CN102154117B - Actinomucor elegans ZGB1 and application thereof in microbial ATP (Adenosine Triphosphate) synthesis - Google Patents
Actinomucor elegans ZGB1 and application thereof in microbial ATP (Adenosine Triphosphate) synthesis Download PDFInfo
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Abstract
The invention provides a novel strain capable of synthesizing ATP (Adenosine Triphosphate), namely an Actinomucor elegans ZGB1 and application thereof. The strain is preserved in China General Microbiological Culture Collection Center (address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Precinct, Beichen West Road, Chaoyang District, Beijing; zip code: 100101), the preservation number is CGMCC No.4313, and the preservation date is November 8th, 2010. By using the strain, adenine can be used as the precursor for ATP synthesis, thereby achieving the advantages of low material cost, abundant material source, simple formula of thallus culture medium, easy thallus separation process and the like; and the conversion efficiency is high: ATP of higher than 10g/L can be produced in the reaction solution containing adenine of higher than 3g/L.
Description
(1) technical field
The present invention relates to a strain and can synthesize the new bacterial strain of ATP--graceful radiation Mucor (Actinomucor elegans) ZGB1, and the application in the synthetic ATP of mikrobe.
(2) background technology
ATP is an atp, as a kind of biochemical drug, has been used to amyotrophy, myocardial infarction, hepatitis etc. and various first aid treatment of conditions or assisting therapy.
The main both at home and abroad at present microbe transformation method production ATP that adopts, it is two kinds of main working methods of precursor raw material that the adenosine of employing and VITAMIN B4 are specifically arranged.The employing VITAMIN B4 of public reported is that the method for the synthetic ATP of microbial transformation of precursor raw material mainly contains: Japan consonance fermentation company adopts and produces ammonia bacillus (Cornebacterium ammoniagenes) and transform synthetic ATP (Biosci.Boitechnol.Biochem.; 65 (3); 644～650,2001); Domestic Zhongshan University adopts cereuisiae fermentum to transform synthetic ATP (Zhongshan University's journal natural science edition, 3,15～26,1975); Zhan Guyu etc. adopt mucor aromaticus Poyah 7 strain mucormycosiss such as (Mucor armaticus) to transform synthetic ATP (Northwest University's journal natural science edition; 02; 118～129; 1980) also be not that precursor raw material adopts graceful radiation mucor strain to transform document or the patent report of synthetic ATP so far etc., with the VITAMIN B4.
VITAMIN B4 has adopted the chemical synthesis mass production at present; Therefore adopting VITAMIN B4 is the working method of precursor raw material; Have that cost of material is lower, the theoretical yield of abundant, the synthetic ATP in source is than advantages such as height; Transforming synthetic ATP with Mucor, also to have a yeast culture based raw material simple, advantages such as thalline separate easily.
(3) summary of the invention
The object of the invention provides the new bacterial strain that can synthesize ATP that a strain screens--graceful radiation Mucor (Actinomucor elegans) ZGB1, and the application in the synthetic ATP of mikrobe.
The technical scheme that the present invention adopts is:
Graceful radiation Mucor (Actinomucor elegans) ZGB1; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Postcode: 100101, deposit number: CGMCC No.4313, preservation date: on November 8th, 2010.
The application contriver shakes bottle through spawn culture and transforms, and measures the method for ATP content in the conversion fluid then with paper electrophoresis method, when screening has the mikrobe of synthetic ATP ability, has screened this bacterial classification.On according to bases such as flat-plate bacterial colony characteristic, mycelia form, conidial fructification and spore shape to its evaluation; In conjunction with 18S rDNA sequential analysis contrast (order-checking company:, confirm that this bacterium is graceful radiation Mucor (Actinomucor elegans) Sangon Biotech (Shanghai) Co., Ltd.).So far do not see that having with the VITAMIN B4 is that precursor raw material adopts graceful radiation mucor strain to transform document or the patent report of synthetic ATP.
The 18S rDNA partial sequence of bacterial strain of the present invention (SEQ ID No.1) is as follows:
TAGGATTCCTCGTTGAGAGCAATAATTACAATGCTCTATCCCCAGCACGATGAAGTTTCAAAAGTTTACCCAGACCTTCCGGCCAAGGTTATAAACTCGCTGACTTCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTAAAACTTCCTTCGGCTTGAAGCCGATAGTCTCTCTAAGAAGCCAGATAGATAAATCTATTCACCAACTAAAAAGTTGGCCTGCCTATTTAGCAGAATAAGGTCTCGTTCGTTATCGGAATTAACCAGACAAATCACTCCACGAACTAAGAACGGCCATGCACCACCACCCATAGAATCTAGAAAGAGCTTTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGTCCCATACTCCCCCCAGAACCCAAAAACTTTACTTTCGCTAAGATGCTGAGCCAGTCATAATAAACAGGCCCAATCTCTAGTCGGCATAGTTTGTGGTTAAGACTACGACGGTATCTAATCGTCTTCGATCCCTTAACTTTAGACCTTGATCAATGAAAACGTCCTGGGTCAAATGCTTTCGCAGTAGTTTGTCTTCGGTCAATCCAAGAATTTCACCTCTAGCGACCAAATACAAATGCCCCCAACCGTTTCTATTCATCATTACTCAAGTTCCTGAACCAACGAAATAGGGACTAGAGTCATATTTCATTATTCCATGCTAACACATTCAAGCTTGAAAGCCTGCTTTAAACACTCTGATTTGCTCATGGTAATCATTTGGCTAGAGACTATAGGCGACCGAAGCCACCCATAGCATAACCAAATACAAAAGCCTCGGCAGAAGCGAGCTTGATCAATGAAGACCAGGCCACCTCGCCTAAAGGCTAAAGTTTGACTACGGACGTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGATCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTGCAAGACCTTAAAGGCCCTGCATTGTTATTTATTGTCACTACCTCACCATGTCGGTATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCCCCGTTACCCGTTGTAGGCATTGTAAGCCTCTATCTTACAATCTCAACCATGATAGGGCAGAAAATCGAGTGGACCGTCGCCAGCACAAGGCCATGCGATCCGCTTAATTATTATGAATCACCAAGGGAAACTGGTTTTACCCAGCGTTGGCTTTATCTAATAAGTGCACCCCTTCCGAAGTCAGGGCTTTTTTGCATGTATTAGCTCTAGAATTACCACGGTTATCCAAGTAGTAAAGAATATGTCACGTAGATCATAACTGATTAATGAGCCATCGCA。
ZGB1 strain morphology characteristic and cultural method:
1. morphological specificity
Colonial morphology (approximate referring to Fig. 1～Fig. 4) with general Mucor, flocculence, aerial hyphae highly surpasses 1cm, the initial stage is a pure white, after transfer lark gradually to, stolon and underdeveloped rhizoid are arranged, sporangiophore is upright, diameter 10.5～26.3 μ m.There is a bigger sporocyst on the major branch top, and it often has 3～8 verticillate branches down, and all there is sporocyst on each branch top.Spherical in shape or the foreign pyriform of sporocyst, surperficial fine hair shape is chocolate after the maturation, not of uniform size, diameter 30～70 μ m.Cyst wall is prone to clear up, and the capsule neck is arranged, columella circle, oval or pyriform, and smooth surface, size is uneven, diameter (25.0～60.0) * (20.5～55.0) μ m, big person can reach 67.3～66.1 μ m.Sporangiospore is short avette and spherical, slightly transparent, smooth surface, and size is uneven, and size is (10.5～21.0) * (7.9～24.0) μ m, and big person can reach 28.9 * 28.9 μ m.Sporangiospore expands when sprouting, and is typical one pole and sprouts.Can form chlamydospore.
28 ℃ of this bacterium optimum growth temperatures, 37 ℃ of growths are suppressed, 40 ℃ of growths hardly.
2. the substratum that adopts
Solid slant culture base 1: yam, glucose agar medium (PDA).
Solid slant culture base 2: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, NaCl 5g/L, agar 20g/L, solvent are water, pH 6.0～6.5.
Liquid seed culture medium: glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, NaCl 5g/L, solvent are water, pH 6.0～6.5.
Fermention medium: glucose 30～50g/L, yeast extract paste 1～6g/L, steeping water 5～20g/L, MgSO
47H
2O 0.5～2g/L, FeSO
47H
2O 0.05～0.5g/L, NH
4Cl 1～5g/L, K
2HPO
43H
2O 1～5g/L, solvent are water, and pH 6.0～6.5.
3. culture condition
The slant strains that 28 ℃ on warp, 3～4d cultivate is processed spore suspension, in the inoculum size access liquid seed culture medium with 10% (V/V), places 28 ℃, cultivates 12h in the rotary shaking table of 160r/min.Inoculum size with 10% (V/V) inserts liquid seeds liquid in the fermention medium, places 28 ℃, cultivates 12h, fermentation ends in the rotary shaking table of 160r/min.
The invention still further relates to the application of described graceful radiation Mucor ZGB1 in the synthetic ATP of mikrobe.
Specifically, said being applied as: with the VITAMIN B4 is substrate, and the thalline that obtains with grace radiation Mucor ZGB1 fermentation is the enzyme source, under 25～35 ℃, carries out conversion reaction 6～24 hours, and ATP product content can reach more than the 10g/L in the conversion fluid of reaction end back.Routinely from hand over, concentrate, separations purification process processing such as crystallization can obtain said product A TP.
Preferably, said application method is following:
(1) graceful radiation Mucor ZGB1 is seeded to fermention medium, cultivates 12～18 hours for 28～30 ℃, and fermented liquid separated and collected thalline cleans, filters and did 40～80 ℃ of freeze-day with constant temperature in back 2～4 hours, obtains graceful radiation Mucor ZGB1 thalline; Said fermention medium final concentration consists of: glucose 30～50g/L, yeast extract paste 1～6g/L, steeping water 5～20g/L, MgSO
47H
2O 0.5～2g/L, FeSO
47H
2O 0.05～0.5g/L, NH
4Cl 1～5g/L, K
2HPO
43H
2O 1～5g/L, pH 6.0～6.5; Fermented liquid after the fermentation ends (is 40～80 ℃ of drying treatment 2～4h) of thermostatic drying chamber through penetratingization processing earlier through filtering the thalline of collecting.Because it is very convenient that mucormycosis filter to be collected thalline, uses this penetratingization treatment process to have characteristics easy and simple to handle, that activity of conversion is high,, also has the advantage of environmental protection owing to need in conversion reaction liquid, not add treatment process such as YLENE;
(2) get step (1) graceful radiation Mucor ZGB1 thalline and substrate preparation conversion reaction liquid, under 30～35 ℃, carry out conversion reaction 6～24 hours, reaction finishes the back conversion fluid and obtains said ATP through aftertreatment; Said conversion reaction liquid initial composition is following: glucose 60～90g/L, Na
2HPO
412H
2O 30～90g/L, MgCl
26H
2O3～6g/L, VITAMIN B4 2～6g/L, Mucor thalline 50～100g/L.Behind the reaction 6h, whenever follow the tracks of the conversion situation that detects, cessation reaction in good time with paper electrophoresis method at a distance from 2h.ATP in the reaction solution can reach more than the 10g/L, and existing mucormycosis transforms the highest ATP productive rate of bibliographical information about 8g/L.
Conversion reaction product analysis and method of calculation:
Sampling 5～20 μ L, point sample are damping fluid with the citric acid-sodium citrate solution of pH 2.5 on the wide common filter paper of 20mm, and voltage 400V, electrophoresis time are 30～60min.After electrophoresis finishes; Dry up filter paper with hair dryer; Under uv analyzer, retouch out the product spot with standard A TP spot same position, spot be cut into the slice about 2mm, with the HCl solution of 5ml 0.01mol/L at 28 ℃; Lixiviate 4h in the rotary shaking table of 160r/min, the mensuration vat liquor is absorbancy (OD at the 260nm place
260), calculate the ATP productive rate by the mole specific absorbance.
ATP calculation of yield formula:
ATP productive rate=OD
260Mr
ATPC/E
ATP
In the formula: Mr
ATPBe the ATP molecular weight, C is an extension rate, E
ATPBe the ATP molar absorptivity.
Transformation efficiency equals molar yield, and numerical value equals the ratio of actual ATP productive rate and theoretical ATP productive rate.
Beneficial effect of the present invention is mainly reflected in: screening obtains the new bacterial strain that a strain can be synthesized ATP--graceful radiation Mucor (Actinomucor elegans) ZGB1; Utilize this bacterial strain to be the synthetic ATP of precursor by VITAMIN B4; Have advantages such as cost of material is lower, the source is abundant, the yeast culture based raw material is simple, thalline separate easily, and transformation efficiency is high: in the reaction solution that contains the above VITAMIN B4 of 3g/L, can produce the ATP more than the 10g/L.
(4) description of drawings
Fig. 1 is a bacterial strain vegetative hyphae electromicroscopic photograph of the present invention (10 * 4 times)
Fig. 2 bacterial strain aerial hyphae of the present invention electromicroscopic photograph (10 * 4 times)
Fig. 3 bacterial strain of the present invention is the branched sporangiophore electromicroscopic photograph of total shape (10 * 10 times)
The sporocyst electromicroscopic photograph (10 * 40 times) that Fig. 4 bacterial strain cyst wall of the present invention begins to melt.
(5) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
Graceful radiation Mucor ZGB1 (CGMCC No.4313) is seeded to the PDA slant medium, cultivates 3～4d for 28 ℃, processes spore suspension (about 10 with the saline water washing
6～10
7Cfu/mL);
Spore suspension inserts liquid seed culture medium (glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L with the inoculum size of 10% (V/V); NaCl 5g/L, solvent are water, and pH 6.0～6.5) in; Place 28 ℃, cultivate 12h in the rotary shaking table of 160r/min, obtain liquid seeds liquid;
Liquid seeds liquid inserts fermention medium (glucose 40g/L, yeast extract paste 3g/L, steeping water 10g/L, MgSO with the inoculum size of 10% (V/V)
47H
2O 1g/L, FeSO
47H
2O 0.2g/L, NH
4Cl 3g/L, K
2HPO
43H
2O 3g/L, solvent are water, and pH 6.0～6.5) in, place 28 ℃, cultivate 12h, fermentation ends in the rotary shaking table of 160r/min.
Fermented liquid is collected thalline through suction filtration, and does with the aseptic water washing filter, and the filter dry mycelium passes through penetratingization of the cell processing of 65 ℃ of dry 3h of thermostatic drying chamber again, and it is subsequent use to get the Mucor thalline.
Conversion reaction liquid is formed: glucose 90g/L, Na
2HPO
412H
2O 90g/L, MgCl
26H
2O 4g/L, VITAMIN B4 3g/L, Mucor thalline 80g/L.
The conversion reaction liquid of above-mentioned preparation carries out conversion reaction in 33 ℃, the rotary shaking table of 160r/min, the output in ATP2Na during 10h is 11.14g/L, and molar yield can reach 91.07%.
Embodiment 2:
Graceful radiation Mucor ZGB1 (CGMCC No.4313) solid slant culture base (glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L; NaCl 5g/L, agar 20g/L, solvent are water; PH 6.0～6.5), cultivate 3～4d for 28 ℃, process spore suspension (about 10 with the saline water washing
6～10
7Cfu/mL);
Spore suspension inserts liquid seed culture medium (glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L with the inoculum size of 10% (V/V); NaCl 5g/L, solvent are water, and pH 6.0～6.5) in; Place 28 ℃, cultivate 12h in the rotary shaking table of 160r/min, obtain liquid seeds liquid;
Liquid seeds liquid inserts fermention medium (glucose 40g/L, yeast extract paste 3g/L, steeping water 10g/L, MgSO with the inoculum size of 10% (V/V)
47H
2O 1g/L, FeSO
47H
2O 0.2g/L, NH
4Cl 3g/L, K
2HPO
43H
2O 3g/L, solvent are water, and pH 6.0～6.5) in, place 28 ℃, cultivate 12h, fermentation ends in the rotary shaking table of 160r/min.
Fermented liquid is collected thalline through suction filtration, and does with the aseptic water washing filter, and the filter dry mycelium passes through penetratingization of the cell processing of 60 ℃ of dry 3h of thermostatic drying chamber again, and it is subsequent use to get the Mucor thalline.
Conversion reaction liquid: glucose 90g/L, Na
2HPO
412H
2O 90g/L, MgCl
26H
2O 4g/L, VITAMIN B4 4g/L, Mucor thalline 80g/L.
Above-mentioned conversion reaction liquid carries out conversion reaction in 33 ℃, the rotary shaking table of 160r/min, ATP2Na output is 13.64g/L during reaction 20h, the molar yield 83.63% of ATP2Na.
SEQUENCE?LISTING
< 110>Zhejiang Polytechnical University
< 120>graceful radiation Mucor ZGB1 and the application in the synthetic ATP of mikrobe thereof
<130>
<160> 1
<170> PatentIn?version?3.4
<210> 1
<211> 1522
<212> DNA
<213> Actinomucor?elegans
<400> 1
taggattcct?cgttgagagc?aataattaca?atgctctatc?cccagcacga?tgaagtttca 60
aaagtttacc?cagaccttcc?ggccaaggtt?ataaactcgc?tgacttcatc?agtgtagcgc 120
gcgtgcggcc?cagaacatct?aagggcatca?cagacctgtt?attgcctaaa?acttccttcg 180
gcttgaagcc?gatagtctct?ctaagaagcc?agatagataa?atctattcac?caactaaaaa 240
gttggcctgc?ctatttagca?gaataaggtc?tcgttcgtta?tcggaattaa?ccagacaaat 300
cactccacga?actaagaacg?gccatgcacc?accacccata?gaatctagaa?agagctttca 360
atctgtcaat?ccttactatg?tctggacctg?gtgagtttcc?ccgtgttgag?tcaaattaag 420
ccgcaggctc?cactcctggt?ggtgcccttc?cgtcaattcc?tttaagtttc?agccttgcgt 480
cccatactcc?ccccagaacc?caaaaacttt?actttcgcta?agatgctgag?ccagtcataa 540
taaacaggcc?caatctctag?tcggcatagt?ttgtggttaa?gactacgacg?gtatctaatc 600
gtcttcgatc?ccttaacttt?agaccttgat?caatgaaaac?gtcctgggtc?aaatgctttc 660
gcagtagttt?gtcttcggtc?aatccaagaa?tttcacctct?agcgaccaaa?tacaaatgcc 720
cccaaccgtt?tctattcatc?attactcaag?ttcctgaacc?aacgaaatag?ggactagagt 780
catatttcat?tattccatgc?taacacattc?aagcttgaaa?gcctgcttta?aacactctga 840
tttgctcatg?gtaatcattt?ggctagagac?tataggcgac?cgaagccacc?catagcataa 900
ccaaatacaa?aagcctcggc?agaagcgagc?ttgatcaatg?aagaccaggc?cacctcgcct 960
aaaggctaaa?gtttgactac?ggacgtttta?actgcaacaa?ctttaatata?cgctattgga 1020
gctggaatta?ccgcggctgc?tggcaccaga?cttgccctcc?aattgatcct?cgttaaggga 1080
tttaaattgt?actcattcca?attgcaagac?cttaaaggcc?ctgcattgtt?atttattgtc 1140
actacctcac?catgtcggta?ttgggtaatt?tgcgcgcctg?ctgccttcct?tggatgtggt 1200
agccgtttct?caggctccct?ctccggaatc?gaaccctaat?tccccgttac?ccgttgtagg 1260
cattgtaagc?ctctatctta?caatctcaac?catgataggg?cagaaaatcg?agtggaccgt 1320
cgccagcaca?aggccatgcg?atccgcttaa?ttattatgaa?tcaccaaggg?aaactggttt 1380
tacccagcgt?tggctttatc?taataagtgc?accccttccg?aagtcagggc?ttttttgcat 1440
gtattagctc?tagaattacc?acggttatcc?aagtagtaaa?gaatatgtca?cgtagatcat 1500
aactgattaa?tgagccatcg?ca 1522
Claims (3)
1. graceful radiation Mucor (Actinomucor elegans) ZGB1; Its preserving number is CGMCCNo.4313; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; The address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, preservation date: on November 8th, 2010.
2. the application of graceful radiation Mucor ZGB1 as claimed in claim 1 in the synthetic ATP of mikrobe; Said being applied as: with the VITAMIN B4 is substrate; The thalline that obtains with grace radiation Mucor ZGB1 fermentation is the enzyme source; Under 25～35 ℃, carry out conversion reaction 6～24 hours, conversion fluid obtains said ATP through aftertreatment.
3. application as claimed in claim 2 is characterized in that said application is following:
(1) graceful radiation Mucor ZGB1 is seeded to fermention medium, cultivates 12～18 hours for 28～30 ℃, and fermented liquid separated and collected thalline cleans, filters and did 40～80 ℃ of freeze-day with constant temperature in back 2～4 hours, obtains graceful radiation Mucor ZGB1 thalline; Said fermention medium final concentration consists of: glucose 30～50g/L, yeast extract paste 1～6g/L, steeping water 5～20g/L, MgSO
47H
2O 0.5～2g/L, FeSO
47H
2O 0.05～0.5g/L, NH
4Cl 1～5g/L, K
2HPO
43H
2O 1～5g/L, pH 6.0～6.5;
(2) get step (1) graceful radiation Mucor ZGB1 thalline and substrate preparation conversion reaction liquid, under 30～35 ℃, carry out conversion reaction 6～24 hours, reaction finishes the back conversion fluid and obtains said ATP through aftertreatment; Said conversion reaction liquid initial composition is following: glucose 60～90g/L, Na
2HPO
412H
2O 30～90g/L, MgCl
26H
2O3～6g/L, VITAMIN B4 2～6g/L, graceful radiation Mucor ZGB1 thalline 50～100g/L.
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