CN110106095B - Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof - Google Patents
Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, a construction method and application thereof, and belongs to Aspergillus niger with a calcium ion channel CchA gene knocked out. The invention also discloses a construction method of the genetic engineering bacteria. Further discloses the application of the genetic engineering bacteria in the production of citric acid. According to the invention, by constructing the Aspergillus niger genetically engineered bacterium with the gene knock-out of the calcium ion channel CchA gene, the yield of a biological membrane is reduced and the hydrophobicity of the Aspergillus niger is reduced in the process of producing citric acid by immobilized fermentation, the agglomeration phenomenon of a carrier is reduced, the yield of citric acid and the sugar conversion efficiency are improved, and the method is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to an Aspergillus niger genetically engineered bacterium for knocking out a calcium ion channel CchA gene, and a construction method and application thereof.
Background
Citric acid is one of the most demanded organic acids worldwide, and is widely used in industries such as food, medicine, daily chemicals and the like. Wherein, 75% of citric acid is used in food industry, mainly used in sour agent, antioxidant, pH regulator in food additive, meanwhile, since citric acid has mild and refreshing sour taste, it is widely used in the manufacture of food such as beverage, cake, wine, dairy products, etc. Another 15% is used in the chemical and textile industries and can be used as buffers, complexing agents, sequestering agents, metal builders, mordants, etc. Also 10% of citric acid is used as anticoagulant, antacid, taste corrigent, cosmetic and feed additive, etc., and is widely used in the pharmaceutical industry and animal husbandry. In recent years, due to the general rise of food prices in China, the cost of raw materials for producing citric acid is greatly increased. Therefore, how to improve the citric acid fermentation method and further reduce the cost becomes the most concerned problem in the whole citric acid industry in China.
The citric acid produced by fermenting aspergillus niger is one of the main ways for producing citric acid by current industrial fermentation, and the fermentation efficiency of aspergillus niger can be greatly improved by a cell immobilization method. At present, there are four main methods for cell immobilization: entrapment, cross-linking, covalent bonding, and adsorption. For filamentous fungi, the adsorption method is simple to operate, the chemical structure is not required to be changed in the fixing process, and the method is theoretically suitable for all filamentous fungi and is more beneficial to industrial popularization and application. The principle of the adsorption method mainly depends on the interaction force between the groups on the surface of the carrier and the surface of the cells, so that the cells are promoted to be adsorbed on the surface of the carrier for growth. And when the cells die, the cells fall off from the carrier, and simultaneously, the active cells are adsorbed to form dynamic balance, so that the whole microenvironment forms an efficient conversion system, and the overall fermentation efficiency is obviously improved compared with the free fermentation. In the process of research on the immobilized fermentation mechanism, microorganisms commonly form a biological membrane on a carrier, and the formed biological membrane is different, so that the performance of immobilized fermentation is greatly different. Therefore, we need further understanding of the mechanism of biofilm formation to really solve the problem of difficult industrial application of immobilization.
Disclosure of Invention
In order to solve the problems of low yield and long fermentation period in the prior industrial production of citric acid by fermenting aspergillus niger, the invention aims to solve the technical problem of providing a calcium ion channel CchA gene-deficient aspergillus niger genetically engineered bacterium.
The invention also aims to solve the technical problem of providing a construction method of the citric acid producing aspergillus niger genetically engineered bacterium.
The invention finally solves the technical problem of providing the application of the Aspergillus niger genetically engineered bacterium in the production of citric acid by fermentation.
In order to solve the technical problems, the invention adopts the following technical scheme:
a calcium ion channel CchA gene inactivated Aspergillus niger genetic engineering bacterium is a genetic engineering bacterium which replaces partial sequence of CchA gene by hyg resistance gene through a double exchange method, and the calcium ion channel CchA gene participates in the polar growth of hypha main axis, sporulation and the integrity of cell wall. The integrity of the cell wall can affect the production of biofilm, and therefore, the calcium channel CchA gene can regulate the production of biofilm, thereby affecting the production of citric acid.
Wherein the Aspergillus Niger is Aspergillus Niger831 (A. Niger831),
the nucleotide sequence of the calcium ion channel CchA gene before inactivation is shown in SEQ ID NO.1, and the nucleotide sequence of the calcium ion channel CchA gene after inactivation is shown in SEQ ID NO. 2.
A method for constructing Aspergillus niger genetically engineered bacteria with calcium ion channel CchA gene inactivated comprises the following steps:
(1) extracting genomic DNA of aspergillus niger a.niger 831;
(2) amplifying an upstream homology arm of the CchA gene by taking the genome obtained in the step (1) as a template and the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers;
amplifying a downstream homology arm of the CchA gene by taking the genome obtained in the step (1) as a template and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers;
amplifying a hyg resistance element by taking the plasmid pan7-1 as a template and nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as primers;
performing PCR amplification by using an upstream homologous arm of a CchA gene, a downstream homologous arm of the CchA gene and a hyg resistance element as templates and nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10 as primers through overlap PCR to obtain a gene knockout fragment;
(3) preparing an Aspergillus niger protoplast;
(4) and (3) introducing the gene knockout recombinant fragment into the protoplast for homologous recombination to obtain the aspergillus niger genetically engineered bacterium with the calcium ion channel CchA gene inactivated.
Wherein, the nucleotide sequence of the upper homologous arm of the Ccha gene is shown as SEQ ID NO.11, the nucleotide sequence of the lower homologous arm of the Ccha gene is shown as SEQ ID NO.12, the nucleotide sequence of the hyg resistance element is shown as SEQ ID NO.13, and the nucleotide sequence of the gene knockout fragment is shown as SEQ ID NO. 14.
The application of the Aspergillus niger genetically engineered bacterium with the calcium ion channel CchA gene inactivated in the preparation of citric acid is within the protection scope of the invention.
Preferably, Aspergillus niger genetically engineered bacteria with calcium ion channel CchA gene inactivated are used as fermentation strains to prepare the citric acid through immobilized fermentation.
The citric acid is prepared by taking a porous fiber material as an immobilization medium through fermentation, wherein the porous fiber material is activated carbon fiber.
The preparation method of the fermentation medium for immobilized fermentation comprises the following steps:
respectively taking 200 g/L-300 g/L of cassava powder and 200 g/L0-300 g/L of corn powder, adding 1-2m L of liquefying enzyme into 1L of fermentation liquor at 60-70 ℃, carrying out enzymolysis for 35-45 min, respectively heating cassava powder liquid and corn powder liquid to 85 ℃, adding 1-2m L of liquefying enzyme into every 1L of fermentation liquor, carrying out enzymolysis for 35-45 min until the iodine solution does not turn blue, filtering the cassava powder liquid to obtain cassava powder liquid supernatant, adding 2-10% by volume of unfiltered corn powder liquid into the cassava powder liquid supernatant, and uniformly mixing to obtain an immobilized fermentation culture medium, wherein the liquefying enzyme contains α -amylase, and the enzyme activity of α -amylase is 60000-70000U/m L.
Wherein the culture conditions of the immobilized fermentation are as follows: the culture temperature is 30-37 ℃, the culture time is 72-120 h, and the rotating speed is 180-330 rpm.
Wherein, the porous fiber material is pretreated by the following method:
soaking the porous fiber material in 1M sodium hydroxide for 1-1.5 h, washing with water until the pH value is neutral, soaking in 1M hydrochloric acid for 1-1.5 h, washing with water until the pH value is neutral, and drying to constant weight to obtain the modified porous fiber material.
The method for producing citric acid by immobilized fermentation of aspergillus niger genetically engineered bacteria with the calcium ion channel CchA gene inactivated specifically comprises the following steps:
(1) preparation of the carrier: soaking porous fiber material in 1M sodium hydroxide for 1h, cleaning with pure water, soaking in 1M hydrochloric acid for 1h, washing with pure water until pH is neutral, and oven drying at 65 deg.C to constant weight. Cutting into carriers with the same size.
(2) And (3) fermenting, namely scraping the activated Aspergillus niger genetically engineered bacterium flat plate by using a sporulation liquid to prepare a spore liquid, inoculating the spore liquid into a sterilized fermentation culture medium containing 0.1g/100m L porous fiber material in an inoculation amount of 0.2 percent (volume fraction), and fermenting to obtain the citric acid.
The fermentation medium comprises the following components of 200-250 g/L corn flour and 200-250 g/L cassava flour.
The fermentation conditions were as follows: the culture temperature is 35 ℃, the culture time is 72-96h, and the rotating speed is 200-250 rpm.
Has the advantages that:
the invention constructs an Aspergillus niger genetically engineered bacterium with a calcium ion channel CchA gene deletion by a gene knockout means. The genetic engineering bacteria reduce the amount of biological membranes in the process of producing citric acid by Aspergillus niger through immobilized fermentation, reduce the phenomenon of cluster, improve the yield and the sugar conversion rate of the citric acid and shorten the fermentation period.
Drawings
FIG. 1 is an electrophoretogram of the genome of Aspergillus Niger 831;
FIG. 2 is a PAN7-1 plasmid map;
FIG. 3 is a PCR electrophoresis of the upstream and downstream homology arms of the Ccha gene, wherein M is DNAmarker D L5000, lane 1 is the upper homology arm of Ccha, lane 3 is the lower homology arm of Ccha, and lane 5 is the hyg resistance expression element;
FIG. 4 is an electrophoretogram of a knockout fragment, where M is marker lane 1;
FIG. 5 is a graph of crystal violet staining;
FIG. 6 is a graph showing the difference in OD values of crystal violet stains;
FIG. 7 shows the results of fermentation of original A.niger and A.niger genetically engineered bacteria.
FIG. 8 is an electron micrograph of a biofilm of the starting Aspergillus niger strain;
FIG. 9 is an electron microscope image of a biological membrane of Aspergillus niger genetically engineered bacteria;
Detailed Description
Example 1: construction of aspergillus niger calcium ion channel cchA gene knock-out bacteria.
Extraction of original Aspergillus niger genome
A kit for extracting a plant genome (takara minitest plant genomic DNA extraction kit) by takara corporation was used, and the following precautions were specifically taken:
1. inoculating 1m L of scraped Aspergillus niger spore liquid into 50m L DP culture medium, and culturing at 35 deg.C and 250r/min for 15 h;
2. centrifuging at 5000r/min for 5min to collect mycelium pellets, washing with normal saline twice, grinding the collected mycelium pellets with liquid nitrogen for 3 times, weighing 100mg of the ground powder, adding the powder into a tube with 500u L of Buffer HS II, mixing uniformly, adding 10u L of RNase A, shaking fully and mixing uniformly, and carrying out water bath at 56 ℃ for 10 minutes.
3. Adding 62.5u L Buffer KAC into step 2, mixing well, standing on ice for 5min, centrifuging at 12000rpm for 5min, collecting supernatant 600u L, adding 600u L Buffer GB, and mixing well.
4. The Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.
5. 500u L of Buffer WA WAs added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate WAs discarded.
6. 700u L of Buffer WB was added to Spin Column, centrifuged at 12000rpm for 1min, and the filtrate was discarded.
7. Repeat step 6 once.
8. Spin Column was mounted on a Collection Tube at 12000rpm and centrifuged for 2 min.
9. Spin Column was mounted on a new 1.5m L centrifuge tube, 40u L of sterilized water at 65 ℃ was added to the center of the Spin Column membrane, left to stand at room temperature for 1 min.12000rpm for 2min, and the dna was eluted.
FIG. 1 shows DNA marker with M D L15000 and the extracted A.niger genome No. 1.
And secondly, amplifying the upper and lower homologous arms of the CchA gene by using a PCR technology.
TABLE 1 PCR reaction System
Amplifying an upper homologous arm by using an original Aspergillus niger genome as a template, using CchA-up-F as an upper primer and using CchA-up-R as a lower primer (shown as SEQ ID NO.3 and SEQ ID NO. 4); the lower homologous arm was amplified using CchA-down-F as the upper primer and CchA-down-R as the lower primer (shown in SEQ ID NO.5 and SEQ ID NO. 6). The reaction system is shown in Table 1, and the reaction conditions are as follows: denaturation at 98 ℃ for 10s, annealing at 55-65 ℃ for 30s, and extension at 68 ℃ for 2min, and repeating the steps for 30 times. After the reaction was completed, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3.
(III) amplification of hyg-resistant expression elements
Amplifying hyg resistance expression element with PAN7-1 plasmid (PAN7-1 plasmid map shown in figure 2, nucleotide sequence shown in SEQ ID NO.17) as template, CchA-hyg-F as upper primer, and CchA-hyg-R as lower primer (shown in SEQ ID NO.7 and SEQ ID NO. 8). The reaction system is shown in Table 1, and the reaction conditions are as follows: denaturation at 98 deg.C for 10s, annealing at 55-65 deg.C for 30s, and extension at 68 deg.C for 3min, and repeating the above steps for 30 times. After the reaction was completed, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 3. Wherein the nucleotide sequence of the hyg resistance expression element is shown in SEQ ID NO: 13.
FIG. 3 shows DNA Marker M D L5000, upper homology arm of Ccha No.1, lower homology arm of Ccha No.3, and hyg resistance expression element No.5, in the case of DNA Marker D L.
(IV) amplification of knockout fragment
Using CchA upstream and downstream homology arms and hyg resistance expression element as template, CchA-F as upper primer, CchA-R as lower primer (shown in SEQ ID NO.9 and SEQ ID NO. 10), CchA gene knockout fragment was amplified by Overlap PCR technique. The reaction system is shown in Table 1, and the reaction conditions are as follows: denaturation at 98 deg.C for 10s, annealing at 55-65 deg.C for 30s, and extension at 68 deg.C for 7min, and repeating the above steps for 30 times. After completion of the reaction, the PCR product was quantified by agarose gel electrophoresis, as shown in FIG. 4. Wherein the nucleotide sequence of the knockout fragment is shown in SEQ ID NO: 14.
FIG. 4 shows a DNA Marker in which M is D L10000, and No.1 is a knockout fragment.
(V) preparation and transformation of Aspergillus niger protoplast
1. Aspergillus niger was inoculated into PDA plates and spores were grown, 3m L scrape buffer was added to the plates, the spores were scraped off with a spreading stick and transferred to sterilized 5m L centrifuge tubes.
2. Inoculating 0.5m L spore liquid to 50m L YPD medium, culturing at 35 deg.C and 250rpm for 9-13 h.
3. After the spores have germinated, they are filtered by Miracloth to leave hypha, and then enzymatic hydrolysate (L lysing enzyme, crashing enzyme, snailase each 0.1g/10m L, cellulase 400 mu L/10 m L) is prepared and sterilized by filtration with a sterile syringe.
4. 2g of mycelia was added to the enzyme solution and subjected to enzymolysis at 30 ℃ and 220rpm for 30 min. The rotation speed is reduced to 150rpm for 4h of culture.
5. After the enzymolysis is finished, filtering with filter paper, taking the filtrate, centrifuging at 4 ℃ and 5000rpm for 10min, removing the supernatant, adding 1M L1M sorbitol (ice water bath), blowing and sucking with a gun, mixing uniformly, adding 15M L sorbitol, centrifuging, removing the supernatant, repeating again, removing the supernatant, adding 1M L Solution5, blowing and sucking with a gun, and mixing uniformly.
6. And (3) sucking 100 mu L of protoplast into a 1.5m L sterilized centrifugal tube, adding the gene knockout fragment constructed in the step (IV) of 10 mu L, mixing uniformly, adding 50 mu L of Solution4, mixing uniformly, placing on ice and timing for 15-30 min.
After 7.20min, adding 900u L Solution4, turning upside down for several times, standing at room temperature for 15-30min, after 15-30min, centrifuging at 6000rpm for 5min, discarding 900u L supernatant, coating the residual thallus on PDA culture medium with sucrose concentration of 1 mol/L and hygromycin concentration of 75 mmol/L, and culturing at normal position to obtain transformant.
8. The specific method comprises the steps of adding a proper amount of transformant into 50u L colony PCR buffer solution (100 mM/L Tris-HCl, 10 mM/L EDTA and 1M/L Kcl), carrying out water bath at 95 ℃ for 10min, adding 0.5u L into a PCR reaction system, setting PCR primers hyg-F and hyg-R (shown as SEQ ID NO.15 and SEQ ID NO. 16), and displaying an amplification band by agarose electrophoresis to indicate that the transformation is successful.
Example 2: crystal violet staining test
Respectively inoculating original Aspergillus Niger (Aspergillus Niger831) and genetically engineered bacteria to PDA plate, adding 3m L spore-scraping buffer solution into the plate, scraping off the spores with a coating rod, transferring to sterilized 5m L centrifuge tube, diluting to 2m L with the spore-scraping buffer solution to obtain spore solution, quantifying with a blood counting chamber, and diluting to 10%6M L, then continuously diluting to 105,104。
Adding 1ml of synthetic culture medium into a 24-pore plate in advance, inoculating 2u L of spore liquid with different concentrations into the culture medium, standing and culturing at 35 ℃ for 36h to enable aspergillus niger to form a film at the bottom of the pore plate, pouring out the culture medium, washing for 2 times by PBS, adding 0.1% crystal violet for dyeing for 15min, pouring out the crystal violet, washing for 2 times by PBS, adding glacial acetic acid, placing for 30min in a shaking instrument to decolor the crystal violet, observing, and detecting by an enzyme labeling instrument, wherein the difference between the crystal violet dyeing graph and the OD value is shown in figures 5 and 6.
TABLE 2 OD values of biofilm crystal violet staining experiments at different spore concentrations
The results in FIGS. 5 and 6 show that the.DELTA.CchA strain was markedly lighter in purple color than the original strain after decolorization, at 105The color of the strain Δ CchA at the concentration was removed and the data was consistent with the color as measured by OD using a microplate reader. Indicating that the biological membrane is reduced after the calcium ion channel CchA gene is inactivated.
Example 3: and (3) performing immobilized fermentation experiments on the genetically engineered bacteria.
1. Preparation of porous fiber material immobilized medium
Soaking porous fiber material (activated carbon fiber) in 1M sodium hydroxide for 1h, cleaning with pure water, soaking in 1M hydrochloric acid for 1h, washing with pure water until pH is neutral, and oven drying at 65 deg.C to constant weight. Cutting into carriers with the same size.
2. Preparation of fermentation Medium
Respectively weighing 200/L-300 g/L of cassava powder and 200 g/L-300 g/L of corn powder, gelatinizing in a 75 ℃ water bath kettle, adding 1-2m L of liquefying enzyme when the temperature of cassava powder liquid and corn powder liquid reaches 65 ℃, liquefying for 40min, then heating the water bath kettle to 95 ℃, adding 1-2m L of liquefying enzyme when the temperature of the cassava powder liquid and the corn powder liquid reaches 85 ℃, liquefying for 40min until the iodine solution does not turn blue, filtering the cassava powder liquid to obtain supernatant, adding 2-10% of unfiltered corn powder liquid as a return material, uniformly mixing, subpackaging every 100m L into 500m L conical bottles containing a proper amount of carriers, sterilizing and cooling for later use.
3. The fermentation method comprises the following steps
(1) Inoculating the frozen aspergillus niger genetically engineered bacteria and the original aspergillus niger spores onto a PDA (personal digital assistant) plate, culturing for 4-5 days at the constant temperature of 35 ℃ in a constant temperature incubator, and filling the spores on the plate.
(2) Scraping spores with a spore scraping buffer solution to obtain a spore suspension, transferring a proper amount of the spore suspension into a 500m L conical flask filled with 100m L immobilized culture medium, culturing for 72-96h at 30-35 ℃ in a shaking table at 250rpm, sampling every 12h in the fermentation process, centrifuging at 12000rpm for 5min, separating the supernatant from the precipitate, and determining the residual sugar concentration and the citric acid yield in the supernatant.
Wherein the NaOH titration method comprises adding a sample of 1M L (diluted to a certain concentration) into a 250M L conical flask, simultaneously adding 50M L pure water, and titrating with 0.1429M NaOH, wherein the consumed NaOH amount is the yield of citric acid.
The DNS method comprises the following steps:
the DNS is prepared by weighing 10g of 3, 5-dinitrosalicylic acid, placing in L m water of about 600m, gradually adding 10g of sodium hydroxide, magnetically stirring and dissolving in 50 ℃ water bath, sequentially adding 200g of sodium methyl tartrate, and,
2g of phenol and 5g of anhydrous sodium sulfite, cooling to room temperature after all the phenol and the anhydrous sodium sulfite are dissolved and clarified, and fixing the volume by pure water
To 1000m L, the product is stored in a brown reagent bottle and used after being placed in the dark for 7 days.
The preparation method of the standard yeast comprises preparing a series of sugar standard solutions with concentration of 0.0-1.0 g/L, adding 0.5m L into 15m L centrifuge tubes, adding 0.5m L DNS solution into each centrifuge tube,
placing the centrifugal tubes in a boiling water bath for reaction for 5min, placing the centrifugal tubes in ice water for cooling, then adding 8m L pure water into each centrifugal tube, uniformly mixing, measuring the light absorption value OD540 under the wavelength of 540nm, taking 0.0 g/L as a reference, taking the concentration of the standard solution as an ordinate, and taking the light absorption value OD540 as an abscissa to draw a standard curve.
The sample measurement method comprises the steps of adding 1m L concentrated sulfuric acid into a 10m L sample after the sample is properly diluted, reacting in a boiling water bath for 15min, placing in ice water for cooling, adjusting the pH to be neutral, fixing the volume to 100m L, and then diluting to the proper concentration, wherein the measurement method is the same as the above, and different sugars correspond to different standard yeasts.
FIG. 7 shows the difference between the Δ CchA strain and the original strain under fermentation conditions, and the results show that after 96h of fermentation, the yield of citric acid produced by fermentation of the Δ CchA strain is 162.8 g/L on average, the yield of citric acid produced by fermentation of the original strain is 153.2 g/L on average, and the yield of citric acid produced by the Δ CchA strain is 6.27% higher than that of the original strain.
Example 4: SEM observation of biofilm
The immobilized carrier after fermentation for 72h was taken out, washed 3 times with PBS to remove adsorbed mycelia. The mixture was placed in a freezer at-80 ℃ overnight and then lyophilized in a lyophilizer. And the carrier is adhered on a point mirror table through conductive glue, and 20mA 30s is subjected to gold spraying. Then, the sample was taken into TM3000 for observation. The electron micrographs of the biofilm are shown in FIGS. 8 and 9. FIG. 8 shows immobilization of the A.niger original strain, and FIG. 9 shows immobilization of the. DELTA.Cch strain. It was found that the Aspergillus niger original bacteria formed a plurality of spheres on the carrier, and these spheres connected to form a film and simultaneously blocked the pore size of the carrier, which affected the oxygen and mass transfer of the inner bacteria. The delta CchA strain forms a small amount of biomembranes at the corners and on the surface of the carrier, does not influence the oxygen and mass transfer of the internal bacteria, and is favorable for producing citric acid by immobilized fermentation.
Sequence listing
<110> Nanjing university of industry
<120> Aspergillus niger genetically engineered bacterium with calcium ion channel CchA gene inactivated, and construction method and application thereof
<160>16
<170>SIPOSequenceListing 1.0
<210>1
<211>6470
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atggcttcaa gtagccatga ccgcggcccc gacgacgata acttccacat cgatcactca 60
atccccttgc aggatctctc atcgccagac catgaacgtg gaggcccagg tggtgttgcg 120
acaaggctgg gccgatcttt gaccggtagg ggtcggacag ggagaaacta tgagcgcata 180
gcggtggatt cacccgttga aacggccaat gcggcgggca atgcgcgccc gcatcctcac 240
atttcccatg aagaagatga agaagcaatc gaagatccgg aggcgtttgc tcaagcaact 300
tcgttcggtc tcagctttga tccctcgccc agtacctctc ttgccccgac gcatagtcga 360
gcaccctcca gcgtcgacct agataccgtg ccgctcgacg gagccgaaca ctacctcgcc 420
cacatcgaca cctacaatga caccgcacga ttgacggaaa cccaaaacgt ccagcccata 480
agcggagcat ccggctcaga caatgaacat cagaacgatc ggggcggcac aagatcggtt 540
caattccctg ctaccggcca ttcggggtca cgattgggtg atgatctaca caatctggag 600
gatggtttcg tggggagatc ccgcggggga agcaatgcag cggacaggtc gcgatcgctg 660
tccccctcgg cctcgggatc tgccttgttg cgggccagtt ctatgatgaa gtccatgtcc 720
cagcgtattg ttaaccttag taacgaacca gaagtggtag agcaatcaat cctcagggag 780
gaatcgcata agaatgcgcg aatggaagag cccccagtac ttccatcttt gccggactat 840
gcgcatgatg cgccgtctac cacttcattg aacagttcag ctcctagaga gaaactgccc 900
tcgagcaata aggcatggcg cggtattagc aatcctttga gagggaagtc gctgggtatc 960
ctaggcccag ataatgctct gcggatgtgg ttgtgtgata tactcgtcca cccgttcaca 1020
gaacccttta ttctgatcgt gatcgtggtc cagacaattt tgctgaccat tgaatctgca 1080
cgttctgtct ggaaatatcc ccgcgctgtc cgttggggag ccaatcccat ggattatccc 1140
tactttgcca tcttcattat ctacacgctc gagattatcg ccaaaatcct tgtgtcagga 1200
ttgatcatca accctgccga atacagcacc atagaccggt cgatgggctt caggaaggca 1260
gtggtcgaga aagggaagaa cctgatcata ccacagcggc aattctcggt gcgaaagtct 1320
gccatgtcag aacaacccca ggcctccatt atacgaactt ttacgggcgg cttgaatcag 1380
ttggagaatc aattggccga tgaccctctt cagaaaagtc gcgtacgact agctcatcgt 1440
gcattcctgc gacactcatt caatcggctc gatttcgtcg ccttagtatc gtactgggtc 1500
tccttcttcc tctcgatata tggagtcgaa actcgccagc agctctatgt tttcagcatg 1560
ttgagctgtc ttcgaattct tcgtctcttg aatctgacca atggtacttc tgtaagtaaa 1620
ccgccgaata ctcttagcca cgtcgttcac tgacatcaga gacaaggtta tcctccgcag 1680
tctcaagaaa gcagcaccct tgcttgcgca cgtagcgttc cttatcggct tcttctggct 1740
tctgtttgct atcgtcggca tccaaagctt caagtcaagc cttcggagaa cctgtgtctg 1800
gcttggcgtt gatggtgaaa gcgattacgc acagaatgat ccaaatggct ctttacagtt 1860
ttgtggaggc tacctcaatg cgaccactaa acaacaaatg ccatggattc agaaggataa 1920
tactccgagt tcgtcttctc cgaaaggcta catctgtcct gcaggctcta tttgtcttga 1980
aggagacaat ccctacaatg ggacgttaag ttttgacaac atcgtgaatt cgctggagct 2040
tgtgtttgta attatgagct cgaacacatt cactgaccta ctctactata ctgccgacag 2100
tgactatctc gcttcttctc ttttcttcat atgcgggctt ctcatattga gtttgtggat 2160
ggtcaatttg ttggttgcag tgatcaccca tgcttttcag gtcattcgag aggaaagtca 2220
gcgcagtgcc tttgccgtta agaagataga cacgactgaa agagaggatt tggcctctcg 2280
gaaggtcagt gcgatcaagc gtctttatga caagaccgaa tggctctggg tttgcatcat 2340
cattttcgat cttgtggtcc aggctctgag gagcgcttcc atgagcgacg atcgggccca 2400
gtttattgac accacagaac ttgttatgac tttcgtattc cttttcgaaa ttatcctacg 2460
atttgcctcg gattggcgca ccttccacaa aaaaacgcgg aattgggtcg atctgggcct 2520
cgtcataata acgtgcatca ttcagatccc ggcgatcaag cgtgaacgag catacgatgt 2580
ccttacgctc tttcaaattc ttcgagtata tcgcgtcgtg ctcgcattta aggtgaccag 2640
ggacctcatt atggttgtct ttcgtaatgc agttggtctg ctgaacctga tcttctttgt 2700
ctttctgatt accttccttg cttccatttt cgcaactcag ctcttccgtg gccagattcc 2760
agaggaagac gcagacggcg ataccataat catcaccttt tccgacatct acaactcttt 2820
tctcgggatg tatcaaatct tgtcaagcga aaattggacg gacatcttgt ataacgctac 2880
cacgtacaca gtgtcataca atacagcttg gatctctgcg gctttcttga tattatggtt 2940
tatcctggcc aacttcattg tcctgaacat gttcattgct gtcatccagg aaagctttga 3000
tgtctcggag gatgaaaaac ggcttcagca ggtcaaagcg ttcttgcagc agaaacaagt 3060
tagcatggcc tcgcagggga acttgtctct ttccaagatt tttaagctgg gcaaggactc 3120
gaaccggtat aaggaccctc ttgaccacgg cccagcggca ctggagatgt tactcaagga 3180
tgctgtcgtc caggagttcc ttgatgagga tgaaccaccc gagcatcggc caggagacaa 3240
cgtcccatta gaacagtcgg ccacggccga gacagctcag ccaggattct tctcgcggat 3300
ctggacgaag ttcaccacgt caatcatgcg tcgagagccg aaccctttct actccaagct 3360
ggatatcccg cgtacatttg atgagctaga cccaaggacg atggcgaagg aattcgtttc 3420
agcagctgag cagaggaaaa gggcccaacg agagtatctc atgcgacacc caaattacaa 3480
caagtcgctg tttatctttg cgccaaacca ccccgttcgg aagctatgcc agcgtatcgt 3540
ggggccgggg cgtggagttc aacgagtgga gggtgtagat ccctacaagc ctgtctggta 3600
tgcattttcc gcgttcattt acgcggccat cgtcgcaatg gtgttgctgg cctgcataac 3660
cacaccaatc taccagaaga atcactttac gacgaacagg gattggttta cctacactga 3720
catgggcttc gcggtcttgt tcaccataga agctatcatc aaagttatag cggatgggtt 3780
tttctggacg cctaatgcgt actttcgcgg ttcctggggc tttcttgatg gtgtggtttt 3840
gatcacgctc tggatcagcg tcggtggatc cctgttcgaa gattggggcg tcacccgagc 3900
gattggagct ttcaaggctc ttagggcttt gagactcttg aacgtcagtg atagcgctaa 3960
gaatactttc cattcagtga tcattgttgg aggatggaag gtcattgctg taagtcccgc 4020
cccctgttga gccccctaga ggcagaatct gatgcgcttg gcaggccgcg gctgtttcga 4080
tgagcttttt gatccccttc gctatctatg gagtaaatct attcgctgga cgaatggttt 4140
catgcaatga tggcgacatt tcaggaagcc tggatcagtg cattggtgag tacaagaaca 4200
cgcctttcaa ttgggatgtt ctttctccgc gagtggccga caattcttat tacgactttg 4260
acaactttgg cgattccctc ttcattctgt tccagattgt ctctcaagaa ggctggatcg 4320
acgtgcagga cagtgctatg agtattactg gtgtggatat gcagccgcag gactacgttg 4380
cgccggagaa tgggctcttc tttatcattt tcaacctgct tggtgccgtc ttcgtcctga 4440
cgctgttcgt atctgtgttc atgcggaact acacagaaga gactggtgta gcgtatctta 4500
ctgctgaaca gcgatcgtgg ctggaattga gaaagttgct ccggcaaatc tccccttcaa 4560
agcgctcctt tgacaataag agccgacaat ggaagatgtg gagttaccga gtcgccgtta 4620
agaaacatgg cccatgggcg agatgcgtga cattcatcct cacactccat ctgttgttgc 4680
tggtcttgga atactatccc gagccggatg tatgggatca gacccgaggt gagtagttct 4740
tcctatggcc ttgcaaacga gcttgttccc gctaatttag tacagagata atattctttg 4800
ccttcaactt tgtctacatt gctaatgttc tgatccgaat gcttggcttg ggttggcacc 4860
ggtttagccg gagttcatgg gatgtgtatt cgttgctctc tgtctccggg acgttcataa 4920
cgacgatttt gagattcgtc tcgtctagcc aggtgatcaa cgagctgaac aagctcttcc 4980
tggtttcgat cactcttctt attatacccc ggaacaacca gcttgaccag ctattcaaaa 5040
ccgccgcagc tagtctgacg tcgatcggaa acctcatcgc cacctggttt gtcctcttcc 5100
tagtgttcgc gatcgccatg aaccaggcct ttggtctcac gaagtttggt tcacaagaga 5160
ccgacaacct caacttccgc gatgttccca aggcgctggt gctgctcttc cggatgagtt 5220
gcggagaagg ctggaacgag atcatggaag attatgccac gatgagtcct ccgatgtgca 5280
cctacgatgg caactttttc ttagatgact gtggcagtgc gccatgggct cggacgctgt 5340
tcattgcttg gaatattatc agcatgtatc tcttcgtctc actgttcacg tccttgatct 5400
tcgagagctt ctcctacgtg tatcagaaga gcagtgggct ctatgcgatc agccgcgagg 5460
atatccgccg tttcaagcat gcgtgggcta catatgaccc ggacggaacc gggtatatca 5520
ccaaagaaca gttcccgcga ctgctgggag agctctcggg ggtgttttcg atgcggatct 5580
acgatggtga gttcactatc ggccaaatca tggaagaatg ccgggtggac aagcgcgact 5640
cgctgcttgc ccatcgcaga gtggtcgacg ggctagactt ggacaagatg gcccgaatcc 5700
ttcgacagat cccaacggac gtggtgcgca accgccggca acggctgaat gcgttctatg 5760
aggaggtgct tgtgtcggcg gacccggtgc gcggcatctc gttccactcc tgtctcatga 5820
tcctggccca ttacaacgtg atcagcgaca gcaagagtct gcgactggaa gagttcctgc 5880
gcagacgggc gcgactgcag cgcgtcgagg aaaccatccg tcggcagacg gtgatcggct 5940
tctttgacac actgtactgg tctcgcgagt tccggcgccg ggttgagcat aagaaatcgg 6000
cccggatgag tggtgttcct cagttctcgg tgccggagat ctttattgat gacggatctc 6060
acgatgagcc ggcggcggtc gaggggccac gagaacaagc acgcgacgcg cttaccggag 6120
aagaatcgtc gcagcagccg atgctgtccc cgacgtcccc gaccgggcga gccacccggt 6180
accagctgcc acccatcgat accagtccgc tgggtcggat ctctgtcctg aactccccgt 6240
cgacggaatg gtccagcatc agcccatcgc tgtcgcctct gcgggagcga gccggcacga 6300
cgtcgtcgta cggcagcgga ggtgatgtgc atgatgagca ggcaagccca gcgcattcgc 6360
ggcagaatag cgcgatgaac gtcaacgatg tgatgcagtc gctgggcgag tcggtatggg 6420
gagagagtat ccgacgcagc tttacacagc gacggcgatc gggggagtga 6470
<210>2
<211>5310
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ctacacgctc gagattatcg ccaaaatcct tgtgtcagga ttgatcatca accctgccga 60
atacagcacc atagaccggt cgatgggctt caggaaggca gtggtcgaga aagggaagaa 120
cctgatcata ccacagcggc aattctcggt gcgaaagtct gccatgtcag aacaacccca 180
ggcctccatt atacgaactt ttacgggcgg cttgaatcag ttggagaatc aattggccga 240
tgaccctctt cagaaaagtc gcgtacgact agctcatcgt gcattcctgc gacactcatt 300
caatcggctc gatttcgtcg ccttagtatc gtactgggtc tccttcttcc tctcgatata 360
tggagtcgaa actcgccagc agctctatgt tttcagcatg ttgagctgtc ttcgaattct 420
tcgtctcttg aatctgacca atggtacttc tgtaagtaaa ccgccgaata ctcttagcca 480
cgtcgttcac tgacatcaga gacaaggtta tcctccgcag tctcaagaaa gcagcaccct 540
tgcttgcgca cgtagcgttc cttatcggct tcttctggct tctgtttgct atcgtcggca 600
tccaaagctt caagtcaagc cttcggagaa cctgtgtctg gcttggcgtt gatggtgaaa 660
gcgattacgc acagaatgat ccaaatggct ctttacagtt ttgtggaggc tacctcaatg 720
cgaccactaa acaacaaatg ccatggattc agaaggataa tactccgagt tcgtcttctc 780
cgaaaggcta catctgtcct gcaggctcta tttgtcttga aggagacaat ccctacaatg 840
ggacgttaag ttttgacaac atcgtgaatt cgctggagct tgtgtttgta attatgagct 900
cgaacacatt cactgaccta ctctactata ctgccgacag tgactatctc gcttcttctc 960
ttttcttcat atgcgggctt ctcatattga gtttgtggat ggtcaatttg ttggttgcag 1020
tgatcaccca tgcttttcag gtcattcgag aggaaagtca gcgcagtgcc tttgccgtta 1080
agaagataga cacgactgaa agagaggatt tggcctctcg gaaggtcagt gcgatcaagc 1140
gtctttatga caagaccgaa tggctctggg tttgcatcat cattttcgat cttgtggtcc 1200
aggctctgag gagcgcttcc atgagcgacg atcgggccca gtttattgac accacagaac 1260
ttgttatgac tttcgtattc cttttcgaaa ttatcctacg atttgcctcg gattggcgca 1320
ccttccacaa aaaaacgcgg aattgggtcg atctgggcct cgtcataata acgtgcatca 1380
ttcagatccc ggcgatcaag cgtgaacgag catacgatgt ccttacgctc tttcaaattc 1440
ttcgagtata tcgcgtcgtg ctcgcattta aggtgaccag ggacctcatt atggttgtct 1500
ttcgtaatgc agttggtctg ctgaacctga tcttctttgt ctttctgatt accttccttg 1560
cttccatttt cgcaactcag ctcttccgtg gccagattcc agaggaagac gcagacggcg 1620
ataccataat catcaccttt tccgacatct acaactcttt tctcgggatg tatcaaatct 1680
tgtcaagcga aaattggacg gacatcttgt ataacgctac cacgtacaca gtgtcataca 1740
atacagcttg gatctctgcg gctttcttga tattatggtt tatcctggcc aacttcattg 1800
tcctgaacat gttcattgct gtcatccagg aaagctttga tgtctcggag gatgaaaaac 1860
ggcttcagca ggtcaaagcg ttcttgcagc agaaacaagt tagcatggcc tcgcagggga 1920
acttgtctct ttccaagatt tttaagctgg gcaaggactc gaaccggtat aaggaccctc 1980
ttgaccacgg cccagcggca ctggagatgt tactcaagga tgctgtcgtc caggagttcc 2040
ttgatgagga tgaaccaccc gagcatcggc caggagacaa cgtcccatta gaacagtcgg 2100
ccacggccga gacagctcag ccaggattct tctcgcggat ctggacgaag ttcaccacgt 2160
caatcatgcg tcgagagccg aaccctttct actccaagct ggatatcccg cgtacatttg 2220
atgagctaga cccaaggacg atggcgaagg aattcgtttc agcagctgag cagaggaaaa 2280
gggcccaacg agagtatctc atgcgacacc caaattacaa caagtcgctg tttatctttg 2340
cgccaaacca ccccgttcgg aagctatgcc agcgtatcgt ggggccgggg cgtggagttc 2400
aacgagtgga gggtgtagat ccctacaagc ctgtctggta tgcattttcc gcgttcattt 2460
acgcggccat cgtcgcaatg gtgttgctgg cctgcataac cacaccaatc taccagaaga 2520
atcactttac gacgaacagg gattggttta cctacactga catgggcttc gcggtcttgt 2580
tcaccataga agctatcatc aaagttatag cggatgggtt tttctggacg cctaatgcgt 2640
actttcgcgg ttcctggggc tttcttgatg gtgtggtttt gatcacgctc tggatcagcg 2700
tcggtggatc cctgttcgaa gattggggcg tcacccgagc gattggagct ttcaaggctc 2760
ttagggcttt gagactcttg aacgtcagtg atagcgctaa gaatactttc cattcagtga 2820
tcattgttgg aggatggaag gtcattgctg taagtcccgc cccctgttga gccccctaga 2880
ggcagaatct gatgcgcttg gcaggccgcg gctgtttcga tgagcttttt gatccccttc 2940
gctatctatg gagtaaatct attcgctgga cgaatggttt catgcaatga tggcgacatt 3000
tcaggaagcc tggatcagtg cattggtgag tacaagaaca cgcctttcaa ttgggatgtt 3060
ctttctccgc gagtggccga caattcttat tacgactttg acaactttgg cgattccctc 3120
ttcattctgt tccagattgt ctctcaagaa ggctggatcg acgtgcagga cagtgctatg 3180
agtattactg gtgtggatat gcagccgcag gactacgttg cgccggagaa tgggctcttc 3240
tttatcattt tcaacctgct tggtgccgtc ttcgtcctga cgctgttcgt atctgtgttc 3300
atgcggaact acacagaaga gactggtgta gcgtatctta ctgctgaaca gcgatcgtgg 3360
ctggaattga gaaagttgct ccggcaaatc tccccttcaa agcgctcctt tgacaataag 3420
agccgacaat ggaagatgtg gagttaccga gtcgccgtta agaaacatgg cccatgggcg 3480
agatgcgtga cattcatcct cacactccat ctgttgttgc tggtcttgga atactatccc 3540
gagccggatg tatgggatca gacccgaggt gagtagttct tcctatggcc ttgcaaacga 3600
gcttgttccc gctaatttag tacagagata atattctttg ccttcaactt tgtctacatt 3660
gctaatgttc tgatccgaat gcttggcttg ggttggcacc ggtttagccg gagttcatgg 3720
gatgtgtatt cgttgctctc tgtctccggg acgttcataa cgacgatttt gagattcgtc 3780
tcgtctagcc aggtgatcaa cgagctgaac aagctcttcc tggtttcgat cactcttctt 3840
attatacccc ggaacaacca gcttgaccag ctattcaaaa ccgccgcagc tagtctgacg 3900
tcgatcggaa acctcatcgc cacctggttt gtcctcttcc tagtgttcgc gatcgccatg 3960
aaccaggcct ttggtctcac gaagtttggt tcacaagaga ccgacaacct caacttccgc 4020
gatgttccca aggcgctggt gctgctcttc cggatgagtt gcggagaagg ctggaacgag 4080
atcatggaag attatgccac gatgagtcct ccgatgtgca cctacgatgg caactttttc 4140
ttagatgact gtggcagtgc gccatgggct cggacgctgt tcattgcttg gaatattatc 4200
agcatgtatc tcttcgtctc actgttcacg tccttgatct tcgagagctt ctcctacgtg 4260
tatcagaaga gcagtgggct ctatgcgatc agccgcgagg atatccgccg tttcaagcat 4320
gcgtgggcta catatgaccc ggacggaacc gggtatatca ccaaagaaca gttcccgcga 4380
ctgctgggag agctctcggg ggtgttttcg atgcggatct acgatggtga gttcactatc 4440
ggccaaatca tggaagaatg ccgggtggac aagcgcgact cgctgcttgc ccatcgcaga 4500
gtggtcgacg ggctagactt ggacaagatg gcccgaatcc ttcgacagat cccaacggac 4560
gtggtgcgca accgccggca acggctgaat gcgttctatg aggaggtgct tgtgtcggcg 4620
gacccggtgc gcggcatctc gttccactcc tgtctcatga tcctggccca ttacaacgtg 4680
atcagcgaca gcaagagtct gcgactggaa gagttcctgc gcagacgggc gcgactgcag 4740
cgcgtcgagg aaaccatccg tcggcagacg gtgatcggct tctttgacac actgtactgg 4800
tctcgcgagt tccggcgccg ggttgagcat aagaaatcgg cccggatgag tggtgttcct 4860
cagttctcgg tgccggagat ctttattgat gacggatctc acgatgagcc ggcggcggtc 4920
gaggggccac gagaacaagc acgcgacgcg cttaccggag aagaatcgtc gcagcagccg 4980
atgctgtccc cgacgtcccc gaccgggcga gccacccggt accagctgcc acccatcgat 5040
accagtccgc tgggtcggat ctctgtcctg aactccccgt cgacggaatg gtccagcatc 5100
agcccatcgc tgtcgcctct gcgggagcga gccggcacga cgtcgtcgta cggcagcgga 5160
ggtgatgtgc atgatgagca ggcaagccca gcgcattcgc ggcagaatag cgcgatgaac 5220
gtcaacgatg tgatgcagtc gctgggcgag tcggtatggg gagagagtat ccgacgcagc 5280
tttacacagc gacggcgatc gggggagtga 5310
<210>3
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
<210>4
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ggtggaggcg gcggatttta agtcgtcaga gtgctaaccg 40
<210>5
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
cccactccac atctccactc gactacacgc tcgagattat cgcc 44
<210>6
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
<210>7
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
cggttagcac tctgacgact taaaatccgc cgcctccacc 40
<210>8
<211>44
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
ggcgataatc tcgagcgtgt agtcgagtgg agatgtggag tggg 44
<210>9
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
<210>10
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
ttcatcctcc gagacatcaa agct 24
<210>11
<211>1956
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
cggcgatgac gacgagacta tggaagatgt tgaaggcgtg gaccatgacc cgaaccacaa 60
ccacggagga aacagcggag gagcaggccc ggatgcgact ggcggtgatg ttcgggcgga 120
aggccaagga tcgagggagg gtggtgccga tggtggaaga gcgtaggtac atatctcgac 180
agttttattt gtctctagtc ttgaacttta ctagccggga tattcaatat gctcggatat 240
tttggtcacg attataggcg ttcctttcca tgaattcaag ctaaccagca tcatttggct 300
cacagggccg ccgctgactc ggatccttca tctgtcgtgc agagcttcct gcagtcgttg 360
catgggggct cacggcaatc ccaagacccc gagagacctt tcactactct gcaagatcta 420
cttacaacct cgactacatt gcctttcctg gaatctgcgg acgaaaaagc ggttgacaat 480
ctcctgagct tcctgccacc atcgctgctt ctcctggcac aggacaatga tctcgacgaa 540
gccgcggcag ctgatcagga cccggacatt gctcaagccg ccctgggatc cctcgaactg 600
acgcaaaaga aagagattct gcgcaaagtt ctacactcac ctcaatttgc gcagagtcta 660
gcaagcttga cgatggccat tagggatggt ggattaccga gcatcagcga ggccctaaaa 720
attccggttg caaatggagg attcatgcgt agagggggag ttccattggg tggtggtgat 780
gctgttgaag cattccttca aggcgttcgg gatcatgtta aggattcggc tcaaggaaac 840
cgtatggaca ccgattgatt ggttccaaat agcaatgatt gtgttatgtt gacagggtcc 900
tttttttaac tttcttatct cttttttact attattatct tcagccagcc attctgtaac 960
tatccactga tgttttagaa atccattagc ggccttgcat ggatgtctca ttccctcctt 1020
agcagagaga gacttgtcat gggttatttt tggcatgagg tgggtggggt aaaaaggcca 1080
gtggccagta ctaggcagca tgataatact ggtaatctta ataccgttaa taataatgtg 1140
tcttggttgt ggcgtatggc ggactccgcc cgttaatctc aaacagaatc aagaattaaa 1200
aaagaaaaaa gtaggggaaa aaagtttccg actcgatgat ctcttcctcc accatggaaa 1260
aaaagaggtg ggacaggtat caaaggcatt gccgtatcgg gagtagacat aagtgatcct 1320
gtcagggacc aaagcaagtc aaatcagtcc gtactaagat acatcatggg gagggggcac 1380
gcatattccc ctcccgggcg cagcaggggg aaaatcccag cttcagaccc cgttccatcc 1440
cagagccatc catatccacc ctcaattatt ggccgtgtgt cgaacgaact cgacccggac 1500
cattcataag gctcggacgt ttagcaatca ctgggccaga gagcctggcc aatgggccat 1560
acagaagccg ccgctgccga atccgcccca gttttggccg ctttggcgcc gtccagccaa 1620
agctccgctg cgcataccga gcaataataa tcatcactgt gatctggaag tcgacacgtt 1680
tgggttcctt tccccatcat gcccgctgct tcttgacggc tgcctggctt gattggatcc 1740
ttgcccacgt ttccgcacgg ggtgccaccg ctaacttaac cagagttcgt cagccctttc 1800
gtactacttt aacttactta gggagtctgt cccatatttt ccccagcgcg cttttacttt 1860
gcctcggtcg tcaacgctcc ccagggccct tctgatcatc tctttttctt ctggaacagc 1920
ctgcgatgtc tgccttcggt tagcactctg acgact 1956
<210>12
<211>1914
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
ctacacgctc gagattatcg ccaaaatcct tgtgtcagga ttgatcatca accctgccga 60
atacagcacc atagaccggt cgatgggctt caggaaggca gtggtcgaga aagggaagaa 120
cctgatcata ccacagcggc aattctcggt gcgaaagtct gccatgtcag aacaacccca 180
ggcctccatt atacgaactt ttacgggcgg cttgaatcag ttggagaatc aattggccga 240
tgaccctctt cagaaaagtc gcgtacgact agctcatcgt gcattcctgc gacactcatt 300
caatcggctc gatttcgtcg ccttagtatc gtactgggtc tccttcttcc tctcgatata 360
tggagtcgaa actcgccagc agctctatgt tttcagcatg ttgagctgtc ttcgaattct 420
tcgtctcttg aatctgacca atggtacttc tgtaagtaaa ccgccgaata ctcttagcca 480
cgtcgttcac tgacatcaga gacaaggtta tcctccgcag tctcaagaaa gcagcaccct 540
tgcttgcgca cgtagcgttc cttatcggct tcttctggct tctgtttgct atcgtcggca 600
tccaaagctt caagtcaagc cttcggagaa cctgtgtctg gcttggcgtt gatggtgaaa 660
gcgattacgc acagaatgat ccaaatggct ctttacagtt ttgtggaggc tacctcaatg720
cgaccactaa acaacaaatg ccatggattc agaaggataa tactccgagt tcgtcttctc 780
cgaaaggcta catctgtcct gcaggctcta tttgtcttga aggagacaat ccctacaatg 840
ggacgttaag ttttgacaac atcgtgaatt cgctggagct tgtgtttgta attatgagct 900
cgaacacatt cactgaccta ctctactata ctgccgacag tgactatctc gcttcttctc 960
ttttcttcat atgcgggctt ctcatattga gtttgtggat ggtcaatttg ttggttgcag 1020
tgatcaccca tgcttttcag gtcattcgag aggaaagtca gcgcagtgcc tttgccgtta 1080
agaagataga cacgactgaa agagaggatt tggcctctcg gaaggtcagt gcgatcaagc 1140
gtctttatga caagaccgaa tggctctggg tttgcatcat cattttcgat cttgtggtcc 1200
aggctctgag gagcgcttcc atgagcgacg atcgggccca gtttattgac accacagaac 1260
ttgttatgac tttcgtattc cttttcgaaa ttatcctacg atttgcctcg gattggcgca 1320
ccttccacaa aaaaacgcgg aattgggtcg atctgggcct cgtcataata acgtgcatca 1380
ttcagatccc ggcgatcaag cgtgaacgag catacgatgt ccttacgctc tttcaaattc 1440
ttcgagtata tcgcgtcgtg ctcgcattta aggtgaccag ggacctcatt atggttgtct 1500
ttcgtaatgc agttggtctg ctgaacctga tcttctttgt ctttctgatt accttccttg 1560
cttccatttt cgcaactcag ctcttccgtg gccagattcc agaggaagac gcagacggcg 1620
ataccataat catcaccttt tccgacatct acaactcttt tctcgggatg tatcaaatct 1680
tgtcaagcga aaattggacg gacatcttgt ataacgctac cacgtacaca gtgtcataca 1740
atacagcttg gatctctgcg gctttcttga tattatggtt tatcctggcc aacttcattg 1800
tcctgaacat gttcattgct gtcatccagg aaagctttga tgtctcggag gatgaaaaac 1860
ggcttcagca ggtcaaagcg ttcttgcagc agaaacaagt tagcatggcc tcgc 1914
<210>13
<211>2740
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
taaaatccgc cgcctccacc atttgtagaa aaatgtgacg aactcgtgag ctctgtacag 60
tgaccggtga ctctttctgg catgcggaga gacggacgga cgcagagaga agggctgagt 120
aataagccac tggccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc 180
gggaccggcc gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga 240
atctattgca tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag 300
aatgtgaagc caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag 360
aagtccaatt gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg 420
tagagcgagt acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc 480
caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc 540
tggccagcgg cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact 600
cgacctgctg aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg 660
tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc720
tctccccacc agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct 780
tcccatccaa gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc 840
ttcccatccc ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg 900
actaacagct accccgcttg agcagacatc accatgcctg aactcaccgc gacgtctgtc 960
gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 1020
gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 1080
agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 1140
ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 1200
tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 1260
ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 1320
gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 1380
tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 1440
gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 1500
cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 1560
acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1620
atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1680
aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 1740
gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 1800
cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 1860
agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1920
cgccccagca ctcgtccgag ggcaaaggaa tagagtagat gccgaccgcg ggatccactt 1980
aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 2040
gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 2100
caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 2160
gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 2220
aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag agctattttc 2280
attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 2340
atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 2400
cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg gttcgcaaag 2460
ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 2520
taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 2580
agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 2640
tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 2700
tcgtgtactg tgtaagcgcc cactccacat ctccactcga 2740
<210>14
<211>6502
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
cgaaccacaa ccacggagga aacagcggag gagcaggccc ggatgcgact ggcggtgatg 60
ttcgggcgga aggccaagga tcgagggagg gtggtgccga tggtggaaga gcgtaggtac 120
atatctcgac agttttattt gtctctagtc ttgaacttta ctagccggga tattcaatat 180
gctcggatat tttggtcacg attataggcg ttcctttcca tgaattcaag ctaaccagca 240
tcatttggct cacagggccg ccgctgactc ggatccttca tctgtcgtgc agagcttcct 300
gcagtcgttg catgggggct cacggcaatc ccaagacccc gagagacctt tcactactct 360
gcaagatcta cttacaacct cgactacatt gcctttcctg gaatctgcgg acgaaaaagc 420
ggttgacaat ctcctgagct tcctgccacc atcgctgctt ctcctggcac aggacaatga 480
tctcgacgaa gccgcggcag ctgatcagga cccggacatt gctcaagccg ccctgggatc 540
cctcgaactg acgcaaaaga aagagattct gcgcaaagtt ctacactcac ctcaatttgc 600
gcagagtcta gcaagcttga cgatggccat tagggatggt ggattaccga gcatcagcga 660
ggccctaaaa attccggttg caaatggagg attcatgcgt agagggggag ttccattggg 720
tggtggtgat gctgttgaag cattccttca aggcgttcgg gatcatgtta aggattcggc 780
tcaaggaaac cgtatggaca ccgattgatt ggttccaaat agcaatgatt gtgttatgtt 840
gacagggtcc tttttttaac tttcttatct cttttttact attattatct tcagccagcc 900
attctgtaac tatccactga tgttttagaa atccattagc ggccttgcat ggatgtctca 960
ttccctcctt agcagagaga gacttgtcat gggttatttt tggcatgagg tgggtggggt 1020
aaaaaggcca gtggccagta ctaggcagca tgataatact ggtaatctta ataccgttaa 1080
taataatgtg tcttggttgt ggcgtatggc ggactccgcc cgttaatctc aaacagaatc 1140
aagaattaaa aaagaaaaaa gtaggggaaa aaagtttccg actcgatgat ctcttcctcc 1200
accatggaaa aaaagaggtg ggacaggtat caaaggcatt gccgtatcgg gagtagacat 1260
aagtgatcct gtcagggacc aaagcaagtc aaatcagtcc gtactaagat acatcatggg 1320
gagggggcac gcatattccc ctcccgggcg cagcaggggg aaaatcccag cttcagaccc 1380
cgttccatcc cagagccatc catatccacc ctcaattatt ggccgtgtgt cgaacgaact 1440
cgacccggac cattcataag gctcggacgt ttagcaatca ctgggccaga gagcctggcc 1500
aatgggccat acagaagccg ccgctgccga atccgcccca gttttggccg ctttggcgcc 1560
gtccagccaa agctccgctg cgcataccga gcaataataa tcatcactgt gatctggaag 1620
tcgacacgtt tgggttcctt tccccatcat gcccgctgct tcttgacggc tgcctggctt 1680
gattggatcc ttgcccacgt ttccgcacgg ggtgccaccg ctaacttaac cagagttcgt 1740
cagccctttc gtactacttt aacttactta gggagtctgt cccatatttt ccccagcgcg 1800
cttttacttt gcctcggtcg tcaacgctcc ccagggccct tctgatcatc tctttttctt 1860
ctggaacagc ctgcgatgtc tgccttcggt tagcactctg acgacttaaa atccgccgcc 1920
tccaccattt gtagaaaaat gtgacgaact cgtgagctct gtacagtgac cggtgactct 1980
ttctggcatg cggagagacg gacggacgca gagagaaggg ctgagtaata agccactggc 2040
cagacagctc tggcggctct gaggtgcagt ggatgattat taatccggga ccggccgccc 2100
ctccgccccg aagtggaaag gctggtgtgc ccctcgttga ccaagaatct attgcatcat 2160
cggagaatat ggagcttcat cgaatcaccg gcagtaagcg aaggagaatg tgaagccagg 2220
ggtgtatagc cgtcggcgaa atagcatgcc attaacctag gtacagaagt ccaattgctt 2280
ccgatctggt aaaagattca cgagatagta ccttctccga agtaggtaga gcgagtaccc 2340
ggcgcgtaag ctccctaatt ggcccatccg gcatctgtag ggcgtccaaa tatcgtgcct 2400
ctcctgcttt gcccggtgta tgaaaccgga aaggccgctc aggagctggc cagcggcgca 2460
gaccgggaac acaagctggc agtcgaccca tccggtgctc tgcactcgac ctgctgaggt 2520
ccctcagtcc ctggtaggca gctttgcccc gtctgtccgc ccggtgtgtc ggcggggttg 2580
acaaggtcgt tgcgtcagtc caacatttgt tgccatattt tcctgctctc cccaccagct 2640
gctcttttct tttctctttc ttttcccatc ttcagtatat tcatcttccc atccaagaac 2700
ctttatttcc cctaagtaag tactttgcta catccatact ccatccttcc catcccttat 2760
tcctttgaac ctttcagttc gagctttccc acttcatcgc agcttgacta acagctaccc 2820
cgcttgagca gacatcacca tgcctgaact caccgcgacg tctgtcgaga agtttctgat 2880
cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2940
tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 3000
tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 3060
agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3120
gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3180
ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3240
cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3300
tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3360
ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3420
cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3480
ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3540
gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3600
tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3660
gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3720
cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3780
ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3840
tccgagggca aaggaataga gtagatgccg accgcgggat ccacttaacg ttactgaaat 3900
catcaaacag cttgacgaat ctggatataa gatcgttggt gtcgatgtca gctccggagt 3960
tgagacaaat ggtgttcagg atctcgataa gatacgttca tttgtccaag cagcaaagag 4020
tgccttctag tgatttaata gctccatgtc aacaagaata aaacgcgttt tcgggtttac 4080
ctcttccaga tacagctcat ctgcaatgca ttaatgcatt gactgcaacc tagtaacgcc 4140
ttncaggctc cggcgaagag aagaatagct tagcagagct attttcattt tcgggagacg 4200
agatcaagca gatcaacggt cgtcaagaga cctacgagac tgaggaatcc gctcttggct 4260
ccacgcgact atatatttgt ctctaattgt actttgacat gctcctcttc tttactctga 4320
tagcttgact atgaaaattc cgtcaccagc ncctgggttc gcaaagataa ttgcatgttt 4380
cttccttgaa ctctcaagcc tacaggacac acattcatcg taggtataaa cctcgaaatc 4440
anttcctact aagatggtat acaatagtaa ccatgcatgg ttgcctagtg aatgctccgt 4500
aacacccaat acgccggccg aaactttttt acaactctcc tatgagtcgt ttacccagaa 4560
tgcacaggta cacttgttta gaggtaatcc ttctttctag aagtcctcgt gtactgtgta 4620
agcgcccact ccacatctcc actcgactac acgctcgaga ttatcgccaa aatccttgtg 4680
tcaggattga tcatcaaccc tgccgaatac agcaccatag accggtcgat gggcttcagg 4740
aaggcagtgg tcgagaaagg gaagaacctg atcataccac agcggcaatt ctcggtgcga 4800
aagtctgcca tgtcagaaca accccaggcc tccattatac gaacttttac gggcggcttg 4860
aatcagttgg agaatcaatt ggccgatgac cctcttcaga aaagtcgcgt acgactagct 4920
catcgtgcat tcctgcgaca ctcattcaat cggctcgatt tcgtcgcctt agtatcgtac 4980
tgggtctcct tcttcctctc gatatatgga gtcgaaactc gccagcagct ctatgttttc 5040
agcatgttga gctgtcttcg aattcttcgt ctcttgaatc tgaccaatgg tacttctgta 5100
agtaaaccgc cgaatactct tagccacgtc gttcactgac atcagagaca aggttatcct 5160
ccgcagtctc aagaaagcag cacccttgct tgcgcacgta gcgttcctta tcggcttctt 5220
ctggcttctg tttgctatcg tcggcatcca aagcttcaag tcaagccttc ggagaacctg 5280
tgtctggctt ggcgttgatg gtgaaagcga ttacgcacag aatgatccaa atggctcttt 5340
acagttttgt ggaggctacc tcaatgcgac cactaaacaa caaatgccat ggattcagaa 5400
ggataatact ccgagttcgt cttctccgaa aggctacatc tgtcctgcag gctctatttg 5460
tcttgaagga gacaatccct acaatgggac gttaagtttt gacaacatcg tgaattcgct 5520
ggagcttgtg tttgtaatta tgagctcgaa cacattcact gacctactct actatactgc 5580
cgacagtgac tatctcgctt cttctctttt cttcatatgc gggcttctca tattgagttt 5640
gtggatggtc aatttgttgg ttgcagtgat cacccatgct tttcaggtca ttcgagagga 5700
aagtcagcgc agtgcctttg ccgttaagaa gatagacacg actgaaagag aggatttggc 5760
ctctcggaag gtcagtgcga tcaagcgtct ttatgacaag accgaatggc tctgggtttg 5820
catcatcatt ttcgatcttg tggtccaggc tctgaggagc gcttccatga gcgacgatcg 5880
ggcccagttt attgacacca cagaacttgt tatgactttc gtattccttt tcgaaattat 5940
cctacgattt gcctcggatt ggcgcacctt ccacaaaaaa acgcggaatt gggtcgatct 6000
gggcctcgtc ataataacgt gcatcattca gatcccggcg atcaagcgtg aacgagcata 6060
cgatgtcctt acgctctttc aaattcttcg agtatatcgc gtcgtgctcg catttaaggt 6120
gaccagggac ctcattatgg ttgtctttcg taatgcagtt ggtctgctga acctgatctt 6180
ctttgtcttt ctgattacct tccttgcttc cattttcgca actcagctct tccgtggcca 6240
gattccagag gaagacgcag acggcgatac cataatcatc accttttccg acatctacaa 6300
ctcttttctc gggatgtatc aaatcttgtc aagcgaaaat tggacggaca tcttgtataa 6360
cgctaccacg tacacagtgt catacaatac agcttggatc tctgcggctt tcttgatatt 6420
atggtttatc ctggccaact tcattgtcct gaacatgttc attgctgtca tccaggaaag 6480
ctttgatgtc tcggaggatg aa 6502
<210>15
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
<210>16
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
Claims (6)
1. The application of Aspergillus niger genetically engineered bacteria with calcium ion channel CchA gene inactivated in preparing citric acid by immobilized fermentation is characterized in that the Aspergillus niger isAspergillusNiger 831;
The nucleotide sequence of the calcium ion channel CchA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the calcium ion channel CchA gene after inactivation is shown in SEQ ID NO. 2;
the immobilized fermentation is to prepare the citric acid by fermenting with porous fiber material as an immobilized medium.
2. The application of claim 1, wherein the Aspergillus niger genetically engineered bacterium is constructed according to the following method:
(1) extraction of Aspergillus nigerAspergillusGenomic DNA of Niger 831;
(2) amplifying an upstream homologous arm of the CchA gene by taking the genomic DNA obtained in the step (1) as a template and taking the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers;
amplifying a downstream homology arm of the CchA gene by taking the genome obtained in the step (1) as a template and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers;
amplifying a hyg resistance element by taking the plasmid pan7-1 as a template and nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 as primers;
performing PCR amplification by using an upstream homologous arm of a CchA gene, a downstream homologous arm of the CchA gene and a hyg resistance element as templates and nucleotide sequences shown in SEQ ID NO.9 and SEQ ID NO.10 as primers through overlap PCR to obtain a gene knockout fragment;
(3) preparing an Aspergillus niger protoplast;
(4) and (3) introducing the gene knockout recombinant fragment into the protoplast for homologous recombination to obtain the Aspergillus niger genetically engineered bacterium with the calcium ion channel CchA gene inactivated, wherein the nucleotide sequence of the calcium ion channel CchA gene inactivated is shown as SEQ ID No. 2.
3. The use according to claim 1, wherein the immobilized fermentation is cultured under the following conditions: the culture temperature is 28-37 ℃, the culture time is 72-120 h, and the rotating speed is 180-330 rpm.
4. The use according to claim 3, wherein the fermentation medium for immobilized fermentation is prepared by:
respectively taking 200 g/L-300 g/L of cassava powder and 200 g/L0-300 g/L of corn powder, adding 1-2m L of liquefying enzyme into every 1L of fermentation liquor at the temperature of 60-70 ℃, carrying out enzymolysis for 35-45 min, respectively heating cassava powder liquid and corn powder liquid to 85 ℃, adding 1-2m L of liquefying enzyme into every 1L of fermentation liquor, carrying out enzymolysis for 35-45 min until the iodine solution does not turn blue, filtering the cassava powder liquid to obtain cassava powder liquid supernatant, adding 2-10% by volume of unfiltered corn powder liquid into the cassava powder liquid supernatant, and uniformly mixing to obtain an immobilized fermentation culture medium, wherein the liquefying enzyme contains α -amylase, and the enzyme activity of α -amylase is 60000-70000U/m L.
5. Use according to claim 1, wherein the porous fibrous material is activated carbon fiber.
6. Use according to claim 5, wherein the porous fibrous material is pre-treated by:
soaking the porous fiber material in 1M sodium hydroxide for 1-1.5 h, washing with water until the pH value is neutral, soaking in 1M hydrochloric acid for 1-1.5 h, washing with water until the pH value is neutral, and drying to constant weight to obtain the modified porous fiber material.
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CN102851328A (en) * | 2012-08-29 | 2013-01-02 | 太仓市茂通化建有限公司 | Method for preparing citric acid through fermenting corn sugar solution by immobilized Aspergillus niger |
CN102864184A (en) * | 2012-08-29 | 2013-01-09 | 太仓市茂通化建有限公司 | Method for producing citric acid by utilizing immobilized aspergillus niger |
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CN102851328A (en) * | 2012-08-29 | 2013-01-02 | 太仓市茂通化建有限公司 | Method for preparing citric acid through fermenting corn sugar solution by immobilized Aspergillus niger |
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《Aspergillus niger CBS 513.88 calcium channel subunit Cch1, mRNA》;NO REPORTED;《NCBI Reference Sequence: XM_001392419.1》;20110303;参见对比文件2序列及其注释 * |
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