CN101037681A - Method for preparing nuclease P1 by ferment process - Google Patents

Method for preparing nuclease P1 by ferment process Download PDF

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CN101037681A
CN101037681A CN 200710063911 CN200710063911A CN101037681A CN 101037681 A CN101037681 A CN 101037681A CN 200710063911 CN200710063911 CN 200710063911 CN 200710063911 A CN200710063911 A CN 200710063911A CN 101037681 A CN101037681 A CN 101037681A
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nuclease
penicillium citrinum
fermentation
culture
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CN100497611C (en
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洪娜
陈慎
黎高沃
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BEIJING YANJING ZHONGKE BIO-TECH Co Ltd
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BEIJING YANJING ZHONGKE BIO-TECH Co Ltd
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Abstract

The invention provides a fermentation method for preparing nuclease P1, which using directly penicillium citrinum sporogon as seed, fermenting by means of fed-batch sugar supplement to improve activity of the nuclease P1, the mehod without first and second order seed culture shorts production cycle greatly and raises efficiency. The invetion also uses obtained enzyme for preparing 5'-mononucleotide with 85-90% rate of enzymatichydrolysis and 15 mg/ml of producing rate of nucleotide. The invention also provides a penicillium citrinum 3.2788-Y01 of nuclease P1, which fungus stroing number is CGMCC NO.1943, the stem can raise one times active unit of the nuclease P1.

Description

A kind of fermentation method prepares nuclease P 1Method
Technical field
The present invention relates to biological technical field, be specifically related to a kind of production and prepare nuclease P 1Method.
Background technology
The degraded product of nucleic acid---Nucleotide and derivative thereof have purposes (Chen Taosheng very widely in fields such as food, medical science, agricultural, makeup and biochemical reagents, the The modern industry microbiology first volume, Science and Technology of Shanghai press, 1979,153～164), and nuclease P 1It is the main enzyme of using that enzymolysis RNA, DNA become 5 '-Nucleotide.
Eighties of last century sixties, Japan just begins to obtain freshener from degradation of rna in order to open up monosodium glutamate market---seasoning Nucleotide (5 '-GMP and 5 '-IMP etc.).Bright grade has screened Penicillium citrinum (Penicillium citrinum) (the Agric Biol Chem 25 that a penicillium belongs in Japanese scholar of the nineteen fifty-seven state, 693 (1961)), can secrete a kind of extracellular enzyme, have the function of phosphodiesterase, enzymolysis RNA generates 5 ' NMP.Behind this enzyme purification, find outside this enzyme decapacitation degradation of rna, can also degradation of dna, but also have the phosphoric acid of cutting 3 '-mononucleotide, promptly the function of 3 '-phosphomonoesterase is different with typical phosphodiesterase, so this enzyme called after nuclease P 1, P wherein 1Be conventional letter (Agric Biol Chem 38:785,1974 of Penicillium citrinum; 39:579,1975).After this, enzymolysis RNA generates 5 '-NMP and adopts Penicillium citrinum production nuclease P 1
At home, Shanghai light industry institute in 1964 and institute of microbiology of academy of sciences etc. have successively studied with Penicillium citrinum and have produced nuclease P 1Method, industrial microorganism institute in Shanghai produces the enzyme higher nuclease P that lives with oak aspergillus (Aspergillus Fumigatus Fresemius) subsequently 1, 1974 to 1975, shanghai food source mill adopted Penicillium citrinum Penicillium citrinum 3.2788 to carry out solid culture and prepares nuclease P 1, (University Of Ningbo's journal 10 (3): 21～28).
Domestic " capital brew-house and the Chinese Academy of Sciences's 824 task groups " also adopt this bacterium Penicillium citrinum 3.2788 bacterial strains to eighties of last century to the beginning of the seventies end of the sixties, and the applying solid culture method is produced nuclease P 1, degradation of rna generates 5 '-NMP.(production of ucleotides material and application, Science Press, 1971)
Guangdong Jiangmen and North China Pharmaceutical Factory once used liquid culture method and produced nuclease P 1But vigor is not high, and statement all is with mycelium inoculation fermentation jar, does not see with spore direct inoculation fermentor tank (internal communication, 1982).Produce nuclease P with respect to plate method 1, liquid culture method possesses the advantage of environmental protection.
About nuclease P 1Separate the nucleic acid aspect, some reports (Agric Biol Chem 38 (9): 1555-1561 is arranged; 785～790 1974) but be mostly to test and prove on the mode of action scheduling theory 38 (11): 2141～2,147 38 (4): to substrate specificity; According to these theories, nuclease P 1To the RNA enzymolysis, should be completely; But in industrial application, to the enzymatic hydrolyzation of RNA only a little more than 70% (Liu Shuhuang, zymin industry, volume two P765, Science Press, 1984).No matter original production method uses solid and liquid culture, and enzyme activity unit is all lower, has only 100～150 units per ml, and fermentation period is long, and production cost is big.
Summary of the invention
First purpose of the present invention is to provide a kind of preparation nuclease P at above-mentioned deficiency 1Method;
Another object of the present invention is to provide a kind of nuclease P that high vigor is arranged at above-mentioned deficiency 1Liquid is to improve the enzymatic hydrolyzation to nucleic acid.
Further object of the present invention provides a kind of nuclease P 1Superior strain.
Fermentative Production nuclease P of the present invention 1Be to utilize the sporophyte of Penicillium citrinum to carry out fermentation culture, thereby shortened the cycle of fermentation culture widely, simultaneously P 1The unit enzyme work of enzyme liquid is improved; Adopt batch feeding ground mode to add substratum and glucose during the fermentation, further improved P 1The unit enzyme of enzyme is lived.Be that the present invention prepares nuclease P below 1Concrete grammar:
1, actication of culture
Get 1 ring bacterium and insert the potato slope substratum, 28～30 ℃ are cultivated 3 days is the first-generation, and the rest may be inferred, switching 3～5 generation slant medium, and routine is preserved standby.
2, enlarged culturing
With the sterilized water thorough washing fresh the 4th generation the inclined-plane, obtain spore suspension, get in the bran mass that 0.1～2.0 milliliter of access prepares, cultivated 3 days for 28～30 ℃.Wheat bran spore counting reaches 10 5～10 9Individual/as to restrain to qualified.
3, fermentation culture
The obtaining liq substratum is pressed 70～80% tinnings of fermentor tank useful volume;
The wheat bran spore that step 2 is obtained to fermentor tank, carries out fermentation culture by 0.35～0.6 grams per liter culture medium inoculated, and fermentation culture adopts the mode of in batches mending sugar to carry out.
Fermentation condition is controlled at pH3.0～6.0,28～30 ℃ of temperature, and tank pressure 0.03～0.1Mpa, air flow 0.5～2.0: 1,100～300 rev/mins of rotating speeds.
Usually fermentation enters logarithmic phase after about 7 hours; Began to enter in 24 hours and produce the enzyme phase.
Fermented about 22～40 hours, 2～6 hours at interval, add liquid nutrient medium 2～6 times in batches, be example with 2 tons of fermentor tanks, add the required siccative of 250L fermentation tank culture medium at every turn.
Ferment about 30～stop preceding two hours, 2～6 hours at interval, add glucose in batches, add altogether 2～6 times, be example with 2 tons of fermentor tanks, each 5～20 kilograms.
Fermented 40～60 hours, and, surpassed 4.5 and 60% respectively and just can stop fermenting when pH value and dissolved oxygen curve are obvious ascendant trend.
4, Plate Filtration
Adopt the method for Plate Filtration to carry out solid-liquid separation, obtain nuclease P 1Liquid.
5, nuclease P 1The preservation of liquid
Deepfreeze is preserved.
6, utilize the resulting nuclease P of above-mentioned fermentation 1Liquid is used for enzymolysis Yeast Nucleic Acid, and enzymatic hydrolyzation is up to 85～90%, and the final concentration of Nucleotide can reach more than the 15mg/ml in the enzymolysis solution of gained.
The present invention also provides a kind of nuclease P 1Superior strain Penicillium citrinum (Penicilliumcitrinum) 3.2788-Y01, this bacterial strain is from Penicillium citrinum 3.2788 bacterial strains, through physical chemistry complex mutations such as too high zinc ion concentration, Sodium Nitrite and ultraviolet rays, and purifying screens, and nearly thousand strain bacterium obtain, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCC No.1943 on February 8th, 2007.
Above involved culture medium prescription be respectively:
Potato slant medium: potato (section) 300 grams, glucose 20 grams, agar 15 grams, water 1000ml)
Wheat bran solid medium: wheat bran: water=1: 1.0～1.5
Liquid fermentation medium: phosphoric acid salt 0.5～5g/L, sal epsom 0.1～1g/L, lime carbonate 0.2～2g/L, zinc sulfate 0.1～0.5g/L, industrial peptone 1～10g/L, cerelose 1～5g/L.
The present invention has following advantage
1, use the spore direct inoculation fermentor tank of cultivating on the wheat bran, do not need I and II seed fermentation jar fermentation culture, reduced equipment cost, simplified technology, fermentation period shortened about 5～6 days of production cycle about 50 hours; In addition, if use mycelium inoculation, fermentation period is more than 60 hours, shortens fermentation period comparatively speaking about 10 hours;
2, adopt Penicillium citrinum 3.2788-Y01 strain fermentation, can make nuclease P 1Vigor increases by 200% on the basis of former bacterial strain, reach 300 units per ml;
3, replace batch fermentation with fed-batch fermentation, can make biomass increase by 50%, the nuclease P 1 vigor is brought up to more than 500 units per ml from 300 units per ml.
4, the nuclease P that adopts Penicillium citrinum 3.2788-Y01 bacterial strain to produce 1Liquid carries out enzymolysis, can obtain enzymatic hydrolyzation up to 85～90%, and the concentration of Nucleotide can reach more than the 15mg/ml.
Description of drawings
Fig. 1 is technological process of production figure of the present invention.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
Embodiment 1 preparation wheat bran spore
1, actication of culture
Get 1 ring bacterium and insert the potato slope substratum, 28～30 ℃ are cultivated 3 days be the first-generation, and the rest may be inferred, transfer four generation slant medium, conventional preservation is standby.
2, enlarged culturing
With the sterilized water thorough washing fresh the 4th generation the inclined-plane, obtain spore suspension, get in the bran mass that 0.1～2.0 milliliter of access prepares, cultivated 3 days for 28～30 ℃.Wheat bran spore counting reaches 10 9Individual/gram.
Embodiment 2 (2 tons of fermentor tanks)
1, culture medium prescription: phosphoric acid salt 15Kg, sal epsom 3Kg, lime carbonate 3Kg, zinc sulfate 1.2Kg, industrial peptone 15Kg, cerelose 9Kg, water complements to 1500L.
2, inoculum size: 900 gram wheat bran spores (embodiment 1);
3, culture temperature: 29～30 ℃;
4, cultivate rotating speed: 150rpm～250rpm, when dissolved oxygen reduces to 40% when following, the adjusting rotating speed;
7, air flow: 1.5m 3/ min;
8, tank pressure: 0.05～0.06Mpa;
9, initial pH value: 6.0
10, supplemented medium:
About 24.1 hours of fermentation culture, the beginning substratum is added the required siccative of 250L fermentation tank culture medium for the first time;
About 29.5 hours of fermentation culture is carried out substratum and is added the required siccative of 250L fermentation tank culture medium for the second time;
About 32 hours of fermentation culture is carried out substratum and is added the required siccative of 250L fermentation tank culture medium for the third time.
11, add glucose:
About 34 hours of fermentation culture carries out mending the first time 5 kilograms of glucose;
About 36 hours of fermentation culture carries out mending the second time 5 kilograms of glucose;
About 38 hours of fermentation culture is mended 5 kilograms of glucose for the third time;
About 43 hours of fermentation culture is carried out the 4th time and is mended 5 kilograms of glucose.
7, stop fermentation:
Fermented 46 hours, pH value and dissolved oxygen curve are obvious ascendant trend, reach 4.7 and 62% respectively.Nuclease P 1Vigor 532 units per ml, 1250 liters of fermentation final volume.
8, enzymolysis (8 tons of jars)
Enzymatic hydrolyzation is 90%, and the final concentration of Nucleotide is 16.2mg/ml.
Embodiment 3 (2 tons of fermentor tanks)
1, culture medium prescription: phosphoric acid salt 3Kg, sal epsom 1.3Kg, lime carbonate 1.3Kg, zinc sulfate 1.5Kg, industrial peptone 30Kg, cerelose 6Kg, water complements to 1500L.
2, inoculum size: 840 gram wheat bran spores;
3, culture temperature: 29～30 ℃;
4, cultivate rotating speed: 150rpm～270rpm, when dissolved oxygen reduces to 40% when following, the adjusting rotating speed;
7, air flow: 1.5m 3/ min;
8, tank pressure: 0.05～0.06Mpa;
9, initial pH value: 5.9
10, supplemented medium:
About 23 hours of fermentation culture, the beginning substratum is added the required siccative of 250L fermentation tank culture medium for the first time;
About 28 hours of fermentation culture is carried out substratum and is added the required siccative of 250L fermentation tank culture medium for the second time;
About 33 hours of fermentation culture is carried out substratum and is added the required siccative of 250L fermentation tank culture medium for the third time;
About 36 hours of fermentation culture is carried out substratum and is added the required siccative of 250L fermentation tank culture medium the 4th time;
11, add glucose:
About 38.5 hours of fermentation culture carries out mending the first time 5 kilograms of glucose;
About 43 hours of fermentation culture carries out mending the second time 15 kilograms of glucose.
About 57 hours of fermentation culture is mended 5 kilograms of glucose for the third time.
11, stop fermentation:
Fermented 59 hours, pH value and dissolved oxygen curve are obvious ascendant trend, reach 4.6 and 60% respectively.Nuclease P 1Vigor 662 units per ml, 1280 liters of fermentation final volume.
12, enzymolysis (8 tons of jars)
Enzymatic hydrolyzation is 88%, and the final concentration of Nucleotide is 16.1mg/ml.
The test example
1, the mensuration of enzyme activity:
Take by weighing 100mg Yeast Nucleic Acid (purity 100%) and be dissolved in 5 ml waters, be 2% standard nucleic acid solution.
Ammonium molybdate-mistake chloric acid reagent: 0.25% ammonium molybdate is dissolved in 2.5% and crosses in the chloric acid.(as joining then adding 70% mistake chloric acid 7ml and 0.5g ammonium molybdate in 193ml water of this reagent of 200ml)
Get the first and second two test tubes, add 2% nucleic acid 1ml and 0.8ml 0.2M pH5 acetate buffer solution respectively.63 ℃ of preheatings 10 minutes, the first pipe adds 0.2ml enzyme liquid then.Continue insulation 30 minutes.First and second liang of pipes add 2ml ammonium molybdate-mistake chloric acid reagent respectively.The second pipe is added 0.2ml enzyme liquid, mixing.Cooling is placed after 15 minutes centrifugal.Get the 0.1ml supernatant liquor respectively and add the 9.9ml deionized water.Survey OD 260Value.Enzyme activity=(OD 260First-OD 260Second) * 100 * enzyme liquid extension rate
2, the mensuration of 5 '-mononucleotide:
Degradation solution sucts clear liquid 0.1 microlitre constant volume to 10 milliliter after treatment.Standard needs before use with 10 times of standard phosphorus solution dilutions, get test tube according to the form below table 1 order (one two three four five) and add sample and reagent, each Guan Fan finishes behind the methylamine reagent 45 ℃ of water bath heat preservations 10 minutes, reorder respectively again 3 milliliters of phosphorus reagent, continuation was 45 ℃ of insulations 20 minutes, after being cooled to room temperature, with blank in contrast, survey OD 650Simultaneously decide the phosphorus value as reference with standard phosphorus measurement result.The difference of first (oxidation) and second (not oxidation) phosphorus content is 5 '-Nucleotide phosphorus.
The mensuration of table 15 '-mononucleotide
Figure A20071006391100101
Figure A20071006391100102
Be reduced to: 5 '-Nucleotide (g)=(first-second) phosphorus content (mg/ml) * 1.097 * cumulative volume (L)
31-phosphorus atom amount;
Four kinds of mononucleotide molecular-weight average of 340-;
100-diluted sample multiple.
4, the calculating of nucleolysis rate:
Figure A20071006391100103

Claims (7)

1, a kind of fermentation method prepares nuclease P 1Method, it is characterized in that, adopt the Penicillium citrinum sporophyte to carry out fermentation culture.
2, the method for claim 1 is characterized in that, adopts the mode of batch feeding to carry out fermentation culture.
3, method as claimed in claim 2 is characterized in that, earlier fermentation is supplemented medium in batches, and back group after group at different time is added glucose.
4, the method for claim 1 is characterized in that, described Penicillium citrinum sporophyte obtains in the following way: get the Penicillium citrinum bacterial strain activated after, get its spore and make the suspension inoculation bran mass, cultivated 3 days for 28～30 ℃.
As each described method of claim 1～4, it is characterized in that 5, described Penicillium citrinum is Penicillium citrinum 3.2788 or Penicillium citrinum 3.2788-Y01.
6, the nuclease P that makes according to the described method of claim 1～5 1Liquid.
7, a kind of nuclease P 1Superior strain Penicillium citrinum (Penicillium citrinum) 3.2788-Y01 CGMCC No.1943.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104560913A (en) * 2014-12-24 2015-04-29 南京工业大学 Method for preparing nuclease P1 through penicillium citrinum semi-continuous fermentation
CN106318926A (en) * 2015-06-19 2017-01-11 安琪酵母股份有限公司 Method for producing nuclease P1 through fermentation of penicillium citrinum
WO2017068012A1 (en) * 2015-10-21 2017-04-27 Novozymes A/S Direct inoculation
CN113980930A (en) * 2021-12-07 2022-01-28 南京工业大学 Preparation method of nuclease P1

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104560913A (en) * 2014-12-24 2015-04-29 南京工业大学 Method for preparing nuclease P1 through penicillium citrinum semi-continuous fermentation
CN104560913B (en) * 2014-12-24 2017-11-03 南京工业大学 A kind of method that Penicillium citrinum semicontinuous fermentation prepares nuclease P 1
CN106318926A (en) * 2015-06-19 2017-01-11 安琪酵母股份有限公司 Method for producing nuclease P1 through fermentation of penicillium citrinum
CN106318926B (en) * 2015-06-19 2019-10-25 安琪酵母股份有限公司 A kind of Penicillium citrinum fermentation produces the production method of nuclease P 1
WO2017068012A1 (en) * 2015-10-21 2017-04-27 Novozymes A/S Direct inoculation
CN108026559A (en) * 2015-10-21 2018-05-11 诺维信公司 Directly it is inoculated with
CN113980930A (en) * 2021-12-07 2022-01-28 南京工业大学 Preparation method of nuclease P1
CN113980930B (en) * 2021-12-07 2023-03-03 南京工业大学 Preparation method of nuclease P1
WO2023103543A1 (en) * 2021-12-07 2023-06-15 南京工业大学 Method for preparing nuclease p1

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