CN113980930A - A kind of preparation method of nuclease P1 - Google Patents
A kind of preparation method of nuclease P1 Download PDFInfo
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- CN113980930A CN113980930A CN202111483356.1A CN202111483356A CN113980930A CN 113980930 A CN113980930 A CN 113980930A CN 202111483356 A CN202111483356 A CN 202111483356A CN 113980930 A CN113980930 A CN 113980930A
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- 101710149004 Nuclease P1 Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000000855 fermentation Methods 0.000 claims abstract description 156
- 230000004151 fermentation Effects 0.000 claims abstract description 156
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 22
- 239000012465 retentate Substances 0.000 claims abstract description 17
- 229920002477 rna polymer Polymers 0.000 claims abstract description 17
- 239000012528 membrane Substances 0.000 claims abstract description 14
- 241000228153 Penicillium citrinum Species 0.000 claims abstract description 10
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 6
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 230000002255 enzymatic effect Effects 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 36
- 108090000790 Enzymes Proteins 0.000 abstract description 35
- 102000004190 Enzymes Human genes 0.000 abstract description 35
- 238000000034 method Methods 0.000 abstract description 26
- 239000002773 nucleotide Substances 0.000 abstract description 15
- 125000003729 nucleotide group Chemical group 0.000 abstract description 15
- 239000002699 waste material Substances 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 20
- 239000002609 medium Substances 0.000 description 20
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- 238000001816 cooling Methods 0.000 description 10
- 239000000843 powder Substances 0.000 description 9
- CQIUKKVOEOPUDV-IYSWYEEDSA-N antimycin Chemical compound OC1=C(C(O)=O)C(=O)C(C)=C2[C@H](C)[C@@H](C)OC=C21 CQIUKKVOEOPUDV-IYSWYEEDSA-N 0.000 description 8
- CQIUKKVOEOPUDV-UHFFFAOYSA-N citrinine Natural products OC1=C(C(O)=O)C(=O)C(C)=C2C(C)C(C)OC=C21 CQIUKKVOEOPUDV-UHFFFAOYSA-N 0.000 description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 239000001888 Peptone Substances 0.000 description 7
- 108010080698 Peptones Proteins 0.000 description 7
- 239000001110 calcium chloride Substances 0.000 description 7
- 229910001628 calcium chloride Inorganic materials 0.000 description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 7
- 235000019341 magnesium sulphate Nutrition 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 description 7
- 235000019319 peptone Nutrition 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 4
- 229920002492 poly(sulfone) Polymers 0.000 description 4
- 230000000087 stabilizing effect Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- MVMSCBBUIHUTGJ-UHFFFAOYSA-N 10108-97-1 Natural products C1=2NC(N)=NC(=O)C=2N=CN1C(C(C1O)O)OC1COP(O)(=O)OP(O)(=O)OC1OC(CO)C(O)C(O)C1O MVMSCBBUIHUTGJ-UHFFFAOYSA-N 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- MVMSCBBUIHUTGJ-LRJDVEEWSA-N GDP-alpha-D-glucose Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=C(NC(=O)C=2N=C1)N)OP(O)(=O)OP(O)(=O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O MVMSCBBUIHUTGJ-LRJDVEEWSA-N 0.000 description 1
- MVMSCBBUIHUTGJ-GDJBGNAASA-N GDP-alpha-D-mannose Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=C(NC(=O)C=2N=C1)N)OP(O)(=O)OP(O)(=O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O MVMSCBBUIHUTGJ-GDJBGNAASA-N 0.000 description 1
- 206010017915 Gastroenteritis shigella Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- LFTYTUAZOPRMMI-CFRASDGPSA-N UDP-N-acetyl-alpha-D-glucosamine Chemical compound O1[C@H](CO)[C@@H](O)[C@H](O)[C@@H](NC(=O)C)[C@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 LFTYTUAZOPRMMI-CFRASDGPSA-N 0.000 description 1
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229920000392 Zymosan Polymers 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013538 functional additive Substances 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
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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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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/30—Endoribonucleases active with either ribo- or deoxyribonucleic acids and producing 5'-phosphomonoesters (3.1.30)
- C12Y301/30001—Aspergillus nuclease S1 (3.1.30.1)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明涉及生物技术领域，具体公开一种核酸酶P1的制备方法，在含发酵促进剂的发酵培养基中，利用桔青霉进行发酵，制备含核酸酶P1的发酵液；其中，所述发酵促进剂的制备方法包括如下步骤：S1.将核糖核酸酶解，得到酶解液；S2.将步骤S1所得酶解液经超滤膜超滤，所得截留物即为发酵促进剂。本发明通过添加发酵促进剂进行发酵，单批发酵时间由55h缩短至18h，酶活由2500U/mL提高至7300‑8200U/mL。该方法可以显著的缩短发酵工时，提高发酵酶活，所用的发酵促进剂添加量少，同时，本发明所用的发酵促进剂为生产核苷酸的过程废弃物，无成本，操作简单，效果显著。The invention relates to the field of biotechnology, and specifically discloses a method for preparing nuclease P1. In a fermentation medium containing a fermentation accelerator, Penicillium citrinum is used for fermentation to prepare a fermentation broth containing nuclease P1; wherein, the fermentation The preparation method of the accelerator includes the following steps: S1. Enzymatic hydrolysis of ribonucleic acid to obtain an enzymatic hydrolysis solution; S2. Ultrafiltration of the enzymatic hydrolysis solution obtained in step S1 through an ultrafiltration membrane, and the obtained retentate is the fermentation accelerator. In the present invention, fermentation is carried out by adding a fermentation promoter, the single batch fermentation time is shortened from 55h to 18h, and the enzyme activity is increased from 2500U/mL to 7300-8200U/mL. The method can significantly shorten the fermentation time, improve the activity of fermentation enzymes, and use a small amount of fermentation accelerator. At the same time, the fermentation accelerator used in the present invention is the waste in the process of producing nucleotides, which has no cost, simple operation and remarkable effect. .
Description
Technical Field
The invention relates to the field of microbial fermentation, and in particular relates to a preparation method of nuclease P1.
Background
With the development of the biotechnology industry and the pharmaceutical industry, research and development of nucleotides and derivatives thereof have been hot and have gradually formed another major industry following amino acids. The nucleotide is a biochemical substance with high added value, has wide application, and supports the development of some larger industries, such as high-end dairy industry, pharmaceutical industry and the like. The milk powder is used as a functional additive in the dairy industry, and the milk powder is added to enable the milk powder to be closer to breast milk, so that the capability of resisting bacillary dysentery of infants can be effectively enhanced, and the occurrence of diarrhea is reduced; in the field of medicine, nucleotides are used as pharmaceutical intermediates as raw materials for producing many nucleotide derivatives, and are used for synthesizing hundreds of compounds such as CDP, UDP, GTP, CDP-choline, SAM, cGMP, CTP, UTP, cAMP, UDP-acetylglucosamine, GDP-mannose, GDP-glucose, UDP-glucose, and the like. The nucleotide medicine has unique curative effect in the aspects of resisting cancer and virus, treating cardiovascular diseases, diabetes, interference induction and the like, and has good application effect and huge application market. The demand of domestic market for nucleotide increases at a rate of 30-50% per year, the global market also increases at a rate of 10-15% per year, and the annual usage amount is more than 2000 tons.
Nucleotide production mainly takes three methods: chemical synthesis method, microbial fermentation method and enzymolysis method. The enzymolysis method can obtain the mixture of four kinds of nucleotides at one time due to degradation of RNA, and has high enzyme reaction yield, so that the method is adopted for the industrial production of nucleotides at home and abroad. The enzyme required in preparing nucleotide by an enzymatic hydrolysis method is nuclease Pl, which is phosphodiesterase capable of hydrolyzing RNA to obtain four 5' -nucleotides, the impurities of the enzymatic hydrolysis product are few, the subsequent separation process is simple, and the method is generally adopted for producing the nucleotide abroad.
Penicillium citrinum belongs to the group of asymmetric penicillium, the subgroup of velvet penicillium and the Penicillium citrinum line, and is an important production strain of nuclease P1. Nuclease P1 produced at home and abroad is mainly produced by penicillium citrinum liquid submerged fermentation, and the existing method consumes longer time and has lower enzyme activity. How to efficiently produce the nuclease P1 and reduce the production cost becomes a primary task of nucleotide production.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a preparation method of nuclease P1 aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a preparation method of nuclease P1, which comprises the step of preparing fermentation liquor containing nuclease P1 by fermenting penicillium citrinum in a fermentation medium containing a fermentation accelerator.
The preparation method of the fermentation promoter comprises the following steps:
s1, hydrolyzing ribonucleic acid to obtain enzymolysis liquid;
s2, performing ultrafiltration on the enzymolysis liquid obtained in the step S1 through an ultrafiltration membrane to obtain a retentate, namely the fermentation accelerator.
In the step S1, performing enzymolysis on 2-6 wt% ribonucleic acid aqueous solution with the pH of 4-6 and nuclease P1 at 50-90 ℃; preferably, the pH5, 4 wt% RNA aqueous solution and nuclease P1 at 70 degrees C for enzymolysis.
Wherein the pH is adjusted by NaOH.
Wherein, after the pH value of the ribonucleic acid aqueous solution is adjusted, the temperature is increased to 70 ℃, and nuclease P1 is added after the ribonucleic acid aqueous solution is stabilized for 30 min.
The dosage of the nuclease P1 is 80-120U/g ribonucleic acid, and preferably 100U/g ribonucleic acid.
Wherein the enzyme activity of the nuclease P1 is 10 ten thousand U/g; nuclease P1 enzyme activity definition: 1.9mL of a substrate solution (containing RNA at a concentration of about 1%; 0.2M of pH5.2 in acetic acid buffer and 0.0005M of ZnSO)4) Adding 0.1mL diluted enzyme solution in 70 deg.C constant temperature water bath for 10min, keeping the temperature at 70 deg.C for 15min, adding 2.0mL nucleic acid precipitant (0.25% ammonium molybdate-2.5 perchloric acid), centrifuging in ice water bath for 20min, and collectingDiluting the supernatant with distilled water by a certain factor, and measuring the absorbance at 260nm as A260. The control was made by adding the precipitant first, and the other operations were the same as before. Under the above conditions, 1 unit of enzyme activity was defined as the difference in absorbance at 260nm between the amounts of nucleotides produced per minute and 1.0.
In the step S2, cooling the enzymolysis liquid obtained in the step S1 to 20 ℃, and performing ultrafiltration through an ultrafiltration membrane to 1/8-1/12 of the original volume to obtain a first cut-off substance; adding water into the obtained first retentate to the original volume, and performing ultrafiltration to 1/8-1/12 of the original volume to obtain a second retentate; adding water into the obtained second retentate to the original volume, and then performing ultrafiltration to 1/8-1/12 of the original volume to obtain a third retentate, wherein the obtained third retentate is the fermentation accelerator; preferably, each time to 1/10 of the original volume.
In step S2, the ultrafiltration is a spiral wound polysulfone ultrafiltration membrane.
In step S2, the molecular weight of the ultrafiltration membrane is 3000-10000 Dalton, preferably 4000-8000 Dalton, more preferably 5000-7000 Dalton, and even more preferably 6000 Dalton.
Wherein the fermentation promoter prepared by the method contains oligonucleotide, short-chain RNA, zymosan, protein and yeast content.
The dosage of the fermentation promoter is 1-20 g/L of fermentation medium, preferably 2-18 g/L of fermentation medium, more preferably 5-15 g/L of fermentation medium, more preferably 8-13 g/L of fermentation medium, and most preferably 10g/L of fermentation medium.
The fermentation medium comprises 1-100 g/L of glucose, 0.1-10 g/L of peptone, 0.02-10 g/L of monopotassium phosphate, 0.02-10 g/L of dipotassium phosphate, 0.02-10 g/L of magnesium sulfate and 0.02-10 g/L of calcium chloride besides a fermentation promoter.
Wherein the solvent of the fermentation medium is water, and the pH value is 5-9.
The preservation number of the penicillium citrinum is CGMCC No.2014, and the strain is described in Chinese patent CN 101067116A.
Wherein the fermentation temperature is 25-35 ℃, and preferably 30 ℃.
The fermentation is semi-continuous fermentation, the semi-continuous fermentation is a fermentation method that penicillium citrinum is fermented in a fermentation tank for a period of time, part of fermentation liquor containing products is discharged periodically, and then fresh culture medium with the same volume is supplemented, and the method specifically comprises the following steps: a, after the citrinin enzyme is fermented for 55 hours at the temperature of 30 ℃ in a fermentation tank, the enzyme activity is stabilized at about 7300-7800U/mL; b, discharging 85% of the volume of the fermentation liquid, taking the fermentation liquid remained in the fermentation tank as the next batch of fermentation seeds, supplementing the same volume of sterile fermentation medium to the fermentation tank for continuous culture, and stabilizing the enzyme activity at about 7300-8200U/mL after fermentation for about 18 hours; c, repeating the step b, circularly feeding for 5 times and stopping the semi-continuous fermentation.
Wherein the time of single batch fermentation is 18 h.
The enzyme activity of the fermentation liquor containing the nuclease P1 is 6500-8500U/mL, preferably 7300-8200U/mL, more preferably 8100-8300U/mL, and even more preferably 8200U/mL.
Has the advantages that: compared with the prior art, the invention has the following technical advantages:
according to the invention, the fermentation promoter is added for fermentation, the single-batch fermentation time is shortened from 55h to 18h, and the enzyme activity is improved from 2500U/mL to 7300 8200U/mL. The method can obviously shorten the fermentation working hours, improve the fermentation enzyme activity, and the used fermentation accelerator has small addition amount, and meanwhile, the used fermentation accelerator is the waste in the process of producing nucleotide, so the method has no cost, simple operation and obvious effect.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The preservation number of the penicillium citrinum in the following embodiments is CGMCC No. 2014.
The nuclease P1 to ribonucleic acid ratio described in the examples below is mass ratio.
Example 1:
dissolving ribonucleic acid powder into 4% concentration (mass ratio) by using deionized water, adjusting pH to 5.0 by using NaOH, heating to 70 ℃, stabilizing for 30min, adding nuclease P1 powder (nuclease P1: ribonucleic acid is 1:100, and the enzyme activity of nuclease P1 is 10 ten thousand U/g) under the condition of stirring for 100 revolutions per minute for enzymolysis, and finishing enzymolysis after 3 h.
Cooling the enzymolysis solution to 20 deg.C, ultrafiltering with a rolled polysulfone ultrafiltration membrane with molecular weight of 3000 daltons to 1/10 of original volume, adding water to original volume, ultrafiltering to 1/10 of original volume, and repeating the water adding and ultrafiltering. The obtained ultrafiltration membrane retentate is the fermentation accelerator 1.
Example 2:
dissolving ribonucleic acid powder into 4% concentration (mass ratio) by using deionized water, adjusting pH to 5.0 by using NaOH, heating to 70 ℃, stabilizing for 30min, adding nuclease P1 powder (nuclease P1: ribonucleic acid is 1:100, and the enzyme activity of nuclease P1 is 10 ten thousand U/g) under the condition of stirring for 100 revolutions per minute for enzymolysis, and finishing enzymolysis after 3 h.
Cooling the enzymolysis solution to 20 deg.C, ultrafiltering the enzymolysis solution with a roll type polysulfone ultrafiltration membrane with molecular weight of 6000 daltons to 1/10 of the original volume, adding water to the original volume, ultrafiltering to 1/10 of the original volume, and repeating the water adding and ultrafiltration. The obtained ultrafiltration membrane retentate is the fermentation accelerator 2.
Example 3:
dissolving ribonucleic acid powder into 4% concentration (mass ratio) by using deionized water, adjusting pH to 5.0 by using NaOH, heating to 70 ℃, stabilizing for 30min, adding nuclease P1 powder (nuclease P1: ribonucleic acid is 1:100, and the enzyme activity of nuclease P1 is 10 ten thousand U/g) under the condition of stirring for 100 revolutions per minute for enzymolysis, and finishing enzymolysis after 3 h.
Cooling the enzymolysis solution to 20 deg.C, ultrafiltering the enzymolysis solution with a roll type polysulfone ultrafiltration membrane with molecular weight of 10000 Dalton to 1/10 of the original volume, adding water to the original volume, ultrafiltering to 1/10 of the original volume, and repeating the water adding and ultrafiltering. The obtained ultrafiltration membrane retentate is the fermentation accelerator 3.
Example 4:
preparation of a fermentation medium: 25g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L calcium chloride, 110 g/L fermentation accelerator, pH6.5, sterilizing at 121 ℃ for 15min, and cooling to about 30 ℃ for later use.
Semi-continuous fermentation: 6L of culture medium is filled into a 10L stirring tank, the sterilized seed liquid of the citrinin strain cultured by the wort culture medium is inoculated into the stirring tank according to the volume ratio of 10 percent for fermentation culture, the enzyme activity is stabilized at about 7300U/mL after the fermentation is carried out for 55 hours in a fermentation tank at the temperature of 30 ℃, the volume of the fermentation liquid is discharged to be 5.1L, the fermentation liquid remained in the fermentation tank is used as the next batch of fermentation seeds, 5.1L of sterile fermentation medium is prepared and injected into the fermentation tank for secondary culture, the enzyme activity is stabilized to 7320U/mL after 18 hours, 5.1L of fermentation liquid is discharged again, and 5.1L of fresh culture medium is supplemented by the same method. Feeding materials for 5 times in such a circulating way, keeping the enzyme activity at about 7310U/mL, and stopping semi-continuous fermentation.
Example 5:
preparation of a fermentation medium: 25g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L calcium chloride, 210 g/L fermentation accelerator, pH6.5, sterilizing at 121 ℃ for 15min, and cooling to about 30 ℃ for later use.
Semi-continuous fermentation: 6L of culture medium is filled in a 10L stirring tank, the sterilized seed liquid of the citrinin mold cultured by the wort culture medium is inoculated according to the volume ratio of 10 percent for fermentation culture, the enzyme activity is stabilized at about 7600U/mL after the fermentation is carried out for 55 hours in a fermentation tank at the temperature of 30 ℃, the volume of the fermentation liquid is discharged to be 5.1L, the fermentation liquid remained in the fermentation tank is used as the next batch of fermentation seeds, 5.1L of sterile fermentation medium is prepared and injected into the fermentation tank for secondary culture, the enzyme activity is stabilized to 8180U/mL after 18 hours, 5.1L of fermentation liquid is discharged again, and 5.1L of fresh culture medium is supplemented in the same method. Feeding materials for 5 times in such a circulating way, keeping the enzyme activity at about 8200U/mL, and stopping semi-continuous fermentation.
Example 6:
preparation of a fermentation medium: 25g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L calcium chloride, 310 g/L fermentation accelerator, pH6.5, sterilizing at 121 ℃ for 15min, and cooling to about 30 ℃ for later use.
Semi-continuous fermentation: 6L of culture medium is filled in a 10L stirring tank, after sterilization, the seed liquid of the citrinin strain cultured by the malt extract culture medium is inoculated into the stirring tank according to the volume ratio of 10 percent for fermentation culture, the enzyme activity is stabilized at about 7500U/mL after fermentation for 55 hours in a fermentation tank at the temperature of 30 ℃, the volume of the fermentation liquid is discharged to be 5.1L, the fermentation liquid remained in the fermentation tank is used as the next batch of fermentation seeds, 5.1L of sterile fermentation medium is prepared and injected into the fermentation tank for secondary culture, the enzyme activity is stabilized to 7620U/mL after 18 hours, 5.1L of fermentation liquid is discharged again, and 5.1L of fresh culture medium is supplemented by the same method. Feeding materials for 5 times in such a circulating way, keeping the enzyme activity about 7600U/mL, and stopping semi-continuous fermentation.
Comparative example 1:
preparation of a fermentation medium: 25g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L calcium chloride, pH6.5, sterilizing at 121 ℃ for 15min, and cooling to about 30 ℃ for later use.
Semi-continuous fermentation: 6L of culture medium is filled into a 10L stirring tank, the sterilized seed liquid of the citrinin mold strain cultured by the wort culture medium is inoculated into the stirring tank according to the volume ratio of 10 percent for fermentation culture, the enzyme activity is stabilized at about 2480U/mL after the fermentation is carried out for 55 hours at the temperature of 30 ℃ in a fermentation tank, the volume of the fermentation liquid is discharged to be 5.1L, the fermentation liquid remained in the fermentation tank is used as the next batch of fermentation seeds, 5.1L of sterile fermentation medium is prepared and injected into the fermentation tank for secondary culture, the enzyme activity is stabilized to 2420U/mL after 18 hours, 5.1L of fermentation liquid is discharged again, and 5.1L of fresh culture medium is supplemented by the same method. Feeding materials for 5 times in such a circulating way, keeping the enzyme activity at about 2500U/mL, and stopping semi-continuous fermentation.
Example 7:
preparation of a fermentation medium: 25g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L calcium chloride, 22 g/L fermentation accelerator, pH6.5, sterilizing at 121 ℃ for 15min, and cooling to about 30 ℃ for later use.
Semi-continuous fermentation: 6L of culture medium is filled in a 10L stirring tank, the sterilized seed liquid of the citrinin mold cultured by the wort culture medium is inoculated according to the volume ratio of 10 percent for fermentation culture, the enzyme activity is stabilized at about 6200U/mL after the fermentation is carried out for 55 hours at the temperature of 30 ℃ in a fermentation tank, the volume of the fermentation liquid is discharged to be 5.1L, the fermentation liquid remained in the fermentation tank is used as the next batch of fermentation seeds, 5.1L of sterile fermentation medium is prepared and injected into the fermentation tank for secondary culture, the enzyme activity is stabilized to 6730U/mL after 18 hours, 5.1L of fermentation liquid is discharged again, and 5.1L of fresh culture medium is supplemented by the same method. Feeding materials for 5 times in such a circulating way, keeping the enzyme activity at about 6700U/mL, and stopping semi-continuous fermentation.
Example 8:
preparation of a fermentation medium: 25g/L glucose, 2g/L peptone, 0.5g/L potassium dihydrogen phosphate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L calcium chloride, 218 g/L fermentation accelerator, pH6.5, sterilizing at 121 ℃ for 15min, and cooling to about 30 ℃ for later use.
Semi-continuous fermentation: 6L of culture medium is filled in a 10L stirring tank, the sterilized seed liquid of the citrinin strain cultured by the malt extract culture medium is inoculated into the seed liquid of the citrinin strain according to the volume ratio of 10 percent for fermentation culture, the enzyme activity is stabilized at about 7620U/mL after the fermentation is carried out for 55 hours in a fermentation tank at the temperature of 30 ℃, the volume of the fermentation liquid is discharged to be 5.1L, the fermentation liquid remained in the fermentation tank is used as the next batch of fermentation seeds, 5.1L of sterile fermentation culture medium is prepared and is injected into the fermentation tank for secondary culture, the enzyme activity is stabilized to 7660U/mL after 18 hours, 5.1L of fermentation liquid is discharged again, and 5.1L of fresh culture medium is supplemented by the same method. Feeding materials for 5 times in such a circulating way, keeping the enzyme activity at about 7700U/mL, and stopping semi-continuous fermentation.
The invention provides a method for preparing nuclease P1, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the invention, and these modifications and decorations should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
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| WO2023103543A1 (en) * | 2021-12-07 | 2023-06-15 | 南京工业大学 | Method for preparing nuclease p1 |
| CN118109448A (en) * | 2024-03-18 | 2024-05-31 | 武汉新华扬生物股份有限公司 | Auxiliary agent for improving nuclease stability and preparation method and application thereof |
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| CN104560913A (en) * | 2014-12-24 | 2015-04-29 | 南京工业大学 | Method for preparing nuclease P1 by semi-continuous fermentation of penicillium citrinum |
| CN107365831A (en) * | 2017-07-18 | 2017-11-21 | 三峡大学 | A kind of Penicillium citrinum nuclease P 1 strain improvement screening technique |
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| CN101654677A (en) * | 2009-09-17 | 2010-02-24 | 南京工业大学 | A gene for highly expressing nuclease P1 and its application |
| CN113980930B (en) * | 2021-12-07 | 2023-03-03 | 南京工业大学 | Preparation method of nuclease P1 |
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| CN101037681A (en) * | 2007-02-14 | 2007-09-19 | 北京燕京中科生物技术有限公司 | Method for preparing nuclease P1 by ferment process |
| CN101067116A (en) * | 2007-05-25 | 2007-11-07 | 南京工业大学 | Penicillium citrinum strain with high nuclease P1 yield and breeding method thereof |
| CN101418327A (en) * | 2008-11-21 | 2009-04-29 | 大连珍奥生物技术股份有限公司 | The new process of production of high purity 5 ' Nucleotide |
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| CN109852596A (en) * | 2019-04-04 | 2019-06-07 | 江苏省生产力促进中心 | A method of nuclease P 1 is prepared using immobilization Penicillium citrinum fermentation |
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| WO2023103543A1 (en) * | 2021-12-07 | 2023-06-15 | 南京工业大学 | Method for preparing nuclease p1 |
| CN118109448A (en) * | 2024-03-18 | 2024-05-31 | 武汉新华扬生物股份有限公司 | Auxiliary agent for improving nuclease stability and preparation method and application thereof |
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