CN114634902B - Culture medium and culture method for producing psicose epimerase and application of culture medium and culture method - Google Patents
Culture medium and culture method for producing psicose epimerase and application of culture medium and culture method Download PDFInfo
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- CN114634902B CN114634902B CN202210298159.0A CN202210298159A CN114634902B CN 114634902 B CN114634902 B CN 114634902B CN 202210298159 A CN202210298159 A CN 202210298159A CN 114634902 B CN114634902 B CN 114634902B
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- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 title claims abstract description 58
- 239000001963 growth medium Substances 0.000 title claims abstract description 26
- 238000012136 culture method Methods 0.000 title abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 100
- 230000004151 fermentation Effects 0.000 claims abstract description 100
- 238000004519 manufacturing process Methods 0.000 claims abstract description 22
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 38
- 239000002609 medium Substances 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 34
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 30
- 235000013379 molasses Nutrition 0.000 claims description 30
- 229910052760 oxygen Inorganic materials 0.000 claims description 30
- 239000001301 oxygen Substances 0.000 claims description 30
- 240000008042 Zea mays Species 0.000 claims description 28
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 28
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 28
- 235000005822 corn Nutrition 0.000 claims description 28
- 238000003756 stirring Methods 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 23
- 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 23
- 239000008103 glucose Substances 0.000 claims description 23
- 229910052757 nitrogen Inorganic materials 0.000 claims description 20
- 244000063299 Bacillus subtilis Species 0.000 claims description 19
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 19
- 150000003839 salts Chemical class 0.000 claims description 19
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 16
- 229910052799 carbon Inorganic materials 0.000 claims description 16
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 235000020958 biotin Nutrition 0.000 claims description 9
- 229960002685 biotin Drugs 0.000 claims description 9
- 239000011616 biotin Substances 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 235000019764 Soybean Meal Nutrition 0.000 claims description 8
- 239000004455 soybean meal Substances 0.000 claims description 8
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000011573 trace mineral Substances 0.000 claims description 7
- 235000013619 trace mineral Nutrition 0.000 claims description 7
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 6
- 230000007423 decrease Effects 0.000 claims description 6
- 235000019192 riboflavin Nutrition 0.000 claims description 6
- 229960002477 riboflavin Drugs 0.000 claims description 6
- 239000002151 riboflavin Substances 0.000 claims description 6
- 235000019157 thiamine Nutrition 0.000 claims description 6
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 6
- 229960003495 thiamine Drugs 0.000 claims description 6
- 239000011721 thiamine Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 229940099596 manganese sulfate Drugs 0.000 claims description 4
- 239000011702 manganese sulphate Substances 0.000 claims description 4
- 235000007079 manganese sulphate Nutrition 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 239000011592 zinc chloride Substances 0.000 claims description 4
- 235000005074 zinc chloride Nutrition 0.000 claims description 4
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 3
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 38
- 102000004190 Enzymes Human genes 0.000 abstract description 30
- 108090000790 Enzymes Proteins 0.000 abstract description 30
- 239000002028 Biomass Substances 0.000 abstract description 8
- 244000005700 microbiome Species 0.000 abstract description 6
- 108090001066 Racemases and epimerases Proteins 0.000 description 48
- 102000004879 Racemases and epimerases Human genes 0.000 description 48
- 238000009472 formulation Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 6
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 description 6
- 229910052698 phosphorus Inorganic materials 0.000 description 6
- 229910052700 potassium Inorganic materials 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 229910052742 iron Inorganic materials 0.000 description 5
- 108030002106 D-psicose 3-epimerases Proteins 0.000 description 4
- 229930091371 Fructose Natural products 0.000 description 4
- 239000005715 Fructose Substances 0.000 description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 235000019750 Crude protein Nutrition 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000084 colloidal system Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 235000010755 mineral Nutrition 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N D-fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 241000192031 Ruminococcus Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 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
- 229930006000 Sucrose Natural products 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- 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
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Abstract
The invention relates to the technical field of industrial microorganisms, and discloses a culture medium and a culture method for producing psicose epimerase and application thereof. The culture medium provided by the invention is particularly suitable for fermentation production of psicose epimerase, and can improve the yield and activity of the psicose epimerase. By matching with the preferred fermentation production method, the yield and the enzyme activity of psicose epimerase can be further improved, the fermentation period can be shortened, the production efficiency can be improved, and the problems of low biomass of strains, low enzyme activity, long fermentation period, high production cost and the like in the existing fermentation enzyme production process can be effectively solved.
Description
Technical Field
The invention relates to the technical field of industrial microorganisms, in particular to a culture medium and a culture method for producing psicose epimerase and application thereof.
Background
D-psicose is taken as a rare sugar which is very few in nature, and has the effects of reducing blood sugar, reducing blood fat, resisting oxidation and the like, so that the D-psicose gradually becomes a research hot spot in the fields of food, health care and medical treatment, and the chemical method for preparing the D-psicose has not been subjected to breakthrough progress due to the reasons of complex product purification steps, serious chemical pollution, a plurality of byproducts and the like. The bioconversion method gradually becomes a main strategy for producing D-psicose due to the advantages of single reaction, simple purification steps and the like. Among them, D-psicose-3-epimerase (DPEase) is an important catalyst for the bioconversion of D-psicose, which is capable of catalyzing the isomerization reaction at the C3 position of D-fructose, and finally producing D-psicose.
Currently, biosynthesis is widely considered to be one of the most suitable methods for industrial production of DPEase. The biosynthesis method generally comprises culturing bioengineering bacteria capable of expressing DPease, collecting bacterial cells, and obtaining DPease through cell disruption, enzyme purification and other steps. In recent years, researchers have developed bioengineering bacteria for industrially producing DPease, such as DPease strains of different clostridium sources established at the university of Jiangnan, and Bacillus subtilis heterologous expression systems into which DPease genes of the genus Ruminococcus are inserted developed by the institute of Tianjin Industrial biotechnology, china academy of sciences. However, the current research on DPease biosynthesis is basically focused on the construction of strains and optimization of expression systems, and there are few reports on the research on the influence of culture media and culture methods on DPease yield and activity and the like. Moreover, the DPease yield and the enzyme activity of the existing DPease strain are generally low, and the requirement of industrialized psicose production is difficult to meet.
Disclosure of Invention
The invention aims to solve the problems of low yield and activity of psicose epimerase produced by a biosynthesis method in the prior art, and provides a culture medium and a culture method for producing psicose epimerase and application thereof. The culture medium provided by the invention is particularly suitable for biosynthesis production of psicose epimerase, and can improve the yield and activity of the psicose epimerase.
In order to achieve the above object, the present invention provides in one aspect a medium for producing psicose epimerase, the medium comprising a carbon source, a nitrogen source, an inorganic salt and a cofactor;
wherein the carbon source comprises glucose and/or molasses;
the nitrogen source comprises at least one of yeast extract, corn steep liquor, peptone and soybean meal;
the inorganic salt is used for providing at least one nutrient element of P, K, S, mn, zn and Fe;
the cofactor comprises at least one of thiamine, riboflavin, and biotin.
In a second aspect the invention provides a method for producing an allose epimerase, the method comprising inoculating an engineering bacterium producing the allose epimerase into a medium as described above for fermentation culture to obtain a fermentation broth containing the allose epimerase.
In a third aspect the invention provides the use of a medium and method as described above in the production of an industrial psicose epimerase.
Through the technical scheme, the invention has the following beneficial effects:
the culture medium provided by the invention can be used for high-density fermentation culture of engineering bacteria for producing psicose epimerase, is especially suitable for high-density fermentation culture of genetic engineering bacteria based on bacillus subtilis, can effectively improve the yield and the enzyme activity of psicose epimerase, and effectively solves the problems of low bacterial strain biomass, low enzyme activity, long fermentation period, high production cost and the like in the general fermentation enzyme production process. In addition, the fermentation period is shorter when the culture medium provided by the invention is used for carrying out psicose epimerase fermentation production, the enzyme activity is high, the biomass of thalli is large, and the large-scale popularization and application are facilitated. In addition, the fermentation efficiency and yield can be further improved by adopting the culture medium provided by the invention and matching the fermentation culture method of the invention to carry out the fermentation production of psicose epimerase.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the present invention, "psicose epimerase", "D-psicose-3-epimerase" and "DPease" are the same in meaning and are used interchangeably.
In the invention, molasses refers to a byproduct of the sugar industry, namely residual viscous liquid which cannot be concentrated and crystallized after repeated concentration and crystallized sugar separation treatment for a plurality of times in the sugar manufacturing process, and is also called waste molasses in industry. Typically brown, tan or black brown. Depending on the raw materials, the types may be specifically classified into various types such as cane molasses, beet molasses, grape molasses, and corn molasses. Unless otherwise indicated, "molasses" and "spent molasses" are synonymous and are used interchangeably.
The inventor of the invention skillfully discovers that the combination of a specific carbon source, a nitrogen source and inorganic salt in a culture medium can effectively improve the growth density of engineering bacteria and the yield of psicose epimerase. Meanwhile, by matching with specific microelements and auxiliary factors, the enzyme activity of psicose epimerase can be improved, and the fermentation period can be shortened.
Based on the above findings, the present invention provides in one aspect a medium for producing psicose epimerase, the medium comprising a carbon source, a nitrogen source, an inorganic salt and a cofactor;
wherein the carbon source comprises glucose and/or molasses;
the nitrogen source comprises at least one of yeast extract, corn steep liquor, peptone and soybean meal;
the inorganic salt is used for providing at least one nutrient element of P, K, S, mn, zn and Fe;
the cofactor comprises at least one of thiamine, riboflavin, and biotin.
According to a preferred embodiment of the present invention, wherein the element C provided by the carbon source in the medium is 2-8g/L, preferably 3-7g/L.
Preferably, the molasses contains 45-55 wt% of total sugar, 2-3 wt% of crude protein, 3-10 wt% of soluble colloid, 10-15 wt% of ash, 3-10 wt% of mineral and 2.5-3.5 wt% of biotin. The molasses also contains a certain amount of moisture, preferably a moisture content of 20-30% by weight.
More preferably, the molasses contains 46-52 wt% of total sugar, 2-3 wt% of crude protein, 3-10 wt% of soluble colloid, 10-15 wt% of ash, 3-10 wt% of mineral, 2.7-3.2 wt% of biotin and 24-26 wt% of water.
In the present invention, the source and kind of the molasses are not particularly limited, and may be any one of common molasses in sugar industry such as cane molasses, beet molasses, grape molasses, corn molasses and the like or a combination of any several of them as long as they have the above-mentioned characteristics.
The inventors of the present invention have also found during the course of the study that when a proper amount of molasses is added as a carbon source to the medium, the biomass of the microorganism for producing psicose epimerase can be increased, and the enzyme activity of the resultant psicose epimerase can also be increased.
According to a preferred embodiment of the invention, wherein the carbon source comprises glucose and molasses.
The specific amounts (proportions) of glucose and molasses in the medium are not particularly limited in the present invention. In order to further increase the biomass of the microorganism used for producing psicose epimerase and the enzyme activity of psicose epimerase, preferably, the weight ratio of glucose to molasses is 1:0.2 to 5.
More preferably, the glucose content in the carbon source is 3 times or more that of molasses. That is, the weight ratio of glucose to molasses is 1:3-5. More preferably 1:4-5. Further preferably 1:4.5-5.
According to a preferred embodiment of the present invention, wherein the N element provided by the nitrogen source in the medium is 3-12/L, preferably 4-10g/L.
The inventors of the present invention have also found during the course of the study that when corn steep liquor is selected as (one of) the nitrogen source, the use of corn steep liquor supernatant can further increase the biomass of the microorganism used for producing psicose epimerase and the enzyme activity of psicose epimerase compared to corn steep liquor (i.e., fully-composed corn steep liquor). The "corn steep liquor supernatant" refers to a liquid phase substance obtained by subjecting (complete component) corn steep liquor to solid-liquid separation (e.g. filtration, centrifugation, etc.), and has a dry solids content of not more than 10% by weight, and mainly contains N, P, fe, mn, mg, ca, K, zn, na and other elements.
The corn steep liquor supernatant used in the invention can be obtained directly and commercially, or can be obtained by carrying out solid-liquid separation on corn steep liquor which is commercially available or prepared by self.
According to a preferred embodiment of the present invention, wherein the nitrogen source comprises at least one of yeast powder, (whole ingredient) corn steep liquor, peptone and soybean meal.
Preferably, the (full ingredient) corn steep liquor has a dry solids content of 40-50%.
According to another preferred embodiment of the present invention, wherein the nitrogen source includes at least one of yeast powder, corn steep liquor supernatant, peptone and soybean meal powder.
Preferably, the dry solid content of the corn steep liquor supernatant is not more than 10 wt%, preferably the corn steep liquor supernatant contains 2-4 wt% of total nitrogen, 1-2 wt% of total phosphorus, 30-40 wt% of iron, 5-7 wt% of manganese, 500-550 wt% of magnesium, 5-10 wt% of calcium, 1-3 wt% of potassium, 22-26 wt% of zinc and 10-20 wt% of sodium.
The amount of the inorganic salt in the medium is not particularly limited in the present invention. According to a preferred embodiment of the present invention, wherein the medium contains 1 to 5g/L, K of the P element, 5 to 10g/L, S of the P element, 0.1 to 0.8g/L, mn of the 5X 10 element -4 -2×10 -3 g/L, zn element is 4×10 -4 -5×10 -3 g/L, fe element is 1×10 -3 -2×10 -2 g/L。
Preferably, in the culture medium, the element P provided by the inorganic salt is 2-4g/L, K element 5-8g/L, S element 0.2-0.6g/L, mn element 8×10 -4 -1.8×10 -3 g/L, zn element is 4.5X10 -4 -4.8×10 -3 g/L, fe element is 1.2X10 -3 -1.5×10 -2 g/L。
According to a preferred embodiment of the invention, wherein the inorganic salts (depending on the amount of inorganic salt) comprise macroelement inorganic salts and/or microelement inorganic salts.
Preferably, the macroelement inorganic salt is used for providing at least two nutrient elements of P, K and S.
More preferably, the macroelement inorganic salt includes at least one of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate and diammonium hydrogen phosphate.
Preferably, the trace element inorganic salt is used to provide at least one nutrient element of Mn, zn and Fe.
More preferably, the trace element inorganic salt includes at least one of manganese sulfate, zinc chloride, iron sulfate, and iron chloride.
In the invention, when the culture medium contains both major inorganic salts and trace inorganic salts, the biomass of engineering bacteria at the fermentation end point, the yield of psicose epimerase, the enzyme activity and the like can be further improved.
In order to obtain a better cultivation effect (e.g. psicose epimerase activity, fermentation end-point microorganism content, etc.), according to a particularly preferred embodiment of the present invention, the formulation of the medium comprises:
carbon source: glucose 1-15g/L, molasses 1-10g/L;
nitrogen source: 5-35g/L yeast powder, 25-85g/L corn steep liquor supernatant, 1-10g/L peptone and 1-5g/L soybean meal powder;
macroelement inorganic salt: 10-15g/L of dipotassium hydrogen phosphate, 1-5g/L of monopotassium hydrogen phosphate and 0.5-3g/L of ammonium sulfate;
inorganic salts of trace elements: manganese sulfate 0.005-0.01g/L, zinc chloride 0.001-0.01g/L, ferric sulfate 0.005-0.05g/L;
cofactor: thiamine 5X 10 -6 -1×10 -3 g/L, riboflavin 1X 10 -5 -2×10 -4 g/L, biotin 1X 10 -6 -5×10 -4 g/L；
The balance being water.
Preferably, the formulation of the medium comprises:
carbon source: glucose 2-12g/L and molasses 2-9g/L;
nitrogen source: 8-32g/L yeast powder, 26-81g/L corn steep liquor supernatant, 2-10g/L peptone and 2-5g/L soybean meal;
macroelement inorganic salt: 11-14g/L of dipotassium hydrogen phosphate, 1.5-3g/L of monopotassium hydrogen phosphate and 0.5-2g/L of ammonium sulfate;
inorganic salts of trace elements: manganese sulfate 0.005-0.01g/L, zinc chloride 0.003-0.01g/L, ferric sulfate 0.005-0.05g/L;
cofactor: thiamine 7X 10 -6 -9×10 -4 g/L, riboflavin 1X 10 -5 -2×10 -4 g/L, biotin 1X 10 -6 -5×10 -4 g/L；
The balance being water.
In the invention, the culture medium can be prepared directly after weighing the components according to the above-mentioned dosage, or can be prepared into aqueous solutions respectively according to the above-mentioned dosage, and the components are added respectively in the fermentation process (at different times) as long as the dosage of the final components meets the above-mentioned requirements.
According to a preferred embodiment of the invention, the trace amounts of inorganic salts and/or cofactors in the culture medium may be formulated (separately or together) separately as an aqueous solution, which is added to the culture system during the fermentation culture.
Preferably, the aqueous solution of the trace inorganic salt and/or the cofactor may be added to the culture system by means of fed-batch addition during fermentation. In the present invention, the specific mode of the fed-batch addition is not particularly limited as long as the total addition amount of the trace inorganic salts and/or cofactors finally meets the requirement of the amount used in the aforementioned medium. For example, the aqueous solution of the trace inorganic salts and/or cofactors may be added to the fermentation system at a uniform rate during the fermentation process, or may be added to the fermentation system in a variable rate fed-batch manner under certain rate-changing conditions.
In a second aspect the invention provides a method for producing an allose epimerase, the method comprising inoculating an engineering bacterium producing the allose epimerase into a medium as described above for fermentation culture to obtain a fermentation broth containing the allose epimerase.
According to a preferred embodiment of the invention, the engineering bacteria are bacillus subtilis.
Preferably, the engineering bacterium is genetically engineered bacillus subtilis, preferably genetically engineered bacillus subtilis 1012.
In order to obtain more engineering bacteria and psicose epimerase with higher enzyme activity at the fermentation end point, according to a preferred embodiment of the present invention, the bacillus subtilis contains a gene with a nucleotide sequence shown in SEQ ID NO. 1.
ATGAACAAGATCGGAGTTCATTTTGGATATTTTAACAGAGATTGGAACACAGATTTCATTAAAAGAATCGAACAGGTTAAGAAGATCGGACTGGATATTCTTGAAGTGGCACCGGCACCGCTGCTTGCACTTACAAAATTTCAGAGAGATGAAATTGCAGCAGCGGCAAAAGCAAATGATATTGAACTGACATTTAGCGTGGGACTTAGCGCAAATCAAGATCTTGCGAGCGAAGATGAAGAAATTAGAAAAAATGGAATCAAGTTCACGACAGATACATTTCAGATTATGTCAGAAATGGGAGGAAAAACATATAGCGGCGTTGATATTGCAGCGTGGAATAAAACATTTATGGAAGGAATTACGGACAAATCAGCAACATGGGAAAGATCAATTAGCGCGGTTAAAGAAATTATGAAAGTTGCAGAAGACAAGGGAATTACATTTGCGGTTGAAGTTGTTAATAGATATGAATCAAGCCTTGTGAATACAGCAGAAGAAGCAGTTAAATATGTGGATGAAGTTGGAAGCCCGAATTGTAAAATTCTTCTTGATACATACCACATGAACATTGAAGAAGATAGCTTTGCGGGCGCGATTAAACTGGTGGGCAATAGACTTGGCCATTTTCATGTTGGAGAAAGCAATAGAAGACCGCCGTGTGAAAATGGAAAAATGCCGTGGAATGAAATTACAAATGCACTTAAAGAGATCGATTACCAAGGAGCGATTGTGATGGAACCGTTTATTAAAATGGGCGGAGAAGTTGGCAGAGATATTAAAGTGTGGAGAGATATTAGCGAAGGCGCGTCAGAAAGCGAAATGGAACAGCTTCTTGCAGATGCAGCAATGATGCTGAGAAAAAAAATGCAAAGACATCATCATCACCATCATTAA(SEQ ID NO:1)
According to a preferred embodiment of the present invention, wherein the conditions of the fermentation culture include: the inoculation amount of the engineering bacteria enables the initial OD of the engineering bacteria in the fermentation system 600 The value is 0.1-0.5, the fermentation temperature is 35-38 ℃, the pH is 7-7.5, the ventilation is 0.8-1.2vvm, the initial dissolved oxygen is 70-90%, the initial stirring rotating speed is 150-250rpm, and the fermentation time is the same as that of the prior art18-25h。
Preferably, the conditions of the fermentation culture include: the inoculation amount of the engineering bacteria enables the initial OD of the engineering bacteria in the fermentation system 600 The value is 0.25-0.35, the fermentation temperature is 35-37 ℃, the pH is 7-7.2, the aeration rate is 0.8-1.2vvm, the initial dissolved oxygen amount is 70-90%, the initial stirring rotating speed is 150-250rpm, and the fermentation time is 15-25h.
Preferably, the method further comprises controlling the amount of dissolved oxygen during the fermentation culture. The purpose of controlling the dissolved oxygen is to keep the dissolved oxygen of the fermentation system within a set range within a period from the beginning of fermentation to the time before the maximum rotation speed is reached (about 0 to 7 hours), thereby improving the biomass of the bacteria and the epimerase activity of psicose.
More preferably, the control method includes: and starting fermentation at an initial stirring rotation speed, and when the dissolved oxygen amount of the culture system naturally decreases and is lower than a dissolved oxygen amount set value, starting to increase the stirring rotation speed, so that the dissolved oxygen amount is maintained within a dissolved oxygen amount set range, wherein the dissolved oxygen amount set value is 20-25%, the dissolved oxygen amount set range is 20-25%, the maximum value of the stirring rotation speed is 800-1000rpm, and when the stirring rotation speed reaches the maximum value, the stirring rotation speed is not increased along with the decrease of the dissolved oxygen amount. The "dissolved oxygen set value" refers to a dissolved oxygen critical value for judging whether to increase the stirring rotation speed, and is any one of determined values in the range of 20-25%. The term "dissolved oxygen amount setting range" means a range of dissolved oxygen amount in the fermentation system maintained by adjusting the stirring rotation speed after starting to increase the stirring rotation speed, which is any range of 20 to 25%, or may be a range of any two values of 20 to 25%.
More preferably, the maximum stirring speed is 600-700rpm different from the initial stirring speed.
According to a preferred embodiment of the invention, the method further comprises the step of feeding during fermentation.
Preferably, the feed is turned on when glucose in the medium is depleted.
Preferably, the feed liquid used for the feed is an aqueous glucose solution containing 0.6-0.8 wt% magnesium sulfate, and preferably the glucose content in the feed liquid is 50-70 wt%.
More preferably, the feed liquid is added in an amount such that the residual amount of glucose in the fermentation system does not exceed 5g/L.
Psicose epimerase prepared by using the culture medium or the method described above also belongs to the protection scope of the present invention.
According to a preferred embodiment of the present invention, wherein the psicose epimerase has an enzyme activity of 300U/mL or more, preferably 300-350U/mL.
In a third aspect the invention provides the use of a medium or method as hereinbefore described in the production of an industrial psicose epimerase.
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only and are not intended to limit the invention.
In the following examples, engineering bacteria were used without specific explanation, which were obtained by inserting the DPease coding gene shown in SEQ ID No. 1 into Bacillus subtilis 1012 by genetic engineering techniques. For a specific preparation method reference is made to CN201911072202.6.
The (sugar cane) molasses used in the examples below was purchased from the midgrain sugar industry division and contained 50.+ -. 5 wt.% total sugar (wherein the weight ratio of sucrose, glucose to fructose was about 3.5:1:1), 2.5.+ -. 0.5 wt.% crude protein, 6.+ -. 3 wt.% soluble colloid, 12.+ -. 2 wt.% ash, 6.+ -. 2 wt.% minerals, 3.+ -. 0.5 wt.% biotin, 25.+ -. 5 wt.% moisture. Corn steep liquor was purchased from midgrain biochemical energy (elm) limited and tested to have a dry solids content of about 45% and a total nitrogen content of about 3.11g/100g. Other reagents were used, unless specifically indicated, were purchased from regular chemical/biological reagent suppliers and were either analytically or chemically pure.
In the following examples, corn steep liquor supernatant was obtained in the following manner: centrifuging corn steep liquor at 8000rpm for 15min, and discarding precipitate to obtain corn steep liquor supernatant. The corn steep liquor supernatant contains 2.78g/100g of total nitrogen, 1.5g/100g of total phosphorus, 36.5mg/kg of iron, 5.95mg/kg of manganese, 510mg/kg of magnesium, 8.45mg/kg of calcium, 1.75g/kg of potassium, 24.7mg/kg of zinc and 16mg/kg of sodium.
In the following examples, the seed liquid is obtained by: inoculating 2 vol.% of Bacillus subtilis engineering bacteria liquid (glycerol tube preservation) into sterilized seed culture medium (yeast powder 5g/L, peptone 10g/L, sodium chloride 10g/L, and water balance) by shake flask fermentation (40 vol%) at 37deg.C for 10 hr at 200rpm to obtain seed liquid (wherein Bacillus subtilis engineering bacteria OD) 600 About 1 to about 1.8).
In the following examples, the enzyme activity of psicose epimerase in the fermentation broth was detected and calculated as follows:
pretreatment of enzyme activity detection: taking a certain amount of fermentation liquor (30-40 mL) and centrifuging at 9000rpm for 10min, discarding the supernatant, weighing and subtracting the empty tube weight to obtain the bacterial mass, adding deionized water with the volume of bacterial mass multiplied by 10 into a centrifuge tube, oscillating on an oscillator to completely resuspension, taking 10mL of resuspension into the centrifuge tube, ultrasonically crushing for 15min, centrifuging at 9000rpm for 10min, and obtaining crushed centrifugal supernatant. Absorbing 4.5mL of fructose solution with the concentration of 300mg/mL, which is added with a trace amount of cobalt chloride (the concentration of cobalt chloride in the added solution is 0.5 ppm), adding 500 mu L of fermentation thalli into the fructose solution, crushing, centrifuging supernatant, immediately placing the test tube into a water bath kettle at 55 ℃ for water bath for 5min, taking out the test tube after the reaction is finished, putting the test tube into boiled water for bacterial inactivation, diluting for 10 times, detecting in a liquid phase, and calculating the enzyme activity value according to the peak area of the liquid chromatography psicose.
Liquid chromatography detection conditions: chromatographic column: waters sugar-Pak I; specification of: 10 mu m6.5mm×300mm; column temperature: 80 ℃; a detector: RID; detecting the temperature: 55 ℃; sample loading amount: 10. Mu.L; mobile phase: water; flow rate: 0.4mL/min; the peak of fructose and psicose is respectively 15min and 22 min.
The enzyme activity calculation method comprises the following steps: let the standard curve of psicose be y=ax+b, Y be the peak area of psicose, X be the concentration of psicose (mg/mL), a, b be constant.
The mass (g) of psicose produced was m=v·k (Y-b)/a (formula 1)
In the formula 1, Y is the peak area of high-yield liquid chromatography corresponding to psicose in enzyme catalysis reaction liquid, v is the volume (mL) of a reaction system (enzyme liquid and substrate), and k is the multiple of solution dilution during high-performance liquid chromatography detection.
Enzyme activity e=z.m/t (2)
In formula 2, z is the dilution factor when the cells are resuspended, t is the reaction time (min, generally 5 min)
Integrating equations 1 and 2 yields: e=z·k·v (Y-b)/a·t (formula 3)
Example 1
This example is intended to illustrate the effect of the selection of carbon and nitrogen sources in the medium on the fermentation results.
Culture medium I:
the carbon source and nitrogen source components and the content are shown in Table 1 in detail
Macroinorganic salts: k (K) 2 HPO 4 13.3g/L、KH 2 PO 4 2g/L、(NH 4 ) 2 SO 4 1g/L
Trace inorganic salts: mnSO 4 0.006g/L、ZnCl 2 0.004g/L、Fe 2 (SO 4 ) 3 0.012g/L
Cofactor: thiamine 4.3X10 -4 g/L, riboflavin 1.4X10 -4 g/L, biotin 3.1X10) -4 The balance of g/L is water.
The psicose epimerase fermentation production is carried out by adopting a 5L fermentation tank (3L of liquid filling amount), and the specific method is as follows: inoculating the seed solution into the fermentation medium I in an amount such that the bacillus subtilis OD in the fermentation system 600 The value was 0.3.+ -. 0.1. After inoculation at 37℃and pH7.2, the initial dissolved oxygen amount is 70-90%, the initial aeration amount is 1vvm, and the initial rotation speed is 200 rpm.
In the fermentation culture process, the dissolved oxygen is controlled through the adjustment of the rotating speed. The specific control mode comprises the following steps: and starting fermentation at an initial stirring rotation speed, and when the dissolved oxygen amount of the culture system naturally decreases and is lower than 20%, starting to increase the stirring rotation speed, so that the dissolved oxygen amount is maintained at a level of 20% -25%, wherein the maximum value of the stirring rotation speed is 800rpm, and when the stirring rotation speed reaches the maximum value, the stirring rotation speed is not increased along with the decrease of the dissolved oxygen amount. The dissolved oxygen in the culture system is continuously reduced along with the proliferation of bacillus subtilis, and finally approaches to 0.
In the fermentation culture process, when sugar (based on glucose) in the culture medium is exhausted, starting feeding, wherein the feeding liquid is 60 wt% glucose aqueous solution (containing 7g/L magnesium sulfate), feeding is fed-batch feeding, and meanwhile, the flow rate of the liquid is regulated, and the residual sugar content of a fermentation system is controlled to be not more than 5g/L. The fermentation time is 20 hours in total, and the fermentation liquor is obtained.
The OD values of Bacillus subtilis in the fermentation broth obtained with the different formulation media were measured by a spectrophotometer (SHIMADZU model UV 1750) and the enzyme activities of psicose epimerase in the fermentation broth were measured and calculated as described above, and the results are shown in Table 1.
TABLE 1 Medium formulations of different carbon sources or Nitrogen sources and fermentation results
Example 2
This example is used to illustrate the effect of trace elements and cofactors in the medium on fermentation efficiency.
Medium II:
carbon source: glucose 11g/L and molasses 2.4g/L
Nitrogen source: 9.5g/L yeast powder, 2.5g/L soybean meal, 75.3g/L corn steep liquor supernatant and 4.5g/L peptone
Macroinorganic salts: k (K) 2 HPO 4 13.3g/L、KH 2 PO 4 2g/L、(NH 4 ) 2 SO 4 1g/L
The contents of trace inorganic salts and cofactors are shown in Table 2, and the balance is water.
The psicose epimerase fermentation production was carried out in accordance with the method in example 1 using medium II for a total of 20 hours to obtain a fermentation broth.
The OD values of Bacillus subtilis in the fermentation broth obtained with the different formulation media were measured by a spectrophotometer (SHIMADZU Co. UV1750 type), and the enzyme activities of psicose epimerase in the fermentation broth were measured and calculated according to the method described above, and the results are shown in Table 2 in detail.
TABLE 2 culture medium formulations of different trace inorganic salts or cofactors and fermentation results
Example 3
This example is to illustrate the effect of the different addition of trace inorganic salts and cofactors on the fermentation effect.
Culture medium III-1: the formulation was the same as the I-3 medium of example 1, except that the medium was formulated without adding corn steep liquor supernatant, trace elements and cofactors.
Medium III-2: an aqueous solution of corn steep liquor supernatant, trace inorganic salts and cofactors was prepared in the amounts used in formulation I-3.
The psicose epimerase fermentation production was performed as in example 1, except that the medium III-1 was fed into the fermenter, and the medium III-2 was fed in the manner shown in Table 3 during the cultivation. The fermentation time is 20 hours in total, and the fermentation liquor is obtained.
The OD values of Bacillus subtilis in the fermentation broth obtained by different addition methods were measured by a spectrophotometer (SHIMADZU Co. UV1750 type), and the enzyme activities of psicose epimerase in the fermentation broth were measured according to the method described above, and the results are shown in Table 3.
TABLE 3 different addition modes of trace inorganic salts and cofactors and fermentation results
Example 4
This example is presented to illustrate the effect of different fermentation/control modes on fermentation results.
The psicose epimerase fermentation production was carried out in the same manner as in example 1 except for the conditions/control manner shown in Table 4 using the medium of the formula II-3 shown in Table 2, and the other conditions and operations in the fermentation were the same as those in example 1.
The OD values of Bacillus subtilis in the fermentation broth obtained with the different formulation media were measured by a spectrophotometer (SHIMADZU Co. UV1750 type), and the enzyme activities of psicose epimerase in the fermentation broth were measured according to the method described above, and the results are shown in Table 4.
TABLE 4 different fermentation/Regulation modes and fermentation results
Comparative example 1
The psicose epimerase fermentation production was carried out in the same manner as in example 1 using a conventional medium, except that the medium was different, and the conditions and operations during the fermentation were the same as those in example 1.
Conventional medium formulation: 24g/L yeast powder, 5g/L peptone, 10g/L dipotassium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 1g/L ammonium sulfate and 10g/L glucose.
(1) Fermenting for 20h to obtain fermentation liquor. The OD value of Bacillus subtilis and the enzyme activity of psicose epimerase in the obtained fermentation broth were measured in accordance with the method in example 1, and the results are shown in Table 5.
(2) Fermenting for 30h to obtain fermentation liquor. The OD value of Bacillus subtilis and the enzyme activity of psicose epimerase in the obtained fermentation broth were measured in accordance with the method in example 1, and the results are shown in Table 5.
TABLE 5 fermentation results on conventional Medium
| Fermentation time | OD value | Enzyme activity (U/mL) |
| 20h | 57.9 | 277.5 |
| 30h | 59.3 | 285.3 |
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
SEQUENCE LISTING
<110> Jilin grain Biochemical Co., ltd
COFCO NUTRITION AND HEALTH RESEARCH INSTITUTE Co.,Ltd.
<120> Medium for producing psicose epimerase and culture method and use thereof
<130> I71387COF
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 897
<212> DNA
<213> artificial sequence
<400> 1
atgaacaaga tcggagttca ttttggatat tttaacagag attggaacac agatttcatt 60
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ctgcttgcac ttacaaaatt tcagagagat gaaattgcag cagcggcaaa agcaaatgat 180
attgaactga catttagcgt gggacttagc gcaaatcaag atcttgcgag cgaagatgaa 240
gaaattagaa aaaatggaat caagttcacg acagatacat ttcagattat gtcagaaatg 300
ggaggaaaaa catatagcgg cgttgatatt gcagcgtgga ataaaacatt tatggaagga 360
attacggaca aatcagcaac atgggaaaga tcaattagcg cggttaaaga aattatgaaa 420
gttgcagaag acaagggaat tacatttgcg gttgaagttg ttaatagata tgaatcaagc 480
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aaagtgtgga gagatattag cgaaggcgcg tcagaaagcg aaatggaaca gcttcttgca 840
gatgcagcaa tgatgctgag aaaaaaaatg caaagacatc atcatcacca tcattaa 897
Claims (9)
1. A method for producing psicose epimerase, characterized in that the method comprises inoculating an engineering bacterium for producing psicose epimerase into a culture medium for fermentation culture to obtain a fermentation broth containing psicose epimerase;
wherein, the formula of the culture medium comprises:
carbon source: glucose 1-15g/L, molasses 1-10g/L;
nitrogen source: 5-35g/L yeast powder, 25-85g/L corn steep liquor supernatant, 1-10g/L peptone and 1-5g/L soybean meal powder;
macroelement inorganic salt: 10-15g/L of dipotassium hydrogen phosphate, 1-5g/L of monopotassium hydrogen phosphate and 0.5-3g/L of ammonium sulfate;
inorganic salts of trace elements: manganese sulfate 0.005-0.01g/L, zinc chloride 0.001-0.01g/L, ferric sulfate 0.005-0.05g/L;
cofactor: thiamine 5.5X10 -5 -9×10 -4 g/L, riboflavin 1X 10 -5 -2×10 -4 g/L, biotin 3.1X10) -5 -5×10 -4 g/L；
The balance being water;
the engineering bacteria are genetically engineered bacillus subtilis 1012, and the bacillus subtilis contains genes with nucleotide sequences shown as SEQ ID NO. 1.
2. The method of claim 1, wherein the fermentation culture conditions comprise: the inoculation amount of the engineering bacteria enables the initial OD of the engineering bacteria in the fermentation system 600 The value is 0.1-0.5, the fermentation temperature is 35-38 ℃, the pH is 7-7.5, the ventilation quantity is 0.8-1.2vvm, the initial dissolved oxygen quantity is 70-90%, the initial stirring rotating speed is 150-250rpm, and the fermentation time is 18-25h.
3. The method of claim 1, wherein the method further comprises controlling the amount of dissolved oxygen during the fermentation culture.
4. A method according to claim 3, wherein the manner of controlling comprises: and starting fermentation at an initial stirring rotation speed, and when the dissolved oxygen amount of the culture system naturally decreases and is lower than a set value of the dissolved oxygen amount, starting to increase the stirring rotation speed so as to maintain the culture system within a set range of the dissolved oxygen amount, wherein the set value of the dissolved oxygen amount is 20-25%, the set range of the dissolved oxygen amount is 20-25%, the maximum value of the stirring rotation speed is 800-1000rpm, and when the stirring rotation speed reaches the maximum value, the stirring rotation speed is not increased along with the decrease of the dissolved oxygen amount.
5. The method of claim 4, wherein the maximum stirring speed is 600-700rpm different from the initial stirring speed.
6. The method of any one of claims 1-5, wherein the method further comprises the step of feeding during fermentation;
7. the method of claim 6, wherein the feed is turned on when glucose in the medium is depleted;
and/or the feed liquid used for the feed is glucose aqueous solution containing 0.6-0.8 wt% of magnesium sulfate.
8. The method according to claim 7, wherein the glucose content of the feed liquid is 50-70 wt%.
9. The method according to claim 7, wherein the feed liquid is added in an amount such that the residual amount of glucose in the fermentation system does not exceed 5g/L.
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| CN107699556A (en) * | 2017-11-10 | 2018-02-16 | 山东百龙创园生物科技股份有限公司 | The method that D psicose epimerases are prepared using bacillus subtilis |
| CN110904132A (en) * | 2019-11-05 | 2020-03-24 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose3-epimerase and application thereof |
| WO2021244005A1 (en) * | 2020-06-03 | 2021-12-09 | 中国科学院天津工业生物技术研究所 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| CN114214251A (en) * | 2021-12-31 | 2022-03-22 | 保龄宝生物股份有限公司 | Bacillus subtilis for producing D-psicose and culture method and application thereof |
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| CN110904132A (en) * | 2019-11-05 | 2020-03-24 | 吉林中粮生化有限公司 | Coding gene, vector and recombinant cell of D-psicose3-epimerase and application thereof |
| WO2021244005A1 (en) * | 2020-06-03 | 2021-12-09 | 中国科学院天津工业生物技术研究所 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| CN114214251A (en) * | 2021-12-31 | 2022-03-22 | 保龄宝生物股份有限公司 | Bacillus subtilis for producing D-psicose and culture method and application thereof |
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