CN107446904A - A kind of lipase and its production method and application - Google Patents
A kind of lipase and its production method and application Download PDFInfo
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- CN107446904A CN107446904A CN201710838182.3A CN201710838182A CN107446904A CN 107446904 A CN107446904 A CN 107446904A CN 201710838182 A CN201710838182 A CN 201710838182A CN 107446904 A CN107446904 A CN 107446904A
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- lipase
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- pseudomonad
- fermentation
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 56
- 102000004882 Lipase Human genes 0.000 title claims abstract description 55
- 239000004367 Lipase Substances 0.000 title claims abstract description 54
- 235000019421 lipase Nutrition 0.000 title claims abstract description 54
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 24
- 108090000790 Enzymes Proteins 0.000 claims abstract description 62
- 102000004190 Enzymes Human genes 0.000 claims abstract description 62
- 230000000694 effects Effects 0.000 claims abstract description 31
- 239000012530 fluid Substances 0.000 claims abstract description 14
- 230000001580 bacterial effect Effects 0.000 claims abstract description 10
- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims description 32
- 230000004151 fermentation Effects 0.000 claims description 32
- 239000001963 growth medium Substances 0.000 claims description 23
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 230000001954 sterilising effect Effects 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 229940041514 candida albicans extract Drugs 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000012138 yeast extract Substances 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 11
- 108010080698 Peptones Proteins 0.000 claims description 11
- 239000001110 calcium chloride Substances 0.000 claims description 11
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 11
- 235000019319 peptone Nutrition 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 235000008504 concentrate Nutrition 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 239000002285 corn oil Substances 0.000 claims description 6
- 235000005687 corn oil Nutrition 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 235000020429 malt syrup Nutrition 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 5
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 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 4
- 239000008103 glucose Substances 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 239000003755 preservative agent Substances 0.000 claims description 4
- 230000002335 preservative effect Effects 0.000 claims description 4
- 239000003381 stabilizer Substances 0.000 claims description 4
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910001562 pearlite Inorganic materials 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 235000010241 potassium sorbate Nutrition 0.000 claims description 3
- 239000004302 potassium sorbate Substances 0.000 claims description 3
- 229940069338 potassium sorbate Drugs 0.000 claims description 3
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 3
- 235000010234 sodium benzoate Nutrition 0.000 claims description 3
- 239000004299 sodium benzoate Substances 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 239000006052 feed supplement Substances 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 239000006071 cream Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 4
- 239000003513 alkali Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 239000000243 solution Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 235000002639 sodium chloride Nutrition 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 4
- 125000001931 aliphatic group Chemical group 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003925 fat Substances 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108010079522 solysime Proteins 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 102100031375 Endothelial lipase Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- -1 long chain fatty acids acyl ester Chemical class 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000003918 potentiometric titration Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to microbial technology field, and in particular to a kind of lipase and its production method and application.The lipase originates from pseudomonad (Pseudomonas sp.) LD 14, and bacterial strain preserving number is CGMCC No.14414.The zymotic fluid enzyme activity of the fermenting and producing lipase of pseudomonad LD 14 reaches 25000U/mL to 26000U/mL；Institute's yielding lipase optimal pH scope is 8.5 10.0, and optimum temperature scope is 30 DEG C 40 DEG C, and remaining enzyme activity is 80% after being incubated 1d at 20 DEG C, has significant lower temperature resistance and alkali resistance, can be widely applied to industrial production.
Description
Technical field：
The invention belongs to microbial technology field, and in particular to a kind of lipase and its fermentation method for producing and application.
Background technology：
Lipase (Triacylglycerol acylhydrolases, EC 3.1.1.3) is to be hydrolyzed on oil-water interfaces
The general name of the class of enzymes of triglycerides, it can hydrolyze triglyceride as aliphatic acid, Diglyceride, monoglyceride and sweet
Oil, its natural substrate are usually long chain fatty acids acyl ester not soluble in water.
Lipase is mainly derived from plant, animal, microorganism, and wherein microbial lipase is widely present in bacterium, yeast
In mould, there is the characteristics of species is more, the cycle is short, breeding is fast, easy generation hereditary variation, and have wider than andvegetable fats enzyme
Operative temperature, action pH and substrate specificity, its can not need coenzyme under conditions of be catalyzed ester type compound hydrolysis,
Alcoholysis, acidolysis, ester exchange and synthesis etc., catalytic condition is gentle, energy consumption is low, accessory substance is few, has efficient, high selectivity, environment
The features such as friendly, change the relatively harsh bars such as high temperature required for traditional esterification or transesterification, strong acid, highly basic
Part.Microbial lipase is suitable for industrialized production, high-purity product can be produced, in fundamental research and practical application
In all there is substantial worth.At present, the production method of lipase mainly includes extraction method and microbe fermentation method, wherein, micro- life
Thing fermentation method is with its higher yield, the microorganism resource of relatively low cost and wide material sources and as the first choice of lipase production
Method.
Lipase and its modified formulation are due to the particularity of itself zymologic property, in food and nutriment processing work
Industry, oil and fat chemical industry, daily-use chemical industry industry, medicine many fields such as synthesize with agriculture, bioactivator and are used widely.
It is widely applied scope, abundant resource, increasingly consequence is shown in industrialized production.
And lipase optimal reactive temperature of the prior art inactivates seriously typically at 40 DEG C or higher below 30 DEG C,
Therefore, being restricted by enzyme activity and enzyme applicable elements, currently available technology fat synthase can not still meet industrial demand, because
This screening is a kind of to have good alkaline-resisting and lower temperature resistance, the lipase of low production cost, to extensive propulsion every profession and trade
Mechanization is significant.
The content of the invention：
In order to solve the above problems, the present invention will provide a kind of high yield lipase and its enzymatic production method and application,
While ensuring pseudomonad fermentation high yield stability of lipase, the good alkaline-resisting and lower temperature resistance of lipase is significantly improved.
The purpose of the present invention is to be achieved through the following technical solutions purpose：
The pseudomonad of one plant height yielding lipase, the bacterial strain are pseudomonad (Pseudomonas sp.) LD-14, bacterium
Strain preserving number is CGMCC No.14414.
Pseudomonad (Pseudomonas sp.) LD-14 is normal by carrying out normal temperature to pseudomonad original strain
Pressure plasma mutagenesis obtains, and described pseudomonad LD-14 is preserved in China Microbiological bacterium on July 13rd, 2017
Kind preservation administration committee's common micro-organisms center (abbreviation CGMCC), preserving number is CGMCC No.14414, and preservation address is：
City of BeiJing, China Chaoyang District North Star West Road 1 institute 3, Institute of Microorganism, Academia Sinica, postcode：100101.
The colony morphology characteristic of the pseudomonad LD-14 is as follows：Circle, milky, periphery be neat, in lens-shaped,
Surface is smooth, has color and luster, be opaque, having stink.
Another object of the present invention is to the enzymatic production method for providing above-mentioned lipase is as follows：
Fermentation tank culture：Ratio access fermentation tank culture medium by seed liquor according to inoculum concentration 6% (v/v), 35 DEG C of constant temperature,
Rotating speed 200r/min, ventilation is set：0 to 5h be 0.25vvm, 5h to 20h be 0.5vvm, 20h to fermentation ends be 1.0vvm,
It is 6.8 that fermentation whole process controls zymotic fluid pH value using supplemented medium, ferments to enzyme activity and increasess slowly, thalline self-dissolving is tied when serious
Beam ferments, and fermentation period 150h, obtains final zymotic fluid；
The zymotic fluid enzyme activity of lipase is reachable：25000U/mL to 26000U/mL；
By final zymotic fluid by extraction and process for purification, lipase production liquid enzyme preparation is obtained.
Further, the preparation method of the seed liquor is as follows：Ratio by secondary seed solution according to inoculum concentration 6% (v/v)
Example is accessed in seed tank culture base, 35 DEG C of constant temperature, and 12-15h is cultivated under the conditions of rotating speed 200r/min；
Further, the preparation method of the secondary seed solution is as follows：A ring is taken to access seed culture medium first order seed
In, 35 DEG C of constant temperature, 48h is cultivated under the conditions of shaking speed 200r/min, obtains secondary seed solution；
Further, the preparation method of the first order seed is as follows：It is oblique that the ring pseudomonad LD-14 of picking one is inoculated in solid
Face culture medium, incubated 36h at 35 DEG C, obtains first order seed；
Further, the extraction of the lipase and process for purification are as follows：
(1) final zymotic fluid is added into 1~5% pearlite filtering aid, press filtration obtains clarifying press filtration enzyme liquid；
(2) the clarification press filtration enzyme liquid is concentrated by ultrafiltration with 20000 molecular weight milipore filters, obtains concentrate；
(3) 20% (m/v) stabilizer, 0.45% (m/v) preservative is added in above-mentioned concentrate, adjusts pH
8.5, filtration sterilization is then carried out, that is, obtains lipase production liquid enzyme preparation.
Preferably, the stabilizer is glycerine, and the preservative is that mass ratio is 1：2 potassium sorbate and sodium benzoate.
The solid slope culture medium (g/L)：Peptone 12, yeast extract 6, agar 15, sodium chloride 1, surplus are water, pH
7.0,121 DEG C of sterilizing 20min；
The seed culture medium (g/L)：Malt syrup 40, peptone 5, yeast extract 5, sodium chloride 0.8, magnesium sulfate 0.5, chlorine
Change calcium 0.1, disodium hydrogen phosphate 0.5, surplus is water, 6.8,121 DEG C of sterilizing 20min of pH；
The seed tank culture base (g/L)：Glucose 80, yeast extract 15, corn steep liquor 5, ammonium sulfate 1, magnesium sulfate 0.5, phosphorus
Acid dihydride potassium 1.5, calcium chloride 0.1, pH 6.8,121-123 DEG C of sterilizing 30min；
The fermentation tank culture medium (g/L)：Maltose 180, peptone 10, yeast extract 5, ammonium sulfate 1.5, magnesium sulfate 0.5,
Corn oil 20, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3, pH 6.8,121-123 DEG C of sterilizing 30min；
The supplemented medium (g/L)：Malt syrup 450, KH2PO45, corn steep liquor 7, corn oil 40, calcium chloride 5, pH
4.0,121-123 DEG C of sterilizing 30min.
The lipase has the property that：
(1) temperature：Optimum temperature scope is 30 DEG C -40 DEG C；
(2)pH：Optimal pH scope is 8.5-10.0；
(3) low temperature tolerance characteristicses：Remaining enzyme activity is 80% after being incubated 1d at 20 DEG C.
Beneficial effect：
The present invention is by carrying out one plant height of normal temperature and pressure plasma mutagenic and breeding to pseudomonad original strain LA-32
The mutant strain LD-14 of yielding lipase, and carrying out feed supplement condition optimizing to pseudomonad LD-14 fermentation process reaches its enzyme activity
25000U/mL is between 26000U/mL, and pseudomonad original strain is 19500U/mL by highest enzyme activity after liquid state fermentation.
It can thus be seen that pseudomonad LD-14 is significantly higher than pseudomonad original strain LA-32 enzyme activity.
Medium component derives from lower-cost raw material in the fermentation process of bacterial strain of the present invention, and is improving fermentation production
While energy, improving production efficiency, greatly reduce fermentation costs, have to production and greatly help.
The lipase optimal pH scope that pseudomonad LD-14 ferments to obtain is 8.5-10.0, and optimum temperature scope is
30 DEG C -40 DEG C, remaining enzyme activity is 80% after being incubated 1d at 20 DEG C, has significant lower temperature resistance and alkali resistance, can extensive use
In industrial production, the industrial applicability of lipase has significantly been expanded, has improved the application value of lipase.
Brief description of the drawings：
Fig. 1：Relative enzyme activity under different pH；
Fig. 2：Relative enzyme activity under different temperatures；
Fig. 3：The relative enzyme activity after 1d is incubated at 20 DEG C.
Embodiment：
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
The various changes carried out on the premise of invention spirit and scope to the material component in these embodiments and dosage or change
Belong to protection scope of the present invention.
The pseudomonad LD-14 of embodiment 1 mutagenic and breeding
Two plants of starting strain LA-32 fresh inclined-plane is taken, with sterile water elution thalline, is made in the test tube vibration for having bead
Thalline disperses, and thalline is collected by centrifugation, and thalline is resuspended with 5% glycerine, and it is 10 to be counted with blood counting chamber up to bacterium is dense7-108Individual/
ML, in this, as the bacteria suspension that sets out.
Normal temperature and pressure plasma system is opened, with alcohol swab wiping operation indoor and outdoor, and opens uviol lamp sterilizing 30min.
To the end of system operatio indoor sterilizing, after take 10 μ L bacteria suspensions to select the mat surface in slide glass, and aseptically by slide glass with
Tweezers are transferred on operating room table top.Helium valves are opened, throughput is set and mutation time carries out mutagenesis.Mutation time is distinguished
It is set as 90s, 120s, 150s, 180s, 210s.Slide glass is placed in containing 990 μ L sterile salines by each mutagenesis after terminating
In EP pipes, whirlpool concussion 1min.It is placed in 30 DEG C of incubators and cultivates after dilution spread.
Enriched medium (g/L)：Glucose 25, tryptone 10, yeast extract 5, sodium chloride 10, olive oil 30, surplus
For water, pH value 6.8；
Flat board secondary screening culture medium (g/L)：Maltose 20, peptone 5, beef extract 2, rhodamine 0.5, agar 20, surplus are
Water, pH 6.8；
Culture medium (g/L)：Maltose 20, peptone 6, beef extract 3, olive oil 30, ammonium sulfate 1, magnesium sulfate 0.8,
Potassium dihydrogen phosphate 0.3, surplus are water, pH 6.8；
The primary dcreening operation of bacterial strain：
Flat board secondary screening culture medium is prepared, picking single bacterium colony is rule, 35 degree of culture 36h, in the uviol lamp of 365nm wavelength
Under, selecting surrounding has the bacterium colony of orange-yellow fluorescence.
Bacterial strain metabolism producing enzyme experiment (primary dcreening operation)：Primary dcreening operation is carried out using tributyrin flat board transparent circle method, first, will be sieved
The inoculation selected is in the 500mL triangular flasks of the culture medium containing 100mL, 35 DEG C, 200r/min shaking table culture 72h,
It is stand-by that 10000r/min centrifugations 20min collects supernatant；Secondly prepared and contained with polyethylene glycol (PVA) solution that volume ratio is 3%
The emulsion of 100g/L tributyrins, tributyrin is added in 180mL Tris-HCl buffer solutions (pH=8.0)
Emulsion 20mL, 1.5% agar is then added, high pressure steam sterilization is down flat plate, then beats diameter 0.8cm's in each flat board again
Aperture, take the 50 above-mentioned supernatants of μ L to react 24h in aperture in 30 degree, survey transparent circle size, take transparent circle person clear greatly to carry out
Enzyme activity determination.
Bacterial strain metabolism producing enzyme experiment (secondary screening)：The bacterial strain obtained in primary dcreening operation is subjected to secondary screening again, every plant connects 5 bottles, is seeded in
In the 500mL triangular flasks of the culture medium containing 100mL, 35 DEG C, 200r/min shaking table culture 72h, zymotic fluid is taken to determine enzyme activity.
5 plants of mutant strains that there is high enzyme to live are selected by shaking flask secondary screening to be listed as follows：
The enzyme activity of mutant strain of the original strain of table 1 with being lived with high enzyme contrasts
| Bacterial strain | Original bacteria | LD-05 | LD-13 | LD-14 | LD-85 | LD-231 |
| Enzyme activity (U/mL) | 2432 | 2571 | 2192 | 3068 | 2605 | 2832 |
It is stable most live high-enzyme strain that LD-14 is selected after shake flask fermentation again.
The pseudomonad LD-14 enzymatic productions of embodiment 2
Pseudomonad LD-14 enzymatic production method, is mainly included the following steps that：
Inclined-plane culture：The ring pseudomonad LD-14 of picking one is inoculated in solid slope culture medium, incubated 36h at 35 DEG C,
Obtain first order seed；
Shaking culture：First order seed is taken in ring access seed culture medium, 35 DEG C of constant temperature, shaking speed 200r/min bars
48h is cultivated under part, obtains secondary seed solution；
Seed tank culture：Secondary seed solution is accessed in seed tank culture base according to the ratio of inoculum concentration 6% (v/v), it is permanent
It is warm 35 DEG C, cultivate 12h under the conditions of rotating speed 200r/min；
Fermentation tank culture：Seed liquor in seeding tank is accessed into fermentation tank culture according to the ratio of inoculum concentration 6% (v/v)
Base, 35 DEG C, rotating speed 200r/min of constant temperature, ventilation is set：0 to 5h be 0.25vvm, 5h to 20h be 0.5vvm, 20h to fermenting
Terminate as 1.0vvm, fermentation is whole to control zymotic fluid pH value to ferment to enzyme activity and increases slowly for 6.8 using supplemented medium, bacterium
Body self-dissolving terminates to ferment when serious, fermentation period 150h, obtains final zymotic fluid；
By final zymotic fluid by extraction and process for purification, lipase production liquid enzyme preparation is obtained.
Solid slope culture medium (g/L)：Peptone 12, yeast extract 6, agar 15, sodium chloride 1, surplus are water, pH 7.0,
121 DEG C of sterilizing 20min；
Seed culture medium (g/L)：Malt syrup 40, peptone 5, yeast extract 5, sodium chloride 0.8, magnesium sulfate 0.5, calcium chloride
0.1, disodium hydrogen phosphate 0.5, surplus is water, 6.8,121 DEG C of sterilizing 20min of pH；
Seed tank culture base (g/L)：Glucose 80, yeast extract 15, corn steep liquor 5, ammonium sulfate 1, magnesium sulfate 0.5, di(2-ethylhexyl)phosphate
Hydrogen potassium 1.5, calcium chloride 0.1, pH 6.8,121-123 DEG C of sterilizing 30min；
Fermentation tank culture medium (g/L)：Maltose 180, peptone 10, yeast extract 5, ammonium sulfate 1.5, magnesium sulfate 0.5, corn
Oil 20, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3, pH 6.8,121-123 DEG C of sterilizing 30min；
Supplemented medium (g/L)：Malt syrup 450, KH2PO45, corn steep liquor 7, corn oil 40, calcium chloride 5, pH 4.0,
121-123 DEG C of sterilizing 30min.
The extraction of lipase and process for purification are as follows：
First by final zymotic fluid, 1% pearlite filtering aid is added, press filtration obtains clarifying press filtration enzyme liquid；
The clarification press filtration enzyme liquid is concentrated by ultrafiltration with 20000 molecular weight milipore filters, obtains concentrate；
Mass percent 20% (m/v) glycerine, 0.15% (m/v) potassium sorbate, 0.3% (m/v) are added in concentrate
Sodium benzoate, pH 8.5 is adjusted, filtration sterilization is then carried out, that is, obtains lipase production liquid enzyme preparation.
The pseudomonad LD-14 fermenting properties of embodiment 3 are verified
50L fermentation tank confirmatory experiments, fermentation period are carried out according to the pseudomonad LD-14 of embodiment 2 enzymatic production method
150h, the enzymatic production situation of its 6 batch, average producing enzyme level are 25558U/mL, and table 2 illustrates bacterial strain not only high yield lipase
And the enzyme activity of its fermenting property and its lipase produced is respectively provided with significant stability.
The enzymatic production situation of the batch high yield lipase strain of table 26
The Assay of lipase activity method of embodiment 4
(1) preparation of enzyme liquid：Zymotic fluid 6000r/min centrifuges 15min, and supernatant is crude enzyme liquid.
(2) enzyme activity defines：To be defined as 1g solid enzyme powders or 1mL liquid enzymes, under the conditions of certain temperature and pH, 1min water
Solve the titratable aliphatic acid that substrate produces 1 μm of ol, as an enzyme activity unit.
(3) enzyme activity determination：
Enzyme sample 1-2mL is drawn, is diluted with the phosphate buffers of pH 7.5, surveys timing controlled enzyme liquid concentration, sample is with compareing
The difference for consuming alkali number is controlled in the range of 1-2mL, during pipette samples, is taken again after should enzyme liquid be shaken up.
Potentiometric titration：Instrumental correction is carried out by pH meter operation instructions；
Two 100mL beakers are taken, substrate dilution 40mL and phosphate buffer are respectively added in blank cup and specimen cup
5mL, 95% ethanol 15mL is added in blank cup, 5 minutes is preheated in 40 0.2 degree water-bath of plus-minus, then in blank cup with
Respectively add enzyme liquid 1mL to be measured in specimen cup, mix timing immediately, accurate response fills into ethanol immediately after 15 minutes in specimen cup
15mL terminating reactions, take out；
One piece of rotor is added in beaker, is placed on magnetic stirrer, side stirring, while with 0.05mol/L sodium hydroxide marks
Until pH 10.3, is titration end-point, record consumes standard solution of sodium hydroxide volume for quasi- solution titration.
X1:The enzyme activity U/mL of sample；
V1:The volume of the standard solution of sodium hydroxide consumed during sample is titrated, unit is milliliter (mL)；
V2：The volume of the standard solution of sodium hydroxide consumed during blank is titrated, unit is milliliter (mL)；
c：Concentration of Sodium Hydroxide Solution Standard, every liter of unit mole (mol/L)；
50：0.05mol/L sodium hydroxide solutions 1.00mL is equivalent to 50 μm of ol of aliphatic acid；
n1：The extension rate of sample；
0.05：Concentration of Sodium Hydroxide Solution Standard conversion coefficient；
Reaction time 15min, in terms of 1 minute；
The most suitable action pH scope of the lipase of embodiment 5
The fermentation broth enzyme vigor 25500U/mL produced using the present invention lipase under the conditions of 35 DEG C, exists respectively as sample
Under different pH value (7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0), enzyme activity is determined, the enzyme activity of measure becomes
Change curve as shown in figure 1, the most suitable action pH scope of enzyme is 8.5-10.0.
The optimum temperature scope of the lipase of embodiment 6
The fermentation broth enzyme vigor 25500U/mL produced using the present invention lipase is sample, under the conditions of pH value is 9.7,
Respectively under different temperatures (20,25,30,35,40,45,50), enzyme activity is determined, the enzyme activity change curve of measure is such as
Shown in Fig. 2, the optimum temperature scope of enzyme is 30 DEG C -40 DEG C.
The lower temperature resistance of the lipase of embodiment 7
On the basis of the fermentation broth enzyme vigor 25500U/mL produced by the present invention lipase, under the conditions of pH value is 9.7,
Remaining enzyme activity is determined after being incubated at 20 DEG C, as shown in figure 3, remaining enzyme activity is 80% after 20 DEG C of insulation 1d, there is well resistance to
Cord blood activity, it can be widely applied in low-temperature industrial production, significantly expanded the industrial applicability of lipase, improve
The application value of lipase.
Claims (8)
1. a kind of lipase, it is characterised in that the lipase comes from pseudomonad (Pseudomonas sp.) LD-14, bacterial strain
Deposit number is CGMCC No.14414.
2. lipase as claimed in claim 1, it is characterised in that the side of lipase is prepared using pseudomonad LD-14 fermentations
Method is as follows：Seed liquor is accessed into fermentation tank culture medium according to the ratio of inoculum concentration 6%, 35 DEG C, rotating speed 200r/min of constant temperature, set
Ventilation：0 to 5h be 0.25vvm, 5h to 20h is that 0.5vvm, 20h to fermentation ends are 1.0vvm, and fermentation whole process utilizes feed supplement
It is 6.8 that culture medium, which controls zymotic fluid pH value, ferments to enzyme activity and increasess slowly, thalline self-dissolving terminates to ferment when serious, fermentation period
For 150h.
3. the lipase described in claim 2, it is characterised in that the fermentation tank culture medium is：Maltose 180g/L, peptone
10g/L, yeast extract 5g/L, ammonium sulfate 1.5g/L, magnesium sulfate 0.5g/L, corn oil 20g/L, calcium chloride 0.1g/L, phosphoric acid hydrogen two
Potassium 0.3g/L, pH 6.8,121-123 DEG C of sterilizing 30min；
The supplemented medium is：Malt syrup 450g/L, KH2PO45g/L, corn steep liquor 7g/L, corn oil 40g/L, calcium chloride
5g/L, pH 4.0,121-123 DEG C of sterilizing 30min.
4. the lipase described in claim 2, it is characterised in that the preparation method of the seed liquor is as follows：By secondary seed solution
Accessed according to the ratio of inoculum concentration 6% in seed tank culture base, 35 DEG C of constant temperature, 12-15h is cultivated under the conditions of rotating speed 200r/min；
The preparation method of the secondary seed solution is as follows：First order seed is taken in ring access seed culture medium, 35 DEG C of constant temperature, shaken
48h is cultivated under the conditions of bed rotating speed 200r/min, obtains secondary seed solution；
The preparation method of the first order seed is as follows：The ring pseudomonad LD-14 of picking one is inoculated in solid slope culture medium, 35 DEG C
Under incubated 36h, obtain first order seed.
5. the lipase described in claim 4, it is characterised in that the solid slope culture medium is：Peptone 12g/L, yeast
Cream 6g/L, agar 15g/L, sodium chloride 1g/L, surplus are water, 7.0,121 DEG C of sterilizing 20min of pH；
The seed culture medium is：Malt syrup 40g/L, peptone 5g/L, yeast extract 5g/L, sodium chloride 0.8g/L, magnesium sulfate
0.5g/L, calcium chloride 0.1g/L, disodium hydrogen phosphate 0.5g/L, surplus are water, 6.8,121 DEG C of sterilizing 20min of pH；
The seed tank culture base is：Glucose 80g/L, yeast extract 15g/L, corn steep liquor 5g/L, ammonium sulfate 1g/L, magnesium sulfate
0.5g/L, potassium dihydrogen phosphate 1.5g/L, calcium chloride 0.1g/L, pH 6.8,121-123 DEG C of sterilizing 30min.
6. the lipase described in claim 2, it is characterised in that the extraction of the lipase and process for purification are as follows：
(1) final zymotic fluid is added into 1~5% pearlite filtering aid, press filtration obtains clarifying press filtration enzyme liquid；
(2) the clarification press filtration enzyme liquid is concentrated by ultrafiltration with 20000 molecular weight milipore filters, obtains concentrate；
(3) 20% is added in above-mentioned concentrate (stabilizer, 0.45% preservative, to adjust pH 8.5, then filtered
It is degerming, that is, obtain lipase production liquid enzyme preparation.
7. the lipase described in claim 6, it is characterised in that the stabilizer is glycerine, and the preservative is that mass ratio is
1：2 potassium sorbate and sodium benzoate.
8. the application of lipase described in claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108128906A (en) * | 2017-12-26 | 2018-06-08 | 重庆精创联合环保工程有限公司 | Hot industry waste water treating agent |
| CN112375699A (en) * | 2020-11-10 | 2021-02-19 | 南昌大学 | Bacillus licheniformis for expressing lipase and fermentation enzyme production method thereof |
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|---|---|---|---|---|
| CN101085984A (en) * | 2007-04-07 | 2007-12-12 | 中国科学院海洋研究所 | Low-temperature lipase and its gene sequence |
| CN102002486A (en) * | 2010-10-22 | 2011-04-06 | 北京理工大学 | Phospholipase B from pseudomonas fluorescens and production method thereof |
| CN104403961A (en) * | 2014-10-30 | 2015-03-11 | 昆明理工大学 | Pseudomonas sp., cold-adapted lipase generated thereby, and gene of cold-adapted lipase |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085984A (en) * | 2007-04-07 | 2007-12-12 | 中国科学院海洋研究所 | Low-temperature lipase and its gene sequence |
| CN102002486A (en) * | 2010-10-22 | 2011-04-06 | 北京理工大学 | Phospholipase B from pseudomonas fluorescens and production method thereof |
| CN104403961A (en) * | 2014-10-30 | 2015-03-11 | 昆明理工大学 | Pseudomonas sp., cold-adapted lipase generated thereby, and gene of cold-adapted lipase |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108128906A (en) * | 2017-12-26 | 2018-06-08 | 重庆精创联合环保工程有限公司 | Hot industry waste water treating agent |
| CN108128906B (en) * | 2017-12-26 | 2020-08-28 | 重庆精创联合环保工程有限公司 | High temperature industrial wastewater treatment agent |
| CN112375699A (en) * | 2020-11-10 | 2021-02-19 | 南昌大学 | Bacillus licheniformis for expressing lipase and fermentation enzyme production method thereof |
| CN112375699B (en) * | 2020-11-10 | 2022-09-16 | 南昌大学 | A strain of Bacillus licheniformis expressing lipase and its fermentation enzyme production method |
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