KR101081745B1 - Staphylococcus saprophyticus BMSZ711 producing poly-Ν-acetylglucosamine and method for producing poly―Ν―acetylglucosamine from Staphylococcus saprophyticus BMSZ711 - Google Patents

Abstract

The present invention is a poly isolated from soil-relates to a novel strain which produces exo-biopolymer containing as a main component acetylglucosamine Staphylococcus four propionic tea coarse BMSZ711 - Ν. Further, from the strain poly- it relates to a acetylglucosamine-Ν-poly production in this method and to produce acetylglucosamine-Ν.

Staphylococcus saprophyteus BMSZ711, poly-Ν-acetylglucosamine producing strain, glycerol, poly-Ν-acetylglucosamine, glucosamine

Description

Staphylococcus saprophyticus BMSZ711 producing poly-Ν-acetylglucosamine and method for producing poly-Ν-acetylglucosamine from Staphylococcus saprophyticus BMSZ711}

The present invention is poly-Ν-Staphylococcus producing exo-biopolymer containing as a main component acetylglucosamine Lactococcus four propionic tea coarse BMSZ711, from the strain of poly-Ν-how to produce acetylglucosamine and from which the produced poly - Ν - it relates to a acetylglucosamine.

Ν - acetylamino saccharide chitin oligosaccharides having the basic structure unit of glucosamine (chitin oligosaccharides) in a known poly - Ν - acetylglucosamine (Poly- Ν -acetyl glucosamine: PNAG) has various biological activities such as anticancer action and induced (elicitor action) Activity has given rise to a multifaceted reputation. Ν-acetyl-glucosamine is Ν-glucose or an analog of (analog) sugar, D- Glucosamine is an amino containing only amino-acetyl-amino-group-containing amine or a glucose analog sugar. When the unit of the (monomer) is polymerized in a linear, poly Ν-acetyl-glucosamine, and poly- form a glucosamine respectively.

Glucosamine or its derivatives Ν -acetyl-glucosamine is a building block of animal and human cartilage and is also found in the fungi's cell wall and in the external skeleton of insects, shrimps and crabs. chitin) building blocks. The microorganism produces an exobiopolymer comprising glucose, glucosamine (chitosan), or Ν -acetylglucosamine (chitin). Glucosamine is produced in all organisms such as bacteria, yeasts, fungi, plants and animals.

Ν -acetyl glucosamine, glucosamine and its epimer galactosamine are large structures called proteoglycans (sugar chains bound to protein cores) and glycoproteins (slightly sugar bound proteins). Found in the molecule. Hyaluronan and chondroitin sulphate ( Ν -acetylgalactosamine and glucuronic acid), known as hyaluronic acid (a very long polymer of Ν -acetyl-D-glucosamine and glucuronic acid) Long polymers) are such structural molecules.

Proteoglycans and glycoproteins are found on the cell surface and extracellular matrix. Proteoglycans function as lubricants and supporting elements in connective tissue. At the cell surface, proteoglycans interact with the extracellular matrix. It absorbs large amounts of water to form a viscoelastic solution. Glycoproteins appear to act as determinants of cell behavior and as lubricants in epithelial tissues. Glycoproteins on the cell surface play a role in communication, cell structure, and self-recognition by the immune system. Many peptide hormones are glycoproteins. For example, follicle stimulating hormone (FSH) is a glycoprotein secreted by the pituitary gland and functions in the gonads. Common sugar units in glycoprotein sugar chains include glucose, galactose, mannose, fructose, N -acetylgalactosamine, N -acetyl-D-glucosamine and the like. The basic structure of a proteoglycan consists of a protein core with one to one hundred sugar chains called glycosaminoglycans covalently linked to specific serine amino acids. It is distinguishable from proteoglycans based on carbohydrates bound to glycoproteins. Glycosaminoglycans consist of repeated disaccharide subunits, whereas sugars bound to glycoproteins lack a series of repeat units. Uronic acid and hexose, most commonly N -acetyl-D-glucosamine or its epimer N -acetylgalactosamine, constitute a disaccharide subunit.

Hyaluronan is an important class of glycosaminoglycans, known as hyaluronic acid, and is widely distributed in most body fluids, such as the synovial fluid of the skin, cartilage and joints, the vitreous fluid of the eye, and the umbilical cord. In the hyaluronic acid synthesis process, N -acetylglucosamine is a structural unit of repeating units.

Chondroitin sulfate is the major glycosaminoglycan of cartilage. It is a major component of the tendons, ligaments, and aorta and has been isolated from brain, kidney and lung tissue. Chondroitin is a N - a copolymer of acetyl galactosyl samin (N -acetylgalactosamine) and gluconic kuron acid (glucuronic acid) going. N -acetylgalactosamine is obtained from N -acetyl-D-glucosamine in a reversible epimerization reaction.

In humans, glucosamine is the precursor of glucosamine-glycans (such as hyaluroic acid, chondroitin sulfate, and keratin sulfate) that are needed to restore or maintain healthy cartilage and joint function. Glucosamine is a component of polymer chains such as proteoglycans and glycosaminoglycans, which are necessary for the repair and maintenance of healthy cartilage, but as they age, they gradually lose their function on proteoglycan production and develop older osteoarthritis. Clinical trials using glucosamine for the treatment of osteoarthritis began in the early 1980s. Since then, the use of glucosamine as a dietary supplement has increased. Poly - Ν - acetylglucosamine, or poly-glucosamine and a variety of functions, such as a semi-medicinal or pharmaceutical agents, or immunomodulators.

First of all, PNAG / PGA functions as an osteoarthritis and joint pain relief agent. Many researchers have looked at the efficacy of glucosamine as a pain-reducing component for osteoarthritis and joint pain. As a result, glucosamine not only inhibits prostaglandin synthesis but also regulates anti-inflammatory activity in animal models, as well as promotes the synthesis of proteoglycans. Muller-Fabbender et al. Have made clinical trials for oral glucosamine and found it to be equally effective in treating knee osteoarthritis. Ibuprofen acted faster than glucosamine but had side effects. Noack et al. Reported that patients with osteoarthritis in the knee had fewer results such as the Lesquene index on the severity of degenerative arthritis in the knee than when placebo was administered orally with glucosamine. I found that. The response to the drug is defined as a 3 point reduction in the response to the Lesquines index for knee degenerative arthritis, with 52% of patients responding to glucosamine compared to 37% of responding to placebo.

In addition, PNAG / PGA is used as a vaccine target. Over the past 25 years, Gram-positive cocci, especially S. aureus and S. epidermidis, are the main isolates of hospital infections. This increase in bacterial infections in hospitals is due to an increase in the antibiotic's antimicrobial resistance, which has given strong attention to alternative strategies such as vaccines to prevent and control infections.

In addition, PNAG can be used as an immune inducer. A series of studies by Azuma et al. Found that some PNAGs can upregulate macrophage function, which is a measure of immune levels (measured by TNF, IL-1, etc.), with 1-10 μm PNAG particles. Innate mice have been shown to have an increased innate immune response. In addition, PNAG has been shown to have specific Th1 adjuvant effects on Th1 immune development against infections such as mycobacterial antigen and tuberculosis.

Commonly known PNAGs, such as chitin, are produced by chemical processes in shrimp, crab, squid and cuttlefish. The basic principle in chitin production is to remove all materials that cannot withstand the treatment of alkali (primarily protein for shrimp and crabs) and diluted acids (primarily minerals that are calcium carbonate). The residue left after washing out unreacted chemicals is chitin. The conversion of chitin to chitosan is achieved by treating alkaline 50%. In general, GlcN is produced by acid hydrolysis of chitin extracted from crab and shrimp shells. Concentrated hydrochloric acid cleaves the polymer and deacetylates GlcNAc to produce glucosamin (GlcN). GlcNAc is produced by acetylating GlcN using acetic anhydride. However, as demand increases, PNAG or GlcNAc production may be limited by raw material supply fluctuations. In addition, PNAGs of crustacean origin are not suitable for people with crustacean allergies. In addition, chemical processes have greater problems such as the generation of environmentally harmful alkali and acid wastes, low yields and high costs.

Thus, this study poly- to separate a possible new strain acetylglucosamine production - Ν. It was confirmed that acetylation can be produced efficiently geulrukosaminreul - is separated from the soil Staphylococcus four propionic Tea Caicos BMSZ711 poly - Ν.

The present invention is poly relates to the star that produces the exo-biopolymer comprising mainly of acetyl glucosamine Philo caucuses four propionic Tea Caicos BMSZ711 (Biological Resource Center Accession Number KCTC 11407BP, deposit date of October 24, 2008) - Ν .

Glucosamine production was checked by HPAEC (High Performance Anion Exchange Chromatography) in strains collected from the soil, and the strains showing the highest productivity were selected, and the selected strains were morphological and physiological as described in Table 1. It has been demonstrated that it is a new strain with glucosamine production ability with chemical and biochemical properties. This was named Staphylococcus saprophyteus BMSZ711 and was deposited in BRC with accession number KCTC 11407BP.

The molecular size of the glucosamine enriched exobiopolymer purified from the media culture of the Staphylococcus saprophyticus BMSZ711 strain of the present invention is about 12 KDa, the smallest size ever known in the literature. In addition, ¹³ C CP / MAS NMR, FTIR and WXRD analysis showed that the purified glucosamine polymers N - polymers, poly-acetylglucosamine (GlcNAc) - Ν - acetyl and glucosamine (PNAG) was found to have a β structure .

The present invention is Staphylococcus four propionic tea coarse BMSZ711 the culture in a medium and polyester from the medium culture - Ν - star, including the step of separating the acetylglucosamine Philo Lactococcus four polyester from propionic tea coarse BMSZ711 - Ν - acetyl A method for producing glucosamine. Staphylococcus four pie Pro from coarse tea BMSZ711 poly - Ν - equal to look at in more detail the process for producing acetylglucosamine.

Poly- N -acetylglucosamine may be prepared by (i) inoculating Staphylococcus saprophyticus BMSZ711 in a culture medium; (ii) centrifuging the culture medium to recover the supernatant; And (iii) filtering and concentrating the recovered supernatant to obtain poly- N -acetylglucosamine.

Strain culture in the step (i) is carried out in a conventional manner under appropriate conditions to optimize the growth of Staphylococcus saprophyticus BMSZ711 and the production capacity of poly- N -acetylglucosamine.

The medium is preferably a liquid medium. In addition, the minimum medium is preferred to the nutrient medium, M1 minimum medium was used in the present invention. Suitable carbon sources for producing poly- N -acetylglucosamine are glucose, fructose, sucrose, gluconate and glycerol, preferably glycerol and fructose, more preferably glycerol. Suitable nitrogen sources for producing poly- N -acetylglucosamine are ammonium chloride and ammonium sulfate, preferably ammonium sulfate.

In addition, in order to increase poly- N -acetylglucosamine production capacity, some amino acids such as yeast extract and valine and glutamic acid may be further included in the medium.

Staphylococcus saprophyticus BMSZ711 of the present invention is the largest poly- N when cultured in a medium containing 100 mM glycerol, 0.3% ammonium sulfate, 1.5 g / l yeast extract, and 2 mM valine. -Shows the ability to produce acetylglucosamine.

Suitable medium pH for the production of poly- N -acetylglucosamine is 6-8, preferably 7, medium temperature 25-37 ° C., preferably 30 ° C., incubation time 60-96 hours, preferably 72 hours. .

Samples obtained through the filtration and concentration of step (iii) may be subjected to further purification, concentration, and the like. For example, additional processes, such as proteinase K treatment and diafiltration, can be performed one to several times.

The present invention also relates to poly- N -acetylglucosamine having a size of 12 KDa derived from Staphylococcus saprophyticus BMSZ711 produced according to the above method.

Glucosamine polymers produced from Staphylococcus saprophyticus BMSZ711 can be processed as such or in a further process and used as main ingredients, adjuvants or additives in foods, dietary supplements, cosmetics, medicines, feeds and the like. For example, the obtained glucosamine polymers can be used in antihypertensives, antihemostatics, anticholesterols, anticancers, antibacterial agents, immune activity enhancers, calcium absorption accelerators, as well as biocompatible surgical sutures, wound healing promoting skin, drugs It can also be used as a carrier and dental material. It can also be used as a raw material for plant growth regulators, biofertilizers and animal feed. It can also be added to a variety of foods.

The poly- N -acetylglucosamine can be produced in high yield by culturing the Staphylococcus saprophyticus BMSZ711 of the present invention under appropriate conditions.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1: Materials and Methods

1-1 Isolation and Characterization of Strain BMSZ711

Soil samples were taken from offshore Sacheon, South Korea, suspended in sterile water, containing 20 mM ammonium sulfate as a nitrogen source and 70 mM fructose as a carbon source (Zamil, SS et al., (2008a) J. Biotechnol.V4-YP-058). After incubation for 2 days at 30 ° C., 180 rmp, the culture solution was diluted 10 ⁵ times. 100 μl of the diluted culture was inoculated into LB solid plate medium and incubated at 30 ° C. for 24 hours. Many single colonies were randomly selected and grown in M1 medium and the production of glucosamine was checked by High Performance Anion Exchange Chromatography (HPAEC). Among them, the bacteria with the highest glucosamine production ability were found.

The isolated strains were characterized using the ID 32 STAPH kit and identified as Staphylococcus saprophyteus by a microbial identification system (Sherlock system software) (% identification [% ID = 97]).

1-2 16s rRNA gene sequencing

The isolated strain was confirmed by 16s rRNA gene sequence homology analysis. Genomic DNA was isolated from bacteria cultured overnight at 30 ° C. in 5 ml LB medium. Genomic DNA isolation was subjected to the Marmur process (Marmur, J. (1961) J. Mol. Biol. 3: 208-218). After genomic DNA isolation, 16s rRNA gene sequencing was performed. Blast search was performed using sequence data and showed 99% sequence homology with S. saprophyticus subsp saprophytiucs ATCC 15305.

1-3 Badge Condition

We used the four S. propionic Atticus BMSZ711 in this study 1% yeast extract, 1.5% Nutrient Broth, was subcultured in NR (nutrient rich) solid medium containing 0.2% of ammonium sulfate and 1.8% agar, 4 Kept at ° C. Seed (seed) inoculated with S. four propionic Atticus BMSZ711 single colonies cultured in 5 ml NR-broth medium and incubated at 30 ℃, 180 rpm 8 hours. 2 ml of spawn cultures containing 200 ml of modified M1 minimal medium of the same composition as previously reported (Zamil, SS et al., (2008a) J. Biotechnol. V4-YP-058). Transfer to a flask.

Effect of Culture Medium, Carbon Source, and Nitrogen Source on 1-4 Glucosamine (glcN) Production

S. saprophyticus BMSZ711 was incubated in LB, NR, Kings B medium, and M1 minimal medium containing fructose (70 mM) and ammonium sulfate (0.3%) as the carbon and nitrogen sources. The initial pH of the medium was adjusted to 7.0. In order to examine the effects of various carbon and nitrogen sources on glcN production, fructose and ammonium sulfate were appropriately replaced. A certain amount of culture medium was taken at appropriate time intervals and cell growth was monitored by measuring optical density at 600 nm (OD ₆₀₀ ). Unless otherwise noted, all cultures were performed by incubating a 1 l flask containing 200 ml medium in a rotary shaker incubator at 30 ° C. and 180 rpm.

Effect of temperature and initial pH on 1-5 glucosamine (glcN) production

examine the effect of temperature on the glcN generated, S. four propionic Atticus BMSZ711 were cultured in minimal medium M1 of the four different temperatures (15, 20, 30 and 37 ℃). To measure PNAG production efficiency, M1 medium containing glycerol (70 mM) and ammonium sulfate (0.3%) as carbon and nitrogen sources, respectively, was used as standard medium. A study was conducted to determine glcN production, bacterial growth rate (OD ₆₀₀ and dry weight) and pH over time.

The effect of pH on glcN production was carried out in a range of pH 2-8 at 30 ° C. using M1 minimal medium containing glycerol (70 mM) and ammonium sulfate (20 mM) as carbon and nitrogen sources, respectively.

Effect of Yeast Extract, Amino Acids and Osmolarity on 1-6 Glucosamine Production

In order to determine the effect of yeast extract, amino acid and molar osmolarity on glucosamine (glcN) production in M1 minimal medium containing glycerol (100 mM) and ammonium sulfate (0.3%), yeast extract, valine, glycine ( Gly), an amino acid such as glutamic acid (GA), and sodium chloride were added so that the final concentrations were 0.5-1.5 g / l, 1-3 mM and 2.5-10 g / l, respectively. Cell growth was measured at an optical density of ₆₀₀ nm (OD ₆₀₀ ) and glcN production was measured by High Performance Anion Exchange Chromatography (HPAEC) method.

1-7 Purification of Glucosamine Rich Exobiopolymer

Glucosamine-containing polymers were purified by slightly modified methods such as Joyce et al. (Joyce, JG (2003) Carbohyd. Res. 338: 903-922). The 72 hour old cell culture was heated at 60 ° C. for 90 minutes and centrifuged at 15000 rpm, 15 minutes. Cell-free centrifuge supernatant (1 L) was collected, filtered using a Suporcap-100 0.8 / 0.2 μm cartridge (Pall corporation, Ann Arbor, Mich.) And 5-KDa molecular weight cut off: (MWCO ) Concentrated to tangential flow filtration (TFF) using a hollow fiber membrane cartridge (Pall corporation, Ann Arbor, MI) to 60 ml.The concentrated sample was diafiltered twice for 1 volume of deionized distilled water. Adjusted to mM Tris-HCl, pH 8, 2 mM CaCl ₂ and 2 mM MgCl _2. Samples were treated with proteinase K (4 mg) for 16 hours at 20 ° C. The samples were treated with 1 volume of 2M NaCl, 1 Diafiltration was performed 1 times and 1 volume of deionized distilled water for a volume of low pH buffer (25 mM sodium phosphate, pH 2.5, 0.1 M NaCl) The sample was 3.5 kDa MWCO with at least 3 exchanges of deionized water. The membrane was filtered for 3 days The sample was lyophilized and used for subsequent analysis. Was used.

Structure Analysis of 1-8 Glucosamine Exobiopolymer

(1) Gel Permeation Chromatography (GPC)

The molecular weight of the BMSZ711 polymer was measured at room temperature by gel permeation chromatography (GPC) using an FPLC system (Amersham Pharmacia Biotech) equipped with a Sephadex-200 column. The column was pre-equilibrated in 0.1M PBS buffer pH 8.0 before sample loading and adjusted using molecular mass standards for GPC. Purified glcN polymer solution (100-500 μl in 1 ml of DW) was GPC analyzed at room temperature. Column normalization was performed using molecular weight standards including ferritin (400 KDa), BSA (67 KDa) and cytochrome (13.7 KDa).

(2) ¹³ C CP / MAS (Cross Polarization / Magic Angle Spinning) NMR Analysis

The solid state ¹³ C CP / MAS NMR spectra were obtained on a Varian Unity NOVA 600 spectrometer, 14.1 T using an 89 mm bore magnet that spins at a magic angle of 7 KHz for a sample weight of about 150 mg. It was.

(3) FTIR spectrum analysis

High resolution (0.1 cm ⁻¹ ) FTIR analysis of the purified samples was performed in vacuo with a VERTEX 80v (Bruker Optics) FTIR spectrophotometer equipped with a DTGS (with KBr window) detector. Freeze-dried samples were mixed with KBr powder, powdered at room temperature and compressed into thin pellets.

(4) Wide-angle X-ray diffraction (WAXD) analysis

Freezing - the two-dimensional space using the co-source radiation (λ = 1.79Å ) over a range 100 (2 θ) - WAXD pattern of the dried sample was 0.05 ° step raengsseu (step length) and the time to 0 of 29.8 s per step Detection was recorded on a General Area Detector X-ray Diffraction System (D8 DISCOVER with GADDS, Bruker AXS (Germany)).

1-9 Determination of Glucosamine and Other Sugars Using HPAEC-PAD

The composition of glucosamine and other sugars in the crude and purified samples was measured after acid hydrolysis. 10 mg of lyophilized sample was treated with 1 ml of 6 N HCl at 100 ° C. for 4 hours. The released sugars were subjected to high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex DX-300 BioLC system with a GP50 gradient pump and an ED40 electrochemical detector. Analyzed. 25 μl of the hydrolyzate was injected into a CarboPac PA1 column (4.6 x 250 mm) equipped with a CarboPac PA1 guard column (4.6 x 50 mm) in front of a borate trap (Dionex) and at 1 ml / min with 16 mM NaOH. Sugars were separated by isocratic elution.

1-10 Electron Microscope Analysis

To visualize the sample, the sample was fixed at 4 ° C. for 3 hours in a solution of glutaraldehyde (Electron Microscopy Sciences) diluted with 0.2 M sodium phosphate buffer (pH 7.2) to a final concentration of 2.5%, followed by fixation of glutaraldehyde. The prepared samples were washed twice with phosphate buffer and fixed for 2 hours at 4 ° C. with 1% osmium-tetra-oxide solution. Osmium-tetra-oxide fixed samples were dehydrated in graded ethanol series (5 minutes each at 50, 60, 70, 80, 90 and 100%). Finally, the samples were coated with gold (fine coat sputter JFC-1100, JEOL) and observed using a field emission scanning electron microscope (FE-SEM) (Philips XL30 S FEG, Netherlands).

Example 2: Characteristics of Isolated Strains

Morphological, biochemical and nutritional characteristics of the isolated strains are listed in Table 1. The cell shape of the strain was coccus and showed positive gram staining. There is activity against oxidase and catalase. Esculine was not hydrolyzed. Glucose, Fructose, Maltose, Lactose, Trehalose, Mannitol, Raffinose, Ribose, Cellulose ), Oz (saccharose as Saccharomyces), N - acetyl-glucosamine (N -acetyl-glucosamine) was for the bacteria to grow. In addition, positive results were observed for sodium pyruvate, pyroglutamique β-naphtylamide and novobiocine. Potassium Nitride, 2-naphtyl-β-D-galactopyranose, L-arginine-β-naphthylamide (L-arginine- Negative results were observed for β-naphtylamide, 2-naphtyl phosphate and acid pyroglutamigue β-naphtylamide. Therefore, from the results obtained using the ID32 STAPH kit, the isolate was identified as Staphylococcus saprophyticus (% identification [% ID = 97]), and the strain was identified as Staphylococcus saprophyte . Named Curse BMSZ711 and deposited with Korean Collection for Type Cultures (KCTC) under the accession number KCTC 11407BP October 24, 2008 Deposited. Strains were also identified by 16S rRNA gene sequence (FIG. 1).

Example 3 Effect of Culture Medium, Carbon Source, and Nitrogen Source on Glucosamine (glcN) Production

Culture media are known to play an important role in the production of biopolymers. The researchers found that different culture media were appropriate for biopolymer production by different bacteria. For PNAG produced by S. aureus , some researchers used TSBG (typtic soy broth 1% glucose) (Cerca, N. et al. (2007) PNAS. 104: 7528-7533). , Enterobacter sp. The medium containing acetic acid was used for BL-2 (Son, MK et al. (2005) J. Microbiol. Biotechnol. 15: 626-632). In this study, LB (luria bertin), NR (nutrient rich) and Kings B medium were used as three kinds of nutrient medium, and M1 medium was used as one kind of minimum nutrient medium. As a result of FESEM visualization, it was found that only M1 medium was capable of producing PNAG, and the nutrient medium did not secrete PNAG (FIG. 2). Therefore, M1 minimal medium was used for this study.

Cell growth and EPS production generally depend on the carbon source which affects the properties, sugar content and / or molecular weight of EPS (Wachenheim, DE et al. (1992) Appl. Environ. Microbiol. 58 (1): 385-391 ). 13 carbon sources were used for the glcN production check. Among them, BMSZ11 was able to produce glucosamine in the presence of five carbon sources (FIG. 3). These are glucose, fructose, sucrose, gluconate and glycerol. As a result of the effect of the carbon source on glucosamine production, glycerol showed the highest production in S. saprophyticus BMSZ711 (˜53.17 mg / l). The second highest production is when fructose is used as a carbon source.

In another study, glycerol produced the highest EPS (Noghabi, KA et al. (2007) Process Biochem. 42: 847-855). Thus, various concentrations of glycerol and fructose were examined to find the optimal concentration (FIGS. 4 and 5). BMSZ711 cells were grown at 30 ° C., pH 7 in three different concentrations of glycerol and fructose-containing M1 medium at 70 mM, 100 mM and 140 mM. When 100 mM glycerol is used as the carbon source (~ 189.31 mg / l), the highest glcN is produced at 72 hours and the second highest production is when 140 mM glycerol is used as the carbon source (~ 170 mg / l). When 70 mM fructose was used as the carbon source, a maximum production of 119 mg / l glcN was observed after 72 hours of cell growth. According to the results obtained from this experiment, 100 mM of glycerol produced the highest glcN at 72 hours of incubation time. Acetylglucosamine-6-phosphate (N -acetyl glucosamine 6 phosphate), uridine 5'-diphosphate - - In a previous study, glycerol N which can play a role in the increased production of glucosamine-N-acetyl galactose amine (uridine 5 '-diphosphate-N-acetyl galactoseamine) and uridine 5'-diphosphate - N - is the acetyl glucose amine (uridine 5'-diphosphate- N -acetyl glucoseamine ) inducing twice was observed (Filer, D. et al. (1977) J. Bacteriol. 131: 751-758).

Inorganic or organic nitrogen addition can affect cell growth and EPS production. Six nitrogen sources were used to test the effect of nitrogen sources on glcN production (FIG. 6). Inorganic nitrogen sources have been observed to be more effective at producing than organic nitrogen sources. The highest glcN was observed when ammonium sulfate was used as the nitrogen source. Similar results were also observed in exobiopolymer production by Pseudomonas fluorescens BM07 (Noghabi, KA et al. (2007) Process Biochem. 42: 847-855). Various concentrations of ammonium sulfate were investigated for glcN production (FIG. 7). Glucosamine production was found to increase slowly with increasing ammonium sulfate, the highest production obtained when 0.9% ammonium sulfate was used as the nitrogen source, but there was no significant difference between glucosamine production when using 0.3% and 0.9% ammonium sulfate. Not observed. It is more economical to use 0.3% ammonium sulfate. From this, it can be seen that glcN production is more dependent on carbon source than nitrogen source. Prasertsan, P, et al. (2008) Carbohyd.Polym. 71: 468-475) shows that low concentrations of nitrogen are more beneficial for EBP production, and Lo et al. Xanthan if the nitrogen concentration in the medium is too high. The production was reported to be bad (Lo, YM et al. (1997) Appl. Microbiol. Biotechnol. 47: 689-694).

Example 4 Effect of pH and Temperature on Glucosamine (glcN) Production

Culture conditions such as pH and temperature affect glucosamine (glcN) or PNAG production. At low pH, glcN production was very low or not produced (FIG. 8). BMSZ711 secreted glcN at pH 4 and increased until pH 7, but then decreased. Cell growth was very slow at low pH, but maximal growth was maximal at pH 7, and cell growth as well as glcN production were inhibited under alkaline conditions. this. Initial media pH was also maintained at 7 for glcN production by other bacteria such as E. coil , S. aureus and S. epidermidis . , T. et al. (2002) Infect. Immun. 70: 4433-4440; Cerca, N. et al. (2007) PNAS. 104: 7528-7533). However, pH 8 was optimal for glcN production by Enterobacter r sp. BL-2 (Son, MK et al. (2005) J. Microbiol. Biotechnol. 15: 626-632) .

Incubation temperature is one of the important conditions for glcN production. In this study, BMSZ711 cells were grown at four different temperatures, such as 15, 20, 30 and 37 ° C. Cell growth was very slow at 15 and 20 ° C. and no glcN was produced, but cells were grown for 72 hours when grown at 30 ° C. in M1 medium containing 70 mM glycerol, 0.3% ammonium sulfate, 0.1% yeast extract and 1% NaCl. Later, glcN maximum production of 131.90 mg / l was observed (FIG. 9). Cell growth was also very fast and a maximum cell dryness of 2.76 g / l was obtained after 72 hours of cell culture. GlcN was also produced when the cells grew at 37 ° C. (FIG. 10). Maximum production of 54.16 mg / l was obtained after 60 hours of cell growth, but production was much lower than 30 ° C. (less than half). At both temperatures glcN is produced in a stationary phase. Many bacteria, such as Enterobacter BL-2, produced EPS at 30 ° C. (Son, M.K. et al. (2005) J. Microbiol. Biotechnol. 15: 626-632).

Example 5: Effect of Yeast Extract, Amino Acids and Molar Osmolarity

Yeast extracts can significantly affect the production of EPS of many microorganisms. The effect of yeast extract on glcN production in BMSZ711 was investigated. When the yeast extract was added in the range of 0.5-1.5 g / l, the yeast extract showed a significant effect on glcN production and also showed cell growth (Fig. 11). Compared to the control medium (without yeast extract added), when the yeast extract was added at 1.5 g / l, glcN production increased by 4 times. In addition, cell growth increased about 7-fold compared to the control sample. Yeast extracts approximately doubled the production of capsular polysaccharides in Streptococcus pneumoniae serotype 23F (Goncalves, VM et al. (2002) Appl. Microbiol. Biotechnol. 59: 713- 717). EPS production from Enterobacter cloace WD7 increased when yeast extract was added at a 0.05% rate (Prasertsan, P, et al. (2008) Carbohyd. Polym. 71: 468-475). In yeast ( Aureobasidium pullulans ) and Kluyveromyces fragilis mixed culture, increased yeast extract concentrations resulted in high pullulan yield (Shin, YC et al (1989) Biotechnol. Bioengin. 33 129-133).

12 and 13 show the effects of three different amino acids, respectively, affecting glcN production and cell growth. Amino acids affect glcN production and cell growth. The highest glcN was produced when valine was inserted at the 2 mM ratio (˜277 mg / l). However, glycine showed a negative effect on glcN production. Glycine significantly reduces glcN production, while glutamic acid has a positive effect on glcN production. In all cases, maximal production occurred in the stationary phase.

In this study, an increase in molar osmolality decreased cell growth and glcN production (FIG. 14).

Example 6: Purification of Glucosamine Rich Exobiopolymer

Glucosamine rich exobiopolymers were purified with a slight modification of the method described by Joyce et al. (Joyce, J.G. et al. (2003) Carbohyd. Res. 338: 903-922). The most important step in the biopolymer purification described in Examples 1-7 is to concentrate the sample using tangential flow filtration (TFF). After this step, the sample contains at least 60% glucosamine. Purified and original samples were analyzed using HPAEC-PAD, and Figures 16 and 17 show that the purified samples contained more than 87% glucosamine. Purified samples were analyzed and characterized using the following techniques.

Example 7: Structural Analysis of Glucosamine Polymer

7-1 Molecular Size of Purified Glucosamine Polymer

The molecular size of the purified polymer was measured by gel permeation chromatography. One distinct peak was obtained. The molecular size of the purified glucosamine rich biopolymer was determined to be about 12 KDa (FIG. 18). Glucosamine polymer size obtained from S. aureus is 300 KDa (Joyce, JG et al. (2003) Carbohyd. Res. 338: 903-922), and S. epidermidis is 460 KDa, 100 KDa, 21 KDa (Maira-Litr, T. et al. (2002) Infect. Immun. 70: 4433-4440) and Enterobater sp BL-2 is 106 KDa (Son, MK et al. (2005) J. Microbiol Biotechnol. 15: 626-632), the smallest glucosamine biopolymer ever reported in glucosamine polymer size.

7-2 Purification of Purified glcN Exobiopolymer ¹³ C CP / MAS NMR Analysis

Solid state NMR (Solid state NMR) is one of the very sophisticated techniques for characterizing solid substrates. ¹³ C CP / MAS NMR of purified glcN exobiopolymers was tested by Tenor et al. (Tanner, S. et al. Macromolecules (1990) 23: 3576-3583) and Cardenas et al. (Crdenas, G. et al. (2004) J. Appl. Poly. Sci. 93: 1876-1885). Chemical shifts are summarized in Table 2. As can be seen in Table 2, the spectra of the purified glucosamine polymer and β chitin were quite similar to each other. For chitin, characteristic peaks of acetyl carbon were observed at 174.5 and 23.8 ppm. Chemical shifts of C-3 and C-5 carbons were observed with a single peak, identical to β chitin. Similar chemical shifts were observed for the glcN polymer, but the chemical shifts of C3 and C5 for α-chitin were different. This is due to the different arrangements of C3 and C5 that form hydrogen bonds. As such, characteristic peaks for acetyl carbon are observed at 174 and 24 ppm only for chitin and GlcNAc alone and these two characteristic peaks are not observed for glucosamine (Webster, A. et al. (2006) Solid State NMR. 30: 150-161), demonstrating that the glucosamine polymer is a polymer of N -acetyl glucosamine (GlcNAc). This result means that the polymer is of β structure, and because the sample is very similar to β chitin, the likelihood of having β (1-4) bonds is very high.

7-3 FTIR Analysis of Purified Glucosamine Exobiopolymers

The most widely known PNAG is chitin and is characterized using FTIR. Therefore, glcN exobiopolymer purified from BMSZ711 was characterized using FTIR, the spectrum is shown in Figure 19, the purified sample is a polymer of N -acetylglucosamine and has a β structure. The α and β structures of PNAG can be distinguished by FTIR spectroscopy because they have different hydrogen bonds. Two absorption peaks for the vibration mode of amide I in α PNAG (chitin) were observed in the 1660-1620 cm ⁻¹ region, while β PNAG (chitin) only one peak in this region. Characteristic peaks such as amides I, II, III were very similar to β PNAG (chitin), although some peaks were not completely similar to β PNAG (chitin). This may be due to the size difference of the biopolymer, which appears to have a simpler FTIR profile in the 3600-3000 cm ⁻¹ region because BMSZ711 PNAG (12 KDa) is much smaller than β PNAG (chitin).

7-4 purified glcN exobiopolymers were analyzed by wide angle X diffraction (WAXD)

Purified glcN exobiopolymer was characterized by WAXD analysis (FIG. 20). A broad peak was observed at the 2θ = 20 position with respect to the purified sample. This is very similar to the chitin peak observed at the 2θ = 19 position for squid chitin or β PNAG (chitin). The broad peak of the purified sample means that the purified polymer is amorphous.

Staphylococcus saprophyticus BMSZ711 of the present invention and the poly- N -acetylglucosamine polymer produced therefrom can be widely used in various fields such as food, dietary supplement, cosmetics, pharmaceuticals, and agriculture.

Figure 1 shows the 16s rRNA gene sequence results of Staphylococcus saprophyticus BMSZ711, a PNAG production isolate.

Figure 2 is a FE-SEM results showing that PNAG is produced in BMSZ711 cultured in M1 medium but not PNAG in LB medium.

Figure 3 is a result showing the effect of the carbon source affects the production of glucosamine. (1 = glucose 70mM, 2 = fructose 70mM, 3 = sucrose 70mM, 4 = lactose 70mM, 5 = galactose 70mM, 6 = glycerol 70 mM, 7 = gluconate 70 mM, 8 = mannitol 70 mM, 9 = sodium acetate 70 mM, 10 = arabinose 70 mM, 11 = xylose 70 mM, 12 = propionic acid Sodium propionate, 13 = mannose 70 mM)

Figure 4 shows the effect of glycerol concentration affecting glucosamine production in M1 medium containing 0.3% ammonium sulfate, 0.1% yeast extract and 1% NaCl, at 30 ° C, pH 7 conditions (1 = 70mM, 2 = 100mM) , 3 = 140 mM).

FIG. 5 shows the effect of fructose concentration affecting glucosamine production in M1 medium containing 0.3% ammonium sulfate, 0.1% yeast extract and 1% NaCl, at 30 ° C., pH 7 (1 = 70 mM, 2 = 100 mM) , 3 = 140 mM)

FIG. 6 shows the effect of nitrogen sources on glucosamine production (1 = potassium nitrate 0.3%, 2 = ammonium chloride 0.3%, 3 = peptone 0.3%, 4 = yeast extract 0.3%, 5 = 0.3% ammonium sulfate, 6 = 0.3% urea).

Figure 7 shows the effect of the concentration of ammonium sulfate affecting glucosamine production in M1 medium containing 100 mM glycerol, 0.1% yeast extract and 1% NaCl, 30 ℃, pH 7 conditions (1 = 0.3%, 2 = 0.6 %, 3 = 0.9%).

Figure 8 shows the effect according to pH affecting glucosamine production (M1 medium, carbon source is glycerol (Gly) 70mM, nitrogen source is ammonium sulfate 20mM, temperature 30 ℃).

FIG. 9 shows the hourly profile of glucosamine production in M1 medium containing Gly 70 mM, 0.3% ammonium sulfate, 0.1% yeast extract and 1% NaCl, at 30 ° C., pH 7 conditions.

FIG. 10 shows the hourly profile of glucosamine production in M1 medium containing Gly 70 mM, 0.3% ammonium sulfate, 0.1% yeast extract and 1% NaCl, 37 ° C., pH 7 conditions.

Figure 11 shows the effect according to the concentration of yeast extract affecting glucosamine production (1 = control: no yeast extract, 2 = yeast extract 0.5g / l, 3 = yeast extract 1 g / l, 4 = yeast extract 1.5 g / l).

12 shows the effect of amino acids on glucosamine production.

13 shows the effect of amino acids on cell growth (1 = valine 1mM, 2 = valine 2mM, 3 = valine 3mM, 4 = glycine 1mM, 5 = glycine 2mM, 6 = glycine 3mM, 7 = glutamic acid 1 mM, 8 = glutamic acid 2 mM, 9 = glutamic acid 3 mM)

14 shows the effect of molar osmolarity (sodium chloride) on glucosamine production and cell growth (1 = NaCl 2.5 g / l, 2 = NaCl 5 g / l, 3 = NaCl 10 g / l).

15 is HPAEC-PAD result showing the sugar composition of the original sample.

16 is HPAEC-PAD results showing the sugar composition of the purified sample.

17 shows the molecular weight of purified glucosamine (glcN) biopolymer measured by FPLC using Sephadex-200. The column was equilibrated with 0.1 M PBS and eluted with the same buffer.

18 shows ¹³ C CP / MAS spectra of purified samples. The peaks at 23.8 and 174.5 ppm are characteristic peaks of (-NH (CO) CH ₃ ) and CO, and the single peak at 75.4 confirms β structure and shows much similarity with squid chitin.

19 is the result of FTIR analysis of purified sample and very similar to β chitin of squid. A single peak at 1652.89 cm ⁻¹ confirms the β structure.

20 is a result of WXRD analysis of the purified sample.

Claims (11)

Staphylococcus saprophytis BMSZ711 (Bio resource Center Accession No. KCTC 11407BP), which produces an exobiopolymer containing poly- N -acetylglucosamine as a main component. Claim 1 Staphylococcus four propionic tea coarse polyacrylic from the culture and the medium culture in a culture medium BMSZ711 - N - star comprising the step of separating the acetylglucosamine Philo Lactococcus four Pro from the pie tea coarse BMSZ711 poly - N - acetyl geulrukosaminreul production How to (KCTC 11407BP). The method of claim 2, wherein the medium is a liquid medium. The method of claim 2 wherein the carbon source of the medium is selected from the group consisting of glucose, fructose, sucrose, gluconate and glycerol. The method of claim 2 wherein the nitrogen source of the medium is selected from the group consisting of ammonium chloride and ammonium sulfate. The method of claim 2 wherein the pH of the medium is 6-8. The method of claim 2 wherein the temperature of the medium is from 25 to 37 ° C. The method of claim 2, wherein the incubation time is between 60 and 96 hours. The method of claim 2, wherein the medium further comprises a yeast extract. The method of claim 3, wherein the medium further comprises an amino acid selected from valine and glutamic acid. delete

Images