate integration of the expression cassette into the chloroplast genome, the expression cassette was flanked by two adjacent sequences identical to the native chloroplast genome sequences: the isoleucyl-tRNA synthetase (trnI) gene and the alanyl-tRNA synthetase (trnA) gene. Shoots emerging from the selection medium were examined for specific integration of the transgene cassette at the trnI and trnA spacer regions, and then Southern blot analysis was used to confirm transformation of all chloroplast genomes in each plant cell (lack of untransformed wild-type chloroplast genome). Therefore, stable integration of all GFP expression cassettes and homoplasmy of the chloroplast genome with the transgene were confirmed before extraction of the fusion proteins. In addition, GFP expression was phenotypically confirmed by visualizing green fluorescence under UV light. The confirmed homoplasmic lines were then transferred to an automated greenhouse for cultivation to increase biomass.

[0055] To scale up the biomass of each GFP-tagged plant leaf material, each homoplasmic line was grown in a temperature- and humidity-controlled greenhouse. Fully grown mature leaves were harvested at midnight to maximize the accumulation of GFP fusion proteins driven by light-regulated regulatory sequences. To further increase the fusion protein content on a weight basis, the frozen leaves were freeze-dried at -40 °C under vacuum. In addition to the protein concentration effect, freeze-drying increased the shelf life of therapeutic proteins expressed in plants at room temperature for more than one year [Daniell et al., 2015;2016]. Thus, in this study, freeze-dried and powdered plant cells expressing GFP fusion tagged proteins were used for oral delivery to mice.

[0056] Expression and purification of GFP-fused antimicrobial peptides from transplastomic tobacco Tobacco leaves expressing GFP-fused antimicrobial peptides RC101 and PG1 were harvested from the greenhouse and subsequently freeze-dried for protein extraction and purification. Based on densitometry, the average expression level of GFP-RC101 was found to be 8.8% of the total protein in the crude extracts, and the expression of GFP-PG1 was 3.8% of the total protein. The difference in expression levels was similar to that reported previously (Lee et al. 2011, Gupta et al. 2015).

[0057] Purification of GFP fused to different antimicrobial peptides (RC101 and PG1) was performed to test the bactericidal activity against both planktonic and biofilm-forming S. mutans. Freeze-dried tobacco material expressing different GFP fusions was used for extraction and subsequent downstream processing (see Figure 8) to obtain the finished purified products, which were then quantified to determine the concentration of the GFP fusion peptides. Quantification of purified GFP-RC101 and GFP-PG1 was performed by both Western blot and native GFP fluorescence methods, with purified GFP-RC101 showing an average purity of 94% with an average yield of 1624 μg GFP (116 μg RC101 peptide) per gm of freeze-dried leaf material (Figure 1A and Figure 1B). For GFP-PG1, both methods (Figure 1C and Figure 1D) show an average purity of 17% with an average yield of 58.8 μg GFP (4.2 μg PG1 peptide) per gm of freeze-dried leaf material. The difference in purity may be due to the difference in the type of tag fused to GFP as seen in previous studies (Xiao et al., 2015, Skosyrev et al., 2003). The enrichment fold of purified GFP-RC101 and purified GFP-PG1 from plant extracts was 10.6 and 4.5, respectively. The Western blot also shows a GFP standard at 27 kDa, corresponding to the monomeric fragment, along with a 54 kDa GFP dimer, with loadings ranging from 6 to 8 ng of GFP. In the GFP-RC101 Western blot, fragments of 29 kDa and 58 kDa, corresponding to the monomeric and dimeric forms of the fusion, are clearly visible (Figure 1A). This may be due to the ability of GFP to form dimers (Ohashi et al., 2007). Western blots of GFP-PG1 (Figure 1D) clearly show a 29 kDa monomer along with a 40 KDa fragment that may result from a mobility shift caused by GFP-PG1 bound to other non-specific plant proteins that may be co-purified as previously described (Morassuttia et al., 2002).The native fluorescence of GFP-RC101 and GFP-PG1 (Figure 1B and 1D) showed multimeric bands, some of which were visible below the 27 kDa GFP standard size, which may be due to the fusion of GFP to a cationic peptide that causes an electrophoretic mobility shift in each GFP fragment as described in a previous study (Lee et al., 2011).

[0058] Antibacterial activity of AMPs We first investigated the antimicrobial activity of GFP-PG1 using a dose-response time-kill study as shown in Figure 2 (A-E). GFP-PG1 exhibited potent antimicrobial activity against Streptococcus mutans, a pathogen documented to form biofilms and cause dental caries, rapidly killing this bacterial cell within 1 hour at low concentrations (Figure 2A). GFP-PG1 also killed the early oral colonizers Streptococcus gordonii and Actinomyces naeslundii, but showed limited antifungal activity against Candida albicans at the concentrations tested (Figure 2E). Time-lapse confocal imaging shows that the viability of S. mutans is affected as early as 10 minutes as shown in Figure 3A compared to the untreated control (Figure 3B). SEM imaging revealed disruption of the S. mutans membrane surface, which caused the release of intracellular components and irregular cell morphology, while untreated bacteria showed an intact and smooth surface without any visible cell lysis or debris (Figure 3C). Having demonstrated the antimicrobial efficacy of GFP-PG1 against S. mutans, we investigated the potential of this antimicrobial peptide to prevent biofilm formation or to disrupt already formed biofilms.

[0059] Inhibition of biofilm initiation by AMPs Prevention of pathogenic oral biofilm formation is difficult because drugs need to exert therapeutic effects after topical application. To determine whether GFP-PG1 can interfere with biofilm initiation, we treated saliva-coated apatite (sHA) surfaces (tooth substitutes) with a single topical treatment of GFP-PG1 for 30 minutes and then incubated with actively growing S. mutans cells under cariogenic (sucrose-rich) conditions. We observed substantial impairment of biofilm formation by S. mutans with minimal accumulation of EPS-matrix on GFP-PG1-treated sHA surfaces (Figure 4B and Figure 4C). Most adherent cell populations were nearly nonviable compared to controls (Figure 4A), demonstrating the strong effect of GFP-PG1 on biofilm initiation, despite localized and short-term exposure.

[0060] Destruction of already formed biofilms by AMP with or without EPS-degrading enzymes The destruction of biofilms formed on surfaces is difficult. The destruction of cariogenic biofilms is particularly difficult because drugs often cannot reach the population of pathogenic bacteria (e.g., S. mutans) due to the surrounding exopolysaccharide (EPS)-rich matrix that entangles and protects the population [Bowen and Koo, 2011]. EPS-degrading enzymes (e.g., dextranase and mutanase) may aid in digesting the matrix of cariogenic biofilms. We first optimized dextranase and / or mutanase required for EPS matrix destruction without affecting cell viability (data not shown). As shown in Figure 5, the combination of dextranase and mutanase can digest EPS (red) and the "open space" (see arrows) between bacterial cell populations (green) and "uncoated" cells (see arrows). Therefore, the combination of GFP-PG1 and EPS-degrading enzymes synergistically enhances the overall anti-biofilm effect.

[0061] To investigate this concept, Streptococcus mutans biofilms were preformed on sHA surfaces and locally treated with GFP-PG1 and EPS-degrading enzymes (Dex / Mut) either alone or in combination. Time-lapse confocal imaging and quantitative computational analysis were performed to analyze EPS-matrix degradation and live / dead bacterial cells within the biofilms (Figure 6A). This enzyme-peptide combination degraded more than 60% of the EPS-matrix while increasing bacterial killing when compared to either GFP-PG or Dex / Mut alone. These findings were further validated by standard culture assays by measuring colony forming units. The antibacterial activity of PG against S. mutans biofilms in combination with Dex / Mut was significantly enhanced compared to either agent alone. Local exposure to Dex / Mut alone showed no effect on biofilm cell viability, whereas GFP-PG1 alone showed limited killing activity (Figure 6B). Taken together, the data demonstrate the potential of this combined approach to synergistically enhance the antimicrobial effect of GFP-PG-1 against established biofilms (Figure 6C).

[0062] Uptake of GFP fused to various tags by human periodontal cells Purified GFP fusion proteins showed interesting results when incubated with human cultured cells (e.g., HPDLS, MMS, SCC-1, GMSC, AGK, and osteoblasts (OBC)). Only one representative image for each cell line is presented, but uptake studies were performed in triplicate and at least 10-15 images were recorded under a confocal microscope. Without the fusion tag, GFP did not enter any of the human cell lines tested. Both CTB-GFP and PTD-GFP efficiently entered all cell types tested, although these localization patterns differed. Upon incubation with CTB-GFP, the GFP signal was mainly localized to the periphery of HPDLSC and MMSC, in SSC-1, in AGK, in uniformly small cytoplasmic puncta in OBC, and in large cytoplasmic foci in GMSC. PTD-GFP was observed as small cytoplasmic foci in MMSCs, as cytoplasmic dots of various sizes in HPDLSCs, GMSCs, AGKs, OBCs, and both in the cytoplasm and periphery of SSC-1 cells. PG1-GFP was the most efficient tag in entering all human cells tested, since GFP could be localized at a 10-fold lower concentration compared to any other fusion protein. PG1-GFP showed cytoplasmic localization exclusively in HPDLCS, SCC-1, GMSC, and AGK cells, and both peripheral and cytosolic localization in MMSCs, but only peripherally in OBCs. RC101-GFP was localized in SSC-1, GMSC, AGK, and OBCs, but was only slightly localized in HPDLSCs and undetectable in MMSC cells.

[0063] Discussion and Conclusion The construction of cariogenic oral biofilms is a prime example of how pathogenic bacteria accumulate on surfaces (teeth) as the extracellular EPS matrix develops. Prevention of cariogenic biofilm formation requires the disruption of bacterial accumulation on tooth surfaces by local treatment. Chlorhexidine (CHX) is considered the “gold standard” of local antimicrobial therapy (Flemmig and Beikler 2011; Marsh et al. 2011; Caufield et al. 2001). Although CHX efficiently suppresses mutans streptococci levels in saliva, CHX has adverse side effects such as tooth staining and calculus formation, and is not recommended for daily preventive or therapeutic use (Autio-Gold 2008). As an alternative, several antimicrobial peptides (AMPs) have been developed and tested against oral bacteria and have shown potential effects against biofilms (albeit with reduced effects against planktonic cells) (reviewed by Silva et al., 2012). Unfortunately, most of these studies tested anti-biofilm efficiency using continuous long-term biofilm exposure (hours) to AMPs rather than topical treatment regimes as used clinically. Furthermore, synthetic AMPs are expensive to produce, making them prohibitively expensive for dental applications. Here, we show that plant-produced AMPs exhibit potent effects in controlling biofilm formation by a single, short-term topical treatment of tooth substitute surfaces.

[0064] The developed cariogenic biofilms are characterized by bacteria embedded in an EPS matrix that makes the biofilm very difficult to treat and remove (Paes Leme et al., 2006; Koo et al., 2013). The EPS-rich matrix facilitates the attachment, aggregation and protection of microorganisms as well as impeding diffusion (Koo et al. 2013; Flemming and Wingender 2010). The EPS matrix creates spatial and microenvironmental heterogeneity in the biofilm (locally modulating pathogen growth and protection against antimicrobial agents) and also creates a highly adhesive scaffold that ensures the firm attachment of the biofilm on the tooth surface (Flemming and Wingender 2010; Peterson et al. 2015). CHX is much less effective against established cariogenic biofilms (Hope and Wilson, 2004; Van Strydonck et al. 2012; Xiao et al., 2012). EPS is mainly composed of a mixture of insoluble (high content of α1,3-linked glucose) and soluble (mostly α1,6-linked glucose) glucans (Bowen and Koo 2011). Therefore, the possibility of using EPS-matrix degrading dextranase or mutanase (of fungal origin) to destroy biofilms and prevent dental caries has been explored and is included in commercial over-the-counter mouthwashes (e.g., Biotene PBF). However, topical application of the enzymes alone has produced clinically modest antibiofilm / anti-caries effects (Hull 1980), possibly due to the lack of antibacterial activity and reduced enzyme activity in the oral cavity (Balakrishnan et al. 2000). Interestingly, recent in vitro studies have shown that chimeric glucanases composed of fused dextranase and mutanase are more effective in destroying dental plaque-biofilms compared to either enzyme alone (Jiao et al. 2014). However, an approach combining antimicrobial agents with both EPS-matrix degrading enzymes in a single treatment system has yet to be developed, possibly due to the cost and difficulties associated with formulation.In this study, we demonstrate that PG1 works synergistically and efficiently with matrix-degrading enzymes to destroy cariogenic biofilms. This feasible and effective topical anti-biofilm approach may use antimicrobial peptides combined with EPS-digesting enzymes to simultaneously disrupt the biofilm matrix scaffold and kill embedded bacteria.

[0065] The retention of high levels of antimicrobial activity by protegrin with GFP fusion opens the door to many clinical applications to promote oral health beyond biofilm destruction. In addition to biofilm destruction, enhanced wound healing in gingival tissues is an important clinical need. We recently reported that both protegrin and retrocyclin can enter human mast cells to induce degranulation, a key step in the wound healing process (Gupta et al. 2015). Thus, the antimicrobial peptides protegrin and retrocyclin play a key role in killing bacteria in biofilms and initiate wound healing via mast cell degranulation. In addition, efficient delivery of growth hormones or other proteins is important to enhance cell attachment and stimulate bone formation, angiogenesis, bone regeneration, osteoblast or endothelial cell differentiation. Already identified cell-penetrating peptides have several limitations. CTB enters all cell types via the ubiquitous GM1 receptor, which requires the pentameric form of CTB. On the other hand, PTD does not invade immune cells (Xiao et al., 2016).

[0066] In this study, we tested the ability of PG1-GFP or RC101-GFP to enter periodontal and gingival cells. PG1-GFP was the most efficient tag in entering periodontal or gingival human cells, because the GFP signal could be detected even at a 10-fold lower concentration compared to any other fusion protein. Although there were some changes in the subcellular localization, PG1-GFP efficiently entered HPDLSC, SCC-1, GMSC, AGK, MMSC, and OBC. In contrast, RC101-GFP entered SCC-1, GMSC, AGK, and OBC, but the localization of RC101-GFP in HPDLSC and MMSC cells was poor or undetectable. Thus, this study identifies a novel role for protegerin in the delivery of drugs to osteoblasts, periodontal ligament cells, gingival epithelial cells, or fibroblasts to promote oral health. It is feasible to release protein drugs synthesized in plant cells by mechanical grinding, and protein drugs bioencapsulated in freeze-dried plant cells embedded in chewing gum provide an ideal mode of drug delivery for their slow and sustained release for a longer duration, overcoming the major limitation of current oral rinse formulations (short duration of antimicrobial contact on the gum / tooth surface).

[0067] Beyond topical application, protein drugs fused with protegrin expressed in plant cells can be orally delivered to deeper tissues of the gingiva in a non-invasive manner to increase patient compliance. Protein drugs bioencapsulated in plants can be stored at room temperature for years without losing their potency (Su et al., 2015; Daniell et al., 2016). The high cost of current protein drugs is due to prohibitively expensive fermentation, purification, transportation / storage at low temperatures, short quality assurance periods and sterile delivery methods. All these challenges may be overcome by using this novel drug delivery concept to promote oral health. The recent FDA approval of plant cells for the production of protein drugs (Walsh 2014) bodes well for the clinical advancement of this novel concept.

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[0069] Example II Construction of chloroplast vectors expressing AMPs, biofilm-degrading enzymes, and their fusion proteins Effective treatment of biofilm-associated infections is problematic because antimicrobial agents often do not reach the microbial population that resides within the surrounding extracellular matrix that enmeshes and protects it. Furthermore, the development of novel therapies for biofilm-associated oral diseases and the maintenance of oral health needs to be cost-effective and readily available.

[0070] To ensure a continuous supply of reagents, dextranase / mutanase and protegrin / retrocyclin are expressed independently and as fusion proteins in the chloroplasts of tobacco and other plants (e.g., lettuce). Proteins will be produced and used in low-cost purification strategies. Tobacco plants produce millions of seeds, making it feasible to scale up production easily. Up to 40 metric tons of biomass can be produced from one acre of tobacco, facilitating low-cost, large-scale production of AMPs, enzymes, and the fusion constructs that encode them. In another approach, these proteins are produced in edible plants such as lettuce.

[0071] Several dextranases (Dex) and mutanases (Mut) have been isolated from fungi and bacteria and characterized for their enzymatic activity. Optimal dextranase and mutanase enzymes should have enzymatic properties suitable for the human oral environment. Based on short-term oral treatments, strong plaque-biofilm binding / retention properties and catalytic activity for both types of EPS (dextran and mutan) are highly desirable. The enzymes added to commercial dextranase-containing mouthwashes (e.g., Biotene) are mainly derived from fungi (Penicillium species and Chaetomium erraticum). However, fungal dextranase shows a higher temperature optimum (50-60°C) compared to bacterial dextranase (35-40°C). Moreover, bacterial dextranase is more stable and more effective at oral temperature (approximately 37°C), making it suitable for caries prevention. Recently, dextranase from Arthrobacter sp. strain Arth410 has shown superior dextran degradation properties at the optimal temperatures (35-45°C) and pH values ​​(pH 5-7) found in the oral cavity and in cariogenic biofilms when compared to fungal dextranase. In addition, topical application of bacterial dextranase is more effective in reducing caries in vivo compared to fungal dextranase. Similarly, bacterial mutanase from Paenibacillus sp. strain RM1 has shown that following an initial incubation with biofilms for 3 min, the biofilms were effectively degraded by incubation for 6 h, even after removal of the mutanase. In addition, RM1 mutanase exhibits enhanced biofilm degrading properties when compared to other microbial species. Notably, the fungal enzyme requires glycosylation, which prevents the expression of the fungal enzyme in chloroplasts. In addition, the immunogenicity of glycoproteins in the human system may raise further regulatory concerns. Thus, the present invention includes the use of bacterial dextranase and bacterial mutanase for expression in chloroplasts.

[0072] To increase production of the Arth410 dextranase and RM1 mutanase proteins in the chloroplast, both sequences have been codon-optimized for chloroplast expression, see Figures 9A and 9B.

[0073] Retrocyclins and protegrin. To maximize synthesis of AMP and reduce toxicity, ten tandem repeats of PG1 or RC101 are used, separated by protease cleavage sites, as shown in FIG. 10. For each copy of the expressed gene, ten functional copies of PG1 or RC101 are made. For this purpose, we have chosen the Tobacco Etch Virus (TEV) protease, which has high specificity and a short cleavage site of seven amino acids. Alternatively, a furin cleavage site may be used. The vector may also be engineered to contain a nucleic acid encoding a biofilm-degrading enzyme. This coding region may be expressed under the promoter utilized to express the AMP, or may be linked to a vector operably linked to a second promoter region. The biofilm-degrading enzyme coding sequence may also contain a TEV protease cleavage site to facilitate release of the enzyme. This approach offers a safer and cleaner alternative to chemical cleavage methods. Most importantly, the individual PG1 peptides in the fusion protein do not form secondary structures prior to cleavage, thus avoiding accumulation of functional peptides that may be lethal to the host production system. Using the methods disclosed in Example I, the antimicrobial activity of cleaved PG1 / RC101, biofilm-degrading enzymes or these fusion proteins can be used to degrade biofilms.

[0074] As mentioned above, the sequence encoding the AMP / bifilm degrading enzyme is optionally codon optimized before insertion into a chloroplast transformation vector such as pLD. Chloroplast transformation relies on double homologous recombination events. Thus, the chloroplast vector contains regions of homology to the chloroplast genome flanking the expression cassette encoding the heterologous protein of interest, which facilitates the insertion of the transgene cassette into the intergenic spacer region of the chloroplast genome without disrupting any functional genes. While any intergenic spacer region may be used to insert the transgene, the most commonly used site for transgene integration is the transcriptionally active intergenic region between the trnI-trnA genes (in the rrn operon), located within the IR region of the chloroplast genome (Figure 10). Due to the similar protein synthesis mechanism between E. coli and chloroplasts, the efficiency of codon optimization can also be evaluated in E. coli and then plants can be generated. Both systems have the potential to be used for expression of AMPs, biofilm-degrading enzymes or their fusion proteins as well as purification and evaluation of AMP or enzyme activity.

[0075] Refining Strategy A hydrophobic interaction column (HIC; TOSOH Butyl Toyopearl 650m) can be used to purify PG1 fused with green fluorescent protein (GFP). GFP selectively binds to HIC and promotes Rc101 / PG1 to a purity of >90%. Despite the use of expensive HIC chromatography methods, the recovery is very poor (<20%). To address this issue and increase the yield, 10 tandem repeats of PG1 with an elastin-like biopolymer (GVGVP (SEQ ID NO: 11); Figure 10) are engineered into a vector. This biopolymer has unique thermal properties such that it precipitates from solution when the temperature is increased above the inverse transition temperature (Tt) of this biopolymer. GVGVP remains in a soluble monomeric state below the Tt and forms insoluble aggregates above the Tt. This phase transition from a soluble to an insoluble state is reversible by changing the temperature of the solution, which facilitates protein purification. The fusion protein is then resolubilized by cooling below the Tt to remove any co-precipitated insoluble contaminants as shown in Figure 11. The process of heating (37°C) and cooling (4°C) is known as inverse transition cycling (ITC), and 3-5 rounds of ITC can be performed to yield highly purified protein (>98% purity, Figure 11).

[0076] In an alternative approach, a signal peptide is fused to dextranase or mutanase for expression in E. coli, which allows the enzyme to be secreted into the extracellular medium. In addition, the secreted protein should pass through the two membrane systems of E. coli while passing through the periplasmic environment where disulfide isomerases, foldases and chaperones are present. Thus, the correct folding and disulfide bond formation of the secreted protein is facilitated by the enzyme, enhancing the biological activity of the protein (ideal for AMPs). Another advantage of this production strategy is the low level of proteolytic activity in the culture medium, which helps to enhance the stability of the recombinant protein. The signal sequence of the secreted protein is cleaved during the transport process, creating the true N-terminus for the native protein. There are several molecules (e.g., TAT-dependent pathway, SRP-dependent pathway or SecB-dependent pathway) that are useful for moving proteins into the extracellular medium. However, instead of functioning independently, the various pathways closely interact with each other. Both the SRP- and SecB-dependent pathways can function together in the targeting of a single protein, and under conditions of Sec deficiency, translocation of Sec pathway substrates can be rescued by the TAT system.

[0077] Among many signal sequences, outer membrane protein A (OmpA) and SeqX (from lacZ) signal peptides show excellent transport function and can transport fusion proteins into the extracellular medium at up to 4 g / L and 1 g / L, respectively. Therefore, these signal sequences are used for efficient transport of Arth 410 Dex and RM1 Mut into the extracellular environment. The accumulation of dextranase and mutanase transported into the medium is measured by protein quantification and enzyme assay.

[0078] These proteins have been successfully expressed in E. coli. See Western blot results shown in Figure 12. Chloroplast vectors containing these sequences are bombarded into tobacco or lettuce leaves to generate plants capable of large-scale production of the extranase / mutanase / AMP proteins. After harvesting large-scale biomass, the leaves are freeze-dried and stored at room temperature. In another approach, clinically proven anti-caries compounds (e.g., fluoride 250 ppm) and the broad-spectrum fungicide chlorhexidine 0.12% may be included to assess whether these agents enhance efficacy.

[0079] The AMP-enzyme combination effectively prevents the development of cariogenic biofilm formation and cavity formation in vivo. Moreover, the AMP-enzyme fusion protein appears to be superior to current chemotherapy for antimicrobial therapy and caries prevention.

[0080] As already mentioned, effective AMP-enzymes (independently or in combination) can be expressed in lettuce chloroplasts under the control of endogenous lettuce regulatory elements for large-scale GLP production and stability evaluation. A key advantage is the lower production cost due to the elimination of prohibitively expensive purification processes. Freeze-dried leaf material expressing the AMPs / enzymes can be stored at ambient temperature for months or years while maintaining their integrity and functionality. See FIG. 13. In addition to long-term storage, increased protein drug concentration and reduced microbial contamination are other advantages. Lettuce leaves after freeze-drying showed a 20-25 fold increase in protein drug concentration when compared to fresh leaves, thereby reducing the amount of material used for oral or topical delivery. After freeze-drying, the plant material can be incorporated into chewing gum to deliver the biofilm-degrading composition contained in the plant material.

[0081] The process for producing AMP / enzyme or their fusions is shown in Figure 12. Lettuce chloroplast vectors useful for expressing the proteins of the invention have been previously described in US patent application Ser. No. 12 / 059,376, which is incorporated herein by reference. Expression levels of up to 70% of the total protein in the case of therapeutic proteins such as proinsulin in lettuce chloroplasts can be achieved using this system.

[0082] The AMP-enzymes expressed in edible plants are preferably delivered orally (topically) when used to treat oral diseases and prevent and inhibit caries formation.To enhance the lysis of plant cells in the oral cavity, the AMP / enzyme expressing plant cells are optionally mixed with plant cells expressing cell wall degrading enzymes as described in U.S. Patent Application Serial No. 12 / 396,382, which is incorporated herein by reference.

[0083] The chewing gum tablet preparation is shown in FIG. 14. Using GFP as an example of a protein of interest, the data shows the amount of GFP that can be incorporated into the chewing gum tablet. GFP levels were assessed by both fluorescence and Western blot. The results are shown in FIG. 15. We evaluated the GFP release kinetics from gum tablets containing GFP using a chewing simulator and artificial saliva as shown in FIG. 16. FIG. 17 shows a graph illustrating the release kinetics over time from gum tablets containing various amounts of GFP present in recombinant lettuce.

[0084] From these data, it is clear that the gum tablets containing the AMP-enzyme fusion protein of the present invention deliver the active substance for a suitable period of time to achieve bacterial killing and plaque or biofilm degradation. However, oral rinses such as Listerine can also be used to deliver the AMP-enzyme fusion protein of the present invention or combinations. Figure 18 shows that crude extracts containing the enzymes of the present invention mixed with Listerine are as effective as commercially produced and purified enzymes, which are very costly to prepare. From this data, it is clear that the dual enzymes in various combinations (both different ratios and amounts) significantly reduced the biomass of S. mutans biofilms in a dose-dependent manner. Among the various combinations, 25U Dex and 5U Mut (5:1, Dex:Mut ratio) was the most effective, with more than 80% of the total biomass degraded within 120 minutes. Further experiments confirmed that a Dex / Mut activity ratio of 5:1 showed the highest effectiveness of Listerine in both EPS degradation and bacterial killing. Excitingly, dual enzyme pretreatment dramatically enhanced the efficacy of Listerine-mediated bacterial killing (>3 log reduction vs. vehicle pretreatment and Listerine). Inclusion of a third enzyme further enhanced overall antibiofilm activity. Furthermore, results from the mixed-species model showed that the dual enzyme combination could not only enhance overall antibacterial activity but also induce a targeted reduction in S. mutans dominance (while increasing the proportion of commensal / probiotic S. oralis) when Listerine was used after enzyme pretreatment. Thus, the enzyme + Listerine strategy should selectively target pathogenic S. mutans while increasing the proportion of commensal S. oralis, thereby preventing microecological imbalance within the mixed-species biofilm.

[0085] In addition to antimicrobial activity, AMPs have the ability to stimulate innate immunity and wound healing. By utilizing the novel mast cell host defense features of this AMP in addition to its antimicrobial properties, the clinical application of this AMP is expanded. Biofilm-associated dental caries is the most challenging model for the development of local therapeutics. If developed, such local drug delivery can be easily adapted to other biofilms, because matrix formation hinders drug efficacy in many other biofilm-associated diseases. Matrix is ​​inherent in all biofilms, so this application goes beyond oral biofilms. The biofilm inhibitor composition described herein can also be used to coat stents, artificial joints, implants, valves and other medical devices inserted into the human body for the treatment of disease.

[0086] As discussed above, the AMPs / enzymes or leaves expressing them can be incorporated into chewing gum for their effective topical application to treat oral diseases. The compositions can also be incorporated into oral rinses such as Listerine. As previously mentioned, such gums or oral rinses may also contain other anti-caries agents such as fluoride or CHX.

[0087] The references in Table 2 below describe many different mutanases derived from a variety of biological sources, each of which is incorporated herein by reference.

[0088] [Table 2]

[0089] Additional biofilm-degrading enzyme coding sequences useful in the practice of the present invention include, but are not limited to, the following:

[0090] I) Paenibacillus humicus NA1123 See also http: / / www.ncbi.nlm.nih.gov / nuccore / AB489092 Genbank AB489092 Total length: 1,146 Mass (Da): 119,007 Reference: Otsuka R, et al.Microbiol Immunol.2015 Jan;59(1):28-36.

[0091] 2. Protein sequence of mutanase from Paenibacillus humicus NA1123 >gi|257153265|dbj|BAI23187.1|putative mutanase [Paenibacillus humicus]

[0092] 3. Structure of the NA1123-strain mRNA of the snail·palaceae (Paenibacillus humicus). >gi|257153264|dbj|AB489092.1|Paenibacillus humicus mut gene for putative mutanase,complete cds 1 aaaggaggat cgccaaccaa tcatcccagc aaagaaggtg atggcagccc aagaattgaa 61 agcgctttga atttggaata tacggatttg gccgacctgc tgattcagtc gtattcaagc 121 gattatgccg cgaaccaatc gaacccgagg aggactaa tgcgtatccg cactaaatat 181 atgaactgga tgttggtgct cgtcctgatc gccgccggct tcttccaggc tgccggcccc 241 atcgctcccg ccaccgctgc aggaggcgcg aatctgacgc tcggcaaaac cgtcaccgcc 301 agcggccagt cgcagacgta cagccccgac aatgtcaagg acagcaatca gggaacttac 361 tgggaagca cgaacaacgc cttcccgcag tggatccaag tcgaccttgg cgccagcacg 421 agcatcgacc agatcgtgct caagcttccg tccggatggg agactcgctc gcaaacgctc 481 tcgatacagg gcagcgcgaa cggctcgacg ttcacgaaca tcgtcggatc ggccgggtat 541 acattcaatc catccgtcgc cggcaacagc gtcacgatca acttcagcgc tgccagcgcc 601 cgctacgtcc gcctgaattt cacggccaat acgggctggc cagcaggcca gctgtcggag 661 cttgagatct acggagcgac ggcgccaacg cctactccca cgcctactcc aacaccaacg 721 ccaacgccaa caccaacgcc aacccctaca gtaacccctg cgccttcggc cacgccgact 781 ccgactcctc cggcaggcag caacatcgcc gtagggaaat cgattacagc ctcttccagc 841 acgcagacct acgtagctgc aaatgcaaat gacaacaata catccaccta ttgggaggga 901 ggaagcaacc cgagcacgct gactctcgat ttcggttcca accagagcat cacttccgtc 961 gtcctcaagc tgaatccggc ttcggaatgg gggactcgca cgcaaacgat ccaagttctt 1021 ggagcggatc agaacgccgg ctccttcagc aatctcgtct ctgcccagtc ctatacgttc 1081 aatcccgcaa ccggcaatac ggtgacgatt ccggtctccg cgacggtcaa gcgcctccag 1141 ctgaacatta cggcgaactc cggcgcccct gccggccaga ttgccgagtt ccaagtgttc 1201 ggcacgccag cgcctaatcc ggacttgacc attaccggca tgtcctggac tccgtcttct 1261 ccggtcgaga gcggcgacat tacgctgaac gccgtcgtca agaacatcgg aactgcagct 1321 gcaggcgcca cgacggtcaa tttctacctg aacaacgaac tcgccggcac cgctccggta 1381 ggcgcgcttg cggcaggagc ttctgcaaat gtatcgatca atgcaggcgc caaagcagcc 1441 gcaacgtatg cggtaagcgc caaagtcgac gagagcaacg ccgtcatcga gcagaatgaa 1501 ggcaacaaca gctactcgaa cccgactaac ctcgtcgtag cgccggtgtc cagctccgac 1561 ctcgtcgccg tgacgtcatg gtcgccgggc acgccgtcgc agggagcggc ggtcgcattt 1621 accgtcgcgc ttaaaaatca gggtacgctg gcttccgccg gcggagccca tcccgtaacc 1681 gtcgttctga aaaacgctgc cggagcgacg ctgcaaacct tcacgggcac ctacacaggt 1741 tccctggcag caggcgcatc cgcgaatatc agcgtgggca gctggacggc agcgagcggc 1801 acctataccg tctcgacgac ggtagccgct gacggcaatg aaattccggc caagcaaagc 1861 aacaatacga gcagcgcgag cctcacggtc tactcggcgc gcggcgccag catgccgtac 1921 agccgttacg acacggagga tgcggtgctc ggcggcggag ctgtcctgag aacggcgccg 1981 acgttcgatc agtcgctcat cgcttccgaa gcatcgggac agaaatacgc cgcacttccg 2041 tccaacggct ccagcctgca gtggaccgtc cgtcaaggcc agggcggtgc aggcgtcacg 2101 atgcgcttca cgatgcccga cacgagcgac ggcatgggcc agaacggctc gctcgacgtc 2161 tatgtcaacg gaaccaaagc caaaacggtg tcgctgacct cttattacag ctggcagtat 2221 ttctccggcg acatgccggc tgacgctccg ggcggcggca ggccgctctt ccgcttcgac 2281 gaagtccact tcaagctgga tacggcgttg aagccgggag acacgatccg cgtccagaag 2341 ggcggtgaca gcctggagta cggcgtcgac ttcatcgaga tcgagccgat tccggcagcg 2401 gttgcccgtc cggccaactc ggtgtccgtc accgaatacg gcgctgtcgc caatgacggc 2461 aaggatgatc tcgccgcctt caaggctgcc gtgaccgcag cggtagcggc cggaaaatcc 2521 ctctacatcc cggaaggcac cttccacctg agcagcatgt gggagatcgg ctcggccacc 2581 agcatgatcg acaacttcac ggtcacgggt gccggcatct ggtatacgaa catccagttc 2641 acgaatccca atgcatcggg cggcggcatc tccctgagaa tcaaaggaaa gcttgatttc 2701 agcaacatct acatgaactc caacctgcgt tcccgttacg ggcagaacgc cgtctacaaa 2761 ggctttatgg acaatttcgg cactaattcg atcatccatg acgtctgggt cgagcatttc 2821 gaatgcggca tgtgggtcgg cgactacgcc catactcctg cgatctatgc gagcgggctc 2881 gtcgtggaaa acagccgcat ccgcaacaat cttgccgacg gcatcaactt ctcgcaggga 2941 acgagcaact cgaccgtccg caacagcagc atccgcaaca acggcgatga cggcctcgcc 3001 gtctggacga gcaacacgaa cggcgctccg gccggcgtga acaacacctt ctcctacaac 3061 acgatcgaga acaactggcg cgcggcggcc atcgccttct tcggcggcag cggccacaag 3121 gctgaccaca actacatcat cgactgtgtc ggcggctccg gcatccggat gaatacggtg 3181 ttcccaggct accacttcca gaacaacacc ggcatcacct tctcggatac gacgatcatc 3241 aacagcggca ccagccagga tctgtacaac ggcgagcgcg gagcgattga tctggaagct 3301 tccaacgacg cgatcaaaaa cgtcaccttc accaacatcg acatcatcaa tgcccagcgc 3361 gacggcgttc agatcggcta tggcggcggc ttcgagaaca tcgtgttcaa caacatcacg 3421 atcgacggca ccggccgcga cgggatatcg acatcccgct tctcgggacc tcatcttggc 3481 gcagccatct atacgtacac gggcaacggc tcggcgacgt tcaacacct ggtgacccgg 3541 aacatcgcct atgcaggcgg caactacatc cagagcgggt tcaacctgac gatcaaatag 3601 gctgcaaaaa aaaggaagct cctcggagct tccttttttt (SEQ ID NO: 13)

[0093] II) Paenibacillus curdlanolyticus MP-1 1. General Information on Mutanase from Paenibacillus curdlanolyticus MP-1 http: / / www.ncbi.nlm.nih.gov / nuccore / HQ640944 Genbank HQ640944; Total length: 1,261; Mass (Da): 131,631 Reference: Pleszczynska M, et al.Protein Expr Purif.2012 Nov;86(1):68-74.

[0094] 2. Protein sequence of mutanase from Paenibacillus curdlanolyticus MP-1 >gi|315201261|gb|ADT91063.1|alpha-1,3-glucanase[Paenibacillus curdlanolyticus]

[0095] 3. Sequence of mRNA derived from Paenibacillus curdlanolyticus MP-1 1 atgcgcaaca agtatgtcac atggacgctc gccctgacga tgctattttc gagcttcttc 61 cttgcagtag gtcccaacaa ggtcgttcac gcagcaggcg gaacgaattt agcgctcggc 121 aaaaacgtta cggcaagcgg ccaatcgcaa acgtatagtc ccaacaatgt aaaagacagc 181 aatcaatcga cgtactggga aagcacgaac aatgcattcc cgcaatggat tcaagtagac 241 ttaggcgcaa cgacgagcat tgaccaaatc gtactgaagc tgcccgctgg atggggtacg 301 cgtacgcaaa cgttagctgt tcaaggaagc acggacggtt cctcgttcac gaatatcgtg 361 ggctccgcag gctatgtatt taatcctgct gttgccaata acgccgttac gattaacttc 421 tctgctgcaa gcacgcgtta tgttcgtctg aacgtaacag cgaacacggc ttggccagca 481 gcgcagctgt ccgaattcga gatttatggc gctggcggca cgacgacgcc tccaacaacg 541 ccagcaggca catatgaagc tgaatccgca gcattgtccg gcggtgcgaa agtgaacacg 601 gatcataccg gctacacggg tacgggcttt gttgacggct actggacaca aggcgcgaca 661 acgacgttca cggctaacgt gtccgcagct ggcaactatg acgttacatt gaaatatgcc 721 aacgcaagcg gcagtgccaa gacgctaagc gtttacgtca acggcacgaa gattcgccag 781 acgacgctgg caagcctggc aaactgggac acttggggca cgaaggttga gacgctgagc 841 ttgaatgccg gcaataatac gattgcatac aagtatgagg ctagcgactc gggcaacgtg 901 aatatcgact ccattgccgt ggcgccatcg acttcgacac cggtagatcc agaaccgccg 961 atcacgccgc caacgggcag caatatcgca atcggcaaag cgatcagcgc atcttcgaat 1021 acgcaagcat tcgtagctgc caacgcgaac gataacgata cgaacacgta ctgggaaggc 1081 ggagctgcat cgagcacgct gacgctggat cttggcgcga accaaaatgt aacctcgatc 1141 gtgctgaagc tgaatccttc ttcggcatgg agcacgcgta cgcaaacgat ccaagtgctt 1201 ggccacaacc aaagcacgac gacgttcagc aatctggtat cttcgcaatc gtatacgttc 1261 aatcctgcaa cgggcaactc cgtgacgatt ccggttacgg caacagttaa gcgcttgcag 1321 ctgagcatta cggcgaactc gggttccggc gctggtcaaa ttgcggaatt ccaagtgtat 1381 ggaacgccgg caccaaaccc agacctgacg atcacaggca tgtcctggac gcctgcttcg 1441 ccaattgaaa cggatgcagt tacgctgaat gcaacggtta aaaacagcgg aaatgcagac 1501 gctcctgcaa cgacggtaaa cttctacctg aacaatgagc tcgtaggctc ctcgccagtt 1561 ggcgcacttg ctgcaggcgc ttcctcgacg gtttcgctga atgttggtac gaaaacggct 1621 gcaacttatg cagttagcgc gaaagtcgat gagagcaatt cgattatcga gcaaaatgat 1681 gcgaacaaca gttatacgaa cgcatcctcg ctcgtcgtcg ctcctgtcgc aagctctgac 1741 ttggttggcg cgacgacgtg gacgcctagc acgccggttg ccggcaatgc aattggcttc 1801 atggtaaatc ttaaaaacca aggaacgatt gcatctgcaa gcggcgcgca tggcattaca 1861 gttgtcgtga aaaatgccgc aggcgctgcg ctccaatcgt tcagcggcac ctacagcgga 1921 gcaatcgcag ctggcgcatc cgttaacgta accctgccag gtacgtggac ggctgtgaat 1981 ggcagctaca cggtaacgac aacggttgct gtcgatgcta acgagctgac gaacaaacaa 2041 gggaacaacg taagcacttc gaacctcgtt gtttatgcac aacgtggcgc aagcatgcct 2101 tacagccgtt atgacacgga agacgctaca cgtggcggcg gtgcaacgct gcaaaccgca 2161 ccaaccttca accaagcgca aatcgcttcg gaagcatccg gacaaagcta tatcgcgctg 2221 ccttcgaacg gctcctccgc acaatggacg gtccgtcaag gacaaggcgg agctggcgtt 2281 acgatgcgct tcacgatgcc ggattcgact gacggtatgg gtttgaacgg ttcgctcgac 2341 gtttatgtca acggcgttaa agtaaaaacg gtatcgctca cgtcctacta cagctggcag 2401 tatttctcgg gcgatatgcc tggcgatgcg ccgtccgctg gccgtccgtt gttccgcttt 2461 gacgaagtac actggaagct tgacacgcct cttcaaccag gcgacacgat caaaatccaa 2521 aaaggcaacg gagatagcct ggaatacggc attgacttcc tcgaaatcga gccggttcca 2581 acagcaatcg ctaaacctgc caactcgctt tccgttacgg agtatggcgc tgtagcaaac 2641 gatggccaag acgaccttgc cgcattcaaa gcaacggtta cggctgcagt tgctgctggc 2701 aaatccgttt acattcctgc tggcacgttc aatctgagca gcatgtggga aatcggatcg 2761 gctaacaaca tgatcaacaa cattacgatt acaggcgcag gctactggca tacgaacatt 2821 caattcacga atccgaatgc agcaggcggc ggcatttcgc tccggatttc cggacagctt 2881 gatttcagca atgtttacat gaactccaac ctgcgttcgc gttatggtca aaatgcgatt 2941 tacaaaggct tcatggacaa cttcggcaca aactccaaaa tccatgacgt atgggttgag 3001 cacttcgagt gcggcatgtg ggtaggcgat tacgcgcata cgccagcgat ctatgcaacg 3061 ggtcttgtcg ttgaaaacag ccggattcgc aacaaccttg cagacggcat caactactcg 3121 caaggcacga gcaattcgat cgtacgcaac agcagtatcc gcaataacgg tgatgacggt 3181 ctggcggttt ggacgagtaa cacgaatggc gcgccagcag gcgtgaacaa cacgttctcg 3241 tacaacacga tcgaaaacaa ctggcgtgca ggcggtatcg cattcttcgg cggcggcggc 3301 cacaaggctg accacaacct gatcgttgat acggttggcg gctccggcat ccggatgaac 3361 acggtattcc caggctacca cttccaaaac aacacgggta ttacgttctc cgacaacacg 3421 ctgatcaaca caggcacaag ccaagatttg tacaacggcg agcgcggtgc gatcgatctc 3481 gaagcatcga acgatgcaat caagaacgtc acgttcacga acatcgacat catcaacacc 3541 cagcgcgatg cgatacaatt cggctacggc ggcggattcg agaacatcgt atttaacaac 3601 attaacatta acggtacggg gcttgacggc gttacaacct cacggtttgc tggaccgcat 3661 aaaggtgctg caatctacac gtacacggggc aatggctctg caacgttcaa taacctgacg 3721 acgagcaacg tggcatatcc aggcttgaat ttcattcagc aaggctttaa tctggtgatc 3781 cagtag (SEQ ID NO: 15)

[0096] III) Paenibacillus sp. strain RM1. 1. General Information about Mutanase Genbank E16590; Total length: 1,291; Mass (Da): 135kD Reference: Shimotsuura I, et al.Appl Environ Microbiol.2008 May;74(9):2759-65.

[0097] 2. Protein sequence of mutanase from Paenibacillus sp. strain RM1 [ka] Deduced amino acid sequence of mutanase RM1. The signal peptide region is underlined and the linker region is boxed. The arrow indicates the cleavage site for the N-terminal domain of the protein. The DNA sequence was deposited under GenBank accession number E16590. (SEQ ID NO: 16)

[0098] 3. Sequence of mRNA derived from Paenibacillus strain RM1 1 cccgggtacc agacctatcg ggaaaaacgc gagcggccct tcgcgcctta tgcgctacgg 61 acggtgctgg cgggcggttt gtttttcatc atcattcccc tgatgatcta cacggcatcg 121 tatatcccgt ttttgctcgt gccgggtccc ggacacgggt tgaaagacgt cgtctccgcc 181 cagaagttca tgttcaatta tcatagccgg cttaacgcca cccacccatt ctcgtcgctg 241 tggtgggagt ggcctctcat ccgcaagccg atctggtatt acggagccgc ggaattggcg 301 ccgggaaaaa tggcgagcat cgtgggcatg ggcaatccgg cggtgtggtg gacgggaacg 361 attgcggtaa tcgcggccct tcgctcggcc tggaagaagc gggaccggag catgaccgtc 421 gtcttcgttg gaatcgcctc gtcttatctt ccgtgggttt tcgtatccag actcaccttt 481 atttatcact ttttcgcttg cgttccgttt ctcgttcttt gcatcgttta ttggattcga 541 aaaatggaat agcgtaagcc gggatatcgg attgcgacgc tcctttacgc aggcgcggtt 601 ctggtgctgt tcattttgtt ttacccgatt ttgtcgggga ccgaaataga cgtttcttac 661 gcggaccgcg ttctgaagtg gttcggcggg tggattttc acgggtaagc gagcgttgga 721 agcaaggag ggaggag cgagcgtctc cttcccgaa tccatccaat atcttgaaat 781 tgcatacatt tttcgtaaga ttgcttctta tctgtctccc tcccctgttc ttataatggg 841 ggtatcccaa cgaaaggagg gtttgtaagc gctgtcagcs tgtttgccga aagttctcgc 901 atttgctgac ctacactttg aggagga atttaatgcg ctgcaaattt gtcgcatggt 961 cgcttgttac agccatgctg atggccagtt tgctgacggc tgtaggaccg ttcggccccg 1021 cttccgccgc gggaggaccg aatctgacgc cgggcaaacc cattacggcg agcggccaat 1081 cccaaaccta cagccctcag aacgtaaaag acggcaatca aatacgtat tgggaaagca 1141 cgaacaacgc gttcccgcaa tggattcaag tggatttggg cgcaagcacg ggcatcgacc 1201 aaattgtgct gaagctgcc gcaagctggg aagcgcgcac gcaaacgctg gccgttcaag 1261 gcagcttgaa cggttcgacg ttcacggaca ttgtcggctc cgccaattat gtattcagtc 1321 cgtctgtcgg cgtctgtcg gttacgatca actttaccgc gaccagcg cgctacgtgc 1381 gcttgtatgt aacggccaac acgggctggc cggcggcgca gctgtccgaa ttcgaaattt 1441 acggctccgg cgaccagacg ccggcgcctg atacgtatca agccgaatcc gcggctctgt 1501 ccggcggcgc gaaagtcaac acggaccatg ccggatatat cggcacgggc tttgttgacg 1561 gttactggac gcaaggcgcg acgacgacct tttcggtcaa cgcgccgacg gcgggcaact 1621 acgatgtaac gctgaggtac ggcaacgcaa ccggcagcaa caaaacggta agcctctacg 1681 tcaatggagc gaagattcgc cagaccacgc tgcccagcct gcctaactgg gattcatgga 1741 gcagcaagac ggagacgctt aacctgaatg caggcagcaa caccattgcg tacaaatacg 1801 acccgggcga ttccggcaac gtcaatcttg accaaatcac ggtcgaagcg tcgacttcaa 1861 cgcctactcc tactccatcc cctactccta cacctacgcc aacgccgacg cctacgccta 1921 cgcctacacc cacacctact ccgaccccga cgcctacgcc tacacctaca cctacaccta 1981 cgccgacgcc tcctccgggc ggcaacatcg ccatcggcaa atcgatttcc gcatcctccc 2041 acacgcagac gtacgttgcg gagaacgcga acgataacga tgtcaacacg tactgggaag 2101 gcggcggcaa tccgagcacg ctgacgctcg atctcggagc gaactacaat attacgtcca 2161 tcgtgctgaa gctgaacccg tcctcgatat gggctgcgcg tacgcaaacg attcaagtgc 2221 2281 tcgatccggc ctccggcaat actgtgacca ttccggttac ggcgacggtg aaacgtttgc 2341 agttgaacat tacgtcgaac tccggcgccc cggccggaca agtcgccgag ttccaggtgt 2401 tcggcacgcc tgcgccgaat ccggacctga cgattaccgg catgtcctgg tcgccttctt 2461 ctccggttga gaccgacgcc attacgctaa acgcaacggt gaaacaac gggaatgcca 2521 gcgccgcggc gaccaccgtc aatttctacc tgaacaacga gctggcgggt tccgcgccgg 2581 tagccgcgct ggcggcaggc gcttcggcaa cggtgccgct gaatgtcggc gcgaaaaccg 2641 ccgcgacata cgcggtcggc gccaaagtag acgagagcaa cgcggtcatc gagctgaacg 2701 agtcgaacaa cagctacacg aatccggctt cactcgttgt ggcccccgtt tccagctcgg 2761 atctggtggg cacggtttcg tggacgccga gcactccgat tgccaacaat gccgtttctt 2821 ttaacgtaaa tcttaaaaat caaggaacga ttgcttccgc cggcgggtct cacggcgtga 2881 cggtcgtgct taaaaatgct tccggttcga ccgttcaaac gttcagcggt tcctataccg 2941 gcagcctggc tccgggagcg tccgtcaaca tcacccttcc ggggacctgg acggcggcag 3001 ccggcagcta cacggtaacg gccaccgttg cggcagacgc caacgaactt ccgatcaagc 3061 aagccaacaa cgcgaacacc gcaagcctga ccgtatattc cgcccgcggc gcgagcatgc 3121 cgtacagccg gtatgacacc gaggacgcca ccctcggcgg cggcgccacg ctgaagtccg 3181 cgccgacatt cgatcaggcg cttacggcat cggaagccac cggccaactc tatgcggcgc 3241 tgccctcgaa cggctcctat cttcaatgga ccgtcagaca gggtcagggc ggcgcaggcg 3301 tgacgatgag atttacgatg cccgactcgg cggacggcat gggattaaac ggttcgctag 3361 acgtttacgt caacggcacc aaagtcaaaa ccgtatcgct gacctcctac tacagctggc 3421 agtatttctc gggcgatatg cccggagacg ctcccagcgc gggccgtccg ctcttccgct 3481 ttgacgaagt gcactggaag ctggatactc cgctcaaacc cggagacacg attcgcatcc 3541 agaagaacaa cggcgacagc ctggaatacg gtgtcgactt tattgaaatc gaaccggttc 3601 cggctgcgat ctcccgtccg gccaactcgg tttccgtaac ggattacggc gctgtgccga 3661 acgacggaca ggacgatctc accgccttta aagccgccgt aaacgcggcg gtcgcatccg 3721 acaagatctt gtacattccg gaaggaacgt tccacctcgg caacatgtgg gagatcggtt 3781 ccgtcagcaa catgatcgat cacattacga ttacgggagc cggtatctgg tatacgaaca 3841 tccagtttac caacgccaat ccggcgtccg gcggcatctc gctccggatt acgggcaagc 3901 ttgatttcag caacgtgtac ctcaactcca atttgcggtc gcggtatggt caaaatgcgg 3961 tttacaaagg ctttatggac aacttcggga ccaattccgt catccgcgac gtctgggtcg 4021 agcacttcga atgcggcttc tgggtcgggg actacgggca tacgccggcg atccgcgcga 4081 gcgggctgct gattgaaaac agccgaatcc gcaacaacct ggccgatggc gtcaacttcg 4141 cccaagggac cagcaattcg accgtacgca acagcagcct gcgcaacaac ggcgacgacg 4201 cccttgccgt atggacgagt aatacgaacg gcgcgcccga aggcgtaaac aataccttct 4261 cgtacacac catcgaaac aactggcgcg cgggaggcat cgccttctc ggaggaagcg 4321 ngacaaggc cgaccacaac tacatcgtcg actgcgtcgg cggttccggc atccggatga 4381 acaccgtgtt cccggatac cacttccaga acataccgg cattgtgttc tcggacacga 4441 ccatcgtcaa ctgcggcacg agcaaagacc tatacaacgg cgaacgcggc gccatcgatc 4501 tggagctc gaacgacgcc atccgaacg tgacgttc cacatcgat attcact 4561 ctcagcgcga tgcgatccag tcggttacg gcggcggctt caccacatc gtgttcaca 4621 acatcacat taacggacc gtcttgacg gcgtaccac ctcgcggttc tcgggaccgc 4681 atctgggc ggggatctc acctataccg gcaacggctc cgccacgttc aacaatctga 4741 ggaccagcaa tatcgcttac cccaatctgt attackacca gagcggttc attctgatca 4801 tcaatatta gatatctggg cccgtctgcg gggaggac tctcggagc tcgaattcgt 4861 aatcatggtc atagctgttt cctgtgtgaa attgttacc gctcacaatt ccacacaca 4921 tacgagccgg aagcataag tgtaagcct ggggtgccta atgagtgagc taactcacat 4981 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa ctgtcgtgcc agctgcatta 5041 atgaatcggc caacgcgcgg ggagaggcsg tttkcgtatt gggcgccctt (SEQ ID NO: 17)

[0099] IV) Trichoderma harzianum (CCM F-470) 1. General information on mutanase from Trichoderma harzianum See also http: / / www.uniprot.org / uniprot / Q8WZM7 Total length: 635 Mass (Da):67,726 Last modified: March 1, 2002 - v1 Checksum: iBB0D864E2F432C58

[0100] 2. Protein sequence of mutanase from Trichoderma harzianum http: / / www.uniprot.org / uniprot / Q8WZM7.fasta) >tr|Q8WZM7|Q8WZM7_TRIHA Alpha-1,3-glucanase OS=Trichoderma harzianum GN=p3 PE=2 SV=1 MLGVFRRLRLGALAAAAALSSLGSAAPANVAIRSLEERASSADRLVFCHFMIGIVGDRGSS ADYDDDMQRAKAAGIDAFALNIGVDGYTDQQLGYAYDSADRNGMKVFISFDFNWWSPGNA VGVGQKIAQYANRPAQLYVDNRPFASSFAGDGLDVNALRSAAGSNVYFVPNFHPGQSSPS NIDGALNWMAWDNDGNNKAPKPGQTVTVADGDNAYKNWLGGKPYLAPVSTWVFNHFGPEV SYSKNWVFPSGPLIYNRWQQVLQQGFPRVEIVTWNDYGESHYVGPLKSKQFHDGNSKWVN DMPHDGFLDLSKPFIAAYKNRDTDISKYVQNEQLVYWYRRNLKALDCDATDTTSNRPANN GSGNYFEGRPDGWQTMDDTVYVAALLKTAGSVTVTSGGTTQTFQANAGANLFQIPASIGQ QKFALTRNGQTVFSGTSLMDITNVCSCGIYNFNPYVGTIPAGFDDPLQADGLFSLTIGLH VTTCQAKPSLGTNPPVTSGPVSSLPASSTTRASSPPPVSSTRVSSPPVSSPPVSRTSSAP PPPGNSTPPSGQVCVAGTVADGESGNYIGLCQFSCNYGYCPPGPCKCTAFGAPISPPASN GRNGCPLPGEGDGYLGLCSFSCNHNYCPPTACQYC(SEQ ID NO:18)

[0101] 3. Sequence of mRNA (Trichoderma harzianum http: / / www.ebi.ac.uk / ena / data / view / AJ243799&display=fasta) >ENA|AJ243799|AJ243799.1 Trichoderma harzianum mRNA for alpha-1,3-glucanase (p3 gene) ATGTTGGGCGTTTTCCGCCGCCTCAGGCTCGGCGCCCTTGCCGCCGCAGCTCTGTCTTCT CTCGGCAGTGCCGCTCCCGCCAATGTTGCTATTCGGTCTCTCGAGGAACGTGCTTCTTCT GCTGACCGTCTCGTATTCTGTCATTTCATGATTGGGATCGTGGGTGACCGTGGCAGCTCG GCAGATTATGATGACGATATGCAACGTGCCAAAGCCGCTGGCATTGACGCCTTCGCCCTG AACATCGGCGTTGACGGCTATACCGACCAGCAGCTCGGCTATGCCTATGACTCTGCCGAT CGTAATGGCATGAAAGTCTTCATTTCATTTGATTTCAACTGGTGGAGCCCCGGCAATGCA GTTGGTGTTGGCCAGAAGATTGCGCAGTATGCCAACCGCCCTGCCCAGCTGTATGTCGAC AACCGGCCATTCGCCTCTTCCTTCGCCGGTGACGGTCTGGATGTAAATGCGTTGCGCTCT GCTGCAGGCTCCAACGTTTACTTTGTGCCCAACTTCCACCCTGGTCAATCTTCCCCCTCC AACATTGATGGCGCCCTTAACTGGATGGCCTGGGATAATGATGGAAACAACAAGGCACCC AAGCCGGGCCAGACTGTCACAGTGGCAGACGGTGACAACGCTTATAAGAATTGGTTGGGT GGCAAGCCTTACCTGGCGCCTGTCTCAACTTGGGTTTTCAACCATTTCGGGCCCGAAGTT TCATATTCCAAGAACTGGGTTTTCCCAAGTGGGCCTCTGATCTATAACCGGTGGCAACAA GTCTTGCAGCAAGGGTTCCCAAGGGTTGAGATCGTTACCTGGAATGACTACGGGGAATCT CACTACGTCGGTCCCCTGAAGTCTAAGCAATTTCATGATGGGAACTCCAAATGGGTCAAT GATATGCCCCACGATGGATTCCTGGATCTTTCGAAGCCGTTCATAGCCGCATATAAAAAC AGGGATACCGACATCTCCAAGTATGTTCAAAATGAGCAGCTTGTTTACTGGTACCGCCGC AACTTAAAGGCACTGGACTGTGACGCCACCGACACAACCTCTAACCGCCCGGCTAACAAT GGAAGCGGCAATTACTTTGAGGGACGCCCCGATGGTTGGCAAACTATGGATGATACGGTT TACGTGGCGGCACTTCTCAAGACTGCCGGTAGCGTCACGGTCACGTCTGGTGGCACCACT CAAACGTTCCAGGCCAACGCCGGAGCCAATCTCTTCCAAATCCCGGCCAGCATCGGCCAG CAAAAGTTTGCTCTGACTCGTAACGGTCAGACCGTCTTTAGCGGAACCTCATTGATGGAT ATCACCAACGTTTGCTCTTGCGGTATCTACAACTTCAACCCATATGTTGGCACCATTCCT GCCGGCTTTGACGACCCTCTTCAGGCTGACGGTCTTTTCTCTTTGACCATCGGATTGCAC GTCACAACTTGTCAGGCCAAGCCATCTCTTGGAACTAACCCTCCTGTCACTTCCGGCCCT GTGTCCTCGCTTCCAGCTTCCTCCACCACCCGCGCATCCTCGCCGCCTCCTGTTTCTTCA ACTCGTGTCTCTTCTCCCCCTGTCTCTTCCCCTCCAGTTTCTCGCACCTCTTCTGCCCCT CCCCCTCCGGGCAACAGCACGCCGCCATCGGGTCAGGTTTGCGTTGCCGGCACCGTTGCC GACGGCGAGTCTGGCAACTACATCGGCCTGTGCCAATTCAGCTGCAACTACGGTTACTGC CCACCAGGACCGTGTAAGTGCACCGCCTTTGGTGCTCCCATCTCGCCACCGGCATCCAAC GGCCGCAACGGCTGCCCTCTGCCGGGAGAAGGCGATGGTTATCTGGGCCTGTGCAGTTTC AGTTGTAACCATAATTACTGCCCGCCAACGGCATGTCAATACTGCTAGGAGGGATCAATC TCAGTATGAGTATATGGAGGCTGCTGAAGGACCAATTAGCTGTTCTTATCGGCAGACGAA ACCCATAGAGTAAGAAGTTAAATAAAATGCAATTAATGTGTTTTCAAAAAAAAAAAAAAA A (SEQ ID NO: 19) (Trichoderma harzianum is a fungus, so it has a polyA tail.)

[0102] V) Trichoderma harzianum 1. General information on mutanase from Trichoderma harzianum See also http: / / www.uniprot.org / uniprot / Q8WZM7;

[0103] 2. Protein sequence of mutanase from Trichoderma harzianum (See http: / / www.uniprot.org / uniprot / Q8WZM7.fasta) >tr|Q8WZM7|Q8WZM7_TRIHA Alpha-1,3-glucanase OS=Trichoderma harzianum GN=p3 PE=2 SV=1 MLGVFRRLRLGALAAAAALSSLGSAAPANVAIRSLEERASSADRLVFCHFMIGIVGDRGSS ADYDDDMQRAKAAGIDAFALNIGVDGYTDQQLGYAYDSADRNGMKVFISFDFNWWSPGNA VGVGQKIAQYANRPAQLYVDNRPFASSFAGDGLDVNALRSAAGSNVYFVPNFHPGQSSPS NIDGALNWMAWDNDGNNKAPKPGQTVTVADGDNAYKNWLGGKPYLAPVSTWVFNHFGPEV SYSKNWVFPSGPLIYNRWQQVLQQGFPRVEIVTWNDYGESHYVGPLKSKQFHDGNSKWVN DMPHDGFLDLSKPFIAAYKNRDTDISKYVQNEQLVYWYRRNLKALDCDATDTTSNRPANN GSGNYFEGRPDGWQTMDDTVYVAALLKTAGSVTVTSGGTTQTFQANAGANLFQIPASIGQ QKFALTRNGQTVFSGTSLMDITNVCSCGIYNFNPYVGTIPAGFDDPLQADGLFSLTIGLH VTTCQAKPSLGTNPPVTSGPVSSLPASSTTRASSPPPVSSTRVSSPPVSSPPVSRTSSAP PPPGNSTPPSGQVCVAGTVADGESGNYIGLCQFSCNYGYCPPGPCKCTAFGAPISPPASN GRNGCPLPGEGDGYLGLCSFSCNHNYCPPTACQYC (SEQ ID NO: 20)

[0104] 3. mRNA sequence (Trichoderma harzianum, further information can be found at http: / / www.ebi.ac.uk / ena / data / view / AJ243799&display=fasta) >ENA|AJ243799|AJ243799.1 Trichoderma harzianum mRNA for alpha-1,3-glucanase(p3 gene) ATGTTGGGCGTTTTCCGCCGCCTCAGGCTCGGCGCCCTTGCCGCCGCAGCTCTGTCTTCT CTCGGCAGTGCCGCTCCCGCCAATGTTGCTATTCGGTCTCTCGAGGAACGTGCTTCTTCT GCTGACCGTCTCGTATTCTGTCATTTCATGATTGGGATCGTGGGTGACCGTGGCAGCTCG GCAGATTATGATGACGATATGCAACGTGCCAAAGCCGCTGGCATTGACGCCTTCGCCCTG AACATCGGCGTTGACGGCTATACCGACCAGCAGCTCGGCTATGCCTATGACTCTGCCGAT CGTAATGGCATGAAAGTCTTCATTTCATTTGATTTCAACTGGTGGAGCCCCGGCAATGCA GTTGGTGTTGGCCAGAAGATTGCGCAGTATGCCAACCGCCCTGCCCAGCTGTATGTCGAC AACCGGCCATTCGCCTCTTCCTTCGCCGGTGACGGTCTGGATGTAAATGCGTTGCGCTCT GCTGCAGGCTCCAACGTTTACTTTGTGCCCAACTTCCACCCTGGTCAATCTTCCCCCTCC AACATTGATGGCGCCCTTAACTGGATGGCCTGGGATAATGATGGAAACAACAAGGCACCC AAGCCGGGCCAGACTGTCACAGTGGCAGACGGTGACAACGCTTATAAGAATTGGTTGGGT GGCAAGCCTTACCTGGCGCCTGTCTCAACTTGGGTTTTCAACCATTTCGGGCCCGAAGTT TCATATTCCAAGAACTGGGTTTTCCCAAGTGGGCCTCTGATCTATAACCGGTGGCAACAA GTCTTGCAGCAAGGGTTCCCAAGGGTTGAGATCGTTACCTGGAATGACTACGGGGAATCT CACTACGTCGGTCCCCTGAAGTCTAAGCAATTTCATGATGGGAACTCCAAATGGGTCAAT GATATGCCCCACGATGGATTCCTGGATCTTTCGAAGCCGTTCATAGCCGCATATAAAAAC AGGGATACCGACATCTCCAAGTATGTTCAAAATGAGCAGCTTGTTTACTGGTACCGCCGC AACTTAAAGGCACTGGACTGTGACGCCACCGACACAACCTCTAACCGCCCGGCTAACAAT GGAAGCGGCAATTACTTTGAGGGACGCCCCGATGGTTGGCAAACTATGGATGATACGGTT TACGTGGCGGCACTTCTCAAGACTGCCGGTAGCGTCACGGTCACGTCTGGTGGCACCACT CAAACGTTCCAGGCCAACGCCGGAGCCAATCTCTTCCAAATCCCGGCCAGCATCGGCCAG CAAAAGTTTGCTCTGACTCGTAACGGTCAGACCGTCTTTAGCGGAACCTCATTGATGGAT ATCACCAACGTTTGCTCTTGCGGTATCTACAACTTCAACCCATATGTTGGCACCATTCCT GCCGGCTTTGACGACCCTCTTCAGGCTGACGGTCTTTTCTCTTTGACCATCGGATTGCAC GTCACAACTTGTCAGGCCAAGCCATCTCTTGGAACTAACCCTCCTGTCACTTCCGGCCCT GTGTCCTCGCTTCCAGCTTCCTCCACCACCCGCGCATCCTCGCCGCCTCCTGTTTCTTCA ACTCGTGTCTCTTCTCCCCCTGTCTCTTCCCCTCCAGTTTCTCGCACCTCTTCTGCCCCT CCCCCTCCGGGCAACAGCACGCCGCCATCGGGTCAGGTTTGCGTTGCCGGCACCGTTGCC GACGGCGAGTCTGGCAACTACATCGGCCTGTGCCAATTCAGCTGCAACTACGGTTACTGC CCACCAGGACCGTGTAAGTGCACCGCCTTTGGTGCTCCCATCTCGCCACCGGCATCCAAC GGCCGCAACGGCTGCCCTCTGCCGGGAGAAGGCGATGGTTATCTGGGCCTGTGCAGTTTC AGTTGTAACCATAATTACTGCCCGCCAACGGCATGTCAATACTGCTAGGAGGGATCAATC TCAGTATGAGTATATGGAGGCTGCTGAAGGACCAATTAGCTGTTCTTATCGGCAGACGAA ACCCATAGAGTAAGAAGTTAAATAAAATGCAATTAATGTGTTTTCAAAAAAAAAAAAAAA A (SEQ ID NO:21) (Trichoderma harzianum is a fungus, so it has a polyA tail.)

[0105] Dextranase (Dex) gene from Penicillium minioluteum GenBank:L41562.1 (http: / / www.ncbi.nlm.nih.gov / nuccore / L41562.1) The mature protein has 574 amino acids with a MW of 67 KD. The optimal reaction conditions are pH 5.5 and 40° C. The pH range is 3-6.

[0106] Amino acid sequence MATMLKLLALTLAISESAIGAVMHPPGNSHPGTHMGTTNNTHCG ADFCTWWHDSGEINTQTPVQPGNVRQSHKYSVQVSLAGTNNFHDSFVYESIPRNGNGR IYAPTDPPNSNTLDSSVDDGISIEPSIGLNMAWSQFEYSHDVDVKILATDGSSLGSPS DVVIRPVSISYAISQSDDGGIVIRVPADANGRKFSVEFKTDLYTFLSDGNEYVTSGGS VVGVEPTNALVIFASPFLPSGMIPHMTPDNTQTMTPGPINNGDWGAKSILYFPPGVYW MNQDQSGNSGKLGSNHIRLNSNTYWVYLAPGAYVKGAIEYFTKQNFYATGHGILSGEN YVYQANAGDNYIAVKSDSTSLRMWWHNNLGGGQTWYCVGPTINAPPFNTMDFNGNSGI SSQISDYKQVGAFFFQTDGPEIYPNSVVHDVFWHVNDDAIKIYYSGASVSRATIWKCH NDPIIQMGWTSRDISGVTIDTLNVIHTRYIKSETVPSAIIGASPFYASGMSPDSRKS ISMTVSNVVCEGLCPSLFRITPLQNYKNFVVKNVAFPDGLQTNSIGTGESIIPAASGL TMGLNISNWTVGGQKVTMENFQANSLGQFNIDGSYWGEWQIS (SEQ ID NO: 22)

[0107] DNA sequence 1 ggcatagtaa tcccgacagc cgagtatgat ggagcttctt cggataatga tagcgccacc 61 agaccttgct tgagctggag agctaaaaca ttaaacgcca cacgaccaac actctcatta 121 gttgcgatag atgatgctcg gagctgttga aactcagaaa ttccttctat gcggggtctc 181 caagatcgat cctgggggat gtgaatacta cggtggacct aattgacgcc ttgacaggtg 241 atgttaagcg aaccaaggaa gaataatctg gggctagatg aagatgttga gctgtaaggt 301 acggtacgtt cctattggct ttatcggagc ttctccgggt tactcagtct ttccgggagc 361 atgatcattt ttgtattgtc caatagtaag cagaaactga gagccaccac aaactcaaaa 421 cctcggtagc gaagtttccc ggaaccagtc aggattctca gaaactgtgc tcgtgttgcg 481 gggaatccgc attctacgtc gtctggagca aggaaatgtt cgtgctggat tgaggaggat 541 aggtaggttg gagaatctct tcagctaacc aatctataag catgctccgg taacctttag 601 agtttcacat tcaacgtaat ttccaagata gccagagcgt ccttgaatta ctatgtagaa 661 atcctaaaat ttcccctgta aaatgcaagt caacgagatg cgtgccctca atgtctctcg 721 gcgctacccc ggaaatgatg cataaggcca agaatgtcac ccggtaactt tttcttcaga 781 atatcctaag atttccatca aacacagtcg aataggtcaa tgctcgcgag agactttctg 841 ccttcactct acgtcctact catagaagtt caacggctca attccggggt aatctagagt 901 ttggacctca agggagatgt tgcaacaaat tgtactagaa cgatgcgctt gctttccaat 961 acagtagttg acttcatata gcttccaaca aaagggatgg ggatgaaggc tctatagcga 1021 gaagtctata agaaagtgtc ctcatacctg tatctctcag tcgttcgaga acaatcccgg 1081 aaactatctt atcttgcgag aaagaagaca atatctcaaa cttatggcca caatgctaaa 1141 gctacttgcg ttgacccttg caattagcga gtccgccatt ggagcagtca tgcacccacc 1201 tggcaattct catcccggta cccatatggg cactacgaat aatacccatt gcggcgccga 1261 tttctgtacc tggtggcatg attcagggga gatcaatacg cagacacctg tccaaccagg 1321 gaacgtgcgc caatctcaca agtattccgt gcaagtgagc ctagctggta caaacaattt 1381 tcatgactcc tttgtatatg aatcgatccc ccggaacgga aatggtcgca tctatgctcc 1441 caccgatcca cccaacagca acacactaga ttcaagtgtg gatgatggaa tctcgattga 1501 gcctagtatc ggccttaata tggcatggtc ccaattcgag tacagccacg atgtagatgt 1561 aaagatcctg gccactgatg gctcatcgtt gggctcgcca agtgatgttg ttattcgccc 1621 cgtctcaatc tcctatgcga tttctcagtc tgacgatggt gggattgtca tccgggtccc 1681 agccgatgcg aacggccgca aattttcagt tgagttcaaa actgacctgt acacattcct 1741 ctctgatggc aacgagtacg tcacatcggg aggcagcgtc gtcggcgttg agcctaccaa 1801 cgcacttgtg atcttcgcaa gtccgtttct tccttctggc atgattcctc atatgacacc 1861 cgacaacacg cagaccatga cgccaggtcc tatcaataac ggcgactggg gcgccaagtc 1921 aattctttac ttcccaccag gtgtatactg gatgaaccaa gatcaatcgg gcaactcggg 1981 gaagttagga tctaatcata tacgtctaaa ctcgaacact tactgggtct accttgcccc 2041 cggtgcgtac gtgaagggtg ctatagagta ttttaccaag cagaacttct atgcaactgg 2101 tcatggtatc ctatcgggtg aaaactatgt ttaccaagcc aatgccggcg acaactacat 2161 tgcagtcaag agcgattcaa ccagcctccg gatgtggtgg cacaataacc ttgggggtgg 2221 tcaaacatgg tactgcgttg gcccgacgat caatgcgcca ccattcaata ctatggattt 2281 caatggaaat tctggcatct caagtcaaat tagcgactat aagcaggtgg gagccttctt 2341 cttccagacg gatggaccag aaatatatcc caatagtgtc gtgcacgacg tcttctggca 2401 cgtcaatgat gatgcaatca aaatctacta ttcgggagca tctgtatcgc gggcaacgat 2461 ctggaaatgt cacaatgacc caatcatcca gatgggatgg acgtctcggg atatcagtgg 2521 agtgacaatc gacacatta atgttatca caccgctac atcaaatcgg agacggtggt 2581 gccttggct atcattgggg cctctccatt ctatgcaagt gggatgagtc ctgattcaag 2641 aaagtccata tccatgacgg tttcaaacgt tgtttgcgag ggtctttgcc cgtccctatt 2701 ccgcatcaca ccccttcaga actacaaaaa ttttgttgtc aaaaatgtgg ctttcccaga 2761 cgggctacag acgaatagta ttggcacagg agaaagcatt attccagccg catctggtct 2821 aacgatggga ctgaatatct ccaactggac tgttggtgga caaaaagtga ctatggagaa 2881 ctttcaagcc aatagcctgg ggcagttcaa tattgacggc agctattggg gggagtggca 2941 gattagctga attccagctc tcggagcgcg tgagtgcttc tacccgctcc tttacccttg 3001 tcgagagata aaggcataag ttagctcatg tgaaggcgat ttcagttcat tctctctttt 3061 tggagcttat ttcctgttcg accaattgtg acaccaactt gcctttcaaa agacgtggac 3121 gatatgtgta cggtaatcag tcaaatgaac gtcaacattc atttaataag gacatttcca 3181 ggtttcctta ctctgtcgat tatgcctaac tcgggttgat gtcttgtcag gatggaaaat 3241 ctcgttgtgt acttccagtg aaatgggcag ggctaagccc taaaccctaa cgcatacaat 3301 ttgtaggcac ctacccatgt aagttcacac ccagtcgact tataagtcta gatatttatg 3361 ctatgcaggc tctggaatga tttacattcc atgctataca tagttatttg caagaatttg 3421 cagacgagat aaaaatcaat ggacgaataa tcacgcatta ctccacaggc tcatgccacg 3481 gagcaagggt tcccccgaat ctaggccaga ccgggatgat attcaaccga ttctttttgc 3541 agtaactatc tccgtacgag ctgcacgagc taaacggatt atataaaggt gctaactgag 3601 cattggatcc gtcagttata tgaaatgca (SEQ ID NO: 23)

[0108] 2. Dextranase (Dex) gene from Penicillium aculeatum (Talaromyces aculeatus strain z01) GenBank:KF999646.1(http: / / www.ncbi.nlm.nih.gov / nuccore / KF999646.1) The optimum pH is about 5. The pH range is 3-6.

[0109] Amino acid sequence MATMLKLLTLALAISESAIGAVLHPPGSSHPSTRTDTTTNNTHCG ADFCTWWHDSGEINTQTPVQPGNVRQSHKYSVQVSLAGANNFQDSFVYESIPRNGNGR IYAPTDPPNSNTLDSSVDDGISIEHSIGLNMAWSQFEYSQDVDIKILAADGSSLGSPS DVVIRPVSISYAISQSDDGGIVIRVPADANGRKFSVEFKNDPYTFLSDGNEYVTSGGS VVGVEPTNALVIFASPFLPSGMIPHMTPDNTQTMTPGPINNGDWGSKSILYFPPGVYW MNQDQSGNSGKLGSNHIRLNSNTYWVYFAPGAYVKGAIEYFTKQNFYATGHGVLSGEN YVYQANAGENYVAVKSDSTSLRMWWHNNLGGGQTWYCVGPTINAPPFNTMDFNGNSGI SSQISDYKQVGAFFFQTDGPEIYPNSVVHDVFWHVNDDAIKIYYSGASVSRATIWKCH NDPIIQMGWTSRDISGVTIDTLNVIHTRYIKSETVPSAIIGASPFYASGMPSPDSSKS ISMTVSNVVCEGLCPSLFRITPLQNYKNFVVKNVAFPDGLQTNSIGTGESIIPAASGL TMGLDISNWSVGGQKVTMQNFQANSLGQFDIDGSYWGEWQIN (SEQ ID NO: 24)

[0110] DNA sequence 1 atggccacaa tgctaaagct acttacgttg gcccttgcaa ttagcgagtc tgccattgga 61 gcagtcctgc acccacctgg cagttctcat cccagtaccc gtacggacac tacgaataat 121 acccattgcg gtgccgactt ctgtacctgg tggcatgatt caggcgagat caacacacag 181 acacctgtcc aaccggggaa cgtgcgccaa tctcacaagt attccgtaca agtgagccta 241 gctggtgcga acaactttca ggactccttt gtatatgaat cgatccctcg gaacggaaat 301 ggtcgcatct atgctcccac cgatccaccc aacagcaaca cactagattc aagtgttgat 361 gatggaatct cgattgaaca tagtattggc ctcaatatgg catggtccca attcgagtac 421 agccaggatg tcgatataaa gatcctggcc gctgatggct catcgttggg ctcaccaagt 481 gatgttgtta ttcgccccgt ctcaatctcc tatgcaattt ctcaatccga cgatggcgga 541 attgtcattc gggtcccagc cgatgcgaac ggccgcaaat tttcagtcga gttcaaaaat 601 gacccgtaca cgttcctctc tgacggcaac gagtacgtca catcgggagg cagcgttgtc 661 ggcgttgagc ctaccaacgc acttgtgatc ttcgcaagcc cgtttcttcc gtcaggcatg 721 attcctcata tgacacccga caacacgcag accatgacac caggacctat caataacggc 781 gactggggct ccaagtcaat tctttatttc ccaccgggcg tatactggat gaaccaagat 841 caatcaggca actcggggaa attaggatct aatcatatac gcctgaactc gaacacctac 901 tgggtctact ttgccccagg tgcgtacgtg aagggtgcta tagagtattt caccaagcag 961 aacttctatg caactggtca tggtgtccta tcgggtgaaa actatgttta ccaagccaat 1021 gctggcgaaa actacgttgc ggtcaagagc gattcgacta gcctccggat gtggtggcac 1081 aataacctgg gaggtggaca aacatggtac tgcgttgggc ctacgatcaa tgcgccgcca 1141 tttaacacaa tggatttcaa tggaaattcc ggtatctcaa gtcaaattag cgactataag 1201 caggtgggag ctttcttctt tcagacggat ggaccagaaa tttatcccaa tagtgtcgtg 1261 cacgacgtct tctggcatgt caatgatgat gcaatcaaaa tctactattc cggagcatct 1321 gtctcgcggg caacgatctg gaaatgtcac aacgatccaa tcatccagat gggatggacg 1381 tctcgggata tcagtggagt gacaatcgac acattgaatg tcatccacac ccgctacatc 1441 aagtcggaga cggtggtgcc ttcggctatc attggggctt ctccattcta tgcaagtggg 1501 atgagtcctg attcaagcaa gtctatatcc atgacggttt caaacgttgt ctgcgaggga 1561 ctttgcccgt ctctgttccg aatcacacct ttacagaact acaagaattt tgttgtcaaa 1621 aatgtggctt tcccagatgg gctacagacg aatagtattg gcacgggaga aagcattatt 1681 ccagccgcat ctggtctaac gatgggactg gatatctcca actggtctgt tggtggtcag 1741 aaggtgacta tgcagaactt tcaagccaat agtctggggc aattcgacat tgacggcagc 1801 tattgggggg agtggcagat taactagctg aataatattg cagctttcag ggcgcatgag 1861 tgcttgtacc cgctccttta cccttgtc (SEQ ID NO: 25)

[0111] 3. Penicillium funiculosum dexA gene for dextranase GenBank:AJ272066.1(http: / / www.ncbi.nlm.nih.gov / nuccore / 7801166) The optimum pH is about 5.5. The optimum temperature is 60°C. The pH range is 5-7.5 (http: / / www.sciencedirect.com / science / article / pii / S0032959298001277)

[0112] Amino acid sequence MATMLKLLALTLAISESAIGAVMHPPGVSHPGTHTTGTTNNTHCG ADFCTWWHDSGEINTQTPVQPGNVRQSHKYSVQVSLAGTNNFHDSFVYESIPRNGNGR IYAPTDPSNSNTLDSSVDDGISIEPSIGLNMAWSQFEYSQDVDIKILATDGSSLGSPS DVVIRPVSISYAISQSNDGGIVIRVPADANGRKFSVEFKNDLYTFLSDGNEYVTSGGS VVGVEPTNALVIFASPFLPSGMIPHMKPHNTQTMTPGPINNGDWGAKSILYFPPGVYW MNQDQSGNSGKLGSNHIRLNSNTYWVYLAPGAYVKGAIEYFTKQNFYATGHGVLSGEN YVYQANAGDNYVAVKSDSTSLRMWWHNNLGGGQTWYCVGPTINAPPFNTMDFNGNSGI SQISDYKQVGAFFFQTDGPEIYPNSVVHDVFWHVNDDAIKIYYSGASVSRATIWKCH NDPIIQMGWTSRDISGVTIDTLNVIHTRYIKSETVPSAIIGASPFYASGMPSPDSSKS ISMTVSNVVCEGLCPSLFRITPLQNYKNFVVKNVAFPDGLQTNSIGTGESIIPAASGL TMGLNISSWTVGGQKVTMENFQANSLGQFNIDGSYWGEWQISRISSSQSA (SEQ ID NO:26)

[0113] DNA sequence 1 atggccacaa tgctaaagct acttgcgttg acccttgcaa ttagcgagtc cgccattgga 61 gcagtcatgc acccacctgg cgtttctcat cccggtaccc atacgggcac tacgaataat 121 acccattgcg gcgccgactt ctgtacctgg tggcatgatt caggggagat caacacgcag 181 acacctgtcc aaccagggaa cgtgcgccaa tctcacaagt attccgtgca agtgagtcta 241 gctggtacaa acaactttca tgactccttt gtatatgaat cgatcccccg gaacggaaat 301 ggtcgcatct atgctcccac cgatccatcc aacagcaaca cattagattc aagcgtggat 361 gatggaatct cgattgagcc tagtatcggc ctcaatatgg catggtccca attcgagtac 421 agccaggatg tcgatataaa gatcctggca actgatggct catcgttggg ctcaccaagt 481 gatgttgtta ttcgccccgt ctcaatctcc tatgcgattt ctcagtccaa cgatggcggg 541 attgtcatcc gggtcccagc cgatgcgaac ggccgcaaat tttcagtcga attcaaaaat 601 gacctgtaca ctttcctctc tgatggcaac gagtacgtca catcgggagg tagcgtcgtc 661 ggcgttgagc ctaccaacgc acttgtgatc ttcgcaagtc cgtttcttcc ttctggcatg 721 attcctcata tgaaacccca caacacgcag accatgacgc caggtcctat caataacggc 781 gactggggcg ccaagtcaat tctttacttc ccaccaggtg tatactggat gaaccaagat 841 caatcgggca actcgggtaa attaggatct aatcatatac gtctaaactc gaacacttac 901 tgggtctacc ttgcccccgg tgcgtacgtg aagggtgcta tagagtattt caccaagcaa 961 aacttctatg caactggtca tggtgtccta tcaggtgaaa actatgttta ccaagccaat 1021 gctggcgaca actatgttgc agtcaagagc gattcgacca gcctccggat gtggtggcac 1081 aataaccttg ggggtggtca aacatggtac tgcgttggcc cgacgatcaa tgcgccacca 1141 ttcaacacta tggatttcaa tggaaattct ggcatctcaa gtcaaattag cgactataag 1201 caggtgggag ccttcttctt ccagacggat ggaccagaaa tctatcccaa tagtgtcgtg 1261 cacgacgtct tctggcacgt caatgatgat gcaatcaaaa tctactattc gggagcatct 1321 gtatcgcggg caacgatctg gaaatgtcac aatgacccaa tcatccagat gggatggaca 1381 tctcgggata tcagtggagt gacaatcgac acattaaatg ttattcacac ccgctacatc 1441 aaatcggaga cggtggtgcc ttcggctatc attggggcct ctccattcta tgcaagtggg 1501 atgagtcccg attcaagcaa gtccatatcc atgacggttt caaacgttgt ttgcgagggt 1561 ctttgcccgt ccctgttccg catcacaccc ctacagaact acaaaaattt tgttgtcaaa 1621 aatgtggctt tcccagatgg gctacagaca aatagtattg gcacaggaga aagcattatt 1681 ccagccgcat ctggtctaac gatgggacta aatatctcca gctggactgt tggtggacaa 1741 aaagtgacaa tggagaactt tcaagccaat agcctggggc agttcaatat tgacggcagc 1801 tattgggggg agtggcagat tagtcgaatt tccagctctc agagcgcgtg agtgcttcta 1861 cccgctcctt tacccttgtc gaaggatcaa ggcataagtt agctcatgtg aaggcgattt 1921 cagttcattc tctctttttt ggagctcatt tccttttcga ccaattgtga caccaaattg 1981 ccatgtgtac tgtaattggt caaatgaacg ttaaccttcg atttaatatg gacatttcca 2041 ggtttcctta ctctgtcgat tatgcctaac tcgggttgat gtcttgtcag gatgaaaatc 2101 tcgttgtcat gtacttcgag tgaaatgggc agggctaacc cctaagccct aacgcccaat 2161 cgacttataa gtctagatgt ttatgctatg caggctctgg aatgatttac attccatgct 2221 ataca (SEQ ID NO: 27)

[0114] Although certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the present invention be limited to such embodiments. Various modifications may be made to such embodiments without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

1. A biofilm-degrading composition comprising a nucleic acid encoding the mutanase enzyme of SEQ ID NO:1 and the mutanase enzyme expressed from said nucleic acid in a pharma- ceutically acceptable carrier.

2. 10. The composition of claim 1, wherein the composition is produced in a plant plastid and comprises a plant residue, the plant residue being freeze-dried.

3. The composition of claim 1 further comprising dextranase.

4. 4. The composition of claim 3, wherein the mutanase and dextranase are produced in plant plastids and are present in plant residues.

5. 3. The composition of claim 2, further comprising at least one biofilm-degrading enzyme selected from dextranase, glucoamylase, glucanase, deoxyribonuclease I, DNAase, disperdin B, glycoside hydrolase, and the enzymes listed in Table 2.

6. 6. The composition of claim 5, wherein the at least one biofilm-degrading enzyme has a sequence selected from SEQ ID NOs: 2, 12, 14, 16, 18, 20, 24 and 26.

7. The composition of claim 3 further comprising an antimicrobial peptide (AMP).

8. The composition of claim 3 further comprising an antimicrobial / antibiotic.

9. 6. The composition of claim 5 further comprising fluoride and / or chlorhexidine (CHX).

10. The composition of claim 3, wherein the carrier is a chewing gum.

11. The composition of claim 3 , wherein the carrier is selected from a candy.

12. The composition of claim 3 , wherein the carrier is an oral rinse.

13. 4. Use of an effective antimicrobial amount of the composition of claim 3 in the manufacture of a medicament for the treatment of a biofilm containing an undesirable microorganism.

14. 14. The use according to claim 13, wherein the biofilm is present in the oral cavity and the medicament is a chewing gum comprising the enzyme.

15. 4. The composition of claim 3, wherein the plant residue is from a tobacco or lettuce plant.

16. 4. The composition of claim 3, wherein the dextranase and mutanase are present in a ratio of 5:

1.

17. 11. A method for inhibiting the deposition of a biofilm on a surface, comprising pretreating said surface with the composition of claim 3, said composition having an antimicrobial effect and inhibiting the formation of said biofilm on said surface, excluding medical procedures on humans.

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