CN101486977A - Bacillus subtilis and method for preparing gamma-polyglutamic acid using the strain - Google Patents

Abstract

A bacillus subtilis and a method for preparing gamma-polyglutamic acid using the strain. The strain is Bacillus substilis XN01, which was deposited in China Center for Type Culture Collection (CCTCC) on March 18, 2008 and is numbered as CCTCC NO: M 208044. The strain is used to prepare gamma-polyglutamic acid through the steps of culture preservation, seed liquid preparation, shake flask liquid fermentation and gamma-polyglutamic acid extraction. The method can efficiently synthesize gamma-polyglutamic acid in a low-cost liquid culture medium, and the method is simple and highly operable.

Description

Bacillus subtilis and method for preparing gamma-polyglutamic acid using the strain

technical field

The invention relates to a bacillus and a method for preparing gamma-polyglutamic acid with the bacillus, in particular to a bacillus subtilis and a method for preparing gamma-polyglutamic acid with the bacillus.

Background technique

γ-Poly glutamic acid (γ-PGA) is a polyamino acid condensed from D-type or L-type glutamic acid monomers with γ-carboxyl and α-amino groups. The biodegradable polymer material has the following structure (Shih I, Van Y. Bioresour Technol, 2001, 79: 207-225):

The relative molecular weight of γ-polyglutamic acid generally ranges from 100,000 to 1 million, and there are a large number of peptide bonds and free carboxyl groups on its main chain, so it has good biodegradability, water solubility and edible properties, and can be used as a thickener agent, humectant, antifreeze, drug carrier, degradable fiber, high-efficiency water-retaining agent, flocculant, feed additive, etc., and has broad application prospects in the fields of food, cosmetics, medicine and water treatment (Shih I, VanY.Bioresour Technol , 2001, 79: 207-225).

，1937，90：304～318)，1942年Bovarnick等研究发现有些芽胞杆菌属细菌能在发酵培养基中累积γ-聚谷氨酸(Bovarnick M.J Biol Chem，1942，145：415～424)。The preparation methods of γ-polyglutamic acid mainly include chemical synthesis and microbial synthesis. The chemical synthesis method adopts the traditional peptide synthesis method to link glutamic acid one by one or combine fragments to form a peptide. This process generally includes steps such as group protection, activation, coupling and deprotection. This method has a long synthetic route and by-products Many, low yield. Gamma-polyglutamic acid was first discovered in the capsule of Bacillus anthracis by Ivánovics et al. in 1937 (Ivánovics G, Bruckner VZ

, 1937, 90:304～318), and in 1942, Bovarnick et al. found that some Bacillus bacteria could accumulate γ-polyglutamic acid in the fermentation medium (Bovarnick MJ Biol Chem, 1942, 145:415～424).

Since the production of polyglutamic acid by microbial fermentation has the advantages of easy control of the production process, stable fermentation yield, high extraction rate, and convenience for large-scale production, microbial synthesis has gradually become the main method for research and production of γ-polyglutamic acid. The γ-polyglutamic acid producing bacteria found so far are mainly concentrated among different strains of Bacillus, including Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. Since the 1990s, research on microbial synthesis of γ-polyglutamic acid has been very active.

Bacillus licheniformis (Bacillus lichemiformis) ATCC 9945 is one of the more researched strains. The bacteria were cultured for 4 days in the optimum medium of glutamic acid 20g/L, glycerol 80g/L, and citric acid 12g/L. , 17-23g/L γ-polyglutamic acid can be obtained (Cromwick A M, Gross R A. Biotechnol Bioeng, 1996, 50: 222-227). Japan has carried out a lot of research on the synthesis of γ-polyglutamic acid by Bacillus subtilis (Bacillus subtilis) IFO 3335. The strain can synthesize 10- 20g/L of γ-polyglutamic acid (Kunioka M, Goto A. Appl Microbiol Biotechnol, 1994, 40: 867-872).

In 1993, Kubota et al reported that using Bacillus subtilis (Bacillus subtilis) F-2-01 as a strain, 48g/L of γ-polymer was obtained through 4 days of fermentation in a liquid culture containing 70g/L glutamic acid. Glutamic acid, which is the highest yield reported abroad (Kubota H, et al. Biosci Biotech Biochem, 1993, 57(7): 1212-1213). In our country, some units have successively carried out the research work on the microbial synthesis of γ-polyglutamic acid, and the production capacity of the strains involved is not the same. CN200410010509.0 introduced that Zhejiang University isolated and bred a high-yielding γ-polyglutamic acid Bacillus subtilis zju-7 from soy sauce production waste, which contained 150g/L glutamic acid and 80g/L peptone 54g/L of γ-polyglutamic acid can be synthesized in culture medium for 24 hours.

At present, although the production capacity of the strain has been greatly improved, its high cost, long cycle time and low production efficiency are obstacles to the industrial scale production of γ-polyglutamic acid. Therefore, finding efficient and low-cost synthetic γ-polyglutamic acid production strains and process conditions is still the direction of future research.

Contents of the invention

The object of the present invention is to provide a kind of bacillus subtilis and the method for preparing gamma-polyglutamic acid with this bacterial strain, it can efficiently synthesize gamma-polyglutamic acid in low-cost liquid medium, and the method is simple and operable Strong.

Technical solution of the present invention is:

A bacterial strain producing γ-polyglutamic acid, characterized in that said bacterial strain is Bacillus subtilis (Bacillus substilis) XN01, said Bacillus subtilis is isolated from soybean food, said Bacillus subtilis was produced in 2008 It was deposited in the China Center for Type Culture Collection (CCTCC) on March 18, and the number is: CCTCC NO: M 208044.

In order to accurately identify the strain, its 16s rRNA gene was sequenced. The amplification primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1527R (5'-AGAAAGGAGGTGATCCAGCC-3'), respectively. The measured sequence was compared with the gene sequence provided by GenBank, and it was confirmed that the microorganism was Bacillus subtilis.

The physiological and biochemical characteristics of the above-mentioned Bacillus subtilis (Bacillus subtilis) XN01 are shown in the table below.

Wherein said list "+" is positive and "-" is negative.

A method for preparing gamma-polyglutamic acid with bacillus subtilis (Bacillus subtilis) XN01, its special feature is that the method comprises the following steps:

1) Bacteria culture and storage

The bacterial species of Bacillus subtilis (Bacillus subtilis) XN01 is cultivated on a solid slope at a constant temperature of 25 to 40°C, and then stored for a short period of time at 2 to 4°C; g/100ml, including peptone 0.8-1.0%, yeast extract 0.5-1.0%, NaCl 1.0-2.0%, agar 1.5-2.0%, and its pH value is 6.0-8.0;

2) Preparation of seed solution

Put about ^1cm2 of the strain on the slope above into a 250ml Erlenmeyer flask filled with liquid culture medium, the liquid volume is 50ml/250ml shaker flask, and cultivate it at a constant temperature of 25-40°C for 10-24 hours, with a rotation speed of 180-220rpm The composition of the liquid medium is in weight and volume percentage, and the unit is g/100ml, wherein glucose is 0.1-1.0%, peptone is 0.8-1.0%, yeast extract is 0.5-1.0%, NaCl is 1.0-2.0%, and the pH value is 6.5～8.0;

3) Shake flask liquid fermentation

The seed liquid is transferred to the sterilized liquid fermentation culture, the inoculum amount is 1-10%, the liquid volume is 50ml/250ml shake flask, and the constant temperature is cultivated at 30-40°C for 12-48 hours, and the rotation speed is 180-220rpm; The composition of the fermentation medium is in terms of weight and volume percentage, and the unit is g/100ml, wherein the carbohydrate carbon source is 1-10%, the nitrogen source is 0.4-3%, and the L-glutamic acid or sodium glutamate is 5-12%. , NaCl 0～5%, MgSO ₄ ·7H ₂ O 0.01～0.1%, MnSO ₄ ·H ₂ O 0.005～0.05%, K ₂ HPO ₄ 0.05～0.5%, CaCl ₂ 0.02～0.2%, pH value 6.5～ 8.0;

4) extract γ-polyglutamic acid

Centrifuge to remove the bacteria in the fermentation broth, add 3 to 4 times the volume of absolute ethanol to the supernatant, refrigerate for 24 hours, centrifuge and collect the precipitate, dialyze to remove small molecules, and freeze-dry to obtain γ-polyglutamic acid.

The sugary carbon source in the above step 3) is preferably glucose, sucrose or maltose.

The content of the nitrogen source in the above step 3) is preferably 0.8-1.5%.

The above step 3) the content of L-glutamic acid or sodium glutamate is preferably 8-12%.

The pH value of the fermented broth in the above step 3) is preferably 7.0-7.5.

The sugary carbon source in the above step 3) is glucose, sucrose, maltose, lactose, fructose or a mixture thereof.

The nitrogen source in the above step 3) is yeast extract, peptone, beef extract, corn steep liquor, (NH ₄ ) ₂ SO ₄ , nitrate or a mixture thereof.

Among them, glucose, sucrose and maltose are suitable carbon sources, and glucose is better from the perspective of cost; nitrogen sources are preferably peptone or a mixed nitrogen source composed of peptone peptone and other nitrogen sources. If peptone is used as a nitrogen source, the preferred content 0.8 to 1.5%; from the perspective of cost and yield, sodium glutamate is better than L-glutamic acid, and the preferred content is 8 to 12%; the pH value of the fermentation broth should be 7.0 to 7.5, and beyond this range will affect Yield and yield of γ-polyglutamic acid.

The present invention has the following advantages:

1) The Bacillus subtilis (Bacillus subtilis) XN01 microbial strain used in the present invention can efficiently synthesize γ-polyglutamic acid in a liquid fermentation medium, and its production rate is about 2.0g/L h, and its higher yield and production The speed meets the requirements of industrial production.

2) The strains used in the present invention have extensive requirements on carbon and nitrogen sources. The carbon source can be a single carbon source or a mixed carbon source, and the nitrogen source can be a single nitrogen source or a mixed nitrogen source.

3) Appropriate addition of NaCl in the fermentation broth can increase the yield of γ-polyglutamic acid, reduce the viscosity of the fermentation broth, increase the dissolved oxygen level of the fermentation broth, and reduce the power consumption during stirring and centrifugation during the production process.

4) In the process of liquid fermentation to synthesize γ-polyglutamic acid, the concentrations of carbon source, nitrogen source and glutamic acid (or sodium glutamate) used are low, especially the concentration of nitrogen source is low, thereby greatly saving the production cost.

Detailed ways

The following examples will further illustrate the present invention, but do not limit the present invention.

The % units in the following examples all represent weight and volume percentages, and the unit is g/100ml.

Example 1

The strain of Bacillus subtilis (Bacillus subtilis) XN01 was cultured on a solid slope at a constant temperature of 37°C for 24 hours, and then preserved at 2-4°C. The slant medium described therein consists of: 1% peptone, 0.5% yeast extract, 1% NaCl, 2.0% agar, and the pH value is 6.0.

Bacillus subtilis (Bacillus subtilis) XN01 of about 1 cm ² preserved on the slant was inserted into the seed medium, and cultured in a shaker flask at 37° C. for 20 hours at a rotation speed of 210 rpm. The composition of the seed culture medium is as follows: 1% glucose, 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.1-7.2, and the filling volume is 50ml/250ml shake flask.

The seed culture medium was inserted into the fermentation medium according to the inoculum amount of 5%, and cultured in a shaker flask at 37° C. for 24 hours at a rotation speed of 210 rpm. The composition of the fermentation medium described therein is: 3% sucrose, 5% L-glutamic acid, 1.0% yeast extract, 0.05% MgSO ₄ ·7H ₂ O, (NH ₄ ) ₂ SO ₄ 1.0%, MnSO ₄ ·H ₂ O 0.01%, K ₂ HPO ₄ 0.1%, pH7.5, liquid volume 50ml/250ml shake flask.

After fermentation, remove the bacteria by centrifugation, add 3 times the volume of cold ethanol to the supernatant, refrigerate overnight, centrifuge and pour off the supernatant, redissolve the precipitate in an equal volume of deionized water, and perform paper chromatography after hydrolysis Method detection, gamma-polyglutamic acid content is 24.5g/L.

The solvent system used in the paper chromatography described therein is n-butanol: 80% formic acid (V/V): water=15:3:2, and the chromogenic agent is 0.5% ninhydrin acetone solution, through 0.1% copper sulfate (CuSO ₄ ·5H ₂ O): 75% ethanol (V/V) = 2:38 The absorbance was measured at 520nm after elution, and the content of γ-polyglutamic acid was calculated by the glutamic acid standard curve.

Example 2

Same as Example 1, the carbon source was changed to maltose with the same mass concentration, and the analysis results showed that the content of γ-polyglutamic acid in the fermentation broth was 30.1 g/L.

Example 3

Bacillus subtilis (Bacillus subtilis) XN01 was activated in the same seed medium as Example 1 for 15 hours, then pressed into the shake flask fermentation medium according to 5% inoculum size, and cultured in the shake flask at 37° C. for 24 hours at a rotation speed of 210 rpm. The composition of the fermentation medium mentioned therein is: glucose 3%, L-glutamic acid 5%, peptone 0.8%, NaCl 1%, MgSO ₄ ·7H ₂ O 0.05%, MnSO ₄ ·H ₂ O 0.01%, K ₂ HPO ₄ 0.1%, the pH value is 7.4-7.5, the filling volume is 50ml/250ml shake flask.

After the fermentation, the content of gamma-polyglutamic acid in the fermentation broth was detected according to the paper chromatography method of Example 1, and the result showed that the content was 48.0 g/L.

Example 4

Bacillus subtilis (Bacillus subtilis) XN01 was activated in the same seed medium as Example 1 for 15 hours, then pressed into the shake flask fermentation medium according to 5% inoculum size, and cultured in the shake flask at 37° C. for 24 hours at a rotation speed of 210 rpm. The composition of the fermentation medium mentioned therein is: glucose 3%, L-glutamic acid 5%, nitrogen source 0.8%, MgSO ₄ ·7H ₂ O 0.05%, MnSO ₄ ·H ₂ O 0.01%, K ₂ HPO ₄ 0.1 %, the pH value is 7.4 to 7.5, and the filling volume is 50ml/250ml shake flask.

The nitrogen source mentioned therein is yeast extract, peptone, (NH ₄ ) ₂ SO ₄ , beef extract, yeast extract + (NH ₄ ) ₂ SO ₄ (equal mass mixed nitrogen source) or peptone + (NH ₄ ) ₂ SO ₄ (equal mass mixed nitrogen sources), after the above-mentioned fermentation medium is fermented by shake flasks, the obtained fermentation broth adopts the paper chromatography method described in Example 1 to detect the content of γ-polyglutamic acid as shown in the table below.

Example 5

The strain of Bacillus subtilis (Bacillus subtilis) XN01 was cultured on a solid slope at a constant temperature of 37°C for 24 hours, and then stored at 2-4°C for a short period of time. The composition of the solid slant medium is as follows: 1% peptone, 0.5% yeast extract, 1% NaCl, 2.0% agar, pH 7.0.

About 1cm2 of Bacillus subtilis (Bacillus subtilis) XN01 was inserted into a 250ml Erlenmeyer flask filled with liquid medium, and incubated at 37°C at a constant temperature of 180-220rpm for 18 hours. The composition of the seed liquid medium is as follows: glucose 1%, peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.1-7.2, liquid volume 50ml/250ml shake flask.

The seed culture medium was inserted into the fermentation medium according to the inoculum amount of 5%, and cultured in a shaker flask at 37° C. for 24 hours at a rotation speed of 210 rpm. The composition of the fermentation medium described therein is: glucose 2%, sodium glutamate 5%, peptone 0.8%, NaCl 1%, MgSO ₄ ·7H ₂ O 0.05%, MnSO ₄ ·H ₂ O 0.01%, K ₂ HPO ₄ 0.1%, pH 7.5, liquid volume 50ml/250ml shake flask.

After the fermentation, the content of γ-polyglutamic acid in the fermentation broth was detected according to the paper chromatography method of Example 1, and the result showed that the content was 21.3 g/L.

Example 6

The seed solution was prepared according to the method of Example 5, and the seed medium was inserted into the fermentation medium according to the inoculation amount of 5%, and cultured in a shake flask at 37° C. for 24 hours at a rotation speed of 210 rpm. The composition of the fermentation medium mentioned therein is: glucose 4%, sodium glutamate 10%, peptone 1.2%, NaCl 1.25%, MgSO ₄ ·7H ₂ O 0.05%, MnSO ₄ ·H ₂ O 0.01%, K ₂ HPO ₄ 0.15%, CaCl ₂ 0.04%, pH 7.5, liquid volume 50ml/250ml shake flask.

After the fermentation, the content of gamma-polyglutamic acid in the fermentation broth was detected according to the paper chromatography method of Example 1, and the result showed that its content was 56.3 g/L.

Example 7

The seed liquid was prepared according to the method of Example 5, and the seed medium was inserted into the fermentation medium according to the inoculum amount of 1%, and cultured in a shake flask at 37° C. for 29 hours at a rotation speed of 210 rpm. The composition of the fermentation medium mentioned therein is: glucose 4%, sodium glutamate 8%, peptone 0.8%, NaCl 1.5%, MgSO ₄ ·7H ₂ O 0.05%, MnSO ₄ ·H ₂ O 0.01%, K ₂ HPO ₄ 0.1%, pH 7.5, liquid volume 50ml/250ml shake flask.

After the fermentation, the content of γ-polyglutamic acid was detected according to the paper chromatography method of Example 1, and the result showed that its content was 36.9 g/L.

Example 8

With example 7, the inoculum size wherein is changed into 2.5%, the initial pH value of fermentation medium is 6.5, after fermentation culture 29 hours, detect the content of gamma-polyglutamic acid according to the paper chromatography of embodiment 1, the result shows that its The content is 39.8g/L.

Example 9

The seed liquid was prepared according to the method of Example 5, and the seed medium was inserted into the fermentation medium according to the inoculation amount of 5%, the culture temperature was 37° C., and the rotation speed was 210 rpm. The composition of the fermentation medium mentioned therein is: glucose 4%, sodium glutamate 8%, peptone 0.8%, NaCl 1.5%, MgSO ₄ ·7H ₂ O 0.025%, MnSO ₄ ·H ₂ O 0.005%, K ₂ HPO ₄ 0.15%, CaCl ₂ 0.04%, pH 7.5, liquid volume 50ml/250ml shake flask.

After 24 hours of fermentation, the content of γ-polyglutamic acid was detected according to the paper chromatography method of Example 1, and the result showed that its content was 37.2 g/L. When the fermentation time increased to 32 hours and 46 hours, the content of γ-polyglutamic acid in the fermentation broth increased to 40.1g/L and 42.7g/L respectively.

Example 10

Same as Example 9, after adjusting the contents of MgSO ₄ .7H ₂ O, MnSO ₄ .H ₂ O and CaCl ₂ to 0.05%, 0.015% and 0.02% respectively, after 24 hours of fermentation, the γ-polyglutamine in the fermentation broth The acid content was 40.3 g/L.

Claims (8)

1. a bacillus subtilis, it is characterized in that: described bacterium is bacillus subtilis (Bacillus substilis) XN01, on March 18, 2008 preserved in China Center for Type Culture Collection (CCTCC), numbering is CCTCC NO: M 208044 . 2. a method for preparing gamma-polyglutamic acid with bacillus subtilis (Bacillus subtilis) XN01 described in claim 1, is characterized in that the method may further comprise the steps: 1) Bacteria culture and preservation The bacterial species of Bacillus subtilis (Bacillus subtilis) XN01 is cultivated on a solid slope at a constant temperature of 25 to 40°C, and then stored for a short period of time at 2 to 4°C; g/100ml, including peptone 0.8-1.0%, yeast extract 0.5-1.0%, NaCl 1.0-2.0%, agar 1.5-2.0%, and its pH value is 6.0-8.0; 2) Preparation of seed solution Put about ^1cm2 of the strain on the slope above into a 250ml Erlenmeyer flask filled with liquid culture medium, the liquid volume is 50ml/250ml shaker flask, and cultivate it at a constant temperature of 25-40°C for 10-24 hours, with a rotation speed of 180-220rpm The composition of the liquid medium is in weight and volume percentage, and the unit is g/100ml, wherein glucose is 0.1-1.0%, peptone is 0.8-1.0%, yeast extract is 0.5-1.0%, NaCl is 1.0-2.0%, and the pH value is 6.5～8.0; 3) Shake flask liquid fermentation The seed liquid is transferred to the sterilized liquid fermentation culture, the inoculum amount is 1-10%, the liquid volume is 50ml/250ml shake flask, and the constant temperature is cultivated at 30-40°C for 12-48 hours, and the rotation speed is 180-220rpm; The composition of the fermentation medium is in terms of weight and volume percentage, and the unit is g/100ml, wherein the carbohydrate carbon source is 1-10%, the nitrogen source is 0.4-3%, and the L-glutamic acid or sodium glutamate is 5-12%. , NaCl 0～5%, MgSO ₄ ·7H ₂ O 0.01～0.1%, MnSO ₄ ·H ₂ O 0.005～0.05%, K ₂ HPO ₄ 0.05～0.5%, CaCl ₂ 0.02～0.2%, pH value 6.5～ 8.0; 4) extract γ-polyglutamic acid Centrifuge to remove the bacteria in the fermentation broth, add 3 to 4 times the volume of absolute ethanol to the supernatant, refrigerate for 24 hours, centrifuge and collect the precipitate, dialyze to remove small molecules, and freeze-dry to obtain γ-polyglutamic acid. 3. The method for preparing γ-polyglutamic acid according to claim 2, characterized in that: the carbohydrate carbon source in the step 3) is glucose, sucrose or maltose. 4. The method for preparing γ-polyglutamic acid according to claim 2, characterized in that: the content of the nitrogen source in the step 3) is 0.8-1.5%. 5. The method for preparing gamma-polyglutamic acid according to claim 2, characterized in that: said step 3) the content of L-glutamic acid or sodium glutamate is 8-12%. 6. The method for preparing gamma-polyglutamic acid according to claim 2, characterized in that: said step 3) the pH value of the fermentation broth is 7.0-7.5. 7. The method for preparing γ-polyglutamic acid according to any one of claims 2 to 6, characterized in that: the carbohydrate carbon source in the step 3) is glucose, sucrose, maltose, lactose, fructose or its mixture. 8. The method for preparing γ-polyglutamic acid according to claim 7, characterized in that: the nitrogen source in the step 3) is yeast extract, peptone, beef extract, corn steep liquor, (NH ₄ ) ₂ SO ₄ , nitrates or mixtures thereof.