KR20060034430A - Process for preparing poly( -??glutamic acid) from bacillus subtilis bs62, and poly( -??glutamic acid) prepared from the same - Google Patents

Abstract

The present invention relates to a method for producing poly (gamma-glutamic acid), which is a natural polymer, from a Bacillus subtilis strain, and to poly (gamma-glutamic acid) prepared therefrom, and more particularly, to Bacillus subtilis strain BS62 (KACC). 91064) and aerobic culture at a temperature of 30 to 39 ℃ in a medium containing L- glutamic acid and glucose; And purifying; Method for producing a poly (gamma-glutamic acid) from Bacillus subtilis BS62, characterized in that the D-type / L-type glutamic acid ratio prepared according to the production method is 40/60 to 60/40, the weight average molecular weight The poly (gamma-glutamic acid) is characterized in that it is about 600 kDa to 1000 kDa, the number average molecular weight is about 200 kDa to 500 kDa, and the polydispersity is 3 to 3.5.

The method for preparing poly (gamma-glutamic acid) using the Bacillus subtilis BS62 prepared according to the present invention has the advantage of producing a large amount of poly (gamma-glutamic acid), thereby increasing the viscosity enhancer in the pharmaceutical, food, and chemical fields. It can be usefully used for the preparation of a coating agent and a hydrogel that is a sustained release material.

Poly (gamma-glutamic acid) (PGA), Cheonggukjang, Bacillus subtilis

Description

Method for producing poly (gamma 산 glutamic acid) derived from Bacillus subtilis US62 and poly (gamma­glutamic acid) produced therefrom {Process for preparing Poly (γ­glutamic acid) from Bacillus Subtilis BS62, and Poly (γ­glutamic acid) prepared from the same}             

1 is a graph showing the growth rate, spore formation rate and poly (gamma-glutamic acid) production rate of bacterial cells measured according to the incubation time produced by Bacillus subtilis BS62 according to the present invention.

FIG. 2 is a graph showing the results of analyzing the ratio of the D / L glutamic acid content of PGA generated during the culturing of Bacillus subtilis BS62 using a medium containing 7% glutamic acid and 8% glucose.

Figure 3 is a graph showing the results of analyzing the change in content ratio of D-type / L-type glutamic acid in poly (gamma-glutamic acid) produced by Bacillus subtilis BS62 over time under each culture medium condition.

(Phase) with 7% L-glutamic acid + 8% glucose containing medium

(Bottom) when using medium containing 3% L-glutamic acid + 2% citric acid

The present invention relates to a method for producing poly (gamma-glutamic acid), which is a natural polymer, from Bacillus subtilis strains, and to poly (gamma-glutamic acid) prepared therefrom.

Poly (gamma-glutamic acid) was discovered in 1937 as an extracellular viscous substance of Bacillus anthracis ( Bacillus mesentericus . Z Immunitats forsch. 90, 304-318, 1937) by Ivanovics et al. It is reported that the main component of natto viscosity is poly (gamma-glutamic acid) ( Nippon Nogeikagaku Kaishi. (1963) 37, 407-411). Poly (gamma-glutamic acid) is a mucous substance that is a gamma-glutamyl conjugated polymer of D-glutamic acid and L-glutamic acid, and is known as an edible, water-soluble, anionic, and biodegradable polymer. It is produced from Bacillus strains hydrolyzed by and isolated from Cheonggukjang and Natto ( Agric. Biol. Chem . 48, 2373-2374, 1984). Poly (gamma-glutamic acid) synthesized by microorganisms is a kind of polypeptide in which the repeating unit of glutamic acid is covalently linked between alpha-amino group and gamma-carboxyl group, but most proteins are composed of gamma-bond as a single amino acid polymer. ( Agric. Chem. Biotech. 40, 173-177, 1997), molecular weight varies from strain to strain, ranging from tens of thousands to millions of kDa. Viscous poly (gamma-glutamic acid) becomes an alpha-helical structure in acidic solution, and in neutral, it changes into a random coil structure with elongated molecules and becomes more viscous.

The production of poly (gamma-glutamic acid) uses different media components depending on the strain used, and the study of the nutrients needed to increase the productivity of poly (gamma-glutamic acid) and the separation of microorganisms that do not require L-glutamic acid. A study for this purpose has also been reported. B. licheniformis and B. subtilis var. Polyglutamicum are composed of glucose and minerals without L-glutamic acid . Medium was used ( J. Bacteriol. 68, 307-315, 1954) and also reported that L-glutamic acid and Mn ²⁺ concentrations in the medium affected the D- and L-glutamic acid ratios of poly (gamma-glutamic acid). ( Appl. Microbiol. Biotechnol. 40, 867-872, 1994).

Poly (gamma-glutamic acid) is used in various fields as a raw material for food, cosmetics, pharmaceuticals, surfactants, and flocculants for wastewater treatment. In addition, research on the use of this functional polymer as a drug carrier, a functional carrier, a membrane, and an electrical material ( Radiat. Phys. Chem. 39 , 485-493, 1992), which is a derivative of poly (gamma-glutamic acid) It has been reported that PGA benzyl ester can be used as a fiber with high vitality ( J. Intell. Mater. Syst. Struct. 4, 533, 1993). Recently, Bacillus subtilis BS62 isolated from Cheonggukjang has been identified and reported for the purification and characterization of gamma-glutamyltranspeptidase (γ-GTP), and the poly by fed-batch culture It has been known that (gamma-glutamic acid) production is found after an increase in cell density in the case of B. licheniformis ( Biotechnol. Letters . 22, 585-588, 2000) . Has been suggested to be used as a treatment for osteoporosis ( J. Microbiol. Biotechnol . 11 , 428-434, 2001).

Many studies have been reported to naturally produce such highly useful poly (gamma-glutamic acid), for example, B. subtilis IFO 3335 is 30 g / l glutamic acid and 20 g / l citric acid. And (NH ₄ ) ₂ SO ₄ 5 g / l to produce 10-20 g / l of poly (gamma-glutamic acid), B. subtilis F-2-01 produces 70 g / l glutamic acid and 1 g of glucose It is known to produce 45.5 g / l of poly (gamma-glutamic acid) in cultures containing / l ( Biosci. Biotech. Biochem. 57, 1212-1213, 1993). In addition, B. subtilis TAM-4 is known to produce 20 g / l PGA and B. licheniform A35 produces PGA 8-12 g / l ( Agric. Biol. Chem. 53 , 2369-2375, 1989).

Meanwhile, as a prior invention related to poly (gamma-glutamic acid), an invention of an efficient method for producing polyglutamic acid has been disclosed in Korean Patent Application No. 1997-3404, and flame resistance isolated from Cheonggukjang in Korean Patent Application No. 2001-1481. Strains Bacillus subtilis strains have been known in the art, Korean Patent Application No. 2001-106025, a technique for producing a high concentration of polyglutamic acid is known, but the production of conventional poly (gamma-glutamic acid) The study on the method is to adjust the fermentation conditions such as pH, stirring conditions, etc., the productivity is low, there is a limit in commercial use.

Therefore, as the need for mass production of hydrogel and its natural raw material poly (gamma-glutamic acid) has recently emerged as an environmentally friendly material, the present inventors have applied a large amount of poly (gamma-glutamic acid) that can be directly applied to industrialization. As a result of intensive research on the development of strains and production methods thereof, the present invention has been completed.

The present inventors have studied to produce high molecular weight poly (gamma-glutamic acid) in a large amount, and as a result, poly (gamma-glutamic acid) using Bacillus subtilis BS62 isolated from Chungkookjang, a traditional Korean food that produces a large amount of viscous substances By selecting the optimum culture conditions suitable for the production and identifying the physicochemical properties of the poly (gamma-glutamic acid) produced from this method, it is possible to prepare natural polymer materials suitable for various industrial fields. Therefore, an object of the present invention is a method for producing poly (gamma-glutamic acid), a novel natural polymer material purified from Bacillus subtilis BS62 and purified with high purity, and poly (gamma-purified with high purity according to the preparation method. Glutamic acid).

The present invention relates to a method for producing poly (gamma-glutamic acid), which is a natural polymer, from a Bacillus subtilis strain, and to poly (gamma-glutamic acid) prepared therefrom, and more particularly, to Bacillus subtilis strain BS62 (KACC). 91064) and aerobic incubation at a temperature of 30 to 39 ℃ in a medium containing L- glutamic acid and glucose; And purifying; Method for producing poly (gamma-glutamic acid) from Bacillus subtilis BS62, characterized in that the D-type / L-type glutamic acid ratio prepared according to the production method is 40/60 to 60/40, the weight average molecular weight The poly (gamma-glutamic acid) is characterized in that it is about 600 kDa to 1000 kDa, the number average molecular weight is about 200 kDa to 500 kDa, and the polydispersity is 3 to 3.5.

The method for preparing poly (gamma-glutamic acid) using the Bacillus subtilis BS62 prepared according to the present invention has the advantage of producing a large amount of poly (gamma-glutamic acid), thereby increasing the viscosity enhancer in the pharmaceutical, food, and chemical fields. It can be usefully used for preparing hydrogel resin as a coating agent and a sustained release material.

Hereinafter, the present invention will be described in detail.

The method for producing poly (gamma-glutamic acid) according to the present invention is selected from the Bacillus subtilis strain BS62 (KACC 91064), a mucus-producing strain isolated from home-made Cheonggukjang, in a medium containing L-glutamic acid and glucose. Culturing aerobicly at a temperature of 40 ° C., preferably at 37 ° C .; And purifying.

The composition of L-glutamic acid and glucose used as the medium is preferably about 5% to 10% of L-glutamic acid and glucose, respectively, based on the culture volume, and in particular, the L-glutamic acid is based on the culture volume. Even more preferred is about 7% content and glucose is about 8% content based on the culture volume.

In the preparation method according to the present invention, it is preferable that the incubation time is about 96 hours, since the production of poly (gamma-glutamic acid) is observed to decrease after about 96 hours.

After culturing in the L-glutamic acid and glucose medium, the culture solution is subjected to centrifugation to obtain the supernatant, and to form a poly (gamma-glutamic acid) precipitate from the supernatant, and to purify the precipitate.

The precipitate was prepared by adjusting the pH of the culture supernatant to 3 to 5 and centrifuging to remove the microbial cells, adjusting the pH to 9 to 11, preferably 10.0, and then adding 3 times cold ethanol to the poly (gamma-glutamic acid. After the precipitate is formed, it is preferable to use a purification process using distilled water.

If necessary, the purified poly (gamma-glutamic acid) may be reprecipitated with ethanol to recover the ethanol precipitate, dissolved in distilled water, and centrifuged again to remove impurities.

The molecular weight, the D-glutamic acid and the L-glutamic acid ratio of the purified poly (gamma-glutamic acid) obtained by the production method according to the present invention were determined to have a D / L glutamic acid ratio of 40/60 to 60/40, and The average molecular weight is about 600kDa to 1000kDa, the number average molecular weight is about 200kDa to 500kDa, the polydispersity is 3 to 3.5, and more specifically, the D / L glutamic acid ratio is about 60/40 and the weight average molecular weight is about 624kDa The number average molecular weight was about 214 kDa, and the polydispersity was found to be 2.91.

The poly (gamma-glutamic acid) product prepared by the method according to the present invention is made into a 10% aqueous solution, followed by methacrylamidopropyl trimethyl ammonium chloride, sodium styrene sulfonate, A hydrogel was prepared by treating with a crosslinking agent such as sodium methacrylate, ethylene glycol diglycidyl ether, and the like. After soaking the hydrogel thus formed in water, and after 24 hours, the weight of the hydrogel was measured by measuring the weight of the hydrogel, and the water absorption of the hydrogel was about 4,320 times that of the dry powder. It can be seen that it has a very high absorption effect compared to the absorption rate of the poly (gamma-glutamic acid) produced by the conventional method is 2000 to 3000 times.

The production method of the present invention can produce a large amount of poly (gamma-glutamic acid), it can be useful in the manufacture of hydrogel resin as a viscosity enhancer, coating agent and sustained release material in the pharmaceutical, food, chemical fields, etc. There is this.

Through the following examples will be described in more detail. In addition, these examples are not intended to limit the scope of the present invention, but only to describe the present invention in more detail.

Example 1

Bacillus strains and culture conditions

Cheonggukjang made at home in Chungnam province of Korea was obtained and used to isolate Bacillus to form spores. Suspension of Cheonggukjang with nutrient medium (NB) was added to the test tube, heated in boiling water for 10 minutes, plated on a NA plate, incubated at 37 ° C, and the viable colonies were separated.

Mucus producing colonies were selected from the NA plate and incubated in 10 ml of nutrient medium. After overnight incubation at 37 ° C, 1.0 ml of culture was injected into a 500 ml flask containing 70 ml of L-glutamic acid, 80 g of glucose, 5 g of yeast extract, 15 g of peptone, 3 g of urea, and ₂ g of K ₂ HPO _4. I was. The pH was adjusted to 7.0 for poly (gamma-glutamic acid) production. The flask culture was agitated at 37 ° C. under rotation of 200 rpm for 4 days.

Viscosity measurement

10 ml of the culture solution was injected into a Canon-Penske capillary viscometer to measure the viscosity, at which time the temperature of the solution was adjusted to 30 ° C. The flow time is measured in seconds and the relative viscosity (η _r ) is calculated by the following equation:

η _r = (ρxt) / (ρ ₀ xt ₀ )

ρ, solution density

ρ ₀ , water density

t, solution flow time (sec)

t ₀ , water flow time (sec)

Example 2

Isolation and Selection of Poly (gamma-glutamic Acid) Production Strains

Biotin requirements in plate and liquid media were also investigated among the various Bacillus isolates selected for poly (gamma-glutamic acid) production. Poly (gamma-glutamic acid) productivity was estimated using the relative viscosity values of the bacterial cultures cultured in the mucus production medium. The results are shown in Table 1.

Most strains except KC-1, KK-1 and P-2 strains were biotin-required, and these strains produced high viscosity poly (gamma-glutamic acid) in the culture. In particular, it can be seen that B. subtilis BS62 shows the maximum productivity among all the isolates.

TABLE 1

Example 3

Bacteria Growth, Spore Formation and PGA Production

As described in Example 1, the B. subtilis BS62 selected in Example 2 was cultured during culture, that is, cell growth, spore formation, pH change, poly (gamma-glutamic acid) production, poly (gamma) -Glutamic acid) was checked for the D- and L-form ratios. As shown in FIG. 1, the growth of B. subtilis BS62 in mucus forming medium reached the end of the logarithmic phase within 12 hours, after which the growth decreased to 120 hours, showing slow growth. Spore formation was the same as the initial spore count of growth curve up to 18 hours, then slowly increased to its highest level at 96 hours and maintained at 120 hours. This was consistent with the time to reach maximum sporulation. B. Viscosity changes of the cultures during subtilis BS62 culture are shown in Table 2. It can be seen that the change in viscosity of the culture broth, which shows higher levels with poly (gamma-glutamic acid) production, is consistent with the production of poly (gamma-glutamic acid) within 96 hours.

Example 4

Purification, Quantification and Molecular Weight Measurement of Poly (Gamma-Glutamic Acid)

After adjusting the pH to 4.0 by adding 4N HCl to the Bacillus subtilis BS62 culture medium selected in Example 2, the cells were precipitated by centrifugation at 6,000 × g at 4 ° C. for 20 minutes. The pH of the supernatant containing poly (gamma-glutamic acid) was adjusted to 10.0, then 3 volumes of ethanol were added and left overnight. It was then centrifuged at 6,000 × g at 4 ° C. for 20 minutes to obtain a precipitate containing poly (gamma-glutamic acid). Precipitated poly (gamma-glutamic acid) was dissolved in distilled water and insoluble contaminants were removed by centrifugation at 6,000 × g for 20 minutes at 4 ° C. The purified poly (gamma-glutamic acid) was dissolved again in a small amount of distilled water and finally lyophilized. Dry poly (gamma-glutamic acid) was weighed and quantified. The poly (gamma-glutamic acid) production from the cultures incubated for 96 hours was about 19 g per liter of culture.

The weight average molecular weight (M _w ) and the number average molecular weight (M _n ) of the poly (gamma-glutamic acid) thus obtained were measured by gel permeation chromatography using a GMPWXL column (Viscotek) and a refractive index detector (Viscotek, Model 410). It was. A calibration curve was constructed using a polydispersity polyethylene oxide standard, from which the molecular weight of poly (gamma-glutamic acid) was calculated. 0.1 M NaNO ₃ was added to the mobile phase.

Table 2 shows the changes in poly (gamma-glutamic acid) concentration, relative viscosity and molecular weight in the cultures during Bacillus subtilis BS62 culture, and the poly (gamma- The weight average molecular weight of the glutamic acid) product was estimated to be 624 kDa, and its polydispersity (M _w / M _n ) was 2.91.

TABLE 2

Comparative Example 1

The production of poly (gamma-glutamic acid) by B. subtilis BS62 when citric acid was used as the carbon source is shown in Table 3. The poly (gamma-glutamic acid) yield was 2.0 g / l using a medium containing 3% glutamic acid and 2% citric acid, whereas 19 g / l using another medium containing 7% L-glutamic acid and 8% glucose. It was. From this it can be seen that citric acid is ineffective for poly (gamma-glutamic acid) production by B. subtilis BS62.

TABLE 3

Example 5

Evaluation of Absorption Rate of Poly (gamma-glutamic Acid) Hydrogel

As described above, the produced poly (gamma-glutamic acid) product prepared by the method of Example 4 was made into a 10% aqueous solution, followed by methacrylamidopropyl trimethyl ammonium chloride, sodium styrene sulfonate A hydrogel was prepared by treating with a crosslinking agent such as (sodium styrene sulfonate), sodium methacrylate, (sodium methacrylate), and ethylene glycol diglycidyl ether. After the hydrogel thus formed was infiltrated with water, the unit moisture absorption amount of the hydrogel was evaluated by measuring the weight increased after 24 hours.

The unit water absorption of the hydrogel prepared from poly (gamma-glutamic acid) having a weight average molecular weight of 624 kDa according to the present invention was about 4,320 times that of the dry powder.

Example 6

D / L composition ratio of poly (gamma-glutamic acid)

Form D and L ratios of glutamic acid of poly (gamma-glutamic acid) produced by B. subtilis BS62 were analyzed by HPLC during the incubation process, and the results are shown in FIGS. In this poly (gamma-glutamic acid), the ratio of D-type and L-type glutamic acid tended to be a composition having a high L-type ratio until the first 12 hours of culture, but then a composition having a high D-type ratio for 18-48 hours. In addition, the D-type ratio was further increased over time (D: L = 60:40). However, when citric acid was used instead of glucose, the high L-type composition ratio appeared up to 120 hours, and this high L-type ratio decreased after 120 hours. As a result, it was confirmed that the D / L composition ratio of poly (gamma-glutamic acid) may be different and change depending on the composition of the medium.

Poly (gamma-glutamic acid) production method according to the present invention is cultured in a medium containing L-glutamic acid and glucose with BS62 as a carbon source, adjusted to pH 10 to form a crude poly (gamma-glutamic acid) by ethanol precipitation In addition, the present invention provides a method for obtaining a large amount of poly (gamma-glutamic acid) with high purity through a purification process using distilled water, and the poly (gamma-glutamic acid) purified with such high purity is related to hydrogel resin fields, that is, pharmaceuticals. It can be widely used in chemicals, food, new material development chemicals, etc.

Claims (4)

Selecting Bacillus subtilis strain BS62 (KACC 91064) and culturing aerobically in a medium containing L-glutamic acid and glucose at a temperature of 30 to 39 ° C; And purifying; Method for producing a poly (gamma-glutamic acid) from Bacillus subtilis BS62, characterized in that it comprises a. The method of claim 1, After the culturing, centrifuging the culture to obtain the supernatant; Forming a poly (gamma-glutamic acid) precipitate from the supernatant and purifying the precipitate; Method for producing a poly (gamma-glutamic acid) from Bacillus subtilis BS62, characterized in that it comprises a. Poly (gamma-glutamic acid) prepared according to the method of claim 1. The method of claim 3, wherein Poly (D) characterized in that the D / L glutamic acid ratio is 40/60 to 60/40, the weight average molecular weight is about 600 kDa to 1000 kDa, the number average molecular weight is about 200 kDa to 500 kDa, and the polydispersity is 3 to 3.5 Gamma-glutamic acid).

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