CN100572546C

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

Info

Publication number: CN100572546C
Application number: CNB2007100681690A
Authority: CN
Inventors: 林建平; 傅维琦; 岑沛霖
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2007-04-20
Filing date: 2007-04-20
Publication date: 2009-12-23
Anticipated expiration: 2027-04-20
Also published as: CN101041839A

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

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

Technical field

The present invention relates to produce the method for 5-amino-laevulic acid with engineering bacteria.

Background technology

(5-aminolevulinic acid ALA) extensively is present in the organism 5-amino-laevulic acid, is first compound in the PBP, also is synthetic protoheme, chlorophyll, vitamins B ₁₂Common precursor Deng tetrapyrrole.(photodynamic agent PDT), has purposes widely in agrochemicals and medical field to ALA as a kind of photodynamics agent.At agriculture field, ALA has weeding, desinsection, increase stress resistance of plant and promotes multiple function such as plant-growth, and the noresidue of easily degrading, to people and animals' nontoxicity, become the nuisanceless green agrochemicals that have development prospect.At medical field, ALA has the effect of selectivity kill cancer cell, be called as s-generation photodynamics medicine (photodynamic medicine), have normal cytotoxic little, patient's lucifuge time is short, distinguishing features such as good effect are used to treat multiple cancers such as skin carcinoma, bladder cancer, colorectal carcinoma and carcinoma of the pancreas.Based on function and the application prospects of ALA, its study on the synthesis has caused unprecedented attention.

The present more employing chemical synthesis of production of ALA, its complex steps, by product is many, productive rate is low and serious environment pollution; And the biological induced-mutation method is produced ALA, mainly is to utilize Rhodobacter sphaeroides mutant strain, and the production peak of the ALA that reports is 27mmol/L, but its culture condition is comparatively complicated, and the production cycle is long, and cost is higher.

Summary of the invention

It is simple to the purpose of this invention is to provide a kind of technology, output height, environment amenable method of producing the 5-amino-laevulic acid with engineering bacteria.

Method of producing the 5-amino-laevulic acid with engineering bacteria of the present invention 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;

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 50-100ml fermention medium 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 2-3L fermention medium carries out fermentation culture, make the initial bacterium liquid density OD on the jar ₆₀₀Be 0.08-0.2, 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 6-10h is at cell density OD ₆₀₀When reaching 8-12, add 5-aminolevulinate dehydratase inhibitor in batches and continue to cultivate;

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

Among the present invention, said deposit number is a CGMCC No.1939 engineering bacteria, preservation date: 2007.1.25, classification name: Chinese colon bacillus by name, the Latin formal name used at school is Escherichia coliRosetta (DE3)-pET28a-R.S.hemA, in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China, depositary institution address: Institute of Microorganism, Academia Sinica.

Among the present invention, 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 to be formed, and its pH value is 6.0-6.3; Contain 15-25g succsinic acid and 10-15g glycine in the said supplemented medium respectively, volume is 800-1000ml; The inhibitor of 5-aminolevulinate dehydratase is a 3-6g/L D-glucose.

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 of drawings

Outer ALA concentration of Fig. 1 born of the same parents and cell density OD ₆₀₀Relation curve with fermentation time.

Embodiment

Further specify the present invention below in conjunction with embodiment

Embodiment

Method with engineering bacteria is produced the 5-amino-laevulic acid may further comprise the steps:

1. from being the glycerine pipe of CGMCC No.1939 engineering bacteria Rosetta (DE3)-pET28a-R.S.hemA, deposit number dips in the bacterium liquid that takes a morsel with inoculating needle, on the LB culture medium flat plate that contains 30 μ g/ml kantlex and 34 μ g/ml paraxin, rule incubated overnight in 37 ℃ of baking ovens;

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

3. get the 1ml first order seed and be inoculated in shaking in the bottle of the 500ml that contains the 100ml fermention medium,, cultivate 3h under the 200rpm condition, obtain secondary seed at 37 ℃.Wherein initial fermention medium is respectively 1% peptone, 0.5% yeast powder, 0.3% succsinic acid, 0.2% glycine and 0.2% glucose by mass volume ratio to be formed, and regulating the pH value with sodium hydroxide is 5.9;

4. the 100ml secondary seed is inoculated in the 5L fermentor tank that contains the above-mentioned fermention medium of 3L, makes the initial bacterium liquid density OD on the jar ₆₀₀Be approximately 0.1, the fermentor tank rotating speed is 600rmp, and air flow quantity is 1.5L/min, and initial culture temperature is 37 ℃, reduces to 28 ℃ behind the 2h, and (IPTG) induces with the 0.1mmol/L isopropyl-;

5. stream adds and contains 21g succsinic acid and 12g glycine behind the inducing culture 4h, and volume is the supplemented medium of 330ml, with the measuring density thalli growth situation of thalline, as cell density OD ₆₀₀Reach at 8 o'clock, beginning is added 4g/L D-glucose as 5-aminolevulinate dehydratase inhibitor every 3h, adds glucose altogether 3 times.6) the initial pH of fermentation culture adopts the dilute sulphuric acid of 10%-20% volume fraction to be controlled at 5.9; Controlling pH by the flow feeding substratum behind the inducing culture 4h is 6.2.The analytical procedure of employing Mauzerall and Granick (with reference to Mauzerall D, Granick S.J.Bio.Chem., 1956, the 219:435-442) concentration of mensuration ALA.Can reach 6.6g/L during the concentration 30h of the outer ALA of born of the same parents as seen from Figure 1.

Claims

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;

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.