KR100250627B1 - Method for producing polyglutamic acid by fed-batch culture of bacillus licheniformis - Google Patents

Abstract

PURPOSE: Provided is a method for producing polyglutamic acid with low production cost through fed batch culture of Bacillus licheniformis by controlling substrates of citric acid and maintaining low viscosity of the acid until the strain grows. CONSTITUTION: A method for producing polyglutamic acid is comprised of the next steps of: i) cultivating Bacillus licheniformis in E medium lacking citric acid, but having 40g/L of glycerol, 125g/L of L-glutamic acid, 250g/L of citric acid, 3g/L of CaCl2 2H2O, 3g/L of MnSO4 5H2O, 500g/L of glycerol, and 3g/L of MgSO4 7H2O at 37 deg.C at pH 6.5-6.7; ii) adding citric acid to the medium; and iii) measuring the production of glutamic acid and cell condensation 9 hours later. Wherein, pH is controlled by 25% ammonia and 2M HCL while dissolved oxygen is changed by increasing the speed of a stirrer from 400 to 1000rpm. And cell growth is measured by spectrophotometer at 660nm.

Description

High concentration production method of polyglutamic acid by fed-batch culture of Bacillus rickeniformis

The present invention relates to a method for producing polyglutamic acid (γ-PGA) at high concentration by fed-batch culture of Bacillus licheniformis through substrate control of citric acid. More specifically, the present invention efficiently grows high concentration γ-PGA by fed-batch culture in which Bacillus rickeniformis strains are grown in high concentration in an initial medium without citric acid, and then fed nutrient medium containing citric acid. It is about how to produce.

Polyglutamic acid is a water-soluble polymer composed of glutamic acid monomers of the D and L forms. When polyglutamic acid is synthesized by microorganisms, it has been found that the carboxyl group at the γ position produces γ-polyglutamic acid (γ-PGA) having an amino bond (see Thorne et al, J. Bacteriol. 68, 307-315). (1954)). γ-PGA is a substitute for non-biodegradable water-soluble polymers and can be used for food, cosmetics, paints, oil removers, synthetic detergents, surfactants, and the like, and also includes biomaterials, functional carriers, membrane materials, and electrical materials. Research on the use as a functional polymer is also in progress.

The research on γ-PGA was active from the early 1950s to the early 1970s. 75 years after γ-PGA was first known, the synthesis pathway and basic substrates of polymer synthesis are not clear. γ-PGA is polymerized by a bond between an α-amino group and a functional group of γ-carboxylic acid, and the polymerization process proceeds differently from general protein synthesis. As a result, γ-PGA is structurally different from general protein. Thorne et al, J. Bacteriol. 68, 307-315 (1954).

Until now, research on γ-PGA synthesis has been carried out mainly by B. anthracis, B. licheniformis, B. megaterium and B. subtilis (natto). These production strains also vary the nutrients needed to produce γ-PGA. Double Bacillus subtilis var. Only polyglutamicum grows in medium containing only carbon sources such as glucose and inorganic salts, producing γ-PGA, and others have been reported to require L-glutamic acid for γ-PGA production (see Thorne et al, J. Bacteriol. 68, 307-315 (1954). This is because γ-PGA is mainly synthesized by γ-D-PGA by L-glutamic acid. In addition, the proportion of L-glutamic acid in γ-PGA has been reported to be affected by the concentration of Mn (II) in culture media (Leonard, CG et al, J. Bacteriol., 76, 499 (1958)). .

Since then, in order to increase the productivity of γ-PGA, studies have been made on the nutrients required and to isolate microorganisms that do not require L-glutamic acid (Morine, RA and Fogovin, P., Biotechnol. 13, 381 (1971). In addition, studies using B. subtilis IFO3335 have been reported to synthesize large amounts of γ-PGA in medium containing L-glutamic acid, citric acid and ammonium sulfate (Goto, A. and Kunioka, Biosci. Biotech Biochem., 56 (7), 1031-1035 (1992).

Previous studies on γ-PGA have been mainly related to synthetic pathways and characteristics (see Goto, A. and Kunioka, Biosci. Biotech. Biochem., 56 (7), 1031-1035 (1992); Kunioka and Goto, A., Appl. Microbiol.Biotechnol., 40, 867-872 (1994); Cromwick and Gross, Can.J. Microbiol., 41: 902-909 (1995); Yokoi et al, J. Fermen.Bioeng , 82 (1), 84-87 (1996)), a study on the production method of γ-PGA is an attempt to improve productivity by adjusting fermentation conditions such as pH or stirring speed, and thus the concentration of γ-PGA produced Was very poor (Cromwick et al, Biotech. Bioeng., 50, 222-227 (1996); Giannos et al, EA Dawes (ed), Novel Biodegradable Microbial Polymers, 457-460 (1990)).

Therefore, there is an urgent need in the art for a mass production method of γ-PGA that can be directly applied to industrialization.

Accordingly, the present inventors have made intensive studies to develop a method capable of efficiently producing high concentration of γ-PGA, and as a result, the selection of γ-PGA production strain, growth characteristics for the selected production strain, γ-PGA synthesis and accumulation characteristics, etc. Through the basic experiments, when cultivation of Bacillus liqueniformis having high production capacity of γ-PGA, strains were removed without citric acid in the initial medium, and when citric acid was supplied during nutrient supply, high concentration of γ-PGA It was confirmed that is produced efficiently, to complete the present invention. In addition, when γ-PGA is produced in this manner, the initial production of γ-PGA is suppressed to maintain a low viscosity until constant cell growth, thereby lowering the agitation speed and aeration rate at the beginning of the process, thereby greatly reducing the production cost. Confirmed that it can.

After all, it is an object of the present invention to provide a method for producing γ-PGA at a high concentration in a fed-batch culture of Bacillus licheniformis by controlling the substrate of citric acid.

1 is a graph showing the effect of each component in the medium in the culture of the flask on cell growth and production of polyglutamic acid.

Figure 2 is a graph showing the concentration of final glycerol (L-glutamic acid) and citric acid (citrate) in the culture medium in the flask culture.

3 is a graph showing the dry cell concentration and the concentration of polyglutamic acid when the cells were grown in an initial medium without citric acid and then fed with a nutrient solution containing citric acid.

4 is a graph showing the concentrations of the final glycerol, glutamic acid and citric acid in the medium when the cells were grown in an initial medium without citric acid and then fed with nutrient solution containing citric acid.

5 is a graph showing the dry cell concentration and the concentration of polyglutamic acid when the cells were grown in an initial medium containing 12 g / L citric acid and then fed with a nutrient solution containing citric acid.

Hereinafter, the present invention will be described in more detail.

As a basic experiment of the present invention, first, a strain that efficiently produces γ-PGA is selected as Bacillus licheniformis, and experiments on growth characteristics, γ-PGA synthesis, and accumulation characteristics of the selected production strains are performed. Was performed. On the basis of the results, the present invention was to considerably improve the productivity of γ-PGA by fed-batch culture with different substrate conditions in the culture in consideration of the synthetic pathway of γ-PGA.

That is, the initial medium without citric acid (L- glutamic acid 20.0 g / L, glycerol 40.0 g / L, NH ₄ Cl 7.0 g / L, K ₂ HPO ₄ 0.5 g / L, MgSO ₄ 7H ₂ O 0.5 g / L, Bacillus rickeniformis strains were grown in high concentration in FeCl ₃ .6H ₂ O 0.04 g / L, CaCl ₂ .2H ₂ O 0.15 g / L, MnSO ₄ .H ₂ O 0.104 g / L), Γ-PGA is produced at high concentration in a fed-batch culture that supplies a nutrient medium containing citric acid.

In the above, the nutrient medium containing citric acid is 125g / L L- glutamic acid, 250 g / L citric acid, CaCl ₂ 2H ₂ O 3 g / L, MnSO ₄ 5H ₂ O 3 g / L, glycerol 500 g / L , MgSO ₄ · 7H ₂ O 3 g / L, when the pH of the fermentation broth is at least 7.0 is added to the fermenter.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

[Effects of Media Components on Cell Growth and γ-PGA Synthesis by Flask Culture]

Bacillus licheniformis ATCC 9945A strain was incubated for 47 hours in E medium without MnSO ₄ and CaCl ₂ (see below) and stored at -70 ° C with 30% glycerol stock. . After the thaw was rapidly thawed in a 37 ° C. water bath, 0.2 ml of the strain solution was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of medium and cultured for 3 days. At this time, the culture was carried out at 37 ℃, 250 rpm, initial pH 6.5.

Through the flask culture, the effect of each medium on cell growth and γ-PGA synthesis was investigated. For this purpose E medium (L-glutamic acid, 20.0 (g / L); citric acid, 12.0 (g / L); glycerol, 80.0 (g / L); NH ₄ Cl, 7.0 (g / L); K ₂ HPO ₄ , 0.5 (g / L); MgSO ₄ 7H ₂ O, 0.5 (g / L); FeCl ₃ 6H ₂ O 0.04 (g / L); CaCl ₂ 2H ₂ O, 0.15 (g / L); MnSO ₄ H ₂ O, 0.104 (g / L) and Ca ⁺⁺ , Mn ⁺⁺ , L-glutamic acid, E medium excluding citric acid, and citric acid were added to E medium so that the concentration of citric acid was 3 or 6 g / L. One medium was used.

The effect of each component on cell growth and γ-PGA production is shown in FIG. 1, and the concentrations of final glycerol, L-glutamic acid and citric acid in the medium are shown in FIG.

In Figures 1 and 2, E is when cells are grown in E medium; E w / o Ca is when the cells were grown in E medium excluding calcium ions; E w / o Mn is when cells were grown in E medium excluding manganese ions; E w / o L-glu was grown in E medium except for L-glutamic acid; E w / o Citrate is when cells were grown in E medium except citric acid; E w / Cit. 3 g / L when the cells were grown in E medium to which 3 g / L citric acid was added; And, E w / Cit. 6 g / L represents the case where the cells were grown in E medium to which 6 g / L citric acid was added.

As shown in FIGS. 1 and 2, γ-PGA production was highest at 3.01 g γ-PGA / L in E medium, and final cell concentration was highest at 2.67 g dry cell / L in E medium without citric acid. . In addition, only a small amount of γ-PGA was produced in a medium excluding L-glutamic acid and citric acid, and a medium containing 3 g / L citric acid, but the final cell concentration was higher than that in the case of culturing in E medium containing all the components. High. This indicates that L-glutamic acid and citric acid are essential for γ-PGA synthesis but do not significantly affect cell growth. The production of γ-PGA and the final cell concentration were inversely proportional to each other, which caused the change of the yield of cells and γ-PGA from the carbon source according to the culture conditions and the difficulty of oxygen transfer as the viscosity in the medium increased as γ-PGA was produced. Because.

Example 2

[Cell Growth and γ-PGA Synthesis by Feeding Cultivation of Citric Acid after Feeding Cells in Citric Acid-Free Medium]

For high-efficiency production of γ-PGA by fed-batch culture, the seed medium culture and the initial medium in the fermenter used a medium to which 40 g / L glycerol was added except for citric acid in the E medium. This was to inhibit the production of γ-PGA at the beginning of the culture to promote the growth of the cells, and later to add the citric acid to the feed nutrient solution to control the timing of γ-PGA production.

The fed-batch culture experiment was performed using a 5 L fermenter (BioFlo 3000 fermentor, New Brunswick Scientific, Edison, NJ, USA), the culture conditions were maintained at 37 ℃, 3 vvm, pH 6.5 ~ 6.7. pH was adjusted with 25% aqueous ammonia and 2M hydrochloric acid. The amount of dissolved oxygen was changed from 400 to 1000 rpm and, if necessary, adjusted to 50% of saturated dissolved oxygen by supplying pure oxygen. The inoculum was inoculated into a fermenter containing 800 ml of the initial medium after incubation in a 1000 ml flask containing 200 ml of medium having the same composition as the initial medium until the initial exponential growth period (37 ° C., 250 rpm). At this time, the growth of the cells was measured by absorbance at 660 nm wavelength using a spectrophotometer.

The initial medium in fed-batch culture was added 40 g / L glycerol to E medium without citric acid, and the composition of the nutrient supply solution was 125 g / L L-glutamic acid, 250 g / L citric acid, CaCl ₂ ㆍ 2H ₂ O 3 g / L, MnSO ₄ 5H ₂ O 3 g / L, glycerol 500 g / L, MgSO ₄ 7H ₂ O 3 g / L. The dry cell concentration was obtained by centrifuging 4 ~ 8 ㎖ samples at 14,000 rpm for 15 minutes to obtain a precipitate, washed with distilled water and dried in an oven at 95 ℃ until no weight loss, the weight of the cells was measured on a balance . Purification of γ-PGA was performed according to conventional methods (Kunioka and Goto, Appl. Microbiol. Biotechnol., 40, 867-872 (1994)). 3 is a graph showing the dry cell concentration and the concentration of γ-PGA when the cells were grown in an initial medium without citric acid and then fed with a nutrient solution containing citric acid.

After 9 hours of inoculation of the strain into the fermenter (pH 7.0, fermentation broth concentration 4.23 g / L) 150 ml of nutrient solution was supplied and γ-PGA production and cell concentration was observed (see Fig. 4). In addition, the concentrations of glycerol, citric acid and L-glutamic acid in the medium were also measured (see Fig. 5). As shown in FIGS. 4 and 5, the point of abrupt γ-PGA production coincides with that of citric acid, indicating that the generated γ-PGA is mostly produced by metabolism of citric acid.

As a result, γ-PGA production was inhibited to 5 g / L or less until the nutrient supply point, and 34.9 g / L of γ-PGA was produced about 17 hours after substrate feeding (26 hours after inoculation). This is 2.5 times higher than that obtained in batch culture (Cromwick et al., Biotech. Bioeng., 50: 222 (1996)). In addition, the productivity was 1.34 g γ-PGA / L / h, which was about five times better than the conventionally reported method of γ-PGA production by fed-batch culture (Cromwick et al, EA Dawes (ed), Novel Biodegradable). Microbial Polymers, 457-460 (1990).

Example 3

[Bacterial Growth and γ-PGA Synthesis When Cells were Grown in an Initial Medium Containing Citric Acid 12 g / L and Feeded with Citric Acid-Containing Liquid]

As a control experiment of Example 2, after growing the cells in the initial medium containing 12 g / L citric acid, cell growth and γ-PGA synthesis was observed when the nutrient solution containing citric acid was supplied (see: Fifth Degree). When the nutrient solution was supplied in the same manner as in Example 2, the dry cell concentration was produced up to 7.20 g / L, γ-PGA up to 5.30 g / L.

This is a significantly lower production compared to the results of Example 2, it can be concluded that it is suitable for the production of γ-PGA after the growth of the cells except citric acid in the initial medium, and supplying nutrient solution containing citric acid.

As described and demonstrated in detail above, once culturing Bacillus rickenomyces cells to the desired concentration under the conditions of inhibiting γ-PGA production, high concentration γ-PGA in the fed-batch culture of the present invention to generate γ-PGA Could be produced efficiently. In addition, in the production of γ-PGA in this manner, the initial production of γ-PGA is suppressed to maintain a low viscosity until a certain cell growth, lowering the agitation speed and the aeration amount of the process to reduce the production cost.

Claims (4)

Production of γ-polyglutamic acid by growth of Bacillus licheniformis strain in high concentration in an initial medium without citric acid, followed by fed-batch culture supplying nutrient medium containing citric acid Way. According to claim 1, the initial medium is L- glutamic acid 20.0 g / L, glycerol 40.0 g / L, NH ₄ Cl 7.0 g / L, K ₂ HPO ₄ 0.5 g / L, MgSO ₄ 7H ₂ O 0.5 g / L , Γ-polyglutamic acid, characterized in that the composition consisting of FeCl ₃ · 6H ₂ O 0.04 g / L, CaCl ₂ ㆍ 2H ₂ O 0.15 g / L, MnSO ₄ ㆍ H ₂ O 0.104 g / L ) Production method. The nutrient medium according to claim 1, wherein the nutrient medium containing citric acid is 125 g / L L-glutamic acid, 250 g / L citric acid, CaCl ₂ 2H ₂ O 3 g / L, MnSO ₄ 5H ₂ O 3 g / L, glycerol Production method of γ-polyglutamic acid (γ-polyglutamic acid), characterized in that the composition consisting of 500 g / L, MgSO ₄ · 7H ₂ O 3 g / L. The method of producing γ-polyglutamic acid according to claim 1, wherein the nutrient medium containing citric acid is added when the pH of the fermentation broth is at least 7.0.

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