CN109929774B - Bacillus and application thereof in preparation of 5-aminolevulinic acid - Google Patents
Bacillus and application thereof in preparation of 5-aminolevulinic acid Download PDFInfo
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- CN109929774B CN109929774B CN201910084426.2A CN201910084426A CN109929774B CN 109929774 B CN109929774 B CN 109929774B CN 201910084426 A CN201910084426 A CN 201910084426A CN 109929774 B CN109929774 B CN 109929774B
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Abstract
The invention discloses a bacillus and application thereof in preparation of 5-aminolevulinic acid. The strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.16179 and the preservation date of 2018, 7 months and 30 days. The invention also discloses a method for preparing 5-aminolevulinic acid by utilizing the strain through pretreated lignocellulose and potato residue waste. Experiments prove that: the strain can be used for synthesizing 5-aminolevulinic acid by using glucose as a carbon source, can also be used for synthesizing 5-aminolevulinic acid by using lignocellulose or cheap fermentable sugar liquor of potato residues as a carbon source, can resist the growth of inhibitors generated in the pretreatment process, not only reduces the cost of the technological process, but also is beneficial to ecological environment protection and realizes sustainable development.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to bacillus and application thereof in preparation of 5-aminolevulinic acid.
Background
5-aminolevulinic acid (ALA) is a precursor substance for synthesizing tetrahydropyrrole compounds (porphyrin, chlorophyll, heme and vitamin B12) in organisms and is widely present in microbial, plant and animal cells.
ALA has wide application in the fields of agriculture and medicine. In the agricultural field, ALA can promote photosynthesis of green plants, regulate respiration of plants, promote differentiation of plant tissues, improve stress resistance of plants and improve quality of agricultural products. The use of low concentrations can significantly improve crop yield, while high concentrations can be used as safe, non-polluting herbicides as well as pesticides. In the medical field, the drug is called a second generation photodynamic drug due to the effect of selectively killing cancer cells. The stimulatory effect of ALA on the photosensitizer protoporphyrin IX is used in photodynamic cancer therapy and tumor localization. Also has wide application in the treatment of skin diseases and the detection of lead poisoning. Based on the function and wide application prospect of ALA, the synthesis research of ALA has attracted unprecedented attention.
A chemical synthesis process taking hippuric acid, succinic acid, furfural and other heterocyclic substances and levulinic acid and other substances as raw materials is a main method for producing ALA at present. However, the chemical synthesis method has the disadvantages of complex process, more byproducts, difficult separation and purification, low yield and serious environmental pollution. The difficulty in chemical synthesis can be effectively relieved by utilizing the microorganism fermentation for producing ALA. Microorganisms capable of producing ALA reported so far are the photosynthetic bacteria Rhodobacter sphaeroides, Rhodopseudomonas palustris, Rhodopseudomonas sp. The photosynthetic bacteria are difficult to culture, the process is complex, the industrialization is limited, the ALA yield is generally low, and high-concentration glucose is required to be used as a carbon source in the culture process, so that the cost is increased. Therefore, the excavation of new strain resources, the improvement of the strain yield and the reduction of the production cost are main research directions of the ALA of the microbial source. No strain capable of efficiently accumulating ALA is reported in Bacillus (Bacillus).
As a big agricultural country, China produces about 6 to 7 million tons of lignocellulose raw materials, such as corncobs, corn straws, potato residues and the like, which are rich in cellulose and hemicellulose substances. The burning and random stacking of the lignocellulose raw materials cause environmental pollution, the agricultural wastes are effectively utilized to change wastes into valuables, and the conversion of the agricultural wastes into other high-added-value products becomes a research hotspot.
Disclosure of Invention
One purpose of the invention is to provide a Bacillus sp.pk9 strain.
The preservation number of the Bacillus sp.PK9 provided by the invention is CGMCC No. 16179.
The Bacillus sp.PK9 provided by the invention is separated from rhizosphere soil of tillers, can be used for preparing 5-aminolevulinic acid (ALA), is classified and named as Bacillus sp, and is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 West Lu No.1 of the sunward area of Beijing, institute of microbiology of China academy of sciences, zip code 100101) in 7 months and 30 days of 2018.
Another object of the present invention is to provide a novel use of Bacillus sp or its suspension or its culture solution or its fermentation solution or a microbial inoculum containing it.
The invention provides application of Bacillus sp or bacterial suspension thereof, or culture solution thereof, or fermentation broth thereof, or a microbial inoculum containing the same in preparation of 5-aminolevulinic acid.
The invention also provides application of the Bacillus sp or the bacterial suspension thereof or the culture solution thereof or the fermentation liquor thereof or the microbial inoculum containing the Bacillus sp or the culture solution thereof in preparing the 5-aminolevulinic acid by taking agricultural wastes as raw materials.
In the above application, the agricultural waste may be lignocellulose agricultural waste or potato residue; further, the lignocellulosic agricultural waste may be corn cobs, sugar cane bagasse, or switchgrass. In one embodiment of the invention, the agricultural waste is corncobs or potato pulp.
The invention also provides application of the Bacillus sp or bacterial suspension thereof or culture solution thereof or fermentation liquor thereof or microbial inoculum containing the Bacillus sp or the bacterial suspension thereof in improving the yield of the 5-aminolevulinic acid.
It is also an object of the present invention to provide a product for the preparation of 5-aminolevulinic acid.
The active ingredient of the product for preparing the 5-aminolevulinic acid provided by the invention is Bacillus sp or bacterial suspension thereof, or culture solution thereof, or fermentation broth thereof, or microbial inoculum containing the same.
The invention finally provides a method for preparing the 5-aminolevulinic acid.
The method for preparing the 5-aminolevulinic acid comprises the following steps: lignocellulose agricultural wastes or potato residues are used as raw materials, and Bacillus sp is used for synthesizing the 5-aminolevulinic acid.
In the method for preparing 5-aminolevulinic acid, the agricultural waste can be lignocellulose agricultural waste or potato residue; further, the lignocellulosic agricultural waste may be corn cobs, sugar cane bagasse, or switchgrass.
The above method for preparing 5-aminolevulinic acid may comprise the steps of:
1) inoculating Bacillus sp to a seed culture medium for culture to obtain a seed solution;
2) inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation product; the fermentation product contains 5-aminolevulinic acid.
In the above method for producing 5-aminolevulinic acid, the seed medium and the fermentation medium each contain an agricultural waste hydrolysate or glucose. The agricultural waste hydrolysate can be fermentable sugar liquid obtained by performing acid treatment on agricultural waste.
The method for performing acid treatment on the lignocellulose agricultural wastes can be specifically performed according to the following steps: collecting 8g lignocellulose agricultural waste sample (such as corn cob, bagasse or switchgrass) by treating with (0.5-1)% dilute sulfuric acid 200ml at 121 deg.C for 30-60 min, cooling, vacuum filtering to collect solid residue, adding 30ml (50-72)% sulfuric acid for treating at 30 deg.C for 30-60 min, adding 840ml water, treating at 121 deg.C for 30-60 min, cooling, vacuum filtering to collect filtrate, and treating with solid Ca (OH)2Adjusting the pH value of the filtrate to 7.0, and filtering to obtain supernatant which is fermentable sugar liquid.
The method for carrying out acid treatment on the potato residues can be carried out according to the following steps: treating 20g potato residue sample with 1.0-1.7% dilute sulfuric acid 200ml (solid-to-liquid ratio 1:10) at 121 deg.C for 90-120 min, cooling, vacuum filtering, collecting filtrate, and treating with solid Ca (OH)2Adjusting the pH value of the filtrate to 7.0, and filtering to obtain supernatant which is fermentable sugar liquid.
Further, in the above-mentioned case,
when the 5-aminolevulinic acid is prepared by taking glucose as a raw material,
the formula of the seed culture medium is as follows: the solvent is deionized water, and the solutes and the concentrations are respectively as follows: glucose (2-4) g/L, tryptone (5-10) g/L, yeast powder (10-15) g/L, potassium dihydrogen phosphate (3 g/L), and initial pH 7.3-7.5.
The formula of the fermentation medium is as follows: the solvent is deionized water, and the solutes and the concentrations 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, and initial pH 7.2-7.3.
When agricultural waste hydrolysate is used as a raw material to prepare the 5-aminolevulinic acid,
the formula of the seed culture medium is as follows: the solvent is deionized water, and the solutes and the concentrations are respectively as follows: 1-5 g/L of fermentable sugar liquid (calculated by glucose), 5-10 g/L of tryptone, 10-15 g/L of beef extract, 1-5 g/L of sodium chloride and 3-5 g/L of dipotassium hydrogen phosphate, and the initial pH value is 7.3-7.5.
The formula of the fermentation medium is as follows: the solvent is deionized water, and the solutes and the concentrations are respectively as follows: 15-20 g/L of fermentable sugar liquid (calculated by glucose), 5-10 g/L of tryptone, 10-15 g/L of beef extract, 1-5 g/L of sodium chloride and 3-5 g/L of dipotassium hydrogen phosphate, and the initial pH value is 7.2-7.3.
Furthermore, when the 5-aminolevulinic acid is prepared by taking glucose as a raw material,
in the step 1), the culture conditions are (28-37) DEG C and (100-200) rpm (12-24) h;
in the step 2), the inoculation amount of the seed solution is (1-5)%, the culture conditions are (28-37) ° C and (100-.
When agricultural waste hydrolysate is used as a raw material to prepare the 5-aminolevulinic acid,
in the step 1), the culture conditions are (28-37) DEG C and (100-200) rpm (12-24) h;
in the step 2), the inoculation amount of the seed solution is (1-5)%, the culture conditions are (30-37) ° C and (100-.
In the method for preparing 5-aminolevulinic acid, the step of inoculating Bacillus sp to an activation medium for activation culture is further included before the step 1).
Further, the formula of the activation medium is as follows: the solvent is deionized water, and the solutes and the concentrations are respectively as follows: tryptone (5-10) g/L, beef extract (3-5) g/L, sodium chloride (5 g/L), agar (18 g/L) and initial pH value (7.2-7.4).
Further, the activation culture is carried out at (28-37) deg.C for 12-24 h.
In the above application or product or method, the Bacillus sp is Bacillus sp.pk9 CGMCC No. 16179.
The invention has the following beneficial effects: the Bacillus sp. CGMCC No.16179 separated and screened from nature can utilize the fermentable sugar liquor of lignocellulose or potato residues with abundant and cheap resources as a carbon source to synthesize the 5-aminolevulinic acid, can resist the good growth of inhibitors generated in the pretreatment process, obtains the maximum yield in 24-48 h of fermentation, and can effectively prevent ALA from decomposing because the fermentation liquor is in an acidic condition.
The invention provides a Bacillus sp.CGMCC No.16179 for high yield of 5-aminolevulinic acid and a method for preparing 5-aminolevulinic acid from lignocellulose agricultural wastes by using the Bacillus sp.CGMCC No. 16179. The strain is used as a strain, lignocellulose or potato residue waste is used as a raw material to synthesize the 5-aminolevulinic acid, so that the cost of the process is reduced, the ecological environment is protected, and sustainable development is realized.
Drawings
FIG. 1 is a standard curve of 5-aminolevulinic acid.
Deposit description
Latin name: bacillus sp.
The strain number is as follows: PK9
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 7 month and 30 days 2018
Registration number of the preservation center: CGMCC No.16179
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The following examples were prepared using corn cob samples as follows: drying corncob (from Yiyang county of Henan province), grinding with a grinder (Bjie multifunctional grinder BJ-150), and sieving with 50 mesh sieve to obtain corncob sample.
The preparation method of potato dregs in the following examples is as follows: cutting potato, putting into water, boiling for 60min, filtering with 4 layers of gauze, and oven drying the residue in oven to obtain potato residue sample.
Example 1, acquisition, identification and preservation of PK9 Strain
First, acquisition of PK9 Strain
Sampling from the rhizosphere soil of the tillers, weighing about 2g of the obtained various soil samples, suspending the soil samples in 0.9 mass percent of NaCl solution, moderately diluting the soil samples, inoculating the soil samples to an LB (Luria Bertani) flat plate, culturing the soil samples at 30 ℃ for 24 hours, selecting single colonies, streaking the LB flat plate to obtain single colonies, performing shake flask fermentation by using glucose as a single carbon source, after fermenting for 36 hours, sampling fermentation liquor, centrifuging and collecting supernatant. The concentration of 5-aminolevulinic acid is quantitatively determined by spectrophotometry. A strain with high ALA yield is obtained by screening, is named as PK9, and has ALA yield of 25.89 mg/L.
II, identification of PK9 strain
1. Physiological and biochemical identification
The PK9 strain screened in the first step is identified according to the physiological and biochemical characteristics of the microbiological experiment course, and the results are shown in the table 1.
TABLE 1 results of physiological and biochemical characterization
| Authentication program | PK9 strain |
| Acid production by glucose | - |
| Methyl Red test | - |
| V-P experiment | - |
| Nitrate reduction | - |
| Producing indole | - |
| Produce H2S | - |
| Liquefaction of gelatin | - |
| Citric acid sodium salt | - |
2. Molecular identification
Extracting genome DNA of the PK9 strain, and carrying out 16S rRNA gene identification on the PK9 strain by adopting a universal primer according to a method of a molecular biology experimental guideline to obtain a 16S rRNA gene sequence, wherein the size of the sequence is 1354bp, and the sequence is shown as a sequence 1 in a sequence table.
The PK9 strain can be determined to belong to Bacillus sp according to the identification result.
III, preservation of PK9 Strain
The classification name of the PK9 strain is Bacillus sp, the strain is preserved in the China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu 1 of Beijing Ind area, microbiological research institute of Chinese academy of sciences, postal code 100101) in 7 months and 30 days in 2018, and the preservation number is CGMCC No. 16179.
Example 2 application of PK9 strain in preparation of 5-aminolevulinic acid by taking glucose as raw material
1. The Bacillus sp.PK9 obtained in example 1 was cultured in an activation medium (the formula of the activation medium is as follows: the solvent is deionized water, the solute and the concentration are respectively 5g/L tryptone, 5g/L beef extract, 5g/L sodium chloride and 18g/L agar, and the initial pH is 7.2) at 37 ℃ for 24h to obtain activated PK 9.
2. And (2) filling 50ml of seed culture medium (the formula of the seed culture medium is as follows: the solvent is deionized water, the solute and the concentration are respectively 2g/L glucose, 10g/L tryptone, 10g/L yeast powder, 3g/L potassium dihydrogen phosphate and initial pH 7.3) into a 250ml triangular bottle, sterilizing, cooling and inoculating the activated PK9 obtained in the step 1 according to a conventional method, and after inoculation, performing shake culture at 37 ℃ and 200rpm for 12 hours to obtain seed fermentation liquid.
3. And (2) filling 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 15g/L glucose, 10g/L tryptone, 10g/L beef extract, 2g/L sodium chloride, 3g/L dipotassium hydrogen phosphate and initial pH 7.2) into a 500ml triangular bottle, sterilizing and cooling by a conventional method, inoculating the seed solution obtained in the step (2) into the fermentation medium according to the inoculation amount of 2%, and after inoculation, performing shake culture at 37 ℃ and 200rpm for 36 hours to obtain the fermentation liquor containing the 5-aminolevulinic acid.
And quantitatively measuring the ALA content in the fermentation liquor containing the 5-aminolevulinic acid by adopting a spectrophotometric method. The method comprises the following specific steps: standard curve of 5-aminolevulinic acid was plotted: 400. mu.l of 5-aminolevulinic acid standard solutions with different concentrations (5 mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L and 50mg/L of 5-aminolevulinic acid standard solutions) are respectively taken, 200. mu.l of acetate buffer solution and 100. mu.l of acetylacetone are added, and boiling water bath is carried out for 15 min. After cooling to room temperature, 700. mu.l of Ehrlich's reagent (Ehrlich's reagent formulation: 1g of p-dimethylaminobenzaldehyde is weighed in 30ml of glacial acetic acid, then 8ml of perchloric acid (70%) is added, made up to 50ml with glacial acetic acid and ready for use) is added, reacted for 20min, and detection is carried out using a spectrophotometer at 554 nm. The concentration of the 5-aminolevulinic acid standard solution is taken as the abscissa, OD554nmValue is longitudinalAnd drawing a standard curve by using the coordinates. The standard curve is shown in figure 1.
And (3) taking 400 mu l of supernatant obtained in the step (3) after the fermentation liquor containing the 5-aminolevulinic acid is centrifuged, adding 200 mu l of acetate buffer solution and 100 mu l of acetylacetone, and carrying out boiling water bath for 15 min. After cooling to room temperature, 700. mu.l Ehrlich's reagent was added, reaction was carried out for 20min, detection was carried out at 554nm using a spectrophotometer, and the obtained OD was measured554nmSubstituting the value into a standard curve, and calculating to obtain the ALA content of the fermentation liquor containing the 5-aminolevulinic acid to be 25.89 mg/L.
The pH value of the fermentation liquor is measured by a HORIBA B-71X pen type pH meter: centrifuging the fermentation liquor, and collecting supernatant; 200 μ l of the supernatant was instilled into the detection cell. The test cell was covered with a lid and left horizontally for about 2 minutes, after which the pH value was measured. The pH value of the fermentation liquor is 4.5.
Example 3 application of PK9 strain in preparation of 5-aminolevulinic acid by taking corncobs as raw materials
1. The Bacillus sp.PK9 obtained in example 1 was cultured in an activation medium (the formula of the activation medium is as follows: the solvent is deionized water, the solute and the concentration are respectively 5g/L tryptone, 5g/L beef extract, 5g/L sodium chloride and 18g/L agar, and the initial pH is 7.2) at 37 ℃ for 24h to obtain activated PK 9.
2. And (2) filling 50ml of seed culture medium (the formula of the seed culture medium is as follows: the solvent is deionized water, the solute and the concentration are respectively 2g/L of corncob hydrolysis fermentable sugar liquid (calculated by glucose), 10g/L of tryptone, 10g/L of beef extract, 5g/L of sodium chloride and 3g/L of dipotassium hydrogen phosphate, adjusting the pH to 7.3) in a 250ml triangular bottle, sterilizing and cooling by a conventional method, inoculating the activated PK9 obtained in the step 1, and culturing for 12 hours in a shaking table at 37 ℃ and 200rpm after inoculation to obtain the seed fermentation liquid.
The preparation method of the corncob hydrolysis fermentable sugar solution comprises the following steps: treating 8g of corncob sample with 200ml of 1% dilute sulfuric acid at 121 ℃ for 60min, cooling, performing suction filtration to collect solid residues, then adding 30ml of 50% sulfuric acid, treating at 30 ℃ for 30min, adding 840ml of water, treating at 121 ℃ for 60min, cooling, performing suction filtration to collect filtrate. With solid Ca (OH)2Adjusting pH of the filtrate to 7.0, filtering to obtainThe supernatant is the corn cob hydrolysis fermentable sugar solution.
3. And (2) filling 100ml of fermentation medium (the formula of the fermentation medium is that deionized water is used as a solvent, the solute and the concentration are respectively 15g/L of corncob hydrolysis fermentable sugar liquid (calculated by glucose), 10g/L of tryptone, 15g/L of beef extract, 5g/L of sodium chloride, 5g/L of dipotassium hydrogen phosphate and initial pH 7.2) into a 500ml triangular bottle, sterilizing and cooling by a conventional method, inoculating the seed liquid obtained in the step (2) into the fermentation medium according to the inoculation amount of 2%, and performing shake culture at 37 ℃ and 200rpm for 36 hours to obtain the fermentation liquid containing the 5-aminolevulinic acid.
The ALA production in the fermentation broth containing 5-aminolevulinic acid obtained in this example was 21.47 mg/L. The pH value of the fermentation liquor is 4.89.
Example 4 application of PK9 strain in preparation of 5-aminolevulinic acid by taking potato dregs as raw material
1. The Bacillus sp.PK9 obtained in example 1 was cultured in an activation medium (the formula of the activation medium is as follows: the solvent is deionized water, the solute and the concentration are 5g/L peptone, 5g/L beef extract, 5g/L sodium chloride, 18g/L agar, initial pH 7.2) at 37 ℃ for 24h to obtain activated PK 9.
2. And (2) filling 50ml of seed culture medium (the formula of the seed culture medium is as follows: the solvent is deionized water, the solute and the concentration are respectively 2g/L of potato residue hydrolysis fermentable sugar liquid (calculated by glucose), 10g/L of tryptone, 10g/L of beef extract, 5g/L of sodium chloride and 3g/L of dipotassium hydrogen phosphate, adjusting the pH to 7.3) in a 250ml triangular bottle, sterilizing and cooling by a conventional method, inoculating the activated PK9 obtained in the step 1, and culturing for 12 hours at 37 ℃ and 200rpm in a shaking table after inoculation to obtain the seed fermentation liquid.
The preparation method of the potato residue hydrolysis fermentable sugar liquid comprises the following steps: treating 20g of potato residue sample with 200ml of 1.0% dilute sulfuric acid (solid-to-liquid ratio of 1:10) at 121 deg.C for 90min, cooling, vacuum filtering, collecting supernatant, and purifying with solid Ca (OH)2Adjusting the pH value of the filtrate to 7.0, and filtering to obtain supernatant which is fermentable sugar liquid.
3. And (2) filling 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 15g/L of potato residue hydrolysis fermentable sugar liquid (calculated by glucose), 10g/L of tryptone, 15g/L of beef extract, 5g/L of sodium chloride, 5g/L of dipotassium hydrogen phosphate and initial pH 7.2) in a 500ml triangular bottle, sterilizing and cooling by a conventional method, inoculating the seed liquid obtained in the step (2) into the fermentation medium according to the inoculation amount of 2%, and after inoculation, carrying out shake culture at 37 ℃ and 200rpm for 36h to obtain the fermentation liquid containing 5-aminolevulinic acid.
The ALA production in the fermentation broth containing 5-aminolevulinic acid obtained in this example was 23.79 mg/L. The pH of the fermentation broth was 4.95.
Sequence listing
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ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtgctagttg aataagctgg 420
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aagtctgatg tgaaagccca cggctcaacc gtggagggtc attggaaact gggagacttg 600
agtgcagaag aggaaagtgg aattccatgt gtagcggtga aatgcgtaga gatatggagg 660
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ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780
agagggtttc cgccctttag tgctgaagtt aacgcattaa gcactccgcc tggggagtac 840
ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900
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Claims (10)
1. A strain of Bacillus (Bacillus sp.) PK9 has a preservation number of CGMCC No. 16179.
2. Application of Bacillus (Bacillus sp.) or bacterial suspension thereof or microbial inoculum containing the same in preparation of 5-aminolevulinic acid;
the Bacillus (Bacillus sp.) is the Bacillus (Bacillus sp.) PK9 with the preservation number of CGMCC No.16179 as claimed in claim 1.
3. Application of Bacillus (Bacillus sp.) or bacterial suspension thereof or microbial inoculum containing the Bacillus sp in preparing 5-aminolevulinic acid by taking agricultural wastes as raw materials;
the Bacillus (Bacillus sp.) is the Bacillus (Bacillus sp.) PK9 with the preservation number of CGMCC No.16179 as claimed in claim 1.
4. Use according to claim 3, characterized in that: the agricultural waste is lignocellulose agricultural waste or potato residue.
5. Use according to claim 4, characterized in that: the lignocellulose agricultural waste is corn cob, bagasse or switchgrass.
6. The application of Bacillus (Bacillus sp.) or its bacterial suspension or microbial inoculum containing it in improving the yield of 5-aminolevulinic acid;
the Bacillus (Bacillus sp.) is the Bacillus (Bacillus sp.) PK9 with the preservation number of CGMCC No.16179 as claimed in claim 1.
7. A microbial inoculum for preparing 5-aminolevulinic acid, which comprises an active ingredient of Bacillus (Bacillus sp.) or a bacterial suspension thereof;
the Bacillus (Bacillus sp.) is the Bacillus (Bacillus sp.) PK9 with the preservation number of CGMCC No.16179 as claimed in claim 1.
8. A process for preparing 5-aminolevulinic acid, comprising the steps of: using lignocellulose agricultural wastes or potato dregs as raw materials, and synthesizing 5-aminolevulinic acid by using Bacillus (Bacillus sp.);
the Bacillus (Bacillus sp.) is the Bacillus (Bacillus sp.) PK9 with the preservation number of CGMCC No.16179 as claimed in claim 1.
9. The method of claim 8, wherein: the lignocellulose agricultural waste is corn cob, bagasse or switchgrass.
10. The method of claim 8, wherein: the method comprises the following steps:
1) inoculating Bacillus (Bacillus sp.) into a seed culture medium for culturing to obtain a seed solution;
2) inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation product; the fermentation product contains 5-aminolevulinic acid;
the seed culture medium and the fermentation culture medium both contain agricultural waste hydrolysate or glucose; the agricultural waste hydrolysate is fermentable sugar liquid obtained by performing acid treatment on agricultural waste.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003018825A2 (en) * | 2001-08-22 | 2003-03-06 | Basf Aktiengesellschaft | Method for producing vitamin b12 |
| CN103180279A (en) * | 2010-08-30 | 2013-06-26 | 阿泽达公司 | Fermentation route for the production of levulinic acid, levulinate esters, valerolactone, and derivatives thereof |
-
2019
- 2019-01-29 CN CN201910084426.2A patent/CN109929774B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003018825A2 (en) * | 2001-08-22 | 2003-03-06 | Basf Aktiengesellschaft | Method for producing vitamin b12 |
| CN1545556A (en) * | 2001-08-22 | 2004-11-10 | �����ɷ� | Method for producing vitamin b12 |
| CN103180279A (en) * | 2010-08-30 | 2013-06-26 | 阿泽达公司 | Fermentation route for the production of levulinic acid, levulinate esters, valerolactone, and derivatives thereof |
Non-Patent Citations (1)
| Title |
|---|
| 产生52氨基乙酰丙酸(ALA)光合细菌生物学研究进展;刘秀艳 等;《生物工程进展》;20000531;第67-71页，参见全文 * |
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