CN116731874B - Aspergillus melidum 01 strain and application thereof - Google Patents
Aspergillus melidum 01 strain and application thereof Download PDFInfo
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- CN116731874B CN116731874B CN202310620224.1A CN202310620224A CN116731874B CN 116731874 B CN116731874 B CN 116731874B CN 202310620224 A CN202310620224 A CN 202310620224A CN 116731874 B CN116731874 B CN 116731874B
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- amp deaminase
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- 241000228212 Aspergillus Species 0.000 title claims description 19
- 102000006267 AMP Deaminase Human genes 0.000 claims abstract description 90
- 108700016228 AMP deaminases Proteins 0.000 claims abstract description 90
- 102000004190 Enzymes Human genes 0.000 claims abstract description 68
- 108090000790 Enzymes Proteins 0.000 claims abstract description 68
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 35
- 241000981399 Aspergillus melleus Species 0.000 claims abstract description 32
- 230000004151 fermentation Effects 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 38
- 239000001963 growth medium Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- 239000000463 material Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000002386 leaching Methods 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 12
- 235000012907 honey Nutrition 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- IGHGOYDCVRUTSU-UHFFFAOYSA-M sodium;2-hydroxypropane-1,2,3-tricarboxylic acid;hydroxide Chemical compound [OH-].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O IGHGOYDCVRUTSU-UHFFFAOYSA-M 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000003825 pressing Methods 0.000 claims description 4
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 claims description 4
- 229940001584 sodium metabisulfite Drugs 0.000 claims description 4
- 235000010262 sodium metabisulphite Nutrition 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 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 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 230000003311 flocculating effect Effects 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 238000012262 fermentative production Methods 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 56
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- 238000012216 screening Methods 0.000 abstract description 7
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- 239000000523 sample Substances 0.000 description 33
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 31
- 238000010438 heat treatment Methods 0.000 description 14
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- 230000008569 process Effects 0.000 description 12
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- 239000000758 substrate Substances 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 240000006439 Aspergillus oryzae Species 0.000 description 5
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 5
- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 235000013902 inosinic acid Nutrition 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
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- 101710149004 Nuclease P1 Proteins 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 235000019634 flavors Nutrition 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical compound [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 description 2
- 241000044446 Melinis Species 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013373 food additive Nutrition 0.000 description 2
- 239000002778 food additive Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000004245 inosinic acid Substances 0.000 description 2
- 229940028843 inosinic acid Drugs 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
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- 239000002773 nucleotide Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- AEOBEOJCBAYXBA-UHFFFAOYSA-N A2P5P Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1OP(O)(O)=O AEOBEOJCBAYXBA-UHFFFAOYSA-N 0.000 description 1
- 241000349731 Afzelia bipindensis Species 0.000 description 1
- 241000351920 Aspergillus nidulans Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- AANLCWYVVNBGEE-IDIVVRGQSA-L Disodium inosinate Chemical compound [Na+].[Na+].O[C@@H]1[C@H](O)[C@@H](COP([O-])([O-])=O)O[C@H]1N1C(NC=NC2=O)=C2N=C1 AANLCWYVVNBGEE-IDIVVRGQSA-L 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000208829 Sambucus Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 235000013409 condiments Nutrition 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- PVBRXXAAPNGWGE-LGVAUZIVSA-L disodium 5'-guanylate Chemical compound [Na+].[Na+].C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP([O-])([O-])=O)[C@@H](O)[C@H]1O PVBRXXAAPNGWGE-LGVAUZIVSA-L 0.000 description 1
- 235000013896 disodium guanylate Nutrition 0.000 description 1
- 235000013890 disodium inosinate Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 235000008995 european elder Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000006180 nutrition needs Nutrition 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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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
- C12N1/145—Fungal isolates
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- C12N1/06—Lysis of microorganisms
- C12N1/063—Lysis of microorganisms of yeast
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
- C12Y305/04017—Adenosine-phosphate deaminase (3.5.4.17)
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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Abstract
The application relates to aspergillus melleus adm01 with a deposit number of GDMCC No:63302. the strain is obtained through ARTP mutation screening, has the characteristic of high yield of 5'-AMP deaminase, and can be used for preparing a 5' -AMP deaminase product through a mode of solid state fermentation and extraction after combination, wherein the fermentation enzyme activity is higher than 31000U/g dry yeast. Meanwhile, the 5' -AMP deaminase produced by the strain has better heat resistance, the optimal reaction temperature reaches 70 ℃, and the 5' -AMP deaminase can be subjected to synchronous high-temperature enzymolysis with the 5' -nuclease in the high I+G production process, so that the preparation process is simplified.
Description
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to aspergillus melleus adm01 and application thereof.
Background
The yeast extract with high I+G (sodium 5 '-inosinate and sodium 5' -guanylate) is a yeast extract which strengthens the content of the flavor nucleotide substances in the product on the basis of the original yeast extract, can be used as a natural and nutritional high-end condiment to replace monosodium glutamate, has the functions of enhancing freshness, improving flavor, reducing salt and the like, and accords with the dietary consumption habit and the nutritional demand trend of modern people. The I+G content of the yeast extract directly affects the flavor quality and the production cost of the product.
In the production of the high I+G yeast extract, single-stranded DNA and RNA are first hydrolyzed with 5 '-nuclease to produce Adenylate (AMP) and Guanylate (GMP), and then reacted with 5' -AMP deaminase to convert the adenylate into inosinic acid (IMP). In the preparation process of the high I+G yeast extract, the 5'-AMP deaminase plays a vital role, and the enzyme activity and the action performance of the 5' -AMP deaminase directly influence the inosinic acid production and the quality of the high I+G yeast extract.
The strains of the 5'-AMP deaminase which accords with the national food additive related regulation requirements are aspergillus melleus and aspergillus oryzae, but the enzyme activity of the 5' -AMP deaminase from the aspergillus melleus is generally lower in the current market, and the enzyme activity of the 5'-AMP deaminase from the aspergillus oryzae is lower than that from the aspergillus melleus, so that the production cost of the 5' -AMP deaminase is high. In addition, the optimal temperature of 5 '-nuclease on the market is 65-70℃at present, whereas the optimal temperature of 5' -AMP deaminase derived from general A.melissus is about 50-55 ℃. Therefore, in the production process of the high I+G yeast extract, two enzymes are required to be subjected to enzymolysis in two steps, 5 '-nuclease enzymolysis is firstly carried out, then the system temperature is reduced to 50-55 ℃ to carry out 5' -AMP deaminase enzymolysis, the process is complicated, and the risk of mixed bacterial pollution is increased due to the reduction of the reaction system temperature. Therefore, it is significant to develop 5' -AMP deaminase which has high enzyme activity, is heat-resistant and meets the requirements of food additives.
At present, the literature on 5' -AMP deaminase is not much reported, and mainly focuses on strain breeding and fermentation condition optimization. Liu Changjun et al, "production of adenylate deaminase by solid state fermentation" discuss the use of Aspergillus oryzae solid state fermentation to obtain fresh yeast with a maximum enzyme activity of 1543.48U/g by strain screening and fermentation process optimization. The section culture and the like of the Aspergillus oryzae adenylate deaminase enzyme production conditions discuss that an optimal fermentation medium and fermentation process parameters for producing the 5'-AMP deaminase by the Aspergillus oryzae solid state fermentation are obtained through experiments, and under the condition, the enzyme activity of the 5' -AMP deaminase in the yeast material is up to 1560.3U/g. Liu Chunjian et al, breeding of Aspergillus melini strain producing AMP deaminase and optimization of solid state fermentation culture medium, discuss that a mutant strain Aspergillus melini UV-26 is obtained by screening by ultraviolet mutagenesis method, and then the solid state fermentation culture medium of the strain is optimized, so that the enzyme activity of 5' -AMP deaminase is improved, and the highest enzyme activity can reach 7553U/g dry yeast. Liu Chunjian et al, "research on solid-state fermentation Process for preparing AMP deaminase by Aspergillus honey" discuss that Aspergillus honey is used as a fermentation strain, and the enzyme activity of 5'-AMP deaminase is improved by optimizing the process parameters such as initial pH value, inoculum size, temperature, moisture, ventilation and time, and the enzyme activity of 5' -AMP deaminase in the yeast material reaches 12000U/g.
Disclosure of Invention
The application mainly aims to provide aspergillus melleus (Aspergillus melleus) adm01 which has the characteristic of high yield of 5'-AMP deaminase and can be used for preparing a 5' -AMP deaminase product. The strain can be used for fermenting to obtain dry yeast with the enzyme activity of 5'-AMP deaminase exceeding 31000U/G, and the produced 5' -AMP deaminase has better heat resistance, the optimal reaction temperature reaches 70 ℃, and the preparation method can simplify the production process when being used for preparing the high I+G yeast extract.
The inventors have conducted intensive studies to achieve the above object, and have completed obtaining the present application by repeating the study demonstration a plurality of times, specifically as follows:
in a first aspect, the application provides an aspergillus melleus (Aspergillus melleus) adm01 deposited at the cantonese microbiological bacterial collection center, address: building 5, no. 59 of Mitsui 100, guangzhou City, guangdong, with the preservation number of GDMCC No. 63302.
The aspergillus melleus adm01 is obtained by screening from soil of agricultural college of agricultural university of south China and then carrying out mutation breeding, and the colony characteristics are as follows: macroscopic morphology: the CYA culture medium is cultured for 7 days at 25 ℃, the colony diameter is 64-69mm, the spore-forming structure is yellow, and the edge is light pink; irregular radial wrinkles on the surface; the back surface is yellow; there was little exudates produced, no soluble pigments. Microscopic morphology: conidiophores occur in the matrix, and the walls of the conidiophores are smooth and transparent, and the diameter is 7-15 mu m; the conidiophore head is nearly spherical and has a diameter of 45-95 μm; the top sac is nearly spherical or flask-shaped, has the diameter of 25-60 mu m, and is full or three-quarters of the surface; bilayer with spore-producing structure, stem base 3-5×2-3 μm, bottle stem 5-9×1.5-2.5 μm; the conidium is nearly spherical, the diameter is 2.5-3.5 mu m, and the surface is slightly rough.
In a second aspect, the application provides a fermentation inoculant, which is characterized by comprising the aspergillus melleus adm01.
In a third aspect, the application provides the use of Aspergillus melinadm 01 in the fermentative production of 5' -AMP deaminase.
In a fourth aspect, the present application provides a method for producing 5' -AMP deaminase, comprising the steps of:
1) Activating strains: inoculating aspergillus melleus adm01 into a culture medium for activation;
2) And (3) performing expansion culture: inoculating activated aspergillus melleus adm01 into a seed culture medium, and culturing for 5-7 days at 26-30 ℃ to obtain solid strains;
3) Fermentation culture: inoculating the solid strain into a fermentation culture medium for culturing, and fermenting for 60-80 h at 26-30 ℃;
4) Leaching of yeast: adding 5-7 times of leaching solution according to the weight of the fermented yeast material, stirring until the yeast material is fully wet by water absorption, standing and soaking for 1.5-2.5 hours to obtain leaching solution;
5) Press filtration of the plate and the frame: adding calcium chloride and dipotassium hydrogen phosphate into the leaching solution, flocculating, and performing plate-frame filter pressing to obtain crude enzyme filtrate;
6) Concentrating: concentrating the crude enzyme filtrate for 7-9 times to obtain enzyme concentrate;
7) And (3) separating and purifying: pre-cooling the enzyme concentrate to 4 ℃, and slowly adding 75% ethanol pre-cooled to 4 ℃ while stirring to ensure that the final concentration of the ethanol reaches 50%. And (3) carrying out alcohol precipitation for 25-35 min, centrifugally separating a supernatant and precipitating the supernatant, and dissolving the precipitate with 0.1mol/L citric acid-NaOH buffer solution to obtain the high-purity 5' -AMP deaminase.
Further, the aspergillus melleus adm01 in the step 1) is cultured for 3 to 5 days at the temperature of 26 to 30 ℃ so as to be activated.
Further, the seed culture medium in the step 2) comprises water and bran, and the mass ratio of the water to the bran is 1:1.
Further, step 3) the fermentation medium comprises, in 100g of water: 100g of bran, 2g of glucose, 2g of peptone, 0.2g of sodium citrate, 0.1g of ammonium sulfate and 0.1g of magnesium sulfate heptahydrate.
Further, the leaching solution of step 4) contains 0.4 to 0.6% sodium metabisulfite.
In a fifth aspect, the application provides a 5'-AMP deaminase characterized in that the 5' -AMP deaminase is obtainable by the process of the present application.
In a sixth aspect, the application provides the use of a 5' -AMP deaminase produced by the method of the application in an enzymatic yeast.
One or more technical solutions provided in the embodiments of the present application at least have the following technical effects or advantages:
1. the aspergillus melleus adm01 provided by the application has the advantage of high yield of 5'-AMP deaminase, and the enzyme activity of the 5' -AMP deaminase in the solid fermentation starter is more than 31000U/g dry starter;
2. the method for preparing the 5' -AMP deaminase by the aspergillus melleus adm01 provided by the application adopts a mode of extraction after solid-state fermentation and combination, so that the production cost of the 5' -AMP deaminase can be reduced, and the large-scale industrial production of the 5' -AMP deaminase can be realized.
3. The 5' -AMP deaminase product prepared by the application has better heat resistance, the optimal reaction temperature reaches 70 ℃, and the product can be subjected to synchronous high-temperature enzymolysis with nuclease in the high I+G production process, so that the preparation process is simplified.
Drawings
FIG. 1 is a graph of colonies of Aspergillus melissus adm 01;
FIG. 2 is a graph showing the results of a test for the optimum reaction temperature of 5' -AMP deaminase;
FIG. 3 is a diagram showing the results of a test for the thermostability of 5' -AMP deaminase;
the aspergillus melleus (Aspergillus melleus) adm01 provided by the application is preserved in the Guangdong microbiological strain collection center at 2023, 3 and 28 days, and has the following addresses: building 5 of Guangzhou City of Guangdong, first, china, no. 100, college, no. 59, with the preservation number of GDMCC No. 63302; the strain was received from the collection at 28 at 3/2023 and registered, and was detected as a viable strain by the collection at 28/2023.
Detailed Description
The present application will be described more fully hereinafter in order to facilitate an understanding of the present application. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Unless otherwise indicated, the reagents, methods and apparatus employed in the present application are conventional food-grade reagents, methods and apparatus in the art.
The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The following examples were used in the medium formulations and methods for detecting the enzymatic activity of 5' -AMP deaminase:
formulation of CYA medium (s.sambucus paste agar medium): 30g of sucrose, 5g of yeast extract powder, 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate heptahydrate, 0.01g of ferrous sulfate heptahydrate and 15g of agar are dissolved in 1L of distilled water, the pH is natural, split charging is carried out, and sterilization is carried out for 20min at 121 ℃. After sterilization, the agar is uniformly shaken to prevent the agar from being deposited at the bottom of the vessel to solidify, and the flat plate is poured for later use.
The formula of the seed culture medium comprises: 100g of bran and 100g of water, and packaging into triangular bottles after uniformly mixing. A250 mL Erlenmeyer flask may be charged with 10g of bran. Sterilizing at 121deg.C for 30min. And cooling for standby.
The formula of the fermentation medium comprises: 100g of bran, 100g of water, 2g of glucose, 2g of peptone, 0.2g of sodium citrate, 0.1g of ammonium sulfate and 0.1g of magnesium sulfate heptahydrate are mixed uniformly and then packaged in a yeast tray. Sterilizing at 121deg.C for 30min. And cooling for standby.
The enzyme activity detection method of the 5' -AMP deaminase comprises the following steps: (1) pretreatment of a yeast sample: 1g of the fermentation starter is ground, added with 0.5% sodium metabisulfite solution to a volume of 100mL, sampled and diluted to a proper concentration with 0.1mol/L citric acid-NaOH buffer solution. (2) preparing a 5' -AMP deaminase reaction substrate: 139mg of adenylate was precisely weighed out and 0.08mol/L NaHCO was used 3 The solution is fixed to 10mL to be used as an original substrate, then 0.5mL is sucked, and 0.1mol/L citric acid buffer solution is used for fixing the volume to 200mL to obtain the final concentration of 0.1 multiplied by 10 -3 mol/L of reaction substrate. (3) 5' -AMP deaminase enzyme activity assay: accurately transferring 3mL of reaction substrate, adding the reaction substrate into a test tube, preserving the temperature at 37 ℃ for 5min, adding 0.1mL of diluted deaminase solution, immediately timing the reaction, and after 15min of reaction, pre-cooling the reaction substrate by ice water, and adding 3mL of 10% perchloric acid solution to terminate the reaction. Control sample: 3mL of the reaction substrate is removed, the temperature is kept at 37 ℃ for 5min, after the same timing is carried out for 15min, 3mL of 10% perchloric acid solution is added to the reaction substrate by chilled water, 0.1mL of the diluted deaminase solution is added to the reaction substrate, and the reaction is stopped. After the reaction was completed, absorbance was measured at 265nm using a quartz cuvette of 10mm in distance with pure water as a reference.
Definition of enzyme activity: the absorbance change per minute under the above conditions was 0.001, defined as one activity unit of the enzyme.
And (3) calculating: enzyme activity of enzyme solution
A-absorbance of sample;
A 0 -blank absorbance;
k-dilution factor of crude enzyme solution;
t is the time of enzyme reaction, min;
in the yeast, 5' -AMP deaminase activity Y (U/g) =100deg.X.
EXAMPLE 1 screening of strains of interest
1. Natural screening
1) 10g of soil powder sample of agricultural college of agricultural university of south China is weighed and added into a triangular flask filled with 90mL of sterile physiological saline and glass beads, and the sample is fully scattered by shaking for 30min at room temperature, and is left to stand to obtain sample suspension. Diluting the sample suspension to 10 in a stepwise 10-fold gradient -2 ～10 -6 Respectively taking 0.1mL of the mixture to be coated on a CYA culture medium plate for culture, wherein the culture condition is 28 ℃ for 48 hours;
2) The single colony of the mould on the flat plate is picked up, added into a test tube filled with 10mL of sterile physiological saline, oscillated for 30min at room temperature to fully break up the sample, and stood to obtain the bacterial liquid. Diluting the bacterial liquid to 10 in a step-by-step 10-time gradient manner -2 ～10 -6 Respectively taking 0.1mL of the mixture to be coated on a CYA culture medium plate for culture, wherein the culture condition is 28 ℃ for 48 hours;
3) 16 single colonies are selected from the flat plate, and the number fm01-fm16 are respectively inoculated into seed culture media prepared from 10g of bran for expansion culture, wherein the culture conditions are 28 ℃ for 72 hours;
4) Inoculating the cultured strain into a fermentation culture medium according to the proportion of 1% for solid state fermentation, wherein the fermentation condition is 28 ℃ for 72 hours;
5) The enzyme activity of deaminase in the fermented koji was measured and recorded in table 1.
TABLE 1 enzyme Activity detection results of bacterial Strain deaminase obtained by Natural screening
| Strain | Enzyme activity (U/g) | Strain | Enzyme activity (U/g) |
| fm01 | 8400 | fm09 | 61 |
| fm02 | 233 | fm10 | 112 |
| fm03 | 172 | fm11 | 960 |
| fm04 | 2378 | fm12 | 2653 |
| fm05 | 825 | fm13 | 282 |
| fm06 | 3491 | fm14 | 1775 |
| fm07 | 476 | fm15 | 47 |
| fm08 | 1413 | fm16 | 2913 |
As is clear from Table 1, the deaminase activity of the strain No. fm01 was the highest, and the strain was selected and stored for subsequent mutagenesis.
2. Mutation breeding
1) Inoculating the fm01 single colony into seed culture medium for expansion culture at 28deg.C
Culturing for 72h;
2) Adding 50mL of sterile physiological saline to elute solid strains into a triangular flask, adding 20 sterile glass beads into the triangular flask, oscillating for 30min at room temperature to fully scatter spores, filtering residues by using sterile gauze, and collecting spore solution;
3) Adding 1mL spore solution into a triangular flask containing 99mL sterile physiological saline, shaking for 1min, and mixing thoroughly to obtain a concentration of about 10 6 ～10 7 cfu/mL spore suspension, and subjecting the spore suspension to ARTP mutagenesis;
4) Taking 1mL of mutagenized spore suspension, and gradually diluting to 10 times in a gradient manner -2 ～10 -6 Respectively taking 0.1mL of bacterial solutions with different concentrations, coating the bacterial solutions on a CYA culture medium plate for culture, wherein the culture condition is 28 ℃ for 48 hours;
5) Selecting 12 single mould colonies, respectively inoculating the single mould colonies with the numbers adm01-adm12 into seed culture mediums prepared from 10g of bran for expansion culture, wherein the culture conditions are 28 ℃ for 72 hours;
6) Inoculating the cultured strain into a fermentation culture medium according to the proportion of 1% for solid state fermentation, wherein the fermentation condition is 28 ℃ for 72 hours;
7) After fermentation, measuring the enzyme activity of deaminase in the fermented yeast material, loading 5g of the yeast material into a 50mL centrifuge tube, sealing, heating in an oven at 70 ℃ for 1h, measuring the enzyme activity of 5' -AMP deaminase in the heated yeast material, and recording the results in Table 2;
TABLE 2 enzyme Activity detection results of Strain 5' -AMP deaminase obtained by mutagenesis breeding
As is clear from Table 2, the fermentation starter of strain No. adm01 was selected as the target strain of the present application, in which the enzyme activity of 5' -AMP deaminase was the highest before and after heating. The strain is sent to China industry microbiological culture collection center for identification (2022.05.20), the identification result is aspergillus melleus (Aspergillus melleus), the identification result is named Aspergillus melleus adm01, and the strain is preserved in Guangdong microbiological culture collection center at the 3 rd month of 2023 and the 28 th day, and the preservation number is GDMCC No. 63302.
Example 2 Aspergillus honey adm01 genetic stability test
Continuously passaging aspergillus melleus adm01 obtained in the example 1 on a CYA culture medium inclined plane for 10 generations, observing and recording the growth condition of each generation of strain, inoculating the 1 st generation, 5 th generation and 10 th generation inclined plane strains into a seed culture medium for expansion culture, and culturing for 7 days at 28 ℃; inoculating the cultured strain into a fermentation culture medium for solid state fermentation, wherein the fermentation temperature is set to be 28 ℃, and the culture time is set to be 72 hours. After the fermentation, the enzyme activity of 5' -AMP deaminase in the fermented material was measured. Judging the genetic stability of the aspergillus melleus adm01 strain according to the enzyme activity of the 5'-AMP deaminase in the yeast material, and if the enzyme activity error of the 5' -AMP deaminase is measured in the range of 10% error in the tenth generation, indicating that the genetic stability of the strain is good. The results of the genetic stability test of the A.melissus adm01 strain are shown in Table 3.
TABLE 3 genetic stability test results for Aspergillus melidum 01 Strain
| Number of passages | Growth conditions | Enzyme activity (U/g) |
| 1 | Colony characterization was normal | 31332 |
| 5 | Colony characterization was normal | 30988 |
| 10 | Colony characterization was normal | 32216 |
As shown in Table 3, the colony characteristics of Aspergillus melleus adm01 strains of different generations are normal, and the enzyme activity error of 5' -AMP deaminase in the fermentation starter is within 10%, which indicates that the genetic stability of the strain is good.
EXAMPLE 3 preparation of 5' -AMP deaminase by Aspergillus honey adm01
(1) Activating strains: transferring the freeze-dried tube storage strain to a CYA slant culture medium, culturing for 4 days at 28 ℃ and activating the strain;
(2) And (3) performing expansion culture: inoculating the activated strain to a 1L triangular flask containing 40g of seed culture medium prepared from bran for expansion culture, and culturing at 28 ℃ for 6 days;
(3) Solid state fermentation culture: weighing 4000g of bran to prepare a fermentation medium according to a proportion; inoculating the cultured strain into a fermentation culture medium according to the proportion of 1% for solid state fermentation, and setting the fermentation temperature to 28 ℃ and the culture time to 72h. After the fermentation, the enzyme activity of 5' -AMP deaminase in the fermented material was measured.
(4) Leaching of yeast: adding 6 times of leaching solution, namely 0.5% sodium metabisulfite solution, according to the weight of the fermented yeast material, stirring for 2 hours until the yeast material is fully absorbed with water and wet, standing and soaking for 2 hours to obtain leaching solution. The extract was sampled and assayed for 5' -AMP deaminase activity.
(5) Flocculation and plate-frame filter pressing: adding 0.5% calcium chloride and 0.5% dipotassium hydrogen phosphate into the leaching solution at normal temperature, flocculating for 30min, and performing plate-frame filter pressing to obtain crude enzyme filtrate. Sampling and measuring the enzyme activity of 5' -AMP deaminase in the filtered clear liquid.
(6) Membrane concentration: the clear solution was concentrated with a 30KD filter membrane with a concentration factor of 8. The concentrated solution was sampled and assayed for 5' -AMP deaminase activity.
(7) And (3) separating and purifying: 250mL of concentrated enzyme solution is pre-cooled to 4 ℃, a certain amount of enzyme solution is taken, 75% ethanol pre-cooled to 4 ℃ is slowly added in the stirring process, and stirring is carried out while adding, the process lasts for 15min, so that the final concentration of the ethanol reaches 50%. And (3) carrying out alcohol precipitation for 30min, and centrifuging the sample to separate supernatant and precipitate. The supernatant volume was approximately 690mL. Then, the precipitate is dissolved by using 90mL of 0.1mol/L citric acid-NaOH buffer solution, and the volume is fixed to 100mL, thus obtaining the high-purity 5' -AMP deaminase sample. Sampling and measuring the enzyme activity of the purified 5' -AMP deaminase.
TABLE 4 preparation of 5' -AMP deaminase by Aspergillus honey adm01 enzyme Activity detection results
| Sample numbering | Sample description | Enzyme activity (U/g) | Enzyme activity (U/mL) |
| Sample 1 | Fermented yeast material | 32044 | - |
| Sample 2 | Yeast extract | - | 5525 |
| Sample 3 | Crude enzyme filtrate | - | 4697 |
| Sample 4 | Concentrated solution | - | 35697 |
| Sample 5 | Purification sample (supernatant) | - | 47 |
| Sample 6 | High purity deaminase sample | - | 70948 |
As shown in Table 4, the enzyme activity of the separated and purified 5' -AMP deaminase sample is more than 7 ten thousand U/mL, and the enzyme activity of the commercially available 5' -AMP deaminase product is most 20000-50000U/g, so that the level of the produced 5' -AMP deaminase is higher, and the method has industrial value.
Example 4 optimum reaction temperature and Heat resistance test of 5' -AMP deaminase prepared from Aspergillus honey adm01
Sample 4 prepared in example 3 was used as a pre-purification sample, and sample 6 was used as a post-deaminase purification sample; a commercially available 5' -AMP deaminase from Aspergillus melis used as a control sample. The enzyme activities of 5' -AMP deaminase of 3 samples were uniformly adjusted to 20000U/mL with 0.1mol/L citric acid-NaOH buffer, and then the optimal reaction temperature and heat resistance were tested.
(1) Testing the optimal reaction temperature: the 5' -AMP substrates were preheated to 35, 40, 45, 50, 55, 60, 65, 70℃respectively, 35 ' -AMP deaminase samples diluted in advance were added, and the enzyme activities of 5' -AMP deaminase at different temperatures were measured, and the results are recorded in FIG. 2 and Table 5.
(2) Heat resistance test: the 3 5' -AMP deaminase samples were incubated at 35, 40, 45, 50, 55, 60, 65, 70℃for 1 hour (the internal temperature reached the set value), rapidly cooled to room temperature, and the enzyme activities at 37℃and pH6 were measured, respectively, and the results are shown in FIG. 3 and Table 6.
Table 5 5' -AMP deaminase optimal reaction temperature test results
Table 6 5' -AMP deaminase thermostability test results
As can be seen from Table 5 and FIG. 2, the optimal reaction temperature of the commercial 5' -AMP deaminase enzyme solution derived from Aspergillus honey is 50 ℃, and the relative enzyme activity is more than 95% when the heating temperature is between 40 and 50 ℃; the optimal reaction temperature of the 5' -AMP deaminase of the sample 4 is 65 ℃, and when the heating temperature is between 55 and 65 ℃, the relative enzyme activity is more than 95 percent; the optimal reaction temperature of the 5' -AMP deaminase of the sample 6 is 70 ℃, and when the heating temperature is between 55 and 70 ℃, the relative enzyme activity is more than 95 percent.
As is clear from Table 6 and FIG. 3, the commercial 5' -AMP deaminase derived from Aspergillus honey has poor heat resistance, the enzyme activity retention rate starts to decrease greatly when the heating temperature reaches 50 ℃, the enzyme activity retention rate is only 35.84% when the heating temperature is 60 ℃, and the enzyme liquid is almost completely deactivated when the heating temperature is 65 ℃; the 5' -AMP deaminase of sample 4 is slightly better in heat resistance, and when the heating temperature reaches 60 ℃, the enzyme activity retention rate starts to decrease, and when the heating temperature rises to 65 ℃, the enzyme activity retention rate is 61.11%; the 5' -AMP deaminase solution of sample 6 is excellent in heat resistance, and when the heating temperature is raised to 65 ℃, the retention rate of the enzyme activity is 85.54%, and when the heating temperature reaches 70 ℃, the retention rate of the enzyme activity is 69.25%.
In conclusion, the optimal reaction temperature of the purified 5' -AMP deaminase enzyme solution derived from aspergillus melleus adm01 is up to 70 ℃, and the heat resistance of the purified 5' -AMP deaminase enzyme solution is obviously better than that of the commercial 5' -AMP deaminase enzyme solution derived from aspergillus melleus.
EXAMPLE 5' -AMP deaminase enzymatic Yeast test prepared by Aspergillus honey adm01
(1) Raw material cracking treatment
10g of dried Saccharomyces cerevisiae and 90g of water are added into a beaker to prepare a yeast solution with the mass ratio of 10%, and the yeast is deactivated after boiling for 10 min. After cooling, the pH value is adjusted to 6 by using 1mol/L HCl or 1mol/L NaOH, and after preheating at 60 ℃,0.1g of papain is added for enzymolysis at 60 ℃ for 3 hours. Heating the enzymolysis solution at 95 ℃ for 30min to inactivate enzyme, cooling to room temperature, and adjusting the pH value to 5.5 by using 1mol/L HCl or 1mol/L NaOH for standby.
(2) Enzymolysis
And (3) carrying out enzymolysis treatment on the yeast lysate sample prepared in the step (1) according to the following different processes:
the first process comprises the following steps: a yeast lysate sample was preheated at 70℃and then 0.15g of 5 '-nuclease (commercially available nuclease P1, 50000U/g) and 0.08g of 5' -AMP deaminase (50000U/g) prepared from Aspergillus honey adm01 were added thereto, followed by enzymatic hydrolysis at 70℃for 12 hours.
And a second process: after preheating the yeast lysate sample at 70℃0.15g of 5 '-nuclease (commercially available nuclease P1, 50000U/g) and 0.08g of commercially available 5' -AMP deaminase (50000U/g) were added and the mixture was subjected to enzymatic hydrolysis at 70℃for 12 hours.
And (3) a process III: after preheating the yeast lysate sample at 70℃0.15g of 5' -nuclease (commercially available nuclease P1, 50000U/g) was added and the mixture was subjected to enzymolysis at 70℃for 7 hours. Then 0.08g of 5' -AMP deaminase (50000U/g) prepared from Aspergillus nidulans adm01 was added thereto, and the mixture was subjected to enzymolysis at 70℃for 5 hours.
And a process IV: after preheating the yeast lysate sample at 70℃0.15g of 5' -nuclease (commercially available nuclease P1, 50000U/g) was added and the mixture was subjected to enzymolysis at 70℃for 7 hours. Then, the temperature of the enzymatic hydrolysate was lowered to 50℃and 0.08g of commercially available 5' -AMP deaminase (50000U/g) was added thereto, followed by enzymatic hydrolysis at 50℃for 5 hours.
(3) Enzymolysis effect detection
And heating the enzymolysis liquid at 95 ℃ for 30min to perform enzyme deactivation treatment. After cooling, the mixture was centrifuged to collect the supernatant. 5g of the supernatant was heated at 105℃for 4 hours, and the mass of the obtained dry solid matter was measured to calculate the dry solid content yield (%) per 1g of dry yeast. 0.5mL of the supernatant was added with 0.5mL of ultrapure water, and after filtration, the contents of the four 5' -nucleotides were determined by HPLC analysis, and the concentrations (w/w%) of IMP and GMP per dry solids content weight were calculated. The results are recorded in table 7.
TABLE 7 enzymatic Yeast test results
| Experimental method | Yield of dry solid (%) | IMP+GMP concentration (w/w%) |
| Enzymolysis technology I | 35.8 | 4.92 |
| Enzymolysis technology II | 34.2 | 3.98 |
| Enzymolysis technology III | 35.4 | 4.81 |
| Enzymolysis technology IV | 35.0 | 4.41 |
As can be seen from table 7, the yields of dry solids and the concentrations of imp+gmp in the samples prepared by the first enzymolysis process were all the highest, while the yields of dry solids and the concentrations of imp+gmp in the samples prepared by the second enzymolysis process were all the lowest; in the production process of the high I+G yeast extract, the purified 5' -AMP deaminase derived from aspergillus melleus adm01 can act with nuclease at high temperature at the same time, so that the production process is shortened.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (8)
1. Aspergillus honey strainAspergillus melleus) adm01, wherein the deposit number of the aspergillus melleus adm01 is GDMCC No. 63302.
2. A fermenting bacteria agent, which comprises aspergillus melleus adm01 according to claim 1.
3. Use of aspergillus melleus adm01 as claimed in claim 1 for the fermentative production of 5' -AMP deaminase.
4. A method for producing 5' -AMP deaminase, comprising the steps of:
1) Activating strains: inoculating the aspergillus melleus adm01 in the culture medium for activation;
2) And (3) performing expansion culture: inoculating the activated strain into a seed culture medium, and culturing for 5-7 days at 26-30 ℃ to obtain a solid strain;
3) Fermentation culture: inoculating the solid strain into a fermentation culture medium for culturing, and fermenting for 60-80 hours at the temperature of 26-30 ℃;
4) Leaching of yeast: adding 5-7 times of leaching solution according to the weight of the fermented yeast material, stirring until the yeast material is fully wet by water absorption, and standing and soaking for 1.5-2.5 hours to obtain leaching solution;
5) Press filtration of the plate and the frame: adding calcium chloride and dipotassium hydrogen phosphate into the leaching solution, flocculating, and performing plate-frame filter pressing to obtain crude enzyme filtrate;
6) Concentrating: concentrating the crude enzyme filtrate for 7-9 times to obtain an enzyme concentrated solution;
7) And (3) separating and purifying: pre-cooling the enzyme concentrate to 4 ℃, and slowly adding 75% ethanol pre-cooled to 4 ℃ while stirring to ensure that the final concentration of the ethanol reaches 50%. And (3) carrying out alcohol precipitation for 25-35 min, centrifugally separating a supernatant and precipitating the sample, and dissolving the precipitate with 0.1mol/L citric acid-NaOH buffer solution to obtain the high-purity 5' -AMP deaminase.
5. The method of claim 4, wherein the aspergillus melleus adm01 of step 1) is cultured at 26-30 ℃ for 3-5 days for activation.
6. The method of claim 4, wherein the seed medium of step 2) comprises water and bran, the mass ratio of water to bran being 1:1.
7. The method of claim 4, wherein step 3) of fermenting the medium comprises, on a 100g water basis: 100g of bran, 2g of glucose, 2g of peptone, 0.2g of sodium citrate, 0.1g of ammonium sulfate and 0.1g of magnesium sulfate heptahydrate.
8. The method of claim 4, wherein the leach solution of step 4) contains 0.4 to 0.6% sodium metabisulfite.
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