CN111471630B - Corynebacterium Ytld-phe09 and application thereof - Google Patents
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Abstract
The invention relates to a Corynebacterium Ytld-phe09 strain and application thereof, belonging to the field of biological engineering, wherein the strain is classified and named as Corynebacterium lucerinis, and is preserved in China general microbiological culture Collection center in 5-15.2020, with the preservation number: CGMCC No. 19832. The strain provided by the invention has higher phenolic substance degradation capability, can degrade various phenolic substances, and can be used for conditioning and repairing phenolic sewage or soil; the strain has the capability of converting and producing protocatechuic acid, the conversion concentration is as high as 9.7g/L, and the industrial application has advantages.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to corynebacterium Ytld-phe09 and application thereof.
Background
The phenol-containing wastewater is one of important pollutants in water and mainly comes from coking or a chemical production process taking phenol or phenolic aldehyde as a raw material. The phenolic substances in the wastewater can directly inhibit the normal growth of organisms in soil and water, and seriously damage the environmental ecosystem. The treatment of phenol-containing wastewater is generally regarded by all countries in the world, and phenol in the wastewater is removed by various physical and chemical methods, such as solvent extraction, adsorption, chemical oxidation, incineration and the like. However, these methods have certain disadvantages, such as high cost, energy consumption, discharge of more toxic by-products, and incomplete removal of phenol. The biodegradation utilizes the microorganisms which grow by taking phenolic substances as carbon sources to degrade phenolic pollutants in the natural environment, has wide adaptability and high efficiency, and is an ideal method for treating the phenolic pollutants at present.
Protocatechuic acid (PCA) widely exists in edible vegetables, fruits, nuts, brown rice, pecans, tea leaves, hibiscus flowers and some Chinese herbal medicines, is a main active ingredient of various traditional Chinese medicines and precursor substances of various medicines, has the biological effects of antioxidation, antibiosis, tumor resistance, anti-inflammation and the like, can be added into food to delay oxidation, and can be absorbed by animals and human beings. The method has wide application in the fields of food and medicine, the demand of the domestic market for protocatechuic acid is increased year by year at present, and the production method mainly adopts a chemical synthesis method and a plant extraction method. Chemical synthesis faces the difficult problems of long reaction time, high energy consumption, toxic by-products and environmental pollution. The highest extraction rate of PCA in the lilac leaves of Zhao et al can only reach 0.0759mg/g in laboratory level, and the highest extraction rate of PCA extracted from the barbed skullcap herb by Yang et al by using supercritical carbon dioxide is 64.094 +/-2.756 mu g/g. Plant extraction is faced with the problems of high cost, low productivity, ecological resource environment and the like. Microbial conversion would be the most promising alternative for the production of protocatechuic acid. Okai et al, using Corynebacterium glutamicum, convert extracellular para-hydroxybenzoic acid to protocatechuic acid using 4-hydroxybenzoic acid hydroxylase under glucose culture conditions in yields of up to 1168.1 + -11.6 mg/L. The Yanxiuqing, etc. recombines p-hydroxybenzoic acid-3-hydroxylase gene in Rhodococcus R04 into Escherichia coli cell for expression, converts p-hydroxybenzoic acid into protocatechuic acid, and the yield can reach 1.156 g/L. The concentration of the product in the research is too low, and a certain distance is left from industrial application. The selection of strains with high activity and high stability is the key point for solving the problems.
Disclosure of Invention
The invention aims to provide a strain for degrading phenolic substances separated from soil, and a method for preparing protocatechuic acid by applying the strain to degrade phenolic pollutants and converting p-hydroxybenzoic acid.
The object of the present invention is achieved as follows.
A Corynebacterium strain Ytld-phe09 is classified and named as Corynebacterium lucricantis, is preserved in China general microbiological culture Collection center (CGMCC) at 5-15 th of 2020, and has the preservation address of Beijing China and the preservation number of CGMCC No. 19832.
The invention also provides application of the strain in degrading phenolic substances.
Further, the application method comprises the following steps: inoculating corynebacterium Ytld-phe09 to a fermentation medium, wherein the components and the final concentration of the fermentation medium are 2-4 g/L of phenolic substances, 3g/L of ammonium sulfate, 1g/L of potassium dihydrogen phosphate, 2g/L of disodium hydrogen phosphate, 1g/L of yeast extract powder, 1g/L of peptone and 1g/L of magnesium sulfate; carrying out shake culture for 36-48 hours at the temperature of 25-40 ℃ and under the condition of 150-240 r/min.
Further, the phenolic substances include phenols such as phenol, catechol, 3-methylphenol, 2-fluorophenol and guaiacol.
The invention also provides application of the strain in preparation of protocatechuic acid by converting p-hydroxybenzoic acid.
Further, the specific application method comprises the steps of firstly inoculating the strain Ytld-phe09 to a solid slant culture medium for culture, inoculating a 1-ring slant strain to a liquid fermentation culture medium at 30 ℃, carrying out shake culture at 220rpm for 24 hours, adding 5-10 g/L of p-hydroxybenzoic acid and 5-40 g/L of glucose, and continuing to culture for 16-24 hours.
Furthermore, the components and final concentration of the solid slant culture medium are 5g/L of yeast extract powder, 10g/L of peptone, 5g/L, NaCl 5g/L of beef extract and 20g/L of agar, and the pH value is 7.0.
Furthermore, the components and final concentration of the liquid fermentation medium are 1-2 g/L of p-hydroxybenzoic acid, 5g/L of glucose, 3g/L of ammonium sulfate, 1g/L of potassium dihydrogen phosphate, 2g/L of disodium hydrogen phosphate, 5g/L of corn steep liquor, 0.5g/L of magnesium sulfate, 110mg/L of vitamin B and 100 mu g/L of D-biotin.
Compared with the prior art, the invention has the beneficial effects that:
1. the Corynebacterium lutescens Ytled-phe 09 provided by the invention has higher phenolic substance degradation capability, can degrade various phenolic substances, and can be used for conditioning and repairing phenolic sewage or soil;
2. the Corynebacterium lucidus Ytled-phe 09 provided by the invention has the capability of producing protocatechuic acid by conversion, the conversion concentration is as high as 9.7g/L, and the industrial application has advantages.
Drawings
FIG. 1 is a microscopic examination of Corynebacterium lucidus Ytled-phe 09.
Detailed Description
To clarify the understanding of the characteristics of the invention, the invention will be further elucidated with reference to some non-limiting embodiments.
Example 1: screening and isolation of Corynebacterium lutescens Ytld-phe09
Sludge samples were collected from the Shandong tobacco pipe. Weighing 1g of sludge sample, adding the sludge sample into a shake flask filled with 100mL of sterile water, shaking uniformly, coating the sludge sample on a screening plate (screening culture medium: 1g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 1g/L sodium chloride, 0.2g/L magnesium sulfate heptahydrate, 0.1g/L calcium chloride, 3g/L ammonium sulfate, 1g/L yeast extract powder, 1g/L peptone, 2g/L phenol and pH6.8) after gradient dilution, culturing for 48h in an incubator at 30 ℃, selecting large colonies with growth advantages, carrying out streak separation and purification, inoculating single colonies grown after streak into a shake flask fermentation culture medium for culturing and determining the degradation rate of phenol, and screening out a strain Ytled-phe 09 capable of efficiently degrading phenol.
Example 2: identification of Corynebacterium lutescens Ytled-phe 09
The suitable growth temperature of the strain Ytld-phe09 is 26-32 ℃, the strain Ytld-phe09 is a milky round colony on an LB culture medium, the surface is smooth, the edge is neat, and the strain Ytld-phe09 is easy to pick up. The bacteriological morphological characteristics are shown in figure 1: the cells were short-rod to small-rod, sometimes slightly bent, blunt-rounded at both ends, single or splayed, approximately (0.5-0.9) μm × (1-2.7) μm in size, positive for gram-staining, no spores, and no movement. The physiological and biochemical indexes are shown in Table 1. The length of the 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing is 1420bp, BLAST comparison is carried out on GenBank, and a phylogenetic tree is drawn. Similarity analysis is carried out by using BLAST in NCBI database, the result shows that the similarity of the 16S rRNA sequence of the strain and the sequence of the Corynebacterium lucerians strain in GenBank is more than 99 percent, the strain is determined to belong to Corynebacterium, is named as Corynebacterium lucerium Ytled-phe 09, and is preserved in China general microbiological culture Collection center at 5-15 th month in 2020, and the preservation number is CGMCC NO. 19832. The address of the depository: beijing in China.
TABLE 1 physiological and biochemical characteristics of Strain Ytld-phe09
The 16S rRNA sequence information is as follows:
cccgatcacttcgaagctccccccaataaaggataggccactggcttcgggtgttaccaactttcatgacgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcagcattgctgatctgcgattactagcgactccgacttcatggggtcgagttgcagaccccaatccgaactaaggccggctttaagcgattagcaccacctcacgatgtagcaacgcgctgtaccgaccattgtagcatgtgtgaagccctggacataaggggcatgatgatttgacgtcatccccaccttcctccgagttaaccccggcagtctctcatgagtccccaccatcacgtgctggcaacataagacaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtacaccaaccacaagggaaagactatctctagcccgatctggtgtatgtcaagcccaggtaaggttcttcgcgttgcatcgaattaatccacatgctccgccgcttgtgcgggcccccgtcaattcctttgagttttagccttgcggccgtactccccaggcggggcgcttaatgcgttagctacggcacaggaaacgtggaagtcccctacacctagcgcccaccgtttacagcatggactaccagggtatctaatcctgttcgctacccatgctttcgctcctcagcgtcagtaactgcccagagacctgccttcgccatcggtgttcctcctgatatctgcgcattccaccgctacaccaggaattccagtctcccctacagcactcaagttatgcccgtatcgcctgcacccccggagttaagccccggacttccacagacgacgcgacaaaccacctacgagctctttacgcccagtaattccggacaacgctcgcaccctacgtattaccgcggctgctggcacgtagttagccggtgcttcttctacaggtaccgtcacttacgcttcgtccctatcgaaaggagtttacaacccgaaggccgtcatcccccacgcggcgtcgctgcatcaggcttccgcccattgtgcaatattccccactgctgcctcccgtaggagtctgggccgtatctcagtcccaatgtggccgtacaccctctcaggccggctacccgtcgacgccttggtaggccattaccccaccaacaagctgataggccgcgagctcatctcataccgcaaaagctttccaccaccatcaccaaacagtggtcctatccggtattagacccagtttcccaagcttatcccgaagtacaaggcagatcacccacgtgttactcacccgttcgccactcgagtaccctgcaagcagggcctttccgttcgactgcatggtaagcacgccgcccagtg
example 3: degradation capability of Corynebacterium lutescens Ytled-phe 09 on different phenolic substances
Respectively adding 2g/L of different phenolic substances into the fermentation culture medium, measuring the residual concentration of the phenolic substances after culturing for 48h, and calculating the degradation rate of the phenolic substances. As can be seen from Table 2, Corynebacterium luteum Ytled-phe 09 has better degradation capability to different phenols, especially phenol, and when the concentration of phenol is as high as 4g/L, the degradation rate is as high as 99.4%.
TABLE 2 substrate specificity
Example 4 production of protocatechuic acid Using transformation of Corynebacterium lucidus Ytld-phe09
Inoculating the strain Ytld-phe09 to a solid slant culture medium (5 g/L yeast extract powder, 10g/L peptone, 5g/L beef extract, 5g/L NaCl, 20g/L agar, pH 7.0) for culturing for 24h, inoculating and picking 1-ring slant strain to a liquid fermentation culture medium (2g/L p-hydroxybenzoic acid, 5g/L glucose, 3g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 2g/L disodium hydrogen phosphate, 5g/L corn steep liquor 0.5g/L magnesium sulfate, 10mg/L vitamin B1, 100 mu g/L D-biotin) 30 ℃, shaking at 220rpm for 24 hr, adding 5g/L p-hydroxybenzoic acid and 10g/L glucose, continuing culturing for 16 hr, and determining protocatechuic acid content to be 5.5 g/L.
Example 5 production of protocatechuic acid Using transformation of Corynebacterium lucidus Ytld-phe09
Inoculating the strain Ytld-phe09 to a solid slant culture medium (5 g/L yeast extract powder, 10g/L peptone, 5g/L beef extract, 5g/L NaCl, 20g/L agar, pH 7.0) for culturing for 24h, inoculating and picking 1-ring slant strain to a liquid fermentation culture medium (2g/L p-hydroxybenzoic acid, 5g/L glucose, 3g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 2g/L disodium hydrogen phosphate, 5g/L corn steep liquor 0.5g/L magnesium sulfate, 10mg/L vitamin B1, 100 mu g/L D-biotin) 30 ℃, shaking at 220rpm for 24 hr, adding 10g/L p-hydroxybenzoic acid and 30g/L glucose, continuing culturing for 20 hr, and determining protocatechuic acid content to be 9.7 g/L.
Sequence listing
<110> university of Ludong
<120> Corynebacterium strain Ytld-phe09 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1420
<212> DNA
<213> Corynebacterium Ytld-phe09(Corynebacterium lucidus Ytld-phe09)
<400> 1
cccgatcact tcgaagctcc ccccaataaa ggataggcca ctggcttcgg gtgttaccaa 60
ctttcatgac gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcagcattgc 120
tgatctgcga ttactagcga ctccgacttc atggggtcga gttgcagacc ccaatccgaa 180
ctaaggccgg ctttaagcga ttagcaccac ctcacgatgt agcaacgcgc tgtaccgacc 240
attgtagcat gtgtgaagcc ctggacataa ggggcatgat gatttgacgt catccccacc 300
ttcctccgag ttaaccccgg cagtctctca tgagtcccca ccatcacgtg ctggcaacat 360
aagacaaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga 420
caaccatgca ccacctgtac accaaccaca agggaaagac tatctctagc ccgatctggt 480
gtatgtcaag cccaggtaag gttcttcgcg ttgcatcgaa ttaatccaca tgctccgccg 540
cttgtgcggg cccccgtcaa ttcctttgag ttttagcctt gcggccgtac tccccaggcg 600
gggcgcttaa tgcgttagct acggcacagg aaacgtggaa gtcccctaca cctagcgccc 660
accgtttaca gcatggacta ccagggtatc taatcctgtt cgctacccat gctttcgctc 720
ctcagcgtca gtaactgccc agagacctgc cttcgccatc ggtgttcctc ctgatatctg 780
cgcattccac cgctacacca ggaattccag tctcccctac agcactcaag ttatgcccgt 840
atcgcctgca cccccggagt taagccccgg acttccacag acgacgcgac aaaccaccta 900
cgagctcttt acgcccagta attccggaca acgctcgcac cctacgtatt accgcggctg 960
ctggcacgta gttagccggt gcttcttcta caggtaccgt cacttacgct tcgtccctat 1020
cgaaaggagt ttacaacccg aaggccgtca tcccccacgc ggcgtcgctg catcaggctt 1080
ccgcccattg tgcaatattc cccactgctg cctcccgtag gagtctgggc cgtatctcag 1140
tcccaatgtg gccgtacacc ctctcaggcc ggctacccgt cgacgccttg gtaggccatt 1200
accccaccaa caagctgata ggccgcgagc tcatctcata ccgcaaaagc tttccaccac 1260
catcaccaaa cagtggtcct atccggtatt agacccagtt tcccaagctt atcccgaagt 1320
acaaggcaga tcacccacgt gttactcacc cgttcgccac tcgagtaccc tgcaagcagg 1380
gcctttccgt tcgactgcat ggtaagcacg ccgcccagtg 1420
Claims (8)
1. A Corynebacterium Ytld-phe09 is classified and named as Corynebacterium lucidus, is preserved in China general microbiological culture Collection center (CGMCC) at 5-15 th of 2020, and has a preservation number of CGMCC No. 19832.
2. Use of the coryneform bacterium Ytld-phe09 according to claim 1 for degrading phenols.
3. The use of the coryneform bacterium Ytld-phe09 according to claim 2 for degrading phenols, characterized in that the specific method for said use is: inoculating corynebacterium Ytld-phe09 to a fermentation medium, wherein the components and the final concentration of the fermentation medium are 2-4 g/L of phenolic substances, 3g/L of ammonium sulfate, 1g/L of potassium dihydrogen phosphate, 2g/L of disodium hydrogen phosphate, 1g/L of yeast extract powder, 1g/L of peptone and 1g/L of magnesium sulfate; carrying out shake culture for 36-48 hours at the temperature of 25-40 ℃ and under the condition of 150-240 r/min.
4. Use according to claim 3, characterized in that the phenolic substance is one or more of phenol, catechol, 3-methylphenol, 2-fluorophenol or guaiacol.
5. Use of the coryneform bacterium Ytld-phe09 according to claim 1 for the transformation of p-hydroxybenzoic acid to protocatechuic acid.
6. The application of claim 5, wherein the specific method of the application comprises the steps of firstly inoculating the strain Ytld-phe09 to a solid slant culture medium for culture, selecting 1-ring slant strain to a liquid fermentation culture medium at 30 ℃ and carrying out shaking culture at 220rpm for 24 hours, adding 5-10 g/L of p-hydroxybenzoic acid and 5-40 g/L of glucose, and continuing the culture for 16-24 hours.
7. The use according to claim 6, wherein the solid slant medium comprises 5g/L yeast extract, 10g/L peptone, 5g/L, NaCl 5g/L beef extract and 20g/L agar, and has a pH of 7.0.
8. The use of claim 6, wherein the liquid fermentation medium comprises 1-2 g/L p-hydroxybenzoic acid, 5g/L glucose, 3g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 2g/L disodium hydrogen phosphate, 5g/L corn steep liquor, 0.5g/L magnesium sulfate, 110mg/L vitamin B, and 100 μ g/L D-biotin.
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