CN109929774A - One bacillus and its preparing the application in 5-ALA - Google Patents

Abstract

The invention discloses a bacillus and its preparing the application in 5-ALA.The bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.16179, and the deposit date is on July 30th, 2018.The invention also discloses the methods that the bacterial strain prepares 5-ALA using pretreated lignocellulosic and soil dynamic test waste material.Experiments have shown that: the bacterial strain not only can synthesize 5-ALA by carbon source of glucose; also abundant available resources, cheap lignocellulosic or soil dynamic test fermentable sugars liquid are that carbon source synthesizes 5-ALA; and it is resistant to the good growth of the mortifier generated in preprocessing process; not only reduce the cost of technical process; and be conducive to ecological environmental protection, realize sustainable development.

Description

A strain of Bacillus and its application in the preparation of 5-aminolevulinic acid

technical field

The invention belongs to the field of biochemical industry, in particular to a Bacillus strain and its application in preparing 5-aminolevulinic acid.

Background technique

5-aminolevulinic acid (ALA) is a precursor substance for the synthesis of tetrahydropyrrole compounds (porphyrin, chlorophyll, heme, vitamin B12) in vivo, and widely exists in microorganisms, plants and animal cells.

ALA has a wide range of uses in agriculture and medicine. In the agricultural field, ALA can promote photosynthesis of green plants, regulate plant respiration, promote plant tissue differentiation, improve plant stress resistance, and improve the quality of agricultural products. Low concentrations can significantly improve crop yields, while high concentrations can be used as safe and non-polluting herbicides and pesticides. In the field of medicine, it is called the second-generation photodynamic drug due to its ability to selectively kill cancer cells. Stimulation of the photosensitizer protoporphyrin IX by ALA is used for photodynamic cancer therapy and tumor localization. It is also widely used in the treatment of skin diseases and the detection of lead poisoning. Based on the functions and broad application prospects of ALA, its synthesis research has attracted unprecedented attention.

The chemical synthesis process using hippuric acid, succinic acid, furfural and other heterocyclic substances and levulinic acid as raw materials is the main method for producing ALA at present. However, the chemical synthesis method is cumbersome, with many by-products, difficult separation and purification, low yield and serious environmental pollution. The use of microbial fermentation to produce ALA can effectively alleviate the difficulties faced by chemical synthesis. Currently reported microorganisms capable of producing ALA include photosynthetic bacteria Rhodobacter sphaeroides, Rhodopseudanonas palustris, Rhodopseudomonas sp., and Propionibacterium riboflauimc. The cultivation of photosynthetic bacteria is difficult, the process is complicated, and the industrialization is limited. Moreover, the yield of ALA is generally not high, and the cultivation process requires high concentration of glucose as a carbon source, resulting in increased costs. Therefore, the mining of new strain resources, improving the yield of strains and reducing the production cost are the main research directions of microbial source ALA. There is no reported strain that can efficiently accumulate ALA in Bacillus.

As a big agricultural country, my country produces about 600-700 million tons of lignocellulose raw materials every year, such as corn cob, corn stalk and potato residue, which are rich in cellulose and hemicellulose. The incineration and random stacking of lignocellulosic raw materials cause environmental pollution. The effective use of these agricultural wastes to turn waste into treasure and convert them into other high value-added products has become a research hotspot.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a strain of Bacillus sp. PK9.

The deposit number of Bacillus sp.PK9 provided by the present invention is CGMCC No.16179.

The Bacillus sp. PK9 provided by the present invention is isolated from the rhizosphere soil of tiller, and can be used to prepare 5-aminolevulinic acid (ALA). It is deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101).

Another object of the present invention is to provide new uses of Bacillus sp. or its bacterial suspension or its culture broth or its fermentation broth or inoculum containing the same.

The present invention provides the application of Bacillus sp. or its bacterial suspension or its culture liquid or its fermentation liquid or a bacterial agent containing the same in the preparation of 5-aminolevulinic acid.

The invention also provides the application of Bacillus sp. or its bacterial suspension or its culture solution or its fermentation broth or its inoculum in preparing 5-aminolevulinic acid by using agricultural wastes as raw materials.

In the above application, the agricultural waste may be lignocellulosic agricultural waste or potato pomace; further, the lignocellulosic agricultural waste may be corncob, bagasse or switchgrass. In a specific embodiment of the present invention, the agricultural waste is corn cob or potato dregs.

The present invention also provides the application of Bacillus sp. or its bacterial suspension or its culture broth or its fermentation broth or a bacterial agent containing the same in improving the production of 5-aminolevulinic acid.

Yet another object of the present invention is to provide a product for preparing 5-aminolevulinic acid.

The active ingredient of the product for preparing 5-aminolevulinic acid provided by the present invention is Bacillus sp. or its bacterial suspension or its culture solution or its fermentation solution or its inoculum.

Finally, the present invention also provides a method for preparing 5-aminolevulinic acid.

The method for preparing 5-aminolevulinic acid provided by the invention comprises the following steps: using lignocellulosic agricultural waste or potato residue as raw materials, and using Bacillus sp. to synthesize 5-aminolevulinic acid.

In the above-mentioned method for preparing 5-aminolevulinic acid, the agricultural waste may be lignocellulosic agricultural waste or potato pomace; further, the lignocellulosic agricultural waste may be corncob, bagasse or Switchgrass.

The above-mentioned method for preparing 5-aminolevulinic acid may comprise the steps:

1) Bacillus sp. is inoculated into the seed medium and cultivated to obtain seed liquid;

2) inoculating the seed liquid into a fermentation medium for cultivation to obtain a fermentation product; the fermentation product contains 5-aminolevulinic acid.

In the above-mentioned method for preparing 5-aminolevulinic acid, the seed medium and the fermentation medium both contain agricultural waste hydrolyzate or glucose. The agricultural waste hydrolyzate may be a fermentable sugar solution obtained by acid-processing agricultural waste.

The method for acid treatment of lignocellulosic agricultural waste can be specifically carried out according to the following steps: take 8g of lignocellulosic agricultural waste samples (such as corn cob, bagasse or switchgrass) first with (0.5-1)% dilute sulfuric acid 200ml, treated at 121°C for (30-60) min, after cooling, the solid residue was collected by suction filtration, then 30ml (50-72)% sulfuric acid was added for treatment at 30°C (30-60) min, then 840ml of water was added, and the mixture was treated at 121°C (30 -60) min, after cooling, the filtrate was collected by suction filtration, and the pH of the filtrate was adjusted to 7.0 with solid Ca(OH) ₂ , and the supernatant obtained after filtration was the fermentable sugar liquid.

The method for acid treatment of potato dregs can be specifically carried out according to the following steps: take 20g of potato dregs sample with (1.0-1.7)% dilute sulfuric acid 200ml (solid-liquid ratio 1:10), 121 ℃ treatment (90-120) min, cooling Then, the filtrate was collected by suction filtration, and the pH of the filtrate was adjusted to 7.0 with solid Ca(OH) ₂ , and the supernatant obtained after filtration was the fermentable sugar solution.

further,

When preparing 5-aminolevulinic acid with glucose as raw material,

The formula of the seed culture medium is as follows: the solvent is deionized water, and the solute and the concentration are as follows: glucose (2-4) g/L, tryptone (5-10) g/L, yeast powder (10-15) g /L, potassium dihydrogen phosphate 3g/L, initial pH 7.3-7.5.

The formula of the fermentation medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: glucose (1-5) g/L, tryptone (5-10) g/L, beef extract (10-15) g /L, sodium chloride (1-5) g/L, dipotassium hydrogen phosphate (3-5) g/L, initial pH 7.2-7.3.

When preparing 5-aminolevulinic acid with agricultural waste hydrolyzate as raw material,

The formula of the seed culture medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: fermentable sugar solution (calculated by glucose) (1-5) g/L, tryptone (5-10) g/L, Beef extract (10-15) g/L, sodium chloride (1-5) g/L, dipotassium hydrogen phosphate (3-5) g/L, initial pH 7.3-7.5.

The formula of the fermentation medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: fermentable sugar solution (calculated by glucose) (15-20) g/L, tryptone (5-10) g/L, Beef extract (10-15) g/L, sodium chloride (1-5) g/L, dipotassium hydrogen phosphate (3-5) g/L, initial pH 7.2-7.3.

Further, when preparing 5-aminolevulinic acid with glucose as raw material,

In the above 1), the culturing conditions are (28-37) °C, (100-200) rpm for (12-24) h;

In the above 2), the inoculation amount of the seed solution is (1-5)%, and the cultivation conditions are (28-37)° C., (100-200) rpm for (24-36) h.

When preparing 5-aminolevulinic acid with agricultural waste hydrolyzate as raw material,

In the above 1), the culturing conditions are (28-37) °C, (100-200) rpm for (12-24) h;

In the above 2), the inoculum amount of the seed solution is (1-5)%, and the cultivation conditions are (30-37)° C., (100-200) rpm for (24-36) h.

In the above-mentioned method for preparing 5-aminolevulinic acid, the step of inoculating Bacillus sp. into an activated medium for activation cultivation is further included before the step 1).

Further, the formulation of the activation medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: tryptone (5-10) g/L, beef extract (3-5) g/L, sodium chloride 5g/L, agar 18g/L, initial pH 7.2-7.4.

Further, the condition of the activation culture is (28-37) °C for (12-24) h.

In the above application or product or method, the Bacillus sp. is Bacillus sp. PK9 CGMCC No.16179.

The beneficial effects of the present invention are as follows: Bacillus sp. CGMCC No. 16179, which is isolated and screened from nature, can use resource-rich and cheap lignocellulose or potato pomace fermentable sugar liquid as carbon source to synthesize 5-aminolevulinic acid Acid, and tolerated the inhibitors produced during the pretreatment process. Good growth, the maximum yield was obtained at the time of fermentation (24-48) h, and the fermentation broth was in acidic conditions at this time, which could effectively prevent the decomposition of ALA.

The invention provides a Bacillus sp. CGMCC No. 16179 high-yielding 5-aminolevulinic acid, and a method for preparing 5-aminolevulinic acid from lignocellulosic agricultural waste by utilizing the strain. Using the strain as a bacterial species, using lignocellulose or potato residue waste as raw materials to synthesize 5-aminolevulinic acid not only reduces the cost of the process, but also is beneficial to ecological environment protection and achieves sustainable development.

Description of drawings

Figure 1 is the standard curve of 5-aminolevulinic acid.

Preservation Instructions

Latin name: Bacillus sp.

Strain number: PK9

Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee

Abbreviation of depositary institution: CGMCC

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: July 30, 2018

Deposit Center Registration Number: CGMCC No.16179

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The preparation method of the corncob sample in the following examples is as follows: after drying the corncob (derived from Yiyang County, Henan Province), use a pulverizer (Baijie multi-functional pulverizer BJ-150) to grind, and pass 50 meshes. sieve to get the corncob sample.

The preparation method of potato dregs in the following examples is as follows: cut potatoes into water, boil for 60 min, filter with 4 layers of gauze, and put the remaining residue into an oven to dry to obtain a potato dregs sample.

Example 1. Acquisition, identification and preservation of PK9 strains

First, the acquisition of PK9 strain

Sampling from the rhizosphere soil of small tiller, weighing about 2g of each soil sample obtained, suspended in NaCl solution with a mass fraction of 0.9%, moderately diluted, inoculated on LB plate, cultured at 30°C for 24h, and single colony was selected. , LB plate streaking to obtain a single colony after using glucose as a single carbon source for shake flask fermentation, after fermentation for 36h, the fermentation broth was sampled, centrifuged, and the supernatant was collected. The concentration of 5-aminolevulinic acid was quantitatively determined by spectrophotometry. A strain with high ALA production was obtained by screening, and the strain was named PK9, and its ALA yield was 25.89 mg/L.

2. Identification of PK9 strains

1. Physiological and biochemical identification

The PK9 strain screened in step 1 was identified according to the physiological and biochemical characteristics of the microbiology experiment tutorial. The results are shown in Table 1.

Table 1. Physiological and biochemical characteristics identification results

Identification project PK9 strain Glucose acid production - Methyl red test - V-P experiment - Nitrate reduction - indole production - produce H₂S - Gelatin Liquefaction - Sodium citrate -

2. Molecular identification

The genomic DNA of the PK9 strain was extracted, and the 16S rRNA gene of the PK9 strain was identified by using universal primers according to the method of molecular biology experiment guide, and the 16S rRNA gene sequence was obtained. The sequence size is 1354bp, as shown in sequence 1 in the sequence table.

Through the above identification results, it can be determined that the PK9 strain belongs to Bacillus sp..

3. Preservation of PK9 strains

The classification name of the PK9 strain is Bacillus sp., which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short) on July 30, 2018. Address: No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101), the deposit number is CGMCC No.16179.

Embodiment 2, the application of PK9 strain in preparing 5-aminolevulinic acid with glucose as raw material

1, the Bacillus sp.PK9 obtained in Example 1 is in the activated medium (the activated medium formula is as follows: the solvent is deionized water, and the solute and the concentration are respectively: tryptone 5g/L, beef extract 5g/L, Sodium chloride 5g/L, agar 18g/L, initial pH 7.2), cultured at 37°C for 24h to obtain activated PK9.

2. Fill 50ml of seed medium in a 250ml conical flask (the recipe of the seed medium is as follows: the solvent is deionized water, the solute and the concentration are: glucose 2g/L, tryptone 10g/L, yeast powder 10g/L, dihydrogen phosphate Potassium 3g/L, initial pH 7.3), sterilize, cool, and inoculate the activated PK9 obtained in step 1 according to conventional methods, and inoculate after inoculation at 37°C, 200rpm shaker for 12h to obtain seed fermentation broth.

3. Use a 500ml triangular bottle to pack 100ml of fermentation medium (the formula of the fermentation medium is as follows: the solvent is deionized water, the solute and the concentration are respectively: glucose 15g/L, tryptone 10g/L, beef extract 10g/L, chlorine Sodium chloride 2g/L, dipotassium hydrogen phosphate 3g/L, initial pH 7.2), sterilize, cool and insert the seed liquid obtained in step 2 into the fermentation medium at 37° C., 200 rpm shaker for 36 h to obtain a fermentation broth containing 5-aminolevulinic acid.

The ALA content in the fermentation broth containing 5-aminolevulinic acid was quantitatively determined by spectrophotometry. The specific steps are as follows: draw the standard curve of 5-aminolevulinic acid: take 400 μl of 5-aminolevulinic acid standard solution of different concentrations (concentrations are 5mg/L, 10mg/L, 20mg/L, 30mg/L, respectively , 40mg/L, 50mg/L 5-aminolevulinic acid standard), add 200 μl acetate buffer and 100 μl acetylacetone, and bath in boiling water for 15 min. After cooling to room temperature, add 700 μl Ehrlich's reagent (Ehrlich's reagent formula is as follows: weigh 1 g of p-dimethylaminobenzaldehyde, add 30 ml of glacial acetic acid, then add 8 ml of perchloric acid (70%), make up to 50 ml with glacial acetic acid, and Prepared for current use), reacted for 20 min, and detected at 554 nm using a spectrophotometer. Draw the standard curve with the concentration of 5-aminolevulinic acid standard solution as the abscissa and the OD _554nm value as the ordinate. The standard curve is shown in Figure 1.

Take 400 μl of the supernatant after centrifugation of the fermentation broth containing 5-aminolevulinic acid obtained in step 3, add 200 μl of acetate buffer and 100 μl of acetylacetone, and bath in boiling water for 15 min. After cooling to room temperature, add 700μl Ehrlich's reagent, react for 20min, use a spectrophotometer to detect at 554nm, and substitute the obtained OD _554nm value into the standard curve to calculate the ALA content in the fermentation broth containing 5-aminolevulinic acid It is 25.89mg/L.

HORIBA B-71X pen pH meter was used to measure the pH value of the fermentation broth: after the fermentation broth was centrifuged, the supernatant was collected; 200 μl of the supernatant was dripped into the detection pool. Close the lid of the detection cell and place it horizontally for about 2 minutes to read the measured pH value. The pH of the fermentation broth was 4.5.

Embodiment 3, the application of PK9 strain in preparing 5-aminolevulinic acid with corn cob as raw material

1, the Bacillus sp.PK9 obtained in Example 1 is in the activated medium (the formula of the activated medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively: tryptone 5g/L, beef extract 5g/L , sodium chloride 5g/L, agar 18g/L, initial pH 7.2) and cultured at 37°C for 24h to obtain activated PK9.

2. Fill 50ml of seed culture medium in a 250ml conical flask (the recipe of the seed culture medium is as follows: the solvent is deionized water, the solute and the concentration are respectively: corncob hydrolyzed fermentable sugar solution (calculated as glucose) 2g/L, tryptone 10g/L. L, beef extract 10g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 3g/L, adjust pH 7.3), sterilize by conventional method, cool, and inoculate the PK9 after the activation that step 1 obtains, after inoculation At 37 ° C, 200 rpm shaker cultured for 12 h to obtain seed fermentation broth.

The preparation method of above-mentioned corncob hydrolyzed fermentable sugar liquid is as follows: take 8g corncob sample and treat 60min with 200ml 1% dilute sulfuric acid at 121 DEG C, collect solid residue by suction filtration after cooling, then add 30ml 50% sulfuric acid at 30 DEG C and treat for 30min, After adding 840 ml of water, the solution was treated at 121 °C for 60 min, and the filtrate was collected by suction filtration after cooling. The pH of the filtrate was adjusted to 7.0 with solid Ca(OH) ₂ , and the supernatant obtained after filtration was the corncob hydrolyzed fermentable sugar solution.

3. Use a 500ml triangular bottle to pack 100ml of fermentation medium (the formula of the fermentation medium is as follows: the solvent is deionized water, the solute and the concentration are respectively: corncob hydrolyzed fermentable sugar solution (calculated as glucose) 15g/L, tryptone 10g/L. L, beef extract 15g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 5g/L, initial pH 7.2), sterilize by conventional method, cool and by 2% of the seed liquid obtained in step 2 The fermentation medium was inserted into the fermentation medium, and then cultured at 37° C. and 200 rpm in a shaker for 36 hours to obtain a fermentation broth containing 5-aminolevulinic acid.

The ALA yield in the 5-aminolevulinic acid-containing fermentation broth obtained in this example was 21.47 mg/L. The pH of the fermentation broth was 4.89.

Embodiment 4, the application of PK9 strain in the preparation of 5-aminolevulinic acid with potato dregs as raw material

1, the Bacillus sp.PK9 obtained in Example 1 is in the activation medium (the activation medium formula is as follows: the solvent is deionized water, and the solute and the concentration are respectively: peptone 5g/L, beef extract 5g/L, chlorine 5g/L sodium chloride, 18g/L agar, initial pH 7.2) were incubated at 37°C for 24h to obtain activated PK9.

2. Fill 50ml of seed medium in a 250ml conical flask (the recipe of the seed medium is as follows: the solvent is deionized water, the solute and the concentration are respectively: potato dregs hydrolyzed fermentable sugar solution (calculated as glucose) 2g/L, tryptone 10g/L L, beef extract 10g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 3g/L, adjust pH 7.3), sterilize by conventional method, cool, and inoculate the PK9 after the activation that step 1 obtains, after inoculation At 37 ° C, 200 rpm shaker cultured for 12 h to obtain seed fermentation broth.

The preparation method of above-mentioned potato dregs hydrolyzed fermentable sugar solution is as follows: take 20g potato dregs sample and treat 90min with 200ml 1.0% dilute sulfuric acid (solid-to-liquid ratio is 1:10) at 121 DEG C for 90min, collect supernatant by suction filtration after cooling, and use solid The pH of the filtrate was adjusted to 7.0 by Ca(OH) ₂ , and the supernatant obtained after filtration was the fermentable sugar solution.

3. Use a 500ml triangular flask to pack 100ml of fermentation medium (the formula of the fermentation medium is as follows: the solvent is deionized water, and the solute and concentration are respectively: potato dregs hydrolyzed fermentable sugar solution (calculated as glucose) 15g/L, tryptone 10g/L. L, beef extract 15g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 5g/L, initial pH 7.2), sterilize by conventional method, cool and by 2% of the seed liquid obtained in step 2 The fermentation medium was inserted into the fermentation medium, and then cultured at 37° C. and 200 rpm in a shaker for 36 hours to obtain a fermentation broth containing 5-aminolevulinic acid.

The ALA yield in the 5-aminolevulinic acid-containing fermentation broth obtained in this example was 23.79 mg/L. The pH of the fermentation broth was 4.95.

sequence listing

<110> China Agricultural University

<120> A strain of Bacillus and its application in the preparation of 5-aminolevulinic acid

<160>1

<170>PatentIn version 3.5

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<211>1354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gagcgaatgg attaagagct tgctcttatg aagttagcgg cggacgggtg agtaacacgt 60

gggtaacctg cccataagac tgggataact ccgggaaacc ggggctaata ccggataaca 120

ttttgaactg catggttcga aattgaaagg cggcttcggc tgtcacttat ggatggaccc 180

gcgtcgcatt agctagttgg tgaggtaacg gctcaccaag gcaacgatgc gtagccgacc 240

tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 300

agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 360

ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtgctagttg aataagctgg 420

caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 480

acgtaggtgg caagcgttat ccggaattat tgggcgtaaa gcgcgcgcag gtggtttctt 540

aagtctgatg tgaaagccca cggctcaacc gtggagggtc attggaaact gggagacttg 600

agtgcagaag aggaaagtgg aattccatgt gtagcggtga aatgcgtaga gatatggagg 660

aacaccagtg gcgaaggcga ctttctggtc tgtaactgac actgaggcgc gaaagcgtgg 720

ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780

agagggtttc cgccctttag tgctgaagtt aacgcattaa gcactccgcc tggggagtac 840

ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900

gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg aaaaccctag 960

agatagggct tctccttcgg gagcagagtg acaggtggtg catggttgtc gtcagctcgt 1020

gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccatca 1080

ttaagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140

caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac ggtacaaaga 1200

gctgcaagac cgcgaggtgg agctaatctc ataaaaccgt tctcagttcg gattgtaggc 1260

tgcaactcgc ctacatgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1320

atacgttccc gggccttgta cacaccgccc gtca 1354

Claims (10)

1. a bacillus Bacillus sp.PK9, deposit number is CGMCC No.16179.

2. prepared by bacillus sp. or its bacteria suspension or its culture solution or its fermentation liquid or the microbial inoculum containing it Application in 5-ALA；

Or, bacillus sp. or its bacteria suspension or its culture solution or its fermentation liquid or containing its microbial inoculum with agriculture Industry waste is that raw material prepares the application in 5-ALA.

3. application according to claim 2, it is characterised in that: the agricultural wastes are lignocellulose agriculture waste Object or soil dynamic test；

Or, the lignocellulose agricultural wastes are corncob, bagasse or switchgrass.

4. bacillus sp. or its bacteria suspension or its culture solution or its fermentation liquid or the microbial inoculum containing it are improving Application in 5-ALA yield.

5. a kind of product for being used to prepare 5-ALA, active constituent is bacillus sp. or its bacterium Suspension or its culture solution or its fermentation liquid or the microbial inoculum containing it.

6. a kind of method for preparing 5-ALA, includes the following steps: with lignocellulose agricultural wastes or soil Bean dregs are raw material, synthesize 5-ALA using bacillus sp..

7. according to the method described in claim 6, it is characterized by: the agricultural wastes are lignocellulose agriculture waste Object or soil dynamic test；

Or, the lignocellulose agricultural wastes are corncob, bagasse or switchgrass.

8. method according to claim 6 or 7, it is characterised in that: described method includes following steps:

1) bacillus sp. is seeded in seed culture medium and is cultivated, obtain seed liquor；

2) seed liquor is seeded in fermentation medium and is cultivated, obtain tunning；Contain in the tunning 5-ALA.

9. according to the method described in claim 8, it is characterized by: containing in described kind of liquid culture medium and the fermentation medium There are agricultural wastes hydrolysate or glucose sugar；

Or, the agricultural wastes hydrolysate is the fermentable sugars liquid for carrying out agricultural wastes to obtain after sour processing.

10. according to product or any institute of claim 6-9 described in claim the 2-4 any application or claim 5 The method stated, it is characterised in that: the bacillus sp. is bacillus described in claim 1 sp.PK9 CGMCC No.16179。