CN103820418A - Method for producing nuclease P1 through large-scale liquid submerged fermentation - Google Patents

Method for producing nuclease P1 through large-scale liquid submerged fermentation Download PDF

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CN103820418A
CN103820418A CN201310507157.9A CN201310507157A CN103820418A CN 103820418 A CN103820418 A CN 103820418A CN 201310507157 A CN201310507157 A CN 201310507157A CN 103820418 A CN103820418 A CN 103820418A
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fermentation
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曾化伟
乔洁
饶胜其
李煜林
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

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Abstract

The invention provides a technology for producing nuclease by adopting penicillium citrinum strain to perform large-scale liquid submerged fermentation. The technology comprises the following steps: firstly, performing slant culture of the strain, secondly, performing strain culture on a shaker, thirdly, performing strain enlarging culture in a fermentation tank with the volume of 500 L, and fourthly, performing large-scale production in the fermentation tank. By the adoption of the fermentation technology, the fermentation production time is only 50 to 70h in a 10-ton fermentation tank, and the activity of nuclease in fermentation liquor reaches 1,180 U/ml. The technology is relatively low in cost; compared with the activity (300 U/ml) of malt sprout extracted enzyme stock solution, the enzyme activity is greatly improved.

Description

A kind of large-scale deep liquid fermentation is produced the method for nuclease P 1
Technical field
The present invention relates to microorganism fermenting enzyme preparation technique field, particularly a kind of large-scale deep liquid fermentation is produced the method for nucleic acid P1.
Technical background
5 '-Nucleotide has purposes widely in food, agricultural and medicine and other fields.In field of food, 5 '-Nucleotide is the basal component of of animal or plant nature taste of food, especially guanosine monophosphate (5 '-Guanosine Monophosphate wherein, be called for short GMP) and inosine monophosphate (5 '-Inosine Monophosphate, be called for short IMP), they present a kind of delicious food of self uniqueness in the time being more than or equal to its thresholding, are used as in a large number flavoring substance.5 '-Nucleotide is used in infant food can obviously improve baby's immunological competence as additive, promote the maturation of enteron aisle, promote the synthetic of lipoprotein and polyunsaturated fatty acid, reduce the generation of the diseases such as baby's flu and diarrhoea, be conducive to baby's normal growth and growth; In agriculture field, 5 '-Nucleotide can be used as plant nutrition additive, after use, can obviously improve the output of the farm crop such as paddy rice, soybean, and the highest volume increase can reach 40%.Raising for animal can improve rate of body weight gain and the feed rate of utilization of animal, and material consumption obviously reduces; In field of medicaments, 5 '-Nucleotide can synthesize many antiviral, antitumor drugs, and its corresponding derivative plays a very important role in aspect diseases such as treatment central nervous system, the recycle system and urinary systems; In field of fine chemical, nucleic acid material is as one of composition of makeup, has skin care, the effect such as wrinkle resistant; At biochemical field, new nucleic acid material, as the appearance of novel oligonucleotide, can be used as the probe of target cell for studying the function of gene, the relevant aspect such as genetic locus and genomic library mapping, even for the Study on mechanism of corresponding anticancer preparation, for molecular biology research provides new ways and means.
Nuclease P 1 (5 '-phosphodiesterase, nuclease P1) 3 in can hydrolysed nucleic acid ', 5 '-phosphodiester bond obtains 4 kind 5 '-Nucleotide, there is lack of raw materials and adjust the expansion of extract production capacity as the yeast of seasonings for 5 of China '-Nucleotide, needs the nuclease P 1 for these productions in a large number for this reason.From current research and industrialization status, nuclease P 1 is mainly to be produced and root of Cornu Cervi Pantotrichum extraction acquisition by Penicillium citrinum (Penicillium citrinum) liquid fermenting, but may contain some toxic substances in root of Cornu Cervi Pantotrichum, the enzyme running water simultaneously extracting is flat lower, the work of original enzyme liquid enzyme is only 300U/ml, and it is larger affected by root of Cornu Cervi Pantotrichum quality, so root of Cornu Cervi Pantotrichum extraction method is abandoned gradually in suitability for industrialized production.The nuclease P 1 that Penicillium citrinum liquid fermenting is produced can be widely used in the industries such as food, and to human body toxicological harmless effect, thereby Penicillium citrinum production nuclease P 1 is more suitable in industrialized production.But the fermentative production of the unrealized nuclease P 1 of domestic enterprise, a kind of method that the invention provides large-scale deep liquid fermentation for this reason and produce this enzyme.
Summary of the invention
The object of the invention is to provide a kind of liquid submerged fermentation and produces the method for nuclease P 1, and requires the method to have higher enzyme activity and lower cost.
The present invention is achieved through the following technical solutions:
Seed culture based formulas is: cerelose 30～50g/L, industrial ox bone peptone 2～5g/L, industrial yeast cream 2～5g/L, K 2hPO 40.5～1.0g/L, KH 2pO 43H 2o0.5～1.0g/L, MgSO 47H 2o0.5～1.0g/L, CaCl 20.5～1.0g/L, bubble enemy 0.5～1.0g/L, all the other are water, pH value 5.5～7.0.
The culture medium prescription of fermentor tank is: cerelose 3～5g/L, industrial sucrose 40～70g/L, industrial ox bone peptone 5～8g/L, industrial corn starch 5～8g/L, wheat bran 5～8g/L, K 2hPO 40.5～1.0g/L, KH 2pO 43H 2o0.5～1.0g/L, MgSO 47H 2o0.5～1.0g/L, CaCl 20.5～1.0g/L, bubble enemy 0.5～1g/L, all the other are water, pH value 5.5～7.0.
Concrete zymotechnique is:
(1) preparation on inclined-plane: the access of Penicillium citrinum bacterial classification is equipped with in the eggplant-shape bottle of PDA solid medium, is placed in 25～28 ℃ of constant incubators and cultivates 4～7d.
(2) preparation of shake-flask seed: the Penicillium citrinum bacterial classification in eggplant-shape bottle is scraped in the triangular flask that seed culture medium is housed, put into 25～28 ℃ of constant-temperature tables, cultivate 1～3d with 100～150rpm/min;
(3) enlarged culturing again of 500L seeding tank: after cooling the stirred-tank fermenter sterilizing that 300L seed culture medium is housed, regulate pH value to 5.5～7.0, by in appropriate secondary shake-flask seed access seeding tank, keeping culture temperature is 25～28 ℃, stirring velocity is 100～150rpm/min, air flow is 1:0.8～1.5V/V.min, and tank pressure 0.03～0.06MPa cultivates through 30～40h; (4) 10 tons of fermentor tanks are produced: pack 7 tons of fermention mediums into fermentor tank, after sterilizing is cooling, regulate pH value to 5.5～7.0, the seed of seeding tank is moved in fermentor tank, keeping culture temperature is 25～28 ℃, and stirring velocity is 50～150rpm/min, and air flow is 1:0.5～1:1.5V/Vmin, tank pressure 0.03～0.06MPa, cultivates through 50～70h.
(7) preparation of enzyme liquid and measure enzyme and live: get fermented liquid 20ml and be added to the centrifuge tube of 50ml, 3000r/min in whizzer, after centrifugal 5min, supernatant liquor is enzyme liquid, and measures enzyme according to following method and live.
Nuclease P 1 measuring method: (add 1.9ml substrate solution (3% yeast rna in test tube, boil 20min, regulate pH to 5.0, with pH be 5.0 acetic acid-sodium-acetate buffer constant volume), be placed in after 68 ℃ of water bath heat preservation 10min, add enzyme liquid 0.1ml, continue after insulation 15min, put in frozen water cooling, add rapidly 2.0ml nucleic acid precipitation agent (0.25% ammonium molybdate-2.5% perchloric acid), continue cooling 10min, in the centrifugal 15min of 3500r/min, get 0.2ml supernatant liquor and add distilled water dilution and be settled to 50ml, in 260nm place mensuration absorbancy.Blank: 1.9ml substrate, at 68 ℃ of insulation 25min, add nucleic acid precipitation agent 2.0ml, more enzyme-added liquid 0.1ml, the same sample determination of remaining step.
Calculation formula is as follows: enzyme (u/mL fermented liquid)=(A × 4 × 50)/(0.1 × 0.2 × 15) alive, in formula: A refers to the light absorption value at 260nm place; It is 4ml that 4 fingers react easy cumulative volume; 50 appointments are dissolved in 50ml volumetric flask; 0.1 finger enzyme liquid amasss as 0.1mi; 0.2 finger is drawn 0.2ml enzymolysis solution; 15 finger reaction total times were 15min.
Advantage of the present invention: this technique culture medium prescription is that industry is conventional, and average price is more cheap, fermentation time is shorter and ferment largerly in addition, and enzyme activity is far longer than the enzyme activity extracting by root of Cornu Cervi Pantotrichum.In general, this technique has good industrial application potentiality.
Embodiment
Example 1:
This example, take the Penicillium citrinum bacterial classification of having preservation by oneself as starting strain ferments, specifically comprises the following steps: be equipped with the access of Penicillium citrinum bacterial classification in PDA solid medium eggplant-shape bottle (1), is placed in 28 ℃ of constant incubators and cultivates 4d; (2) 2 Penicillium citrinum eggplant-shape bottle bacterial classifications are all scraped and are equipped with in 8L first order seed substratum, and be divided in the triangular flask of 4 5L, put into 28 ℃ of constant-temperature tables, under rotating speed 150rpm/min, cultivate 2d (3) and 300L seed culture medium will be housed after the sterilizing of 500L stirred-tank fermenter is cooling, regulate pH value to 6.5, by in 8L seed access seed fermentation tank, keeping culture temperature is 28 ℃, stirring velocity is 100rpm/min, air flow is 1:0.8V/V.min, tank pressure 0.4MPa, cultivates culture transferring through 35h; (4) pack 7 tons of fermention mediums into fermentor tank, after sterilizing is cooling, regulate pH value to 6.5, the seed of seeding tank is moved in fermentation culture tank, keeping culture temperature is 28 ℃, stirring velocity is 100rpm/min, air flow is 1: 0.8V/Vmin, tank pressure 0.04MPa finishes fermentation after 60h cultivates.Fermented liquid ion measurement enzyme is lived as 1180U/ml.
Example 1 shake-flask seed substratum used and fermentor tank seed culture medium are: cerelose 50g/L, industrial ox bone peptone 2g/L, yeast extract paste 2g/L, K 2hPO 40.5g/L, KH 2pO 43H 2o0.5g/L, MgSO 47H 2o0.5g/L, CaCl 20.5g/L, bubble enemy 0.5g/L, all the other are water, pH value 6.5.
Example 1 10 tons of tank fermention mediums used are: cerelose 3g/L, industrial sucrose 60g/L, industrial ox bone peptone 8g/L, industrial corn starch 5g/L, wheat bran 5g/L, K 2hPO 40.5g/L, KH 2pO 43H 2o0. 5g/ L, MgSO 47H 2o0.5g/L, CaCl 20.5g/L, bubble enemy 0.5g/L, all the other are water, pH value 6.5.
It should be noted that: although the present invention only lifts a specific examples, obviously the invention is not restricted to above example, can also have many distortion.Those of ordinary skill in the art can be easy to it to make some modifications or improvements.Therefore, any modification or the improvement made without departing from theon the basis of the spirit of the present invention, all should think the scope of protection of present invention.

Claims (3)

1. liquid submerged fermentation is produced a method for nuclease, and its feature comprises the following steps:
(1) preparation on inclined-plane: the access of Penicillium citrinum bacterial classification is equipped with in the eggplant-shape bottle of PDA solid medium, is placed in 25～28 ℃ of constant incubators and cultivates 4～7d;
(2) preparation of shake-flask seed: scraping slant strains is inoculated in the triangular flask that seed culture medium is housed, puts into 25～28 ℃ of constant-temperature tables, cultivates 1～3d with 100～150rpm/min;
(3) enlarged culturing of 500L seeding tank: after cooling the stirred-tank fermenter sterilizing that 300L seed culture medium is housed, regulate pH value to 5.5～7.0, by in appropriate shake-flask seed access seeding tank, keeping culture temperature is 25～28 ℃, stirring velocity is 50～150rpm/min, air flow is 1:0.8～1:1.5V/V.min, and tank pressure 0.03～0.06MPa cultivates culture transferring through 30～40h; (4) 10 tons of fermentor tanks are produced: pack 7 tons of fermention mediums into fermentor tank, after sterilizing is cooling, regulate pH value to 5.5～7.0, the seed of seeding tank is moved in fermentor tank, keeping culture temperature is 25～28 ℃, and stirring velocity is 50～150rpm/min, and air flow is 1:0.5～1: 1.5V/V.min, tank pressure 0.03～0.06MPa, cultivates and finishes to put tank through 50～70h.
2. fermentation process according to claim 1, the seed culture based formulas described in its feature is: cerelose 30～50g/L, industrial ox bone peptone 2～5g/L, industrial yeast cream 2～5g/L, K 2hPO 40.5～1.0g/L, KH 2pO 43H 2o0.5～1.0g/L, MgSO 47H 2o0.5～1.0g/L, CaCl 20.5～1.0g/L, bubble enemy 0.5～1.0g/L, all the other are water, pH value 5.5～7.0.
3. fermentation process according to claim 1, the fermentor cultivation based formulas described in its feature is: cerelose 3～5g/L, industrial sucrose 40～70g/L, industrial ox bone peptone 5～8g/L, industrial corn starch 5～8g/L, wheat bran 5～8g/L, K 2hPO 40.5～1.0g/L, KH 2pO 43H 2o0.5～1.0g/L, MgSO 47H 2o0.5～1.0g/L, CaCl 20.5～1.0g/L, bubble enemy 0.5～1g/L, all the other are water, pH value 5.5～7.0.
CN201310507157.9A 2013-10-23 2013-10-23 Method for producing nuclease P1 through large-scale liquid submerged fermentation Withdrawn CN103820418A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

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Application publication date: 20140528