CN107058137A - 一种渥曼青霉及其生产渥曼青霉素的方法 - Google Patents
一种渥曼青霉及其生产渥曼青霉素的方法 Download PDFInfo
- Publication number
- CN107058137A CN107058137A CN201710447621.8A CN201710447621A CN107058137A CN 107058137 A CN107058137 A CN 107058137A CN 201710447621 A CN201710447621 A CN 201710447621A CN 107058137 A CN107058137 A CN 107058137A
- Authority
- CN
- China
- Prior art keywords
- wortmannin
- culture
- mould
- wet
- seed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- QDLHCMPXEPAAMD-UHFFFAOYSA-N wortmannin Natural products COCC1OC(=O)C2=COC(C3=O)=C2C1(C)C1=C3C2CCC(=O)C2(C)CC1OC(C)=O QDLHCMPXEPAAMD-UHFFFAOYSA-N 0.000 title claims abstract description 50
- QDLHCMPXEPAAMD-QAIWCSMKSA-N wortmannin Chemical compound C1([C@]2(C)C3=C(C4=O)OC=C3C(=O)O[C@@H]2COC)=C4[C@@H]2CCC(=O)[C@@]2(C)C[C@H]1OC(C)=O QDLHCMPXEPAAMD-QAIWCSMKSA-N 0.000 title claims abstract description 50
- 238000004519 manufacturing process Methods 0.000 title description 16
- 238000000034 method Methods 0.000 claims abstract description 13
- 241001136485 Talaromyces wortmannii Species 0.000 claims abstract description 8
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 24
- 239000002609 medium Substances 0.000 claims description 20
- 239000000843 powder Substances 0.000 claims description 15
- 238000011218 seed culture Methods 0.000 claims description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 12
- 235000010216 calcium carbonate Nutrition 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 235000012054 meals Nutrition 0.000 claims description 12
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 12
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 12
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 10
- 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 claims description 10
- 235000012343 cottonseed oil Nutrition 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 235000002639 sodium chloride Nutrition 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 244000068988 Glycine max Species 0.000 claims description 8
- 235000010469 Glycine max Nutrition 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 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 7
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 235000011187 glycerol Nutrition 0.000 claims description 7
- 239000008101 lactose Substances 0.000 claims description 7
- 229940099596 manganese sulfate Drugs 0.000 claims description 7
- 239000011702 manganese sulphate Substances 0.000 claims description 7
- 235000007079 manganese sulphate Nutrition 0.000 claims description 7
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- 235000003276 Apios tuberosa Nutrition 0.000 claims description 2
- 244000105624 Arachis hypogaea Species 0.000 claims description 2
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 2
- 235000010744 Arachis villosulicarpa Nutrition 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 229920002261 Corn starch Polymers 0.000 claims description 2
- 229920002774 Maltodextrin Polymers 0.000 claims description 2
- 239000005913 Maltodextrin Substances 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 235000015278 beef Nutrition 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 239000008120 corn starch Substances 0.000 claims description 2
- 229940099112 cornstarch Drugs 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 229960005150 glycerol Drugs 0.000 claims description 2
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 229940035034 maltodextrin Drugs 0.000 claims description 2
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 claims description 2
- 229940073769 methyl oleate Drugs 0.000 claims description 2
- 235000019198 oils Nutrition 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- -1 sorbierite Chemical compound 0.000 claims description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 2
- 235000011046 triammonium citrate Nutrition 0.000 claims description 2
- 239000001393 triammonium citrate Substances 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229960001763 zinc sulfate Drugs 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 235000009508 confectionery Nutrition 0.000 claims 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 229960004793 sucrose Drugs 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 28
- 230000001580 bacterial effect Effects 0.000 description 19
- 238000000855 fermentation Methods 0.000 description 18
- 230000004151 fermentation Effects 0.000 description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 102000038030 PI3Ks Human genes 0.000 description 6
- 108091007960 PI3Ks Proteins 0.000 description 6
- 241000228143 Penicillium Species 0.000 description 6
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 241000228168 Penicillium sp. Species 0.000 description 3
- 239000007857 degradation product Substances 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- QDLHCMPXEPAAMD-ZGSWIPFCSA-N LSM-1318 Chemical compound C1([C@]2(C)C3=C(C4=O)OC=C3C(=O)O[C@@H]2COC)=C4C2CCC(=O)[C@@]2(C)C[C@H]1OC(C)=O QDLHCMPXEPAAMD-ZGSWIPFCSA-N 0.000 description 2
- 102100035044 Myosin light chain kinase, smooth muscle Human genes 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 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 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000013530 defoamer Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 229960001866 silicon dioxide Drugs 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Natural products O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Natural products O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- 101710198035 Myosin light chain kinase, smooth muscle Proteins 0.000 description 1
- 108010074596 Myosin-Light-Chain Kinase Proteins 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 241001199619 Penicillium sp. SGE10 Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- LRFVTYWOQMYALW-UHFFFAOYSA-N Xanthine Natural products O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 1
- UGAPHEBNTGUMBB-UHFFFAOYSA-N acetic acid;ethyl acetate Chemical compound CC(O)=O.CCOC(C)=O UGAPHEBNTGUMBB-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- PRORZGWHZXZQMV-UHFFFAOYSA-N azane;nitric acid Chemical compound N.O[N+]([O-])=O PRORZGWHZXZQMV-UHFFFAOYSA-N 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- HEDRZPFGACZZDS-MICDWDOJSA-N deuterated chloroform Substances [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011081 inoculation Methods 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
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000015424 sodium Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 229920001221 xylan Polymers 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种渥曼青霉(Penicillium wortmannii)1507‑N13，以及通过培养它制备渥曼青霉素的方法。渥曼青霉(Penicillium wortmannii)1507‑N13于2017年01月09日保藏于“中国微生物菌种保藏管理委员会普通微生物中心”，保藏编号为CGMCC NO.13366。
Description
技术领域
本发明涉及一种新的渥曼青霉，通过发酵培养该菌产生渥曼青霉素的方法。
背景技术
渥曼青霉素(Wortmannin)的分子式为C23H24O8，分子量为428.43，其结构如下式所示：
渥曼青霉素是一类甾醇类抗生素，它主要对磷脂酰肌醇-3激酶(PI3K)有抑制作用，因此关于它治疗磷脂酰肌醇-3激酶依赖性疾病的研究很多，特别是对肿瘤的治疗作用。
Wortmannin可以通透细胞，和PI3K的110kD催化亚基相结合，特异性抑制PI3K，抑制PI3K/Akt信号途径，包括常见的抑制Akt磷酸化等。同时Wortmannin也可以抑制myosinlight chain kinase(肌球蛋白轻链激酶，MLCK)等。Wortmannin无明显的抗细菌作用，但具有很强的抑制真菌活性。但因其对细胞的毒性作用目前还都限于体外研究，尚未发展到临床应用。
现有技术中，P.W.Brian等报道了渥曼青霉素最初是从Penicillium wormanninKlocker的发酵培养物中分离得到的天然产物，并对渥曼青霉素生产菌的形态进行了详细的描述(Trans.Brit.mycol.Soc.40(3),365-368(1957))；US5378725和CN1111127A公开了渥曼青霉素生产菌A24603.1的发酵培养和渥曼青霉素的分离和纯化，A24603.1产渥曼青霉素的效价为151μg/mL，由于其效价低、渥曼青霉素产量小，现有的渥曼青霉素产生菌存在不适合工业化生产的问题；一些文献中例如WO2007120364和WO2003024183仅报道了渥曼青霉素是从真菌Penicillium wormannin的发酵培养基中分离得到的，而对于产生菌的生理生化特征、培养条件、固体、种子及发酵培养基的具体组成及含量并未有新的报道。由此可见，公开的专利及文献中渥曼青霉素的生产菌比较原始，发酵水平低，那么开发新菌株及发酵技术很有必要，因此寻找一种新的高效的渥曼青霉素生产菌的工作仍然在继续进行中。
发明内容
本发明的目的之在于提供一种新的高效的渥曼青霉素生产菌，该菌为渥曼青霉(Penicillium wortmannii)1507-N13，其保藏编号为CGMCC No.13366。
本发明渥曼青霉1507-N13的微生物菌种于2017年01月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所)，保藏编号为CGMCC No.13366，分类命名为渥曼青霉Penicillium wortmannii，并登记入册，证明存活。
本发明的目的还在于提供了一种渥曼青霉1507-N13(CGMCC No.13366)在制备渥曼青霉素或者含有渥曼青霉素的药物组合物中的应用。
本发明还提供了一种渥曼青霉素的制备方法，该方法包括采用渥曼青霉1507-N13(CGMCC No.13366)在含有可同化的碳源和/或氮源的营养培养基里，进行发酵培养的步骤。
优选的实施方案中，上述可同化的碳源选自葡萄糖、乳糖、甘露醇、山梨醇、蔗糖、麦芽糊精、玉米淀粉、甘油、豆油、油酸甲酯之一或上述物质的组合。
优选的实施方案中，上述可同化的氮源选自酵母抽提粉、酵母粉、牛肉浸膏、大豆蛋白胨、蛋白胨、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、麸皮之一或上述物质的组合。
优选的实施方案中，所述营养培养基还包括无机盐，所述无机盐选自氯化铵、硝酸铵，柠檬酸三铵、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或上述物质的组合。
优选的实施方案中，所述营养培养基含有甘油10.0～50.0g/L、乳糖5.0～50.0g/L、蔗糖5.0～50.0g/L、黄豆饼粉5.0～20.0g/L、酵母粉5.0～20.0g/L、棉籽饼粉5.0～30.0g/L、磷酸二氢钾0.5～5.0g/L、硫酸铵1.0～5.0g/L、碳酸钙1.0～5.0g/L、硫酸锰0.01～1.0g/L。
优选的实施方案中，所述发酵培养的温度为20℃-30℃，优选24℃-26℃；培养基pH为5.0-8.0，优选6.0-7.0；培养时间为24-240小时，优选72-192小时。
优选的实施方案中，所述渥曼青霉1507-N13(CGMCC No.13366)是通过种子液接种至所述培养基中进行所述发酵培养的；
其中，所述种子液是将渥曼青霉1507-N13(CGMCC No.13366)进行种子培养得到的；所述种子培养的条件为：种子培养的温度为20℃-30℃，优选24℃-26℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-80小时，优选24-60小时。
优选的实施方案中，所述的种子培养基含有蔗糖0～20.0g/L、葡萄糖10.0～50.0g/L、棉籽饼粉10.0～30.0g/L、酵母粉5.0～15.0g/L、磷酸二氢钾1.0～10.0g/L、硫酸铵0.5～1.5g/L、碳酸钙0.5～2.0g/L。
本发明渥曼青霉素通过以下方法进行检测：
色谱柱：C18分析柱(5μm,4.6×150mm).
进样量：10μL
流动相：甲醇：水：甲酸＝50：50：0.1(v/v/v)
流速：1mL/min
检测波长：254nm
本发明渥曼青霉(Penicillium wortmannii)1507-N13(CGMCC No.13366)生产能力高；且，其产生渥曼青霉素的能力比现有技术中其他菌种有了大幅度的提高，有利于实现工业化生产。
本发明渥曼青霉(Penicillium wortmannii)1507-N13(CGMCC No.13366)的主要生物学特征为：孢子呈椭圆形，菌落颜色是黄色，边缘呈白色，反面白色，平铺，无褶皱，有水珠产生，直径1.0～2.0cm，不易挑取。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例1：菌株来源
本发明渥曼青霉1507-N13(CGMCC No.13366)系从中国东南沿海的浙江省台州市九峰山土壤中分离得到的原始菌株，再通过诱变得到的突变菌株。
原始菌株在PDA斜面培养基中30℃培养12天后，在无菌条件下用接种铲将菌丝体刮下，在磨口试管中磨碎菌丝体并悬浮于无菌水中，制得菌悬液，供NTG(亚硝基胍)诱变处理。
称取NTG晶体2mg，溶解在2mL无菌Tris缓冲液(pH6.0)中。用移液管吸取1ml NTG溶液加入到1ml菌悬液，于30℃振荡处理40min。其余具体步骤如下：
(1)菌丝体的制备与培养
固体培养基配方：PDA，经121℃灭菌30分钟后，冷却到50-60℃倒入平板，将处理过的菌悬液经适当稀释，吸取0.1mL菌悬液涂布于PDA平板上，未作诱变处理的菌悬液亦经适当稀释涂布于PDA平板作为对照，置于30℃恒温培养箱培养，菌落培养7天，菌丝成熟。
(2)种子液的制备与培养
种子培养基配方(g/L)：蔗糖10.0g/L、葡萄糖25.0g/L、棉籽饼粉10.0g/L、酵母粉15.0g/L、磷酸二氢钾5.0g/L、硫酸铵1.0g/L、碳酸钙1.0g/L，pH6.0。摇瓶装液量为20mL/250mL，121℃灭菌30分钟。每个种子摇瓶接入0.5个菌落左右，培养温度20～30℃，摇瓶机振幅5cm，转速250rpm，培养36小时。
(3)渥曼青霉素发酵培养基的制备与培养
甘油20.0g/L、乳糖40.0g/L、蔗糖30.0g/L、黄豆饼粉10.0g/L、酵母粉15.0g/L、棉籽饼粉10.0g/L、磷酸二氢钾0.5g/L、硫酸铵2.0g/L、碳酸钙2.0g/L、硫酸锰0.05g/L，pH6.5，摇瓶装液量为25mL/250mL，121℃灭菌30分钟。将种子液以10％(体积百分比)的接种量接入发酵培养基，培养温度25℃，摇瓶机振幅5cm，转速250rpm，培养98小时。
挑选经过NTG诱变后的单菌落1000株，进行摇瓶发酵，HPLC检测渥曼青霉素的产量。筛选出产渥曼青霉素最高产的突变菌株即渥曼青霉1507-N13(CGMCC No.13366)。其主要生物学特征为：孢子呈椭圆形，菌落颜色是黄色，边缘呈白色，反面白色，平铺，无褶皱，有水珠产生，直径1.0～2.0cm，不易挑取。
实施例2：菌种鉴定
参照《普通真菌学》、《常见细菌系统鉴定手册》、《分子克隆实验指南》及《中国药典》(2015版)等书中的有关内容进行实验；颜色判断参照RAL K7色卡系列的颜色进行对照。
1.菌种编号：1507-N13(CGMCC No.13366)。
2.培养特征：采用查氏、PDA、SDA、MEA和MEPG 5种培养基，28℃培养5～7天后，观察菌丝体的颜色及色素情况。菌株的培养特征见表1。
3.生理生化试验：除温度实验外，均为28℃培养5～7天。
a)碳源的利用：采用ISP9作为基础培养基，各种碳源的终浓度均为1.0％，见表2。
b)无机氮源的利用：采用ISP9作为基础培养基，硝酸钾和硫酸铵的浓度均为0.1％，见表2。
c)降解试验和NaCl耐受实验采用基础培养基为GYEA(pH6.8)，各种降解物的浓度及降解试验结果见表3；NaCl耐受实验结果见表7。
d)氧化酶和过氧化氢酶试验、pH试验和温度试验均采用PDA培养基。氧化酶和过氧化氢酶试验结果见表4，pH试验结果见表5，温度试验结果见表6。
e)M.R、V-P和脲酶实验采用《常见细菌系统鉴定手册》方法，其结果见表4。
4.28S rDNA序列分析：收集PDA培养的新鲜菌体，采用液氮破壁方法提取总DNA模板，采用Fungi Identification PCR Kit(TaKaRa)进行28S rDNA基因ITS区域扩增，PCR产物经琼脂糖电泳检测后，直接进行序列测定，PCR产物的纯化与测序由南京金斯瑞生物科技有限公司进行。菌株1507-N13所测的28S rDNA基因ITS区域序列经校对后，经BLAST比较确定其同源性。
实验结果
1.培养特征：菌株1507-N13(CGMCC No.13366)的培养特征见表1。
表1菌株1507-N13(CGMCC No.13366)在5种培养基上的培养特征
2.生理生化特征：见表2～表7。
表2菌株1507-N13(CGMCC No.13366)的碳源和氮源的利用情况
表3菌株1507-N13(CGMCC No.13366)的降解试验结果
| 降解物 | 降解物浓度 | 结果* | 降解物 | 降解物浓度 | 结果 |
| 腺嘌呤 | 0.5％ | 4，- | 酪蛋白 | 1.0％ | 4，- |
| 鸟嘌呤 | 0.5％ | 4，- | 酪氨酸 | 1.0％ | 4，- |
| 黄嘌呤 | 0.4％ | 4，- | Tween-40 | 1.0％ | 3，- |
| 木聚糖 | 0.4％ | 4，- | Tween-60 | 1.0％ | 3，- |
| 次黄嘌呤 | 0.4％ | 4，- | Tween-80 | 1.0％ | 3，- |
表4菌株1507-N13(CGMCC No.13366)主要的生理生化特征
| 试验项目 | 结果 | 试验项目 | 结果 | 试验项目 | 结果 |
| 明胶液化 | + | 牛奶胨化 | - | 纤维素利用 | - |
| 淀粉水解 | - | 硝酸盐还原 | - | 过氧化氢酶 | - |
| 牛奶凝固 | - | 硫化氢产生 | - | ||
| V.P实验 | - | M.R实验 | + |
表5菌株1507-N13(CGMCC No.13366)生长的pH试验
| pH | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| 生长情况 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
表6菌株1507-N13(CGMCC No.13366)生长的温度试验
| 温度(℃) | 7 | 14 | 28 | 37 | 45 |
| 生长情况 | 0 | 4 | 4 | 0 | 0 |
表7菌株1507-N13(CGMCC No.13366)对NaCl的耐受性
| NaCl浓度 | 1％ | 4％ | 7％ | 10％ |
| 菌株生长情况 | 4 | 0 | 0 | 0 |
*注：表2-7中：0，无生长；1，生长很弱；2，能生长，有少量孢子；3，生长良好，有大量孢子；4，生长最好，有丰富孢子；+，阳性；-，阴性。
3.28S rDNA序列分析：菌株1507-N13(CGMCC No.13366)的28S ITS序列与GenBank中相关序列进行BLAST比较，结果见表8(表中只列出同源性较高的菌株)。
表8菌株1507-N13(CGMCC No.13366)和相关菌株的同源性
菌种鉴定结果
通过对菌株1507-N13(CGMCC No.13366)的28S rDNA ITS区域测序，与GenBank数据库中相关种、属的序列进行同源序列BLAST比较，结果表明：菌株1507-N13(CGMCCNo.13366)和青霉属(Penicillium sp.SGE10)有很高同源性，最高的达99.6％，同时对菌株1507-N13(CGMCC No.13366)的表观特征的试验，发现该菌株和青霉属(Penicillium sp.)分类相关参数非常接近，故将菌株1507-N13鉴定为青霉属(Penicillium sp.)菌株。
本发明渥曼青霉1507-N13(CGMCC No.13366)和其他渥曼青霉素产生菌的比较如下：
P.W.Brian等报道(Trans.Brit.mycol.Soc.40(3),365-368(1957)Penicilliumwormannin Klocker(1952年以Penicillium sp.保藏)在査氏培养基上菌落相当小，菌丝体主要是浅黄色的，菌落反面呈橙红色或者浅棕色，有褶皱，微微高耸；而本发明渥曼青霉1507-N13(CGMCC No.13366)在査氏培养基上菌落直径10mm，菌落呈亮黄色的，菌落反面呈白色，无褶皱，平铺。
US5378725和CN1111127A的渥曼青霉素生产菌A24603.1产渥曼青霉素的效价为151μg/mL；而本发明渥曼青霉1507-N13(CGMCC No.13366)产渥曼青霉素的效价高达3500μg/mL。
结合前述本发明的渥曼青霉1507-N13(CGMCC No.13366)的形态学、培养特征、生理生化特征和DNA序列鉴定结果可知，本发明的渥曼青霉1507-N13(CGMCC No.13366)属于渥曼青霉Penicillium wortmannii，且它不同于已知的渥曼青霉素产生菌，故本发明渥曼青霉1507-N13(CGMCC No.13366)是一株全新的菌种。
实施例3：制备渥曼青霉素
(1)固体培养基配方及培养
固体培养基配方：PDA，经121℃灭菌30分钟后，冷却待用，接入0.1mL菌悬液培养7天。
(2)种子培养基配方及培养
种子培养基配方(g/L)：蔗糖10.0g/L、葡萄糖25.0g/L、棉籽饼粉10.0g/L、酵母粉15.0g/L、磷酸二氢钾5.0g/L、硫酸铵1.0g/L、碳酸钙1.0g/L，pH6.0。摇瓶装液量为20mL/250mL，121℃灭菌30分钟。每个种子摇瓶接入0.5个菌落左右，培养温度20～30℃，摇瓶机振幅5cm，转速250rpm，培养36小时。
(3)渥曼青霉素发酵培养基的制备与培养
甘油40.0g/L、乳糖50.0g/L、酵母粉25g/L、棉籽饼粉10.0g/L、磷酸二氢钾0.5g/L、硫酸铵2.0g/L、碳酸钙2.0g/L、硫酸锰0.05g/L，pH6.5，摇瓶装液量为25mL/250mL，121℃灭菌30分钟。将种子液以10％(体积百分比)的接种量接入发酵培养基，培养温度25℃，摇瓶机振幅5cm，转速250rpm，培养96小时。经HPLC检测，渥曼青霉素效价为3156μg/mL。
实施例4：制备渥曼青霉素
(1)固体培养基配方及培养条件等同实施例3中步骤(1)
(2)种子培养基配方及培养条件等同实施例3中步骤(2)
(3)渥曼青霉素发酵培养基的制备与培养
甘油20.0g/L、乳糖50.0g/L、蔗糖30.0g/L、黄豆饼粉15.0g/L、酵母粉15.0g/L、棉籽饼粉10.0g/L、磷酸二氢钾0.8g/L、硫酸铵1.5g/L、碳酸钙1.5g/L、硫酸锰0.05g/L，pH6.5，摇瓶装液量为25mL/250mL，121℃灭菌30分钟。将种子液以10％(体积百分比)的接种量接入发酵培养基，培养温度25℃，摇瓶机振幅5cm，转速250rpm，培养100小时。经HPLC检测，渥曼青霉素效价为3344μg/mL。
实施例5制备渥曼青霉素
(1)固体培养基配方及培养条件等同实施例3中步骤(1)
(2)种子液的制备与培养
种子培养基配方：葡萄糖35.0g/L、棉籽饼粉10.0g/L、酵母粉15.0g/L、磷酸二氢钾3.0g/L、硫酸铵1.0g/L、碳酸钙1.0g/L，pH6.0。摇瓶装液量为20mL/250mL，121℃灭菌30分钟。培养温度20～30℃，摇瓶机振幅5cm，转速250rpm，培养36小时。
(3)发酵培养基配方及培养条件等同实施例3中步骤(3)
经HPLC检测，渥曼青霉素效价为3271μg/mL。
实施例6：制备和分离纯化渥曼青霉素
(1)种子罐种子液的制备
在15L种子罐中投入10L的种子培养基(种子培养基的配方同实施例3中步骤(2))，同时添加0.05％的PPG作为消泡剂)，灭菌采用蒸汽灭菌，121℃条件下30分钟，待冷却后，接入200ml摇瓶种子液，培养温度25±1℃，搅拌转速100～500rpm，通气量0.5～1.0vvm，溶氧20～70％，培养36小时。
(2)发酵罐培养基的配制与培养
发酵培养基的配方与前述实施例3中步骤(3)发酵配方相同，但要添加0.05％PPG作为消泡剂，发酵罐装量为30L/50L，pH7.0，于121℃下蒸汽灭菌30分钟，冷却后，接入约3.5L种子罐培养液，发酵温度25±1℃，搅拌转速200～500rpm，通气量为0.5～1.0vvm，发酵培养103小时。经HPLC检测，渥曼青霉素效价为3510μg/mL。
(3)发酵液萃取
发酵得到30L发酵液，用等体积乙酸乙酯萃取2次，将所得乙酸乙酯萃取液在45℃条件下减压浓缩去除乙酸乙酯直至干，得到76g油状物质。
(4)硅胶柱柱层析纯化
将所得的油状物用5L氯仿溶解上硅胶柱(粒径100-200目)进行柱层析，用氯仿:乙酸乙酯＝85:15(V/V)洗脱，并收集含渥曼青霉素的洗脱液。
(5)粗品的纯化和纯品的结构鉴定
将含渥曼青霉素的洗脱液浓缩至干，然后用乙酸乙酯500mL溶解，滤纸过滤后加入1000mL正己烷，混合均匀后转入4℃冰箱中冷藏。2小时后，将冷藏液过滤，得到的针状晶体转入真空干燥箱，50℃真空干燥5小时。
上述晶体通过NMR、MS等波谱分析，确定其为渥曼青霉素，其结构如下：
渥曼青霉素的理化性质如下：
性状:白色针状晶体
溶解性:易溶解于氯仿、丙酮、甲醇，不溶于水
分子式:C23H24O8
ESIMS m/z:427.12[M-H]-
表9渥曼青霉素在CDCl3中的核磁数据(氢谱，400MHz；碳谱，100MHz)
上述结果表明，本发明渥曼青霉1507-N13(CGMCC No.13366)产渥曼青霉素的效价高达3500μg/mL(CGMCC No.13366)，本发明渥曼青霉1507-N13(CGMCC No.13366)产渥曼青霉素的能力较其他菌种有了大幅度的提高，有利于实现渥曼青霉素工业化生产。
Claims (10)
1.一种渥曼青霉(Penicillium wortmannii)1507-N13，其保藏编号为CGMCCNo.13366。
2.根据权利要求1所述的渥曼青霉1507-N13在制备渥曼青霉素或者含有渥曼青霉素的药物组合物中的应用。
3.一种渥曼青霉素的制备方法，其特征在于：包括采用如权利要求1所述的渥曼青霉1507-N13在含有可同化的碳源和/或氮源的营养培养基里，进行发酵培养的步骤。
4.根据权利要求3所述的方法，其特征在于：所述可同化的碳源选自葡萄糖、乳糖、甘露醇、山梨醇、蔗糖、麦芽糊精、玉米淀粉、甘油、豆油、油酸甲酯之一或上述物质的组合。
5.根据权利要求3所述的方法，其特征在于：所述可同化的氮源选自酵母抽提粉、酵母粉、牛肉浸膏、大豆蛋白胨、蛋白胨、黄豆饼粉、棉籽饼粉、花生饼粉、麸质粉、玉米浆干粉、麸皮之一或上述物质的组合。
6.根据权利要求3-5任一项所述的方法，其特征在于：所述营养培养基还包括无机盐，所述无机盐选自氯化铵、硝酸铵，柠檬酸三铵、磷酸二氢钾、磷酸氢二钾、硫酸铵、碳酸钙、硫酸亚铁、硫酸锌、硫酸铜、氯化钠、氯化钾、氯化钙、硫酸镁、氯化铁、硫酸锰之一或上述物质的组合。
7.根据权利要求3-5任一项所述的方法，其特征在于：所述营养培养基含有甘油10.0～50.0g/L、乳糖5.0～50.0g/L、蔗糖5.0～50.0g/L、黄豆饼粉5.0～20.0g/L、酵母粉5.0～20.0g/L、棉籽饼粉5.0～30.0g/L、磷酸二氢钾0.5～5.0g/L、硫酸铵1.0～5.0g/L、碳酸钙1.0～5.0g/L、硫酸锰0.01～1.0g/L。
8.根据权利要求3-5任一项所述的方法，其特征在于：所述发酵培养的温度为20℃-30℃，优选24℃-26℃；培养基pH为5.0-8.0，优选6.0-7.0；培养时间为24-240小时，优选72-192小时。
9.根据权利要求3-8任一项所述的方法，其特征在于：所述渥曼青霉1507-N13是通过种子液接种至所述营养培养基中进行所述发酵培养的；
其中，所述种子液是将权利要求1所述的渥曼青霉1507-N13在种子培养基里进行种子培养得到的；所述种子培养的条件为：种子培养的温度为20℃-30℃，优选24℃-26℃；培养基pH为5.0-8.0，优选5.0-7.0；培养时间为24-80小时，优选24-60小时。
10.根据权利要求9所述的方法，其特征在于：所述的种子培养基含有蔗糖0～20.0g/L、葡萄糖10.0～50.0g/L、棉籽饼粉10.0～30.0g/L、酵母粉5.0～15.0g/L、磷酸二氢钾1.0～10.0g/L、硫酸铵0.5～1.5g/L、碳酸钙0.5～2.0g/L。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710447621.8A CN107058137B (zh) | 2017-06-14 | 2017-06-14 | 一种渥曼青霉及其生产渥曼青霉素的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710447621.8A CN107058137B (zh) | 2017-06-14 | 2017-06-14 | 一种渥曼青霉及其生产渥曼青霉素的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107058137A true CN107058137A (zh) | 2017-08-18 |
| CN107058137B CN107058137B (zh) | 2020-02-07 |
Family
ID=59594503
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710447621.8A Active CN107058137B (zh) | 2017-06-14 | 2017-06-14 | 一种渥曼青霉及其生产渥曼青霉素的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058137B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182147A (zh) * | 2018-10-13 | 2019-01-11 | 浙江海正药业股份有限公司 | 一种青霉及其生产烟曲霉素的方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03215423A (ja) * | 1990-01-18 | 1991-09-20 | Kyowa Hakko Kogyo Co Ltd | 血管拡張剤 |
| US5378725A (en) * | 1993-07-19 | 1995-01-03 | The Arizona Board Of Regents | Inhibition of phosphatidylinositol 3-kinase with wortmannin and analogs thereof |
-
2017
- 2017-06-14 CN CN201710447621.8A patent/CN107058137B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03215423A (ja) * | 1990-01-18 | 1991-09-20 | Kyowa Hakko Kogyo Co Ltd | 血管拡張剤 |
| US5378725A (en) * | 1993-07-19 | 1995-01-03 | The Arizona Board Of Regents | Inhibition of phosphatidylinositol 3-kinase with wortmannin and analogs thereof |
Non-Patent Citations (3)
| Title |
|---|
| BY P. W.等: "WORTMANNIN, AN ANTIBIOTIC PRODUCED BY PENICILLIUM WORTMANNI", 《TRANS. BRIT. MYCOL. SOC.》 * |
| HAI-E. DING等: "Secovironolide, a novel furanosteroid scaffold with a five-membered B ring from the endophytic fungus Talaromyces wortmannii LGT-4", 《TETRAHEDRON LETTERS》 * |
| SATOSHI NAKANISHI等: "Wortmannin, a Microbial Product Inhibitor of Myosin Light Chain Kinase", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182147A (zh) * | 2018-10-13 | 2019-01-11 | 浙江海正药业股份有限公司 | 一种青霉及其生产烟曲霉素的方法 |
| CN109182147B (zh) * | 2018-10-13 | 2020-07-14 | 浙江海正药业股份有限公司 | 一种青霉及其生产烟曲霉素的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107058137B (zh) | 2020-02-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676757A (zh) | 一株链霉菌属菌株及其产星孢菌素的应用 | |
| CN103642734B (zh) | 一种甜菜内生微杆菌及其用于甜菜病害生物防治中的应用 | |
| CN110699304B (zh) | 一种生防链霉菌 | |
| CN106148215B (zh) | 一种链霉菌及其生产米尔贝霉素a4的方法 | |
| CN104342390A (zh) | 一种苜蓿中华根瘤菌株及其组合物与应用 | |
| CN101245362B (zh) | 发酵法生产多肽类抗生素安来霉素的方法 | |
| CN106148216B (zh) | 一种链霉菌及其生产米尔贝霉素a3的方法 | |
| CN109182147B (zh) | 一种青霉及其生产烟曲霉素的方法 | |
| CN107815478A (zh) | 利用小金色链霉菌bjx007发酵生产春雷霉素的方法 | |
| CN114934002B (zh) | 一株放线菌新物种及其在植物抗旱促生中的应用 | |
| CN102399702B (zh) | 一种黑曲霉及其应用及发酵制备柠檬酸的方法 | |
| CN104560766B (zh) | 一种游动放线菌菌株及其应用 | |
| CN102174414B (zh) | 一种新的肋脉羊肚菌m8-13液体发酵物在保健品及医药开发中的应用 | |
| CN108823110B (zh) | 一株产灰黄霉素的菌株及其应用 | |
| CN103555636B (zh) | 一种深红紫链霉菌及其应用 | |
| WO2023016387A1 (zh) | 一种解淀粉芽孢杆菌及其在制备1-脱氧野尻霉素中的应用 | |
| EP0547898A2 (en) | Microbial process for the production of trans-4-hydroxy-L-proline | |
| CN120536288A (zh) | 一株碱土中华根瘤菌及其应用 | |
| CN110713956B (zh) | 一株赖氨酸芽孢杆菌s12及其应用 | |
| CN108841889A (zh) | 利用微生物发酵生产松刚霉素主份——灰黄霉素的方法 | |
| CN107058137A (zh) | 一种渥曼青霉及其生产渥曼青霉素的方法 | |
| CN113337432B (zh) | 一种产吡咯喹啉醌的食甲基菌及其应用 | |
| CN106190854A (zh) | 一种荒漠拟孢囊菌和奥利万星中间体的制备方法 | |
| CN105018410B (zh) | 一种诱导三孢布拉氏霉老化菌株快速大量产孢的方法 | |
| CN107488609A (zh) | 一种链霉菌及其代谢物的制备方法与应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20171122 Address after: 311404 Shing village of Fuyang District, Hangzhou City, Zhejiang Province Applicant after: Zhejiang Hisun Pharmaceutical Co., Ltd. Address before: Jiaojiang District of Taizhou City, Zhejiang province 318000 road outside No. 46 Applicant before: Zhejiang Hisun Pharmaceutical Co., Ltd. Applicant before: Zhejiang Hisun Pharmaceutical Co., Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |