KR20050007670A - Novel Bacillus subtilis E2 highly producing surfactin and the method for the surfactin production - Google Patents

Abstract

PURPOSE: A microorganism Bacillus subtilis E2 and a method for producing the surfactin using the same microorganism are provided, which microorganism highly produces surfactin which is a lipoproteinic biosurfactant useful for environment, food, cosmetic and medicine fields. CONSTITUTION: The microorganism Bacillus subtilis E2(KCTC 10389BP) highly producing surfactin is provided, wherein Bacillus subtilis E2(KCTC 10389BP) is isolated from the polluted soil. The method for producing the surfactin comprises culturing Bacillus subtilis E2(KCTC 10389BP) in a medium containing glucose and bean flour as carbon sources under conditions of pH 5 to 10, temperature of 20 to 45 deg. C, air flow rate of 0.01 to 1.0 vvm and agitation rate of 50 to 800 rpm.

Description

Novel Bacillus subtilis E2 highly producing surfactin and the method for the surfactin production}

The present invention relates to a bacterium Bacillus subtilis E2 having excellent suctinin production ability and a method for producing suctin using the same, more specifically, Bacillus subtilis E2 ( Bacillus subtilis E2) [ KCTC 10389BP], and glucose and soy flour as carbon sources to isolate the strain, and a method for mass production of suctin, a lipoprotein biosurfactant.

Surfactin is a representative lipoprotein biosurfactant produced by bacteria.

Biosurfactant refers to a biodegradable surfactant produced by microorganisms such as yeast, mold, bacteria, etc., and is produced extracellularly or intracellularly depending on the strain. Biosurfactants are non-toxic and biodegradable than chemical synthetic surfactants, so they do not become secondary pollutants when they are used, and they can be used for special purposes due to the complex chemical structures that are difficult to synthesize by conventional methods. In addition, since the surface tension lowering ability, the stability to the temperature, pH and the like of the surfactant in terms of physical and chemical performance of the conventional chemical synthesis surfactant almost shows the same result, the biosurfactant can be very useful.

The global market for surfactants was $ 2 billion in 1988 and about $ 9.4 billion in 1994, growing more than 400% in just six years, and demand is growing year after year. However, most of them have been reported as chemical synthetic surfactants that are not biodegradable by themselves and cause environmental pollution.

Thus, by replacing these problems with non-toxic, biodegradable, environmentally friendly microbial surfactants, environmental pollution can be reduced, and furthermore, biosurfactants are secondary to cosmetics, pharmaceuticals, foods, detergents, pulp and paper, and crude oil. It can be widely applied in various fields such as recovery and environmental purification.

As a prior art for such a method of producing a serpentine, Cooper et al. Derived an optimal base medium (Cooper's medium) through the result that iron and manganese ions promote the production of the serpentine [Copper DG et al. , Appl. Environ. Microbiol. 42, 408 (1981)], Sandrin et al. Optimized the medium for the production of suptin, in which L-glutamic acid was contained at a concentration of 5 g / l as an organic nitrogen source [Sandrin C et al. , Biotechnol. Appl. Biochem. 12, 370 (1990)], reports that sustained production of suspectin for at least 36 generations through continuous operation [Sheppard and Cooper, Appl. Microbiol. Biotechnol. 35, 72 (1991). However, the yield of the serpentine is very low at a concentration of 0.2 to 1.0 g / l.

In addition, the study on the development of high-peptide microorganisms using UV mutation method [de Roubin MR et al., Can. J. Microbiol. 35, 854 (1989), and the like, but in the case of fermentation for the production of the suspectin, there is a problem that the mass culture is difficult due to the disadvantage that the foam is severe during operation.

Therefore, in order to realize the practical use of the biosurfactant suspectin, it is necessary to study a method for reducing the occurrence of bubbles while dramatically increasing the production yield for mass production.

In order to solve the above problems, the present inventors conducted a study by separating microorganisms capable of decomposing hydrophobic environmental contaminants, and as a result, isolates the mycobacteria excellent in the production of suctin, a biosurfactant, from soil. The present invention has been completed by establishing fermentation conditions for high production of suspectin.

Accordingly, an object of the present invention is to provide a bacterium Bacillus subtilis E2 [KCTC 10389BP] having an excellent ability to produce suspectin and a method for producing suspectin using the new strain.

Figure 1 shows the amount of suctin (surfactin) production in accordance with the incubation time in the fermentor.

Figure 2 shows the TLC results of the suctinin (surfactin) standard and the new strain culture medium according to the present invention.

Figure 3 shows the FAB-mass results for the suspectin produced from the new strain according to the present invention.

Figure 4 shows the surface tension according to the concentration of the suspectin produced from the new strain according to the present invention.

The present invention is characterized by a bacterium Bacillus subtilis E2 [KCTC 10389BP] having excellent ability to produce suspectin and a method for producing suspectin using the bacterium.

Referring to the present invention in more detail as follows.

The present invention relates to a method for separating the new strain Bacillus subtilis E2 [KCTC 10389BP] from contaminated soil and using this strain to mass-produce a lipoprotein biosurfactant, suspectin, using glucose and soy flour as carbon sources.

First, in order to screen the new strains, firstly isolate the viable strains from soil samples using a medium containing oil and hexadecane as a carbon source, and then use the isolated strain as a carbon source to add glucose, hexadecane, and soybean oil. In the surface tension is reduced a lot, and the emulsion having excellent emulsification is separated secondarily. Secondly isolated strains were cultured in a liquid medium containing glucose, hexadecane, soybean oil, and oil as carbon sources, respectively, to finally select strains having low surface tension and excellent emulsifying ability.

The selected strains were Gram-positive bacteria, bacilli with motility and spore-forming ability, having catalase and oxidase, gelatin, starch, casein, and citrate. It was estimated to be a strain of the genus Bacillus of aerobic strains with resolution but no urea resolution. In addition, as a result of sequencing of the 16S rDNA, it was confirmed that the selected strain had a high homology with Bacillus subtilis (Accession No. AF549498) through "The BLAST search" on the 647 bp base sequence was determined. . Based on the above characteristics, the selected strain was identified as Bacillus subtilis , named Bacillus subtilis E2, and deposited on November 28, 2002 at the Korea Biotechnology Research Institute Gene Bank, accession number KCTC 10389BP. Was granted.

The method for producing suptin from the new strain Bacillus subtilis E2 [KCTC 10389BP] is as follows.

Use glucose and soybean powder as a carbon source for Bacillus subtilis E2 [KCTC 10389BP], Initial pH 5 to 10 (preferably pH 8), Temperature 20 to 45 ° C (preferably 30 ° C), Aeration rate 0.01 to 1.0 vvm (Preferably 0.1 vvm) and incubation for 20 to 100 hours at the conditions of a stirring speed of 50 to 800 rpm (preferably 100 rpm) to produce a serpentine. At this time, the optimal medium is glucose 40.0 g / L, NH ₄ HCO ₃ 13.5 g / L, yeast extract 0.5 g / L, K ₂ HPO ₄ 10.5 g / L, MgSO ₄ 7H ₂ O 0.5 g / L, MnSO ₄ 0.05 g / l of 4H ₂ O, CaCl ₂ 0.05 g / l of 2H ₂ O is preferred.

The biosurfactant produced from this, serpentin, is composed of 7 amino acids combined to form a circular peptide. The minimum surface tension is 28 dyne / cm, and micelle formation concentration (CMC) is 40 μM. Activity.

In addition, supectin has antitumor activity [Kameda et al., Chem. Pharm. Bull. 22, 938 (1974)], inhibits fibrin clot formation [Arima et al., Biochem. Biophys. Res. Commun. 31,488 (1974)], recently anti-HIV activity [Itokawa et al., Chem. Pharm. Bull. 42, 604 (1994), which is a biological material that can be usefully used for high value-added medicine.

Therefore, the suptin produced from the new strain Bacillus subtilis E2 [KCTC 10389BP] according to the present invention is applied to soil or wastewater contaminated with oil or used as an environmentally friendly detergent, renal emulsifier, natural antibacterial agent, thrombolytic agent, antivirus / It is expected to be very useful in various fields such as antibacterial agents. In addition, in the present invention solved the problem of foaming, which had previously been a problem in the production of the suspectin by using the new strain, was solved by using soy flour as a carbon source, it was confirmed that the production also increased significantly.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1: Biosurfactant Production Microbial Isolation and Screening

First, isolate the viable strains from soil samples using medium containing oil and hexadecane as carbon source, and then culture the isolated strains in liquid medium containing glucose, hexadecane, and soybean oil as carbon source for 3 days, and then collapse the oil film. Oil film-collapsing ability, surface tension, the emulsification capacity for the hydrocarbons (hydrocarbons) was measured, and then the surface tension was greatly reduced, and the strains excellent in emulsification capacity were separated secondarily. The surface tension was measured by liquid culture of the secondary isolates as a carbon source in production medium containing glucose, hexadecane, soybean oil, and oil. The strain was lowered, and the strain having excellent emulsification capacity was finally selected.

Example 2: Identification of Selected Microbial Strains

The results of examining the morphological, culture, and physiological characteristics of the strains selected in Example 1 are shown in Table 1 below. Selected strains were Gram-positive bacteria, bacilli with motility and spore-forming ability, with catalase and oxidase, and gelatin, starch, casein, and citrate degradability. However, it was estimated to be a strain of the genus Bacillus of aerobic strains without urea resolution. In addition, nitrate was reduced to N ₂ , no indol test from tryptophan was observed, and acetoine production from pyruvate (VP test) was positive. Seemed. As a result of the carbon fermentation ability, acid was produced in all sugars such as glucose, arabinose, mannitol, xylose and the like, and no gas production was observed from glucose [Table 1]. In addition, as a result of performing sequencing of 16S rDNA by molecular genetic method, the novel strain of the present invention is Bacillus subtilis (Accession No. AF549498) through "The BLAST search" on 647 bp base sequence determined It was confirmed that it had high homology (99%). By combining the above morphological, physiological and molecular genetic characteristics, the selected strain was identified as Bacillus subtilis and named Bacillus subtilis E2 and deposited on November 28, 2002 with the Korea Biotechnology Research Institute Gene Bank. The number KCTC 10389BP was assigned.

division Screened strains Bacillus subtilus Morphological classification Gram strain + + Shape rod rod Width of rod 0.6-0.8㎛ 0.7-0.8 ㎛ Length of rod 1.4-2.3㎛ 2-3㎛ Spore + + Motility + + Acid fast - - Culture classification Growth in air + + Growth anaerobically - - Growth at 40 ℃ + + Growth at 65 ℃ - - Growth at pH 5.7 + + Growth in 7% NaCl + + Physiological Classification Catalase + + Oxidase + d Glucose (acid) + d Oxidation-Fermentation F Carbohydrates, acid form glucose + + arabinose + + mannitol + + xylose + + Hydrolysis of gelatin + + starch + + casein + + Utilzation of citrate + + Voges-Proskauer reaction + + Nitrate reduction + + Indole - - Urease - d β-galactosidase + d Arginine dihydrolase - - Lysine decarboxylsae - - d: different reaction in different strains

Example 3: Influence of Carbon Sources on Biosurfactant Production

The choice of raw materials for the production of biosurfactants is important when considering the economics of the production process. In particular, the choice of carbon source is a very important part of biosurfactant production, and a wide range of raw materials are used. The raw materials mainly used as a carbon source can be divided into three types: carbohydrate, hydrocarbon, and vegetable oil. Some strains produce biosurfactants only in hydrocarbons, while others produce biosurfactants only in carbohydrates, while others require complex combinations of two or more substrates. To investigate which carbon source is required for biosurfactant production by Bacillus subtilis E2, glucose was used as carbohydrate, hexadecane as hydrocarbon, and soybean oil as vegetable oil.

As shown in Table 2 below, Bacillus subtilis E2 reduced the surface tension of the culture medium to 28 dyne / cm in a medium containing glucose as a carbon source, and was also excellent in emulsifying ability to hydrocarbon and emulsifying ability to oil. Was only observed in glucose medium. At this time, the cell concentration was 1 g / L. When soybean oil was incubated with a substrate, the emulsifying ability to hydrocarbon was excellent, but no emulsifying ability to oil was observed, and the surface tension was also lowered to 43 dyne / cm as measured by diluting the culture solution 10-fold. When carbon was used as the carbon source, the production of biosurfactants as well as the biomass was very low, and the mass production was the highest at 1.58 g / l when glucose and soybean oil were added at a weight ratio of 1: 1. However, the emulsifying ability and the surface tension lowering ability were very low. From these results, subsequent carbohydrate glucose was used as a carbon source in the production of biosurfactants by Bacillus subtilis E2.

Effect of Carbon Sources on the Production of Biosurfactants by Bacillus Subtilis E2 Carbon source Cell concentration (g / l) Surface tension (dyne / cm) Emulsifying capacity (unit / ml of culture broth Culture 10-fold dilution of culture hydrocarbon Crude oil None 0.25 32.1 59.3 3.5 - Glucose 1.06 28.2 30.1 138.8 683.8 Soybean oil 0.39 45.3 43.2 133.8 - Hexadecane 0.28 50.9 58.7 1.0 - Glucose + Soybean Oil 1.58 41.3 52.1 3.0 - Glucose + hexadecane 1.19 41.5 53.2 2.5 -

Example 4 Establishment of Culture Conditions for Biosurfactant Production

For the biosurfactant production of Bacillus subtilis E2, glucose was added as a carbon source and then cultured, and the result showed the highest biosurfactant production at a glucose concentration of 40 g / l. At glucose concentrations above 50 g / l, cell growth and biosurfactant production may be delayed or inhibited.

Examination of the initial pH effect produced the best cell growth and biosurfactant at pH 8.0. Below pH 6.0, cell growth was reduced and the production of biosurfactants was also reduced.

Cell growth and biosurfactant productivity were investigated by adding 0.1% of inorganic nitrogen sources, respectively, and NH ₄ HCO ₃ showed the highest biosurfactant productivity. This result is thought to increase productivity by preventing the pH of the culture from lowering as can be observed at the final pH. In addition, the yeast extract was added to the medium containing 1.0 g / l and 2.0 g / l of NH ₄ HCO ₃ as inorganic nitrogen source by concentration, and the result of investigation of cell growth and biosurfactant productivity was calculated as NH ₄ HCO ₃ was 1.0. At g / l, yeast extract 1.0 g / l showed the highest biosurfactant production, and as the concentration of inorganic nitrogen source NH ₄ HCO ₃ increased, the requirement of organic nitrogen source decreased.

To investigate the effect of inorganic salts on cell growth and biosurfactant production of Bacillus subtilis E2 strain, MnSO ₄ ㆍ 4H ₂ O, NaCl and CaCl ₂ ㆍ 2H ₂ O were removed from the basal medium, and then After incubation for 3 days, the effect of each component on cell growth and biosurfactant production was investigated. Na ⁺ ions had no effect on the removal of the media, and when Ca ²⁺ ions were removed, the biosurfactant production was slightly decreased, and the cell concentration was also decreased. On the other hand, Mn ²⁺ ions showed a great effect not only on cell growth but also on biosurfactant production, and were investigated by MnSO ₄ · 4H ₂ O concentration. As a result, when MnSO ₄ · 4H ₂ O was added at a concentration of 20 mg / l or more, the cell concentration was about 1.8 g / l, and the maximum biosurfactant was obtained at a concentration of 2.16 g / l at a concentration of 50 mg / l. Produced.

In the above experiments, the growth of biomass and biosurfactant production was investigated by varying the ratio of carbon source and nitrogen source to the adjusted media components. When glucose concentration was 40 g / l, the highest C / N ratio was adjusted to 3 Surfactant production was shown.

In the above results, the optimum medium composition was glucose 40.0 g / l, NH ₄ HCO ₃ 13.5 g / l, yeast extract 0.5 g / l, K ₂ HPO ₄ 10.5 g / l, MgSO ₄ .7H ₂ O 0.5 g / l, 0.05 g / L of MnSO ₄ 4H ₂ O, 0.05 g / L of CaCl ₂ 2H ₂ O, initial pH 8.0, and incubation temperature of 30 ° C. were determined.

Example 5: Substrate Selection for Biosurfactant Production

The culture of Bacillus subtilis E2 with homogeneous soybean flour or uneven soybean meal resulted in a biosurfactant production concentration that was approximately 5 times higher than that of cultured with only single substrate. In addition, the amount of foam that was a problem in the production of suctin was significantly reduced. However, in consideration of the production cost side, in subsequent experiments were incubated using non-uniform soy flour.

division Glucose (4%) + Uneven Bean Flour (5%) Glucose (4%) + Uniform Soy Flour (5%) Glucose (4%) Suspectin Production (g / ℓ) 10.60 10.85 1.95

The non-uniform soy flour was added at a concentration of 5% in the medium condition in Example 4, and the culture temperature was 30 ° C. in a 5 L fermenter, and the aeration was 0.1 vvm, and the stirring speed was 100 rpm. The biosurfactant is produced, it was confirmed that about 7.4 g / L of suctin is produced after 72 hours of culture [Fig. 1].

This result is described in Arima et al. , US Pat. No. 3,687,926, Biochem. Biophs. Res. Commun. , 31, 488 (1968)] produced 0.05-0.1 g / L of suctin using Bacillus subtilis strains, and Mulligan et al. [ Appl. Microbiol. Biotechnol. , 31, 486 (1989)] showed a 10-fold increase in production after 40 hours of incubation compared to 0.56 g / l of serpentin.

Example 6: Analysis of Biosurfactant Extracted from New Strains

After incubating Bacillus subtilis E2, extracting the biosurfactant with an extraction solvent mixed with chloroform and methanol 2: 1, and then distilled under reduced pressure to develop a sample obtained by TLC (F ₂₅₄ ), The same R _f value as pectin was confirmed [FIG. 2], and measured by FAB-MS, the [M + H] ⁺ peak values of the samples were 1022 and 1036, and the [M + Na] ⁺ peak values were 1044 and 1058. Since it shows the same value, it was confirmed that the biosurfactant produced by Bacillus subtilis E2 was suspectin [FIG. 3].

Example 7 Change of Surface Tension with Biosurfactant Concentration

In order to measure the surface tension lowering activity of the purified biosurfactant, the surface tension of water was measured for each concentration. The minimum surface tension of the purified biosurfactant was 28 dyne / cm and the critical micelle concentration (CMC) was 40 μM [FIG. 4].

As described above, it was confirmed that the new strain Bacillus subtilis E2 [KCTC 10389BP] according to the present invention has a production capacity of 10 times or more than the amount of suctin produced from the existing Bacillus subtilis. Therefore, it is expected that the serpentine produced from the new strain is widely used in the environmental field, food, cosmetic field, medicine field and the like.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel Bacillus subtilis E2 highly producing surfactin and the          method for the surfactin production <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 647 <212> DNA <213> Bacillus subtilis E2 <400> 1 cataatggcg ccgcccatgc ttccggccgc catggcggcc gcgggaattc gattagtttg 60 atcctggctc aggacgaacg ctggcggcgt gcctaataca tgcaagtcga gcggacagat 120 gggagcttgc tccctgatgt tagcggcgga cgggtgagta acacgtgggt aacctgcctg 180 taagactggg ataactccgg gaaaccgggg ctaataccgg atggttgttt gaaccgcatg 240 gttcaaacat aaaaggtggc ttcggctacc acttacagat ggacccgcgg cgcattagct 300 agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag agggtgatcg 360 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcaagt agggaatctt 420 ccgcaatgga cgaaagtctg acggagcaac gcccgcgtga gtgatgaagg ttttcggatc 480 gtaaagctct gttgttaggg aagaacaagt accgttcgaa tagggcggta ccttgacggt 540 acctaaccag aaagccacgg ctaactacgt gccagcagcc cgcggttata caatcactag 600 tgaattcgcg ggcgcttgag gtttgaccat attgggtcct agtttga 647

Claims (2)

Bacillus subtilis E2 (KCTC 10389BP) with excellent surfactin production capacity. Add glucose and soybean powder to Bacillus subtilis E2 [KCTC 10389BP] as a carbon source, and have an initial pH of 5 to 10, a temperature of 20 to 45 ° C., an aeration rate of 0.01 to 1.0 vvm, and a stirring speed of 50 to 800 rpm. Method for producing a suspectin, characterized in that cultured in.