CN100572546C - Produce the method for 5-amino-laevulic acid with engineering bacteria - Google Patents

Abstract

The present invention relates to produce the method for 5-amino-laevulic acid with engineering bacteria.This method may further comprise the steps: be CGMCC No.1939 engineering bacteria Rosetta (DE3)-pET28a-R.S.hemA with LB culture medium flat plate activation deposit number 1); 2) mono-clonal on the flat board is inoculated in the LB culture media shaking vase,, obtains first order seed 37 ℃ of incubated overnight; 3) get first order seed and be inoculated in shaking in the bottle of LB substratum and cultivate, obtain secondary seed; 4) secondary seed is inoculated in carries out fermentation culture in the fermentor tank that contains fermention medium, induce, continue to cultivate and carry out feed supplement after for some time and cultivate with the isopropyl-cooling.Technology of the present invention is simple, controllability is high, cost is low, fermenting process is environmentally friendly, and the outer ALA output height of the born of the same parents of gained has wide industrial prospect.

Description

Method for producing 5-aminolevulinic acid with engineering bacteria

technical field

The invention relates to a method for producing 5-aminolevulinic acid with engineering bacteria.

Background technique

5-Aminolevulinic acid (5-aminolevulinic acid, ALA) widely exists in organisms, is the first compound in the biosynthesis pathway of porphyrin, and is also a common precursor for the synthesis of tetrapyrrole compounds such as heme, chlorophyll, and vitamin _B12 . body. As a photodynamic agent (photodynamic agent, PDT), ALA has a wide range of uses in the fields of agrochemicals and medicine. In the field of agriculture, ALA has many functions such as weeding, insecticide, increasing plant stress resistance and promoting plant growth, etc., and is easy to degrade without residue, non-toxic to humans and animals, and has become a very promising pollution-free green agrochemical. In the field of medicine, ALA has the effect of selectively killing cancer cells. It is called the second-generation photodynamic medicine (photodynamic medicine). It is used in the treatment of various cancers including skin cancer, bladder cancer, colon cancer and pancreatic cancer. Based on the function and wide application prospect of ALA, its synthesis research has attracted unprecedented attention.

At present, the production of ALA mostly adopts the chemical synthesis method, which has cumbersome steps, many by-products, low yield and serious environmental pollution; while the biological mutagenesis method mainly uses the mutant strain of Rhodobacter sphaeroides to produce ALA, and the reported ALA is the highest. The output is 27mmol/L, but its culture condition is relatively complicated, the production cycle is long, and the cost is high.

Contents of the invention

The purpose of the present invention is to provide a kind of technique simple, high yield, the method for environment-friendly production 5-aminolevulinic acid with engineering bacterium.

The method for producing 5-aminolevulinic acid with engineering bacteria of the present invention comprises the following steps:

1) Use an inoculation needle to dip the bacterial solution from the glycerin tube with the preservation number of CGMCC No.1939 engineering bacterium Rosetta(DE3)-pET28a-R.S.hemA. After streaking the chloramphenicol LB medium plate, place it in a 37°C oven for overnight culture;

2) inoculate the monoclonal on the plate in a 250ml shake flask containing 30-50ml LB medium, the rotation speed is 200-220rmp, and cultivate overnight at 37°C to obtain first-class seeds;

3) Take 1ml-5ml primary seeds and inoculate them in a 500ml shake flask containing 50-100ml fermentation medium and cultivate them for 3-4h to obtain secondary seeds;

4) Inoculate 50-100ml of secondary seeds into a 5L fermenter containing 2-3L of fermentation medium for fermentation and cultivation, so that the initial bacterial liquid density _OD600 on the tank is 0.08-0.2, and the rotation speed of the fermenter is 400-600rpm, The air flow rate is 1-2L/min, the initial culture temperature is 37°C, and after 2 hours, it is lowered to 26-30°C, and then induced with 0.05-0.2mmol/L isopropyl-β-D-thiogalactoside;

5) After 6-10 hours of induction culture, the feeding medium was fed, and when the cell density _OD600 reached 8-12, the 5-aminolevulinic acid dehydratase inhibitor was added in batches to continue the culture;

6) The initial fermentation culture uses 10%-20% volume fraction of dilute sulfuric acid to control the pH at 5.8-6.0; after 4-8 hours of induction culture, the pH is controlled at 6.1-6.3 by fed-batch feeding medium.

In the present invention, said preservation number is CGMCC No.1939 engineering bacteria, preservation date: 2007.1.25, classification and naming: Chinese name is Escherichia coli, and Latin literary name is Escherichia coli Rosetta (DE3)-pET28a-R.S.hemA , in the depository unit designated by the Chinese Patent Office, the General Microbiology Center of the China Committee for the Preservation of Microorganisms, and the address of the depository unit: Institute of Microbiology, Chinese Academy of Sciences.

In the present invention, said initial fermentation medium is composed of 0.5%-2% peptone, 0.25%-1% yeast powder, 0.3%-1% succinic acid, 0.2%-0.4% glycine and 0.1% -0.5% glucose composition, its pH value is 6.0-6.3; Contain 15-25g succinic acid and 10-15g glycine respectively in the said feeding medium, and volume is 800-1000ml; 5-aminolevulinic acid dehydratase The inhibitor is 3-6g/L D-glucose.

The invention has the advantages of simple process, high controllability, low cost, environment-friendly fermentation process, high yield of extracellular ALA, and broad industrialization prospect.

Description of drawings

Fig. 1 The relationship curves of extracellular ALA concentration, cell density OD ₆₀₀ and fermentation time.

Detailed ways

Further illustrate the present invention below in conjunction with embodiment

Example

The method for producing 5-aminolevulinic acid with engineering bacteria comprises the following steps:

1. Use an inoculation needle to dip a small amount of bacterial solution from the glycerol tube of CGMCC No.1939 engineering bacterium Rosetta (DE3)-pET28a-R.S.hemA, in the presence of 30 μg/ml kanamycin and 34 μg/ml chloramphenicol Streak on the LB medium plate and cultivate overnight in a 37°C oven;

2. Pick a single clone and inoculate it in a 250ml shake flask containing 50ml LB medium at a rotation speed of 200rmp, and culture overnight at 37°C to obtain first-grade seeds;

3. Take 1ml of primary seeds and inoculate them in a 500ml shake flask containing 100ml of fermentation medium, culture at 37°C and 200rpm for 3h to obtain secondary seeds. Wherein the initial fermentation medium is composed of 1% peptone, 0.5% yeast powder, 0.3% succinic acid, 0.2% glycine and 0.2% glucose in mass volume ratio, and the pH value is adjusted to 5.9 with sodium hydroxide;

4. Inoculate 100ml of secondary seeds into a 5L fermenter containing 3L of the above-mentioned fermentation medium, so that the initial bacterial liquid density _OD600 on the tank is approximately 0.1, the rotation speed of the fermenter is 600rmp, and the air flow rate is 1.5L/min. The initial culture temperature was 37°C, and it was lowered to 28°C after 2 hours, and induced with 0.1mmol/L isopropyl-β-D-thiogalactoside (IPTG);

5. Add 21g succinic acid and 12g glycine after 4 hours of induction culture, with a volume of 330ml, and monitor the growth of the bacteria with the density of the bacteria. When the OD ₆₀₀ of the bacteria reaches 8, start every 3 hours Add 4 g/L D-glucose as a 5-aminolevulinate dehydratase inhibitor, and add glucose 3 times in total. 6) The initial pH of the fermentation culture was controlled at 5.9 with 10%-20% volume fraction of dilute sulfuric acid; the pH was controlled at 6.2 by fed-batch feed medium after 4 hours of induction culture. The concentration of ALA was determined by the analytical method of Mauzerall and Granick (refer to Mauzerall D, Granick SJ Bio. Chem., 1956, 219: 435-442). It can be seen from Figure 1 that the concentration of extracellular ALA can reach 6.6g/L in 30 hours.

Claims (4)

1. produce the method for 5-amino-laevulic acid with engineering bacteria, it is characterized in that may further comprise the steps:

1) dips in from deposit number is the glycerine pipe of CGMCC No.1939 engineering bacteria Rosetta (DE3)-pET28a-R.S.hemA with inoculating needle and get bacterium liquid, after line on the LB culture medium flat plate that contains 30-50 μ g/ml kantlex and 30-50 μ g/ml paraxin, place 37 ℃ of baking oven incubated overnight, deposit number is the CGMCCNo.1939 engineering bacteria, the classification name: Chinese colon bacillus by name, the Latin formal name used at school is Escherichiacoli Rosetta (DE3)-pET28a-R.S.hemA;

2) mono-clonal on the flat board is inoculated in the 250ml that contains 30-50ml LB substratum and shakes in the bottle, rotating speed is 200-220rmp, 37 ℃ of incubated overnight, obtains first order seed;

3) get the 1ml-5ml first order seed and be inoculated in the shaking of 500ml that contains the initial fermention medium of 50-100ml and cultivate 3-4h in the bottle, obtain secondary seed;

4) the 50-100ml secondary seed is inoculated in the 5L fermentor tank that contains the above-mentioned fermention medium of 2-3L carries out fermentation culture, make the initial bacterium liquid density OD on the jar ₆₀₀Be 0.08-0.15, the fermentor tank rotating speed is 400-600rpm, and air flow quantity is 1-2L/min, and initial culture temperature is 37 ℃, reduces to 26-30 ℃ behind the 2h, induces with the 0.05-0.2mmol/L isopropyl-again;

5) flow feeding substratum behind the inducing culture 4-8h is at cell density OD ₆₀₀When reaching 8-12, add the inhibitor of glucose as the 5-aminolevulinate dehydratase in batches;

6) the initial pH of fermentation culture adopts the dilute sulphuric acid of 10%-20% volume fraction to be controlled at 5.8-6.0; Controlling pH by the flow feeding substratum behind the inducing culture 4-8h is 6.1-6.3.

2. method of producing the 5-amino-laevulic acid with engineering bacteria according to claim 1, it is characterized in that said initial fermention medium is respectively 0.5%-2% peptone, 0.25%-1% yeast powder, 0.3%-1% succsinic acid, 0.2%-0.4% glycine and 0.1%-0.5% glucose by mass volume ratio and forms, its pH value is 6.0-6.3.

3. according to the described method of producing the 5-amino-laevulic acid with engineering bacteria of claim 1, it is characterized in that containing in the said supplemented medium 15-25g succsinic acid and 10-15g glycine, volume is 300-400ml.

4. method of producing the 5-amino-laevulic acid with engineering bacteria according to claim 1, the inhibitor that it is characterized in that said 5-aminolevulinate dehydratase is a 3-6g/L D-glucose.

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