CN101508962A

CN101508962A - Non-glutamic acid dependent form gamma-polyglutamic acid synthesis bacterium and fermentation method

Info

Publication number: CN101508962A
Application number: CNA2008100539007A
Authority: CN
Inventors: 宋存江; 刘莉; 王淑芳; 曹名锋; 刘娜; 胡丹; 代森; 耿伟涛
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2008-07-21
Filing date: 2008-07-21
Publication date: 2009-08-19
Anticipated expiration: 2028-07-21
Also published as: CN101508962B

Abstract

The invention provides a synthetic strain of non-glutamic acid dependent gamma-polyglutamic acid, Bacillus amyloliquefaciens NK-LL3. The invention further provides a fermentation and culture method of the strain and an applicable culture medium thereof. The stain of gamma-polyglutamic acid provided in the invention has simple nutritional requirements, stable culture transfer and simple and easy culture method. The Bacillus amyloliquefaciens NK-LL3 is used as zymogenic strain, sugar material is used for shake flask fermentation, without adding L-glutamic acid to fermentation material, thereby efficiently reducing the production cost and having good industrial application prospect compared with the generally used microbial fermentation needing glutamic acid as raw material.

Description

One non-glutamic acid dependent form gamma-polyglutamic acid synthesis bacterium and fermentation process thereof

Technical field

The present invention relates to the production that biological process prepares the macromolecule polymer material gamma-polyglutamic acid-, relate to bacterial strain and cultivation and fermentation process that a strain non-glutamic acid dependent form produces gamma-polyglutamic acid-particularly.

Background technology

Contain gamma-polyglutamic acid-[Poly (γ-glutamicacid) in the discovery Bacillus anthraciss such as nineteen thirty-seven Ivanovics, γ-PGA), it is the amido linkage that is formed at γ-hydroxyl and alpha-amino group by single L-glutamic acid molecule and the polymer that connects into, be water miscible amide compound, γ-PGA average molecular weight range is generally at 10-1, between 0,000,000.Its structural formula is as shown in the figure:

Polyglutamic acid contains a large amount of carboxyls, is weak anionic type polymerization macromole, and contains chiral carbon atom in the monomer.γ-PGA and derivative thereof have splendid film-forming properties, become fibering, resistance oxygen, plasticity-, cohesiveness, moisture retention and biodegradable physics and chemistry and the biological characteristics that waits uniqueness.Because the character of polyglutamic acid uniqueness, it is extensively applied to various fields, comprises foodstuffs industry, wetting Agent for Printing Inks, pharmaceutical carrier, biological flocculant, biological adhesive etc.Therefore, the research of polyglutamic acid and suitability for industrialized production thereof has at present obtained numerous investigators' concern.

According to the situation of needs L-glutamic acid whether in the substratum, γ-PGA can be produced bacterium and be divided into two classes, a class produces γ-PGA (I type) for needs L-glutamic acid, and does not need to add L-glutamic acid (II type) in the another kind of substratum.The former comprises many microorganisms, as B.subtilis ATCC9945, B.subtilis IFO3335, these class bacterium such as B.subtilis F-2-01 and B.licheniformisNK-03 generate the γ-PGA of more amount usually, this class is produced the production aspect that bacterium not only is widely used in γ-PGA, and has all played very important effect aspect the γ of whole genus bacillus-PGA synthesis mechanism illustrating.Back one class comprises B.subtilis TAM-4, B.licheniformis A35, because need not in the substratum to add to provide L-glutamic acid, can significantly reduce production costs, but for glutamic acid dependent form production bacterium comes, their kinds are less, and the γ that accumulates in substratum-PGA amount is produced the bacterium height not as glutamic acid dependent form, so also be not a lot of to their understanding at present.

Summary of the invention

One. the technical problem that solve

The purpose of this invention is to provide strain non-glutamic acid dependent form gamma-PGA synthesis bacterium, another purpose of the present invention provides the cultivation and the fermentation process of this bacterial strain.

Two. technical scheme

The present invention screens from food and obtains strain non-glutamic acid dependent form gamma-PGA synthesis bacterium, bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3), this bacterial strain is preserved in Chinese typical culture collection center on July 14th, 2008, and deposit number is: CCTCC No:M208109.

This bacterial strain obtains the gene segment of 1431bp by 16S rDNA pcr amplification, send commercial company's order-checking with amplified production, and login GenBank registration sequencing result obtains registration sequence EU755383.

The morphological feature of bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) is:

Thalli morphology is shaft-like, and several thalline can be linked to be chain, produces gemma in the cell growth later stage.

Colony characteristics is that bacterium colony is moistening, circular, opaque, and smooth surface has the thickness sense when provoking, and long-time the cultivation has inversion phenomenon (be inverted when cultivating because bacterium colony viscosity is excessive, bacterium colony secretory product occurred and dripped phenomenon in the below);

The cultural characteristic of bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) is:

Aerobic, aerobic conditions could be grown down.Suitable growth temperature is 28-37 ℃, and appropriate pH is 6.5-7.2, and lag phase is shorter in fermention medium, can enter logarithmic phase after two hours, enters the later stage of logarithmic phase after 5 hours, obtains maximum biomass about 17 hours.

The separation method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) is:

Scuppit with the bacterium of going out adopts aseptic technique that leavened food is inserted in the sterile physiological water, under-4 ℃ of conditions, stirs 30 minutes on magnetic stirring apparatus, changes 100 ℃ then over to and handles 15 minutes.Carry out gradient dilution again, select the bacteria suspension of suitable gradient to be coated on the screening flat board, the some strains of the bacterium colony that the picking surface becomes sticky are gone down to posterity and purifying, have finally obtained γ of the present invention-PGA synthesis bacterium----bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) by the fermentation lab scale.The bacterial strain that obtains behind the purifying is preserved in-70 ℃ the glycerine pipe.

The cultural method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) is:

1. the activation of bacterial classification: inoculation bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) was cultivated 24 hours down at 37 ℃ to the culture dish solid medium;

2. seed culture: dress 100ml liquid seed culture medium in the triangular flask of 500ml sterilization, then the activatory bacterial classification is connected in the seed culture medium, 37 ℃,, make seed liquor with 180-220rpm shaking culture 24 hours;

3. fermentation culture: the inoculum size with volume ratio 1.0-5.0% inserts seed liquor in the fermention medium, and 37 ℃, 180-220rpm shaking culture 48-72 hour.

Wherein used substratum composition is respectively:

(1) culture dish solid medium: peptone 8.0-12.0g/L, yeast powder 3.0-8.0g/L, NaCl9.0-11.0g/L, agar 15.0-25.0g/L, pH6.5-7.2;

(2) liquid seed culture medium: glucose 20.0g/L, K ₂HPO ₄10.0-15.0g/L, KH ₂PO ₄5.0-8.0g/L, MgSO ₄0.1-0.4g/L, (NH ₄) ₂SO ₄2.0-6.0g/L trace element solution is (with FeSO ₄4H ₂O, CaCl ₂2H ₂O, MnSO ₄4H ₂O and ZnCl ₂Obtained aqueous solution makes each constituent concentration be 1.0mM) 1.0-3.0mL pH6.5-7.2;

(3) fermention medium: glucose 20.0-50.0g/L, K ₂HPO ₄12.0g/L, KH ₂PO ₄6.0g/L, MgSO ₄0.3g/L, (NH ₄) ₂SO ₄4.0g/L trace element is (with FeSO ₄4H ₂O, CaCl ₂2H ₂O, MnSO ₄4H ₂O and ZnCl ₂Obtained aqueous solution makes each constituent concentration be 1.0mM) 1.0-3.0mL, pH6.5-7.2.

Three. beneficial effect

γ provided by the invention-PGA synthesis bacterium bacillus amyloliquefaciens (Bacillus amyloliquefaciensNK-LL3) separates from a kind of leavened food and obtains.Need not to add L-L-glutamic acid in the substratum in the process of the synthetic γ-PGA of this strain fermentation, can be directly from the synthetic γ-PGA of saccharine material.This compares as the microbial fermentation of raw material with the L-glutamic acid that needs that adopts in the past, has reduced production cost effectively, has the industrial applications prospect.

Non-glutamic acid dependent form gamma of the present invention-PGA synthesis bacterium bacillus amyloliquefaciens (Bacillusamyloliquefaciens NK-LL3) is preserved in Chinese typical culture collection center on July 14th, 2008, and deposit number is: CCTCC No:M208109.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

Embodiment 1

The separation screening of non-glutamic acid dependent form gamma-PGA synthesis bacterium

Scuppit with the bacterium of going out adopts aseptic technique that the leavened food access is equipped with in the 250ml triangular flask of 100ml stroke-physiological saline solution, under-4 ℃ of conditions, stirs 30 minutes on magnetic stirring apparatus, makes bacteria suspension, handles 15 minutes for 100 ℃.Carry out gradient dilution again, make and diluted 10 ¹-10 ⁹The bacteria suspension of different gradients.Selection has diluted 10 ³, 10 ⁵, 10 ⁷, 10 ⁹Bacteria suspension is doubly coated on the screening flat board that does not contain L-glutamic acid, cultivates, and the some strains of the bacterium colony that the picking colony surface becomes sticky are uploaded to be commissioned to train at the culture dish solid medium and supported and purifying, obtain single bacterium colony of purifying.Finally obtained γ of the present invention-PGA synthesis bacterium----bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) by the fermentation lab scale.The single bacterium colony that obtains is cultivated in liquid seed culture medium, and the concentration with 15% is kept in the glycerine pipe ,-70 ℃ of preservations.

Using such method screening of the present invention obtains strain non-glutamic acid dependent form gamma-PGA synthesis bacterium-----bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3).

Embodiment 2

The cultivation of bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) and γ-PGA detects

(1) actication of culture: the bacillus amyloliquefaciens (Bacillus amyloliquefaciensNK-LL3) of-70 ℃ of preservations is seeded on the activation culture ware solid plate with transfering loop, the activation culture based component is: peptone 8.0-12.0g/L, yeast powder 3.0-8.0g/L, NaCl 9.0-11.0g/L, agar 15.0-25.0g/L, pH6.5-7.2; The culture dish solid plate is inverted in 37 ℃ to be cultivated 24 hours down;

(2) seed culture: will activate good bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) ring and be inoculated in the seed culture medium and (contain the 100ml seed culture fluid in the 500ml triangular flask).The seed culture fluid composition is: glucose 20.0g/L, K ₂HPO ₄10.0-15.0g/L, KH ₂PO ₄5.0-8.0g/L, MgSO ₄0.1-0.4g/L, (NH ₄) ₂SO ₄2.0-6.0g/L trace element solution is (with FeSO ₄4H ₂O, CaCl ₂2H ₂O, MnSO ₄4H ₂O and ZnCl ₂Obtained aqueous solution makes each constituent concentration be 1.0mM) 1.0-3.0mL pH6.5-7.2, the seed culture fluid that inoculation is good places 37 ℃ of constant-temperature shaking culture casees, and 180-220rpm shaking culture 24 hours makes seed liquor;

(3) fermentation culture: the cultured seed nutrient solution is got 1.0-5.0ml insert fermentation culture concentrated (containing the 100ml fermentation culture in the 500ml triangular flask), the fermentation culture based component is: glucose 20.0-50.0g/L, K ₂HPO ₄12.0g/L, KH ₂PO ₄6.0g/L, MgSO ₄0.3g/L, (NH ₄) ₂SO ₄4.0g/L trace element is (with FeSO ₄4H ₂O, CaCl ₂2H ₂O, MnSO ₄4H ₂O and ZnCl ₂Obtained aqueous solution makes each constituent concentration be 1.0mM) 1.0-3.0mL, pH6.6-7.2.The fermention medium that inoculation is good placed 37 ℃ of constant-temperature shaking culture case 180-220rpm shaking culture 48 hours.

(4) mensuration of γ-PGA output: fermented liquid 6000rpm was collected supernatant in centrifugal 10 minutes, with the long-pending ethanol sedimentation supernatant liquor of tetraploid, the centrifugal precipitation of obtaining, throw out is dissolved in deionized water adds the long-pending ethanol of tetraploid once more, the centrifugal precipitation of obtaining, dialysis in deionized water behind the gained resolution of precipitate (hold back scope: 8000-15000) 12 hours, changed deionized water one time in per 2 hours, last lyophilize is also weighed.

(5) detection and result:

Employing FT-IR, ¹HNMR, gas-matter coupling etc. detect the hydrolyzate that adopts above-mentioned medium component and cultural method fermentative production γ-PGA, confirm as γ-PGA.

Claims

The bacterial strain----bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) of gamma-polyglutamic acid-is produced in 1 one strains.
2 cultural methods according to the described bacterial strain of claim 1 are:

(1) activation of bacterial classification: inoculation bacillus amyloliquefaciens (Bacillus amyloliquefaciens NK-LL3) was cultivated 24 hours down at 37 ℃ to the culture dish solid medium;

(2) seed culture: dress 100ml liquid seed culture medium in the triangular flask of 500ml sterilization, then the activatory bacterial classification is connected in the seed culture medium, 37 ℃,, make seed liquor with 180-220rpm shaking culture 24 hours;

(3) fermention medium: the inoculum size with volume ratio 1.0-5.0% inserts seed liquor in the fermention medium, and 37 ℃, 180-220rpm shaking culture 48-72 hour.
3 according to right 2 described cultural methods, it is characterized in that used substratum consists of:

(1) culture dish solid medium: peptone 8.0-12.0g/L, yeast powder 3.0-8.0g/L, NaCl 9.0-11.0g/L, agar 15.0-25.0g/L, pH6.5-7.2;

(2) liquid seed culture medium: glucose 20.0g/L, K ₂HPO ₄10.0-15.0g/L, KH ₂PO ₄5.0-8.0g/L, MgSO ₄0.1-0.4g/L, (NH ₄) ₂SO ₄2.0-6.0g/L trace element solution is (with FeSO ₄4H ₂O, CaCl ₂2H ₂O, MnSO ₄4H ₂O and ZnCl ₂Obtained aqueous solution makes each constituent concentration be 1.0mM) 1.0-3.0mLpH6.5-7.2;

(3) fermention medium: glucose 20.0-50.0g/L, K ₂HPO ₄12.0g/L, KH ₂PO ₄6.0g/L, MgSO ₄0.3g/L, (NH ₄) ₂SO ₄4.0g/L trace element is (with FeSO ₄4H ₂O, CaCl ₂2H ₂O, MnSO ₄4H ₂O and ZnCl ₂Obtained aqueous solution makes each constituent concentration be 1.0mM) 1.0-3.0mL, pH6.5-7.2.