CN117802070A - Composite biological enzyme catalyst and preparation method and application thereof - Google Patents
Composite biological enzyme catalyst and preparation method and application thereof Download PDFInfo
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- CN117802070A CN117802070A CN202410223706.8A CN202410223706A CN117802070A CN 117802070 A CN117802070 A CN 117802070A CN 202410223706 A CN202410223706 A CN 202410223706A CN 117802070 A CN117802070 A CN 117802070A
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- lipase
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- penicillium expansum
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 70
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 70
- 239000003054 catalyst Substances 0.000 title claims abstract description 65
- 239000002131 composite material Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 22
- 108090001060 Lipase Proteins 0.000 claims abstract description 78
- 102000004882 Lipase Human genes 0.000 claims abstract description 77
- 239000004367 Lipase Substances 0.000 claims abstract description 76
- 235000019421 lipase Nutrition 0.000 claims abstract description 76
- 241001123663 Penicillium expansum Species 0.000 claims abstract description 53
- IEJIGPNLZYLLBP-UHFFFAOYSA-N dimethyl carbonate Chemical compound COC(=O)OC IEJIGPNLZYLLBP-UHFFFAOYSA-N 0.000 claims abstract description 53
- 238000000855 fermentation Methods 0.000 claims abstract description 42
- 230000004151 fermentation Effects 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 41
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 241000223250 Metarhizium anisopliae Species 0.000 claims abstract description 19
- KMTRUDSVKNLOMY-UHFFFAOYSA-N Ethylene carbonate Chemical compound O=C1OCCO1 KMTRUDSVKNLOMY-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000000284 extract Substances 0.000 claims abstract description 15
- 241000589513 Burkholderia cepacia Species 0.000 claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 239000002609 medium Substances 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 14
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- 239000000843 powder Substances 0.000 claims description 11
- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 2
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- 238000005809 transesterification reaction Methods 0.000 abstract description 18
- 230000000694 effects Effects 0.000 description 24
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- 238000012360 testing method Methods 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 13
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- 239000011734 sodium Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
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- 230000035484 reaction time Effects 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000006136 alcoholysis reaction Methods 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 239000003254 gasoline additive Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 239000011942 biocatalyst Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- SPFUWSBRIIKPOR-UHFFFAOYSA-N carbonyl dichloride;methanol Chemical compound OC.ClC(Cl)=O SPFUWSBRIIKPOR-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002608 ionic liquid Substances 0.000 description 2
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- GEYJUFBPCGDENK-UHFFFAOYSA-M sodium;3,8-dimethyl-5-propan-2-ylazulene-1-sulfonate Chemical compound [Na+].CC(C)C1=CC=C(C)C2=C(S([O-])(=O)=O)C=C(C)C2=C1 GEYJUFBPCGDENK-UHFFFAOYSA-M 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- URAYPUMNDPQOKB-UHFFFAOYSA-N triacetin Chemical compound CC(=O)OCC(OC(C)=O)COC(C)=O URAYPUMNDPQOKB-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 102100031375 Endothelial lipase Human genes 0.000 description 1
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- -1 diglycerides Chemical class 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000001087 glyceryl triacetate Substances 0.000 description 1
- 235000013773 glyceryl triacetate Nutrition 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 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 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- RUOJZAUFBMNUDX-UHFFFAOYSA-N propylene carbonate Chemical compound CC1COC(=O)O1 RUOJZAUFBMNUDX-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229960002622 triacetin Drugs 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 239000012138 yeast extract 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
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- 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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- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/141—Feedstock
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a composite biological enzyme catalyst, a preparation method and application thereof, and belongs to the field of bioengineering. The composite biological enzyme catalyst is prepared from penicillium expansum lipase, metarhizium anisopliae fermentation extract and pseudomonas cepacia lipase according to the weight ratio of (4-6): (1-3): and (2-4) are compounded according to the proportion. The composite biological enzyme catalyst is applied to the process of producing the dimethyl carbonate by the transesterification method, and can stably improve the yield of the dimethyl carbonate and the conversion rate of the ethylene carbonate.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a composite biological enzyme catalyst and a preparation method and application thereof.
Background
Dimethyl carbonate (DMC) is an environmentally friendly compound and is a basic organic synthetic raw material that has been widely used in recent years. Dimethyl carbonate is not only a nontoxic, environment-friendly and widely used chemical raw material, but also an important organic synthesis intermediate; in addition, the catalyst can be used as a gasoline additive to replace methyl tertiary butyl ether, has wide development prospect. As a gasoline additive, the dimethyl carbonate can obviously reduce the emission of hydrocarbon, carbon monoxide and formaldehyde in automobile exhaust; in addition, dimethyl carbonate has relatively poor water solubility, so that gasoline can be prevented from being easily dissolved in water and polluting underground water as a gasoline additive.
With the increasing demand of dimethyl carbonate, synthetic routes for dimethyl carbonate have been developed in recent years, including phosgene-methanol process, urea alcoholysis synthesis process, one-step process and transesterification process. Wherein, the raw materials used by the phosgene-methanol method are extremely toxic and pollute the environment, and are eliminated; the single-pass yield of the urea alcoholysis method is too low, and the urea alcoholysis method is mainly carried out in an autoclave, so that the equipment investment is large and the competitiveness is poor; the process for synthesizing the dimethyl carbonate by the one-step method of the carbon dioxide and the methanol prevents the large-scale industrial application of the method due to the problems of low raw material conversion rate, great thermodynamic limit and the like. The transesterification method has the advantages of simple operation, short reaction time, easy separation of products, most mature technology and the like, so the transesterification method is still the most commonly used synthesis method in the current industrial production. At present, the synthetic route for synthesizing the dimethyl carbonate by the transesterification method has the defects of poor catalyst selectivity, low activity, low yield of the dimethyl carbonate and the like.
As a biocatalyst, the enzyme has been widely used in the fields of food production and detection, environmental protection technology, biotechnology, biological medicine and the like in recent years. Lipase (EC 3.1.1.3) which is the most widely studied and used at present is a biocatalyst capable of catalyzing grease and short-chain alcohol to carry out transesterification reaction to generate fatty acid methyl ester, and the Lipase-mediated reaction has the advantages of mild reaction condition, small alcohol consumption, easy collection and purification of products, no pollutant emission and the like, and is suitable for being used as a catalyst for preparing dimethyl carbonate. The invention patent CN103525798B discloses a biological enzyme catalyst for preparing dimethyl carbonate and a preparation method thereof. According to the method, ionic liquid and lipase are used as catalysts, and the obtained catalysts have high catalytic efficiency in the process of preparing the dimethyl carbonate through esterification. However, the conversion rate of propylene carbonate in the catalytic reaction of the catalyst is unstable, and the problems of low activity of the biological enzyme catalyst and low yield of dimethyl carbonate are not solved yet. Therefore, development of a bio-enzyme catalyst with high activity and high yield for preparing dimethyl carbonate by transesterification is needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a composite biological enzyme catalyst and a preparation method and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a composite biological enzyme catalyst is provided, which is prepared from penicillium expansum lipase, metarhizium anisopliae fermentation extract and pseudomonas cepacia lipase according to the weight ratio (4-6): (1-3): and (2-4) are compounded according to the proportion.
In a second aspect of the present invention, there is provided a method for preparing the above-mentioned composite bio-enzyme catalyst, comprising the steps of:
(1) Inoculating the penicillium expansum seed solution to a lipase fermentation medium, and fermenting to obtain a fermentation liquor; centrifuging the fermentation liquor, collecting supernatant, concentrating and drying the supernatant to obtain penicillium expansum lipase;
(2) Mixing penicillium expansum lipase, metarhizium anisopliae fermentation extract and pseudomonas cepacia lipase to obtain a composite biological enzyme catalyst;
the lipase fermentation medium consists of the following components in percentage by weight: 4-8% of soybean cake powder, 0.5-3% of corn starch, 1-4% of chamomile powder, 1-3% of birch bark and 0.2-0.6% of NaNO 3 ，0.1-0.3%Na 2 HPO 4 ，0.015-0.2%K 2 SO 4 ，0.0015-0.1%MgSO 4 ，0.015-0.1%FeSO 4 ，0.025-0.03%CaCO 3 ，0.001-0.0015%Na 2 CO 3 The balance being water.
Preferably, the preparation method of the chamomile powder comprises the following steps:
the chamomile flower is dried, liquid nitrogen is added, chamomile flower powder is obtained by grinding, and the weight ratio of the liquid nitrogen to the chamomile flower is (1-3): (8-10).
Preferably, in step (1), the fermentation conditions are: the seed liquid of Penicillium expansum has inoculation amount of 5-12%, and is fermented at 25-35deg.C and 150-250rpm for 24-48 hr.
In a third aspect of the invention, the application of the composite biological enzyme catalyst in synthesizing dimethyl carbonate is provided.
Preferably, the method for synthesizing dimethyl carbonate comprises the following steps:
mixing ethylene carbonate and methanol, and then adding a composite biological enzyme catalyst for reaction to obtain dimethyl carbonate, wherein the weight ratio of the composite biological enzyme catalyst to the ethylene carbonate is (0.01-0.02): (1-1.5).
Preferably, the reaction conditions are: reacting at 40-50 deg.C under 1-1.5atm for 60-72h.
Preferably, the molar ratio of ethylene carbonate to methanol is (0.5-2): (2-6).
The invention has the beneficial effects that:
(1) The invention combines the penicillium expansum lipase, the metarhizium anisopliae fermentation extract and the pseudomonas cepacia lipase to obtain the composite biological enzyme catalyst, and the composite biological enzyme catalyst is applied to the process for producing the dimethyl carbonate by the transesterification method, so that the yield of the dimethyl carbonate and the conversion rate of the ethylene carbonate can be stably improved.
(2) According to the invention, chamomile powder and birch bark are added into a lipase fermentation medium, and the penicillium expansum lipase obtained by inoculating penicillium expansum into the medium has higher enzyme activity, and the obtained penicillium expansum lipase is used as a biological enzyme catalyst, so that the problem of low enzyme catalyst activity in the process of producing dimethyl carbonate by a transesterification method is further solved.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described above, in the prior art, the transesterification method is the most widely used method for industrial production at present by virtue of the advantages of simple operation, short reaction time, easy separation of products, the most mature technology and the like. However, the existing transesterification method for producing dimethyl carbonate has the problems of low conversion rate and low yield caused by unstable catalyst and low activity.
Based on the above, the invention provides a composite biological enzyme catalyst, and a preparation method and application thereof. The composite biological starter has higher enzyme activity and stability, and can improve the conversion rate of raw materials and the yield of products in the process of synthesizing the dimethyl carbonate by the transesterification method when being applied to the process of synthesizing the dimethyl carbonate by the transesterification method. The inventors have first selected lipases capable of catalyzing esterification reactions and industrially used them widely. After looking up the data, the inventor knows that there is a technology of catalyzing the synthesis of dimethyl carbonate by using penicillium lipase as a catalyst, wherein the catalytic effect of the catalyst obtained by compounding penicillium expansum lipase with an ionic liquid catalyst is not stable, and the problems of low activity of the biological enzyme catalyst and low product yield are not solved yet. Therefore, the inventor develops a biological enzyme catalyst with high activity, stable catalytic effect and high product yield based on the penicillium expansum lipase.
To achieve the purpose, on one hand, the inventor adds birch bark and chamomile powder into a lipase fermentation medium of the penicillium expansum to improve the enzyme activity of the penicillium expansum lipase; wherein the active ingredients in the chamomile comprise sodium azulene sulfonate, and after the sodium azulene sulfonate contacts lipase in a culture medium, the enzyme activity decay of the lipase can be effectively inhibited; the birch bark has higher carbon content, can provide a large amount of carbon sources for the fermentation of the penicillium expansum and provide nutrition for the preparation of the extended lipase.
On the other hand, after improving the enzyme activity of the penicillium expansum lipase, the inventor compounds the penicillium expansum lipase, the metarhizium anisopliae fermentation extract and the pseudomonas cepacia lipase to obtain the composite biological enzyme catalyst. The metarhizium anisopliae fermentation extract and the pseudomonas cepacia lipase can enhance the stability of the penicillium expansum lipase, thereby improving the catalytic effect of the obtained composite biological enzyme catalyst and the yield of the dimethyl carbonate and the conversion rate of raw materials.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
In the following examples and comparative examples, the sources or preparation methods of the materials used are as follows:
penicillium expansum is purchased from China Center for Type Culture Collection (CCTCC) AF 93007; metarhizium anisopliae is purchased from China Center for Type Culture Collection (CCTCC) CF 2008439; pseudomonas cepacia lipase (. Gtoreq.30U/mg) was purchased from Sigma Aldrich Biochemical technology Co., ltd., product number 62309.
The preparation method of the metarhizium anisopliae fermentation extract comprises the following steps:
(1) Inoculating Metarhizium anisopliae in PDA culture medium, culturing at 25deg.C for 7d, and activating after spore production; inoculating spore powder of the activated strain to a seed culture medium, and culturing at 25 ℃ and 15rpm for 48 hours to obtain metarhizium anisopliae fermentation liquor;
the composition of the PDA medium was as follows: 1L of potato juice with the mass concentration of 20%, 20g of glucose and 20g of agar;
the composition of the seed medium was as follows: 20g/L yeast extract, 20g/L glucose, 1g/L potassium dihydrogen phosphate, 1g/L magnesium sulfate heptahydrate, 0.01g/L ferric sulfate and 50ml/L glyceryl triacetate.
(2) Centrifuging the metarhizium anisopliae fermentation liquid in the step (1) at 5 ℃ and 5000rpm for 5min to obtain supernatant, concentrating and drying the supernatant to obtain metarhizium anisopliae fermentation extract.
Preparation of penicillium expansum seed liquid: penicillium expansum is inoculated into PDB culture medium and cultured at 25 deg.C and 150rpm for 24 hr to obtain penicillium expansum seed liquid. Preparation of PDB Medium: 200g of potato is weighed, boiled in water for 20min, filtered while the double-layered gauze is hot, 20g of glucose is added into the filtrate, distilled water is fixed to 1000ml, and the mixture is autoclaved at 121 ℃ for 20min.
Example 1: and (3) preparation of dimethyl carbonate.
1. And (3) preparing the composite biological enzyme catalyst.
(1) Inoculating the penicillium expansum seed solution into a lipase fermentation culture medium according to an inoculum size of 7%, fermenting for 36 hours at 27 ℃ and 200rpm to obtain a fermentation liquor, centrifuging the fermentation liquor at 5000rpm to obtain a supernatant, concentrating and drying the supernatant to obtain the penicillium expansum lipase.
The lipase fermentation medium consists of the following components: 5 parts of soybean cake powder, 1 part of corn starch, 2 parts of chamomile powder, 2 parts of birch bark and 0.4 part of NaNO 3 0.2 part of Na 2 HPO 4 0.1 part of K 2 SO 4 0.05 part of MgSO 4 0.002 parts of FeSO 4 0.01 part of CaCO 3 0.0013 part of Na 2 CO 3 。
(2) The penicillium expansum lipase, the metarhizium anisopliae fermentation extract and the pseudomonas cepacia lipase are mixed according to the weight ratio of 3:2:3, and obtaining the composite biological enzyme catalyst after mixing the components in proportion.
2. Dimethyl carbonate is prepared by transesterification.
Ethylene carbonate and methanol were mixed in a molar ratio of 1.5:4, and then placing the composite biological enzyme catalyst into a reaction device, and reacting 65 under the conditions of 1.25atm and 45 ℃ to obtain the dimethyl carbonate. The weight ratio of the composite biological enzyme catalyst to the ethylene carbonate is 0.015:1.125.
example 2: and (3) preparation of dimethyl carbonate.
1. And (3) preparing the composite biological enzyme catalyst.
(1) Inoculating penicillium expansum seed solution into lipase fermentation culture medium according to an inoculum size of 5%, fermenting at 25deg.C and 150rpm for 24 hr to obtain fermentation liquor, centrifuging the fermentation liquor at 5000rpm to obtain supernatant, concentrating and drying the supernatant to obtain penicillium expansum lipase.
The lipase fermentation medium consists of the following components: 4 parts of soybean cake powder, 0.5 partCorn starch 1 part, chamomile flower powder 1 part, birch bark 1 part, naNO 0.2 part 3 0.1 part of Na 2 HPO 4 0.015 part of K 2 SO 4 0.0015 part of MgSO 4 0.015 part of FeSO 4 0.025 part of CaCO 3 0.001 part of Na 2 CO 3 。
(2) The penicillium expansum lipase, the metarhizium anisopliae fermentation extract and the pseudomonas cepacia lipase are mixed according to the weight ratio of 4:1:2, and obtaining the composite biological enzyme catalyst after mixing the components in proportion.
2. Dimethyl carbonate is prepared by transesterification.
Ethylene carbonate and methanol were mixed in a molar ratio of 0.5:2, then placing the composite biological enzyme catalyst into a reaction device, and reacting for 60 hours at the temperature of 1atm and 40 ℃ to obtain the dimethyl carbonate. The weight ratio of the composite biological enzyme catalyst to the ethylene carbonate is 0.01): 1.
example 3: and (3) preparation of dimethyl carbonate.
1. And (3) preparing the composite biological enzyme catalyst.
(1) Inoculating the penicillium expansum seed solution into a lipase fermentation culture medium according to 12% of inoculum size, fermenting for 48 hours at 35 ℃ and 250rpm to obtain a fermentation liquor, centrifuging the fermentation liquor at 5000rpm to obtain a supernatant, concentrating and drying the supernatant to obtain the penicillium expansum lipase.
The lipase fermentation medium consists of the following components: 8 parts of soybean cake powder, 3 parts of corn starch, 4 parts of chamomile powder, 3 parts of birch bark and 0.6 part of NaNO 3 0.3 part of Na 2 HPO 4 0.2 part of K 2 SO 4 0.1 part of MgSO 4 0.1 part of FeSO 4 0.03 part of CaCO 3 0.0015 part of Na 2 CO 3 。
(2) The penicillium expansum lipase, the metarhizium anisopliae fermentation extract and the pseudomonas cepacia lipase are mixed according to the weight ratio of 6:3:4, and obtaining the composite biological enzyme catalyst after mixing the components in proportion.
2. Dimethyl carbonate is prepared by transesterification.
Ethylene carbonate and methanol were mixed in a molar ratio of 2:6, then placing the composite biological enzyme catalyst into a reaction device, and reacting for 72 hours at the temperature of 50 ℃ under 1.5atm to obtain the dimethyl carbonate. The weight ratio of the composite biological enzyme catalyst to the ethylene carbonate is 0.02:1.5.
comparative example 1: and (3) preparation of dimethyl carbonate.
The complex biological enzyme catalyst in example 1 was replaced with penicillium expansum lipase, and dimethyl carbonate was prepared; the preparation method of the penicillium expansum lipase is the same as that of example 1, and the rest preparation methods and conditions are the same as those of example 1.
Comparative example 2: and (3) preparation of dimethyl carbonate.
A complex bio-enzyme catalyst and dimethyl carbonate were prepared in accordance with the method of example 1, except that the complex bio-enzyme catalyst of this comparative example did not contain Pseudomonas cepacia lipase.
Comparative example 3: and (3) preparation of dimethyl carbonate.
A complex bio-enzyme catalyst and dimethyl carbonate were prepared according to the method of example 1, except that the complex bio-enzyme catalyst of this comparative example did not contain Metarhizium anisopliae fermentation extract.
Comparative example 4:
a composite bio-enzyme catalyst was prepared according to the method of example 1, except that the lipase medium of this comparative example did not contain chamomile powder and birch bark.
Comparative example 5:
a composite bio-enzyme catalyst was prepared according to the method of example 1, except that the lipase medium of this comparative example did not contain birch bark.
Comparative example 6:
a composite bio-enzyme catalyst was prepared according to the method of example 1, except that the lipase medium of this comparative example did not contain chamomile powder.
Test example 1: yield of dimethyl carbonate and conversion of the reactants.
(1) The test method comprises the following steps:
the measurement methods for calculating the yield of dimethyl carbonate and the conversion rate of ethylene carbonate prepared in examples 1 to 3 and comparative examples 1 to 6 were described in the "research for preparing dimethyl carbonate with high activity by transesterification" of the "Shuoshi paper.
(2) Test results:
the yield of dimethyl carbonate and the conversion of ethylene carbonate are shown in table 1:
table 1: yield and conversion of dimethyl carbonate produced by transesterification
As shown in Table 1, the conversion rate of the ethylene carbonate is higher than 88% and the yield of the dimethyl carbonate is higher than 86%, which indicates that the composite biological enzyme catalyst provided by the invention has higher stability and catalytic capability for the reaction for producing the dimethyl carbonate; comparing the data of examples 1-3 with comparative examples 1-3, it is known that the addition of metarhizium anisopliae fermentation extract and Pseudomonas cepacia lipase on the basis of penicillium expansum lipase has a synergistic effect on improving catalyst stability, ethylene carbonate conversion and dimethyl carbonate yield.
Test example 2: and (5) measuring the enzyme activity of the penicillium expansum lipase.
Lipases hydrolyze triglycerides into fatty acids, diglycerides, monoglycerides and glycerol under certain conditions. The released fatty acid can be subjected to neutralization titration with a standard alkaline solution, the end point of the reaction is indicated by a phenolphthalein indicator, and the enzyme activity is calculated according to the amount of the consumed alkaline.
Definition of enzyme activity units in this comparative example: at 45 ℃ and pH value of 7.5, 1 mu mol of free fatty acid released by the hydrolyzed fat of the sample per minute is 1 lipase activity unit (U).
(1) The test method comprises the following steps:
test groups 1-6 were each set, and 4mL of an emulsion of olive oil and PVA was added to 5mL of a phosphate buffer solution (pH 7.5) of 25mmol/L, and the mixture was preheated in a water bath at 45℃for 5 minutes to obtain a mixed solution. The treatment method for each test group was as follows:
taking the supernatant obtained in the preparation of the penicillium expansum lipase in example 1 and comparative examples 4-6 respectively, and dispersing each supernatant into an enzyme solution of the penicillium expansum lipase with the same mass concentration by using water;
test group 1: adding 1ml of the enzyme solution of the penicillium expansum lipase obtained in example 1 to the mixed solution;
test group 2: adding 1ml of the enzyme solution of the penicillium expansum lipase obtained in comparative example 4 to the mixed solution;
test group 3: adding 1ml of the enzyme solution of the penicillium expansum lipase obtained in comparative example 5 to the mixed solution;
test group 4: adding 1ml of the enzyme solution of the penicillium expansum lipase obtained in comparative example 6 to the mixed solution;
the test groups 1-4 are respectively provided with a control group, which is marked as the control group 1-4, and the operation method of the control group 1-4 is as follows: 15mL of stop solution and 1mL of corresponding penicillium expansum lipase enzyme solution were added.
The test and control groups were reacted in a 45℃water bath for 15 minutes, and 15mL of 95% ethanol was added to terminate the reaction. The amount of NaOH consumed at 0.05mol/L was calculated by titrating the fatty acids generated in the samples and controls with 0.05 mol/LNaOH.
The calculation formula of the enzyme activity is as follows: enzyme Activity (U/mL) = (B-A). Times.C/0.05X1.15 XN
Wherein:
b: the volume of NaOH standard solution is consumed in titration of test group samples, and the volume is mL;
a: the volume of NaOH standard solution is consumed in titration of the control group sample, and the volume is mL;
c: naOH standard solution concentration min/L;
0.05: concentration conversion coefficient of NaOH standard solution;
50: 1mL of 0.05mol/LNaOH standard solution corresponds to 50mM of fatty acid;
n: dilution factor of enzyme solution;
1/15: the reaction time is 15min, and the reaction time is converted into a coefficient of 1 min;
(2) Test results:
the enzyme activities of the penicillium expansum lipases in each test group are shown in table 2.
Table 2: enzyme activity of penicillium expansum lipase
As is clear from the data in the table, the enzyme activity of the penicillium expansum lipase in test group 1 was 2834U/ml, which indicates that the penicillium expansum lipase obtained in example 1 had a higher enzyme activity at 45 ℃. Further, comparing the data of test group 1 with the data of test groups 4 to 6, it can be seen that the enzyme activity of the obtained penicillium expansum lipase can be significantly improved after the birch bark and the chamomile flower powder are added into the culture medium, and meanwhile, the birch bark and the chamomile flower powder play a synergistic effect of 1+1 > 2 in improving the enzyme activity of the obtained penicillium expansum lipase.
The foregoing description of the preferred embodiment of the invention is not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (8)
1. A composite biological enzyme catalyst is characterized by comprising penicillium expansum lipase, metarhizium anisopliae fermentation extract and pseudomonas cepacia lipase according to the weight ratio (4-6): (1-3): and (2-4) are compounded according to the proportion.
2. The method for preparing the composite biological enzyme catalyst according to claim 1, which is characterized by comprising the following steps:
(1) Inoculating the penicillium expansum seed solution to a lipase fermentation medium, and fermenting to obtain a fermentation liquor; centrifuging the fermentation liquor, collecting supernatant, concentrating and drying the supernatant to obtain penicillium expansum lipase;
(2) Mixing penicillium expansum lipase, metarhizium anisopliae fermentation extract and pseudomonas cepacia lipase to obtain a composite biological enzyme catalyst;
the lipase fermentation culture medium consists ofThe composition of the components in percentage by weight is as follows: 4-8% of soybean cake powder, 0.5-3% of corn starch, 1-4% of chamomile powder, 1-3% of birch bark and 0.2-0.6% of NaNO 3 ，0.1-0.3%Na 2 HPO 4 ，0.015-0.2%K 2 SO 4 ，0.0015-0.1%MgSO 4 ，0.015-0.1%FeSO 4 ，0.025-0.03%CaCO 3 ，0.001-0.0015%Na 2 CO 3 The balance being water.
3. The method for preparing the composite biological enzyme catalyst according to claim 2, wherein the method for preparing chamomile flower powder comprises the following steps:
the chamomile powder is obtained by adding liquid nitrogen into the dried chamomile, and grinding the dried chamomile, wherein the weight ratio of the liquid nitrogen to the chamomile is (1-3): (8-10).
4. The method for preparing a composite biological enzyme catalyst according to claim 2, wherein in the step (1), fermentation conditions are as follows: the seed liquid of Penicillium expansum has inoculation amount of 5-12%, and is fermented at 25-35deg.C and 150-250rpm for 24-48 hr.
5. Use of the composite biological enzyme catalyst according to any one of claims 1-4 for synthesizing dimethyl carbonate.
6. The use according to claim 5, characterized in that the method for synthesizing dimethyl carbonate comprises the steps of:
mixing ethylene carbonate and methanol, and then adding a composite biological enzyme catalyst for reaction to obtain dimethyl carbonate, wherein the weight ratio of the composite biological enzyme catalyst to the ethylene carbonate is (0.01-0.02): (1-1.5).
7. The use according to claim 6, wherein the reaction conditions are: reacting at 40-50 deg.C under 1-1.5atm for 60-72h.
8. Use according to claim 6, characterized in that the molar ratio of ethylene carbonate to methanol is (0.5-2): (2-6).
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| CN101712951A (en) * | 2009-10-15 | 2010-05-26 | 福建师范大学 | Immobilization method-based lipase fixing method and application of lipase in ferulic acid esterification |
| CN103525798A (en) * | 2012-07-03 | 2014-01-22 | 深圳市绿微康生物工程有限公司 | Biological enzyme catalyst for preparing dimethyl carbonate and preparation method thereof |
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