CN115044577B - An immobilized Rhizopus oryzae lipase and a controllable preparation method and application thereof - Google Patents
An immobilized Rhizopus oryzae lipase and a controllable preparation method and application thereof Download PDFInfo
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- 101000966369 Rhizopus oryzae Lipase Proteins 0.000 title claims abstract description 161
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 73
- 108090000790 Enzymes Proteins 0.000 claims abstract description 73
- 230000000694 effects Effects 0.000 claims abstract description 47
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 claims description 17
- 239000004246 zinc acetate Substances 0.000 claims description 17
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000011942 biocatalyst Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 36
- 108090001060 Lipase Proteins 0.000 abstract description 35
- 239000004367 Lipase Substances 0.000 abstract description 35
- 102000004882 Lipase Human genes 0.000 abstract description 35
- 235000019421 lipase Nutrition 0.000 abstract description 35
- 239000012621 metal-organic framework Substances 0.000 abstract description 32
- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical group [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 abstract description 23
- 238000005538 encapsulation Methods 0.000 abstract description 17
- 230000003197 catalytic effect Effects 0.000 abstract description 14
- 238000011065 in-situ storage Methods 0.000 abstract description 11
- 238000003860 storage Methods 0.000 abstract description 9
- 230000003100 immobilizing effect Effects 0.000 abstract description 8
- 238000013459 approach Methods 0.000 abstract description 3
- 239000013110 organic ligand Substances 0.000 abstract description 3
- 239000002184 metal Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 25
- 239000000463 material Substances 0.000 description 20
- 239000013154 zeolitic imidazolate framework-8 Substances 0.000 description 18
- 239000011148 porous material Substances 0.000 description 12
- 230000002829 reductive effect Effects 0.000 description 12
- 230000003301 hydrolyzing effect Effects 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 7
- 239000011701 zinc Substances 0.000 description 7
- 108010093096 Immobilized Enzymes Proteins 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Substances C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 230000007423 decrease Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 240000005384 Rhizopus oryzae Species 0.000 description 2
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000005452 bending Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 150000002500 ions Chemical group 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000003313 weakening effect Effects 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 description 2
- OTLNPYWUJOZPPA-UHFFFAOYSA-M 4-nitrobenzoate Chemical compound [O-]C(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-M 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101001003495 Pseudomonas fluorescens Lipase Proteins 0.000 description 1
- 101001064559 Pseudomonas fluorescens Lipase Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000004993 emission spectroscopy Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009616 inductively coupled plasma Methods 0.000 description 1
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000012924 metal-organic framework composite Substances 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000013259 porous coordination polymer Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002411 thermogravimetry Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses an immobilized rhizopus oryzae lipase, a controllable preparation method and application thereof, and belongs to the technical field of immobilized lipase preparation. According to the invention, an amorphous ZIF-8 structure is prepared by adjusting the concentration of a metal organic ligand, and the immobilized lipase ROL@aZIF-8 is prepared by immobilizing the lipase ROL on a MOF carrier aZIF-8 by adopting an in-situ self-encapsulation method. The results show that compared with free lipase ROL, the enzyme activity of the immobilized lipase ROL@aZIF-8 is improved, and the immobilized lipase ROL@aZIF-8 has higher heat stability, pH tolerance and storage time stability. Meanwhile, when the concentration of the added enzyme is 4mg/mL, the immobilized lipase shows the maximum enzyme activity of 5.69U/mg. The invention is helpful to provide a potential approach for immobilized lipase, and deserves to be expanded in the catalytic application and industrial biocatalysis of different MOFs and lipases.
Description
Technical Field
The invention belongs to the technical field of immobilized lipase preparation, relates to a method for immobilizing rhizopus oryzae lipase by an amorphous ZIF-8 carrier material, and in particular relates to an immobilized rhizopus oryzae lipase, a controllable preparation method and application thereof.
Background
The rhizopus oryzae source lipase (Rhizopus oryzae lipase, ROL) has good triacylglycerol sn-1.3-position catalytic specificity, can produce structural grease with certain special functions, is an excellent catalyst of biodiesel, and has important application in the aspects of food, chemical industry, biological energy sources and the like. However, although ROL has the advantages of high catalytic efficiency, mild reaction conditions, environmental friendliness and the like, free ROL has the disadvantages of poor stability, easy deactivation, incapability of continuous operation and the like, and the disadvantages limit the practical application of the ROL in the food industry. Compared with free enzyme, the lipase has the advantages that the stability is greatly improved after immobilization treatment, the tolerance to environments such as heat, pH and the like is improved, the sensitivity to inhibitors is reduced, the utilization efficiency of the lipase is remarkably improved, and the production cost is greatly reduced. Thus, finding biocompatible carriers and immobilization methods suitable for lipases has been a difficult task and challenge that researchers have to face.
Metal-organic frameworks (MOFs) have received great attention because of their unique performance advantages of large specific surface area, high porosity, pore channel controllability, ease of functionalization, ease of modification, and the like. Zeolite-imidazole framework-8 (zeolitic imidazolate framework-8, zif-8) is a second generation porous coordination polymer, is a MOFs material formed by self-assembly of Zn 2+ ions and imidazole linkers through coordination bonds and has high specific surface area, excellent chemical and thermal stability and negligible cytotoxicity. The morphology and performance of ZIF-8 can be changed by changing the concentration ratio of Zn 2+ ions to imidazole connectors. The preparation of the immobilized lipase by the ZIF-8 material has the characteristics of easy recovery and reuse, high tolerance, high pH value, good thermal stability and the like in the production process.
Currently, four means of a surface adsorption method, a covalent crosslinking method, a pore encapsulation method and an in-situ self-encapsulation method are generally adopted to prepare the immobilized lipase based on the ZIF-8 material. The pore encapsulation method and the in-situ self-encapsulation method are to seal the enzyme molecules inside the ZIF-8, put the ZIF-8 armor on the enzyme molecules, and effectively improve the stability of the enzyme by means of the rigid frame of the ZIF-8 structure. The pore encapsulation method is to incubate lipase molecules with the synthesized ZIF-8 carrier to make the enzyme molecules pass through the ZIF-8 pore canal and enter the carrier to prepare immobilized enzyme, and the in-situ self-encapsulation method is to add the enzyme molecules into the reaction liquid synthesized by the ZIF-8 carrier, and directly seal the enzyme molecules in MOFs while self-assembling to form the carrier. Research shows that compared with immobilized lipase prepared by a surface adsorption method and a covalent crosslinking method, the immobilized lipase based on the ZIF-8 structure prepared by a pore encapsulation method and an in-situ self-encapsulation method has better stability because enzyme molecules are positioned in a carrier, and in the process of preparing the immobilized lipase by the pore encapsulation method, the enzyme molecules are easy to develop a protein structure when passing through a pore channel of the ZIF-8 carrier, so that the enzyme molecules lose activity, and the in-situ self-encapsulation method can effectively avoid the problem of inactivation of the enzyme molecules caused by the change of the protein structure.
However, the catalytic activity of the immobilized enzyme prepared by the in-situ self-encapsulation method is lost to some extent at present because of mass transfer resistance generated when a substrate or a product passes through the immobilized enzyme pore canal due to the small pore canal volume of the ZIF-8 carrier. Chinese patent CN108396023B discloses a method for preparing magnetic MOF material by grinding and fixing enzyme, the scheme shows that the material is prepared first, and enzyme is immobilized after the material is formed, but since enzyme is always on the surface of the material, stability is affected to a certain extent, and the process adds magnetic substance Fe 3O4 in the process of preparing material, enzyme load of immobilized enzyme may be reduced, and mass transfer resistance of substrate or product may be increased. Chinese patent CN111471663a discloses a method for immobilizing pseudomonas fluorescens lipase with a metal organic framework material, which also comprises preparing the material first and then immobilizing the lipase, i.e. immobilizing the lipase on the surface of Uio-66 (Zr), so that the stability is also affected.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the immobilized rhizopus oryzae lipase, and the controllable preparation method and the application thereof, which can effectively solve the problems of low catalytic activity and influenced stability of the immobilized enzyme prepared by the prior art.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
The invention discloses a controllable preparation method of immobilized rhizopus oryzae lipase, which comprises the steps of adding a zinc acetate solution into a 2-methylimidazole solution, adding a solution containing rhizopus oryzae lipase, stirring and reacting for 20-40 min, washing, collecting precipitate, and performing vacuum freeze-drying treatment to obtain the immobilized rhizopus oryzae lipase, namely immobilized lipase ROL@aZIF-8.
Preferably, the concentration of the zinc acetate solution is 20mmol/L, the concentration of the 2-methylimidazole solution is 33.8mmol/L, and the concentration of the solution containing Rhizopus oryzae lipase is 5mg/mL.
Further preferably, the volume ratio of zinc acetate solution, 2-methylimidazole solution and rhizopus oryzae lipase-containing solution is 1:1:0.08.
Preferably, the stirring reaction is carried out at a rotational speed of 300-500 r/min.
Preferably, the precipitate is washed 3-5 times by deionized water, and the collected precipitate is subjected to vacuum freeze drying treatment at-80 ℃ for 12 hours.
The invention also discloses the immobilized rhizopus oryzae lipase prepared by the controllable preparation method.
Preferably, the immobilized rhizopus oryzae lipase has higher enzyme activity than free enzyme when the temperature range is 30-70 degrees C, pH and the value is 7.5-10.0.
Preferably, the maximum enzyme activity of the immobilized rhizopus oryzae lipase reaches 5.69U/mg when the concentration of the added enzyme is 4 mg/mL.
The invention also discloses application of the immobilized rhizopus oryzae lipase in preparation of industrial biocatalysts.
Compared with the prior art, the invention has the following beneficial effects:
The immobilized rhizopus oryzae lipase and the controllable preparation method thereof disclosed by the invention are characterized in that 2-methylimidazole is mixed with zinc acetate solution, then the solution containing rhizopus oryzae lipase is added immediately for stirring reaction, compared with ZIF-8 material, the prepared amorphous MOF carrier aZIF-8 material has smaller mass transfer resistance for substances to enter and exit the carrier, and the immobilized lipase ROL@aZIF-8 prepared by immobilizing Rhizopus Oryzae Lipase (ROL) by adopting an in-situ self-encapsulation method is adopted.
Further, the ZIF-8 structure can be prepared controllably by a certain concentration ratio of 2-methylimidazole to zinc acetate, so that the zinc acetate solution with the concentration of 20mmol/L and the zinc acetate solution with the concentration of 33.8 mmol/L2-methylimidazole are preferable, and the zinc nitrate solution can be used by test, but the enzyme activity of the protoenzyme cannot be kept maximally after fixation, and the structure is changed from a complete structure to an amorphous structure by adjusting the concentration, and then the immobilization concentration suitable for rhizopus oryzae lipase is found, so that the enzyme activity is obviously improved after the zinc acetate solution with the concentration and the ZIF-8 structure immobilization enzyme prepared from the 2-methylimidazole solution are selected.
Compared with free lipase ROL, the immobilized rhizopus oryzae lipase prepared by the method provided by the invention has the advantages that the enzyme activity can be improved, the immobilized lipase ROL@aZIF-8 has higher thermal stability, pH tolerance and storage stability, namely, the enzyme activity is higher than that of the free enzyme when the temperature range is 30-70℃, pH and the temperature range is 7.5-10.0, the residual activity of the immobilized lipase ROL@aZIF-8 is reduced to a smaller extent within the storage time range of 0-30 days, the residual activity of the free lipase ROL is reduced to a larger extent, and the enzyme activity of the immobilized lipase ROL@aZIF-8 is higher than that of the free lipase ROL. Meanwhile, when the concentration of the added enzyme is 4mg/mL, the immobilized lipase shows the maximum enzyme activity of 5.69U/mg.
Based on the activity characteristics of the immobilized lipase, a potential approach is provided for immobilized lipase, and the immobilized lipase is worthy of expanding in the catalytic application and industrial biocatalysis of different MOFs materials and lipases.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) image of the MOF vector aZIF-8 and immobilized lipase ROL@aZIF-8 prepared by the invention, wherein A is MOF vector aZIF-8;B and is immobilized lipase ROL@aZIF-8;
FIG. 2 is a Transmission Electron Microscope (TEM) image of the MOF vector aZIF-8 and immobilized lipase ROL@aZIF-8 prepared by the invention, wherein A is MOF vector aZIF-8;B and is immobilized lipase ROL@aZIF-8;
FIG. 3 is a scanning transmission electron microscope-energy spectrum element surface scanning analysis chart of the MOF carrier aZIF-8 and the immobilized lipase ROL@aZIF-8, wherein A is the MOF carrier aZIF-8;B and is the immobilized lipase ROL@aZIF-8;
FIG. 4 is an X-ray diffraction (XRD) spectrum of the immobilized lipase ROL@aZIF-8, MOF vector aZIF-8 and simulated ZIF-8 prepared by the present invention;
FIG. 5 is a graph showing N2 adsorption and desorption of the immobilized lipase ROL@aZIF-8 and MOF vector aZIF-8 prepared by the present invention;
FIG. 6 is a Thermogravimetric (TGA) analysis of the immobilized lipase ROL@aZIF-8 and MOF vector aZIF-8 prepared according to the present invention;
FIG. 7 is a chart of Fourier Transform Infrared (FTIR) analysis of free lipase ROL, immobilized lipase ROL@aZIF-8 and MOF vector aZIF-8 prepared by the present invention;
FIG. 8 is a graph showing water contact angle analysis of the immobilized lipase ROL@aZIF-8 and the MOF vector aZIF-8 prepared by the method, wherein A is the MOF vector aZIF-8;B and is the immobilized lipase ROL@aZIF-8;
FIG. 9 is a graph showing the effect of temperature on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8;
FIG. 10 is a graph showing the effect of pH on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8;
FIG. 11 is a graph showing the effect of shelf life on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8;
FIG. 12 is a graph showing the effect of different amounts of added enzyme on the catalytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
According to the invention, amorphous ZIF-8 material, namely MOF carrier aZIF-8, is prepared by a certain concentration ratio of metal ions and organic ligands, rhizopus oryzae source lipase is immobilized in MOF carrier aZIF-8 by an in-situ self-encapsulation method, and immobilized lipase ROL@aZIF-8 is obtained. Under the same conditions, the immobilized lipase ROL@aZIF-8 has stronger hydrolysis capability to p-nitrobenzoate (p-NPB), and in further research, compared with the free lipase ROL, the immobilized lipase ROL@aZIF-8 has improved enzyme activity, and the immobilized lipase ROL@aZIF-8 has higher thermal stability, pH tolerance and storage time stability. Namely, when the temperature range is 30-70 degrees C, pH and the value is 7.5-10.0, the enzyme activity is higher than that of the free enzyme, the residual activity of the immobilized lipase ROL@aZIF-8 is reduced slightly within the storage time range of 0-30 days, the residual activity of the free lipase ROL is reduced relatively greatly, and the enzyme activity of the immobilized lipase ROL@aZIF-8 is higher than that of the free lipase ROL. Meanwhile, when the concentration of the added enzyme is 4mg/mL, the immobilized lipase ROL@aZIF-8 shows the maximum enzyme activity of 5.69U/mg. The result shows that the immobilized lipase prepared by the in-situ self-encapsulation method based on aZIF-8 carrier can effectively improve the enzyme activity and stability of ROL.
The invention is described in further detail below with reference to the attached drawing figures:
1. Immobilization of Lipase
1ML of 20mmol/L zinc acetate solution was added dropwise to 1mL of 33.8 mmol/L2-methylimidazole, followed by 80. Mu.L of 5mg/mL ROL solution, and reacted at 300r/min for 30min. After the reaction, the precipitate was washed 3 times with deionized water, collected, freeze-dried (-80 ℃ for 12 h), and stored at 4 ℃ to obtain immobilized lipase ROL@aZIF-8.
Meanwhile, aZIF-8 prepared without adding enzyme under the same condition is used as a blank control, and the specific method comprises the following steps:
1mL of 20mmol/L zinc acetate solution was added dropwise to 1mL of 33.8 mmol/L2-methylimidazole and reacted at 300r/min for 30min. After the reaction was completed, the precipitate was washed 3 times with deionized water, collected, freeze-dried (-80 ℃ for 12 hours), and stored at 4 ℃ to obtain MOF support aZIF-8, the TEM image of which is shown in FIG. 2A.
The immobilized lipase ROL@ZIF-8 prepared by not changing the concentration ratio of 2-methylimidazole to zinc acetate is used as a control, and the specific method comprises the following steps:
1mL of 20mmol/L zinc acetate solution was added dropwise to 1mL of 80 mmol/L2-methylimidazole, followed by 80. Mu.L of 5mg/mL ROL solution, and reacted at 300r/min for 30min. After the reaction, the precipitate was washed 3 times with deionized water, collected, freeze-dried (-80 ℃ for 12 h), and stored at 4 ℃ to obtain immobilized lipase ROL@ZIF-8, the TEM image of which is shown in FIG. 2B.
Immobilized lipase ROL@aZIF-8 (1:3) prepared by different concentration ratios of 2-methylimidazole to zinc acetate is used as a control, and the specific method comprises the following steps:
1mL of 20mmol/L zinc acetate solution was added dropwise to 1mL of 60 mmol/L2-methylimidazole, followed by 80. Mu.L of 5mg/mL ROL solution, and reacted at 300r/min for 30min. After the reaction, the precipitate was washed 3 times with deionized water, collected, freeze-dried (-80 ℃ for 12 h), and stored at 4 ℃ to obtain immobilized lipase ROL@aZIF-8 (1:3).
2. Determination of enzyme Activity of immobilized Lipase ROL@aZIF-8, ROL@ZIF-8 and ROL@aZIF-8 (1:3)
The immobilized lipases ROL@aZIF-8, ROL@ZIF-8 and ROL@aZIF-8 (1:3) prepared were weighed, 1mL of 2mg/mL of p-NPB solution in isopropanol and 1mL of 0.01mol/L (pH 7.5) phosphate buffer (phosphate buffer saline, PBS) were added, homogenized at 13000r/min for 30s, and reacted at 200r/min for 5min. After the reaction was completed, 1mL of a stop solution (NaOH 40g, EDTA 93.5g in 1L of deionized water) was added, and the supernatant was collected by centrifugation at 4℃and absorbance at 405nm was measured. One unit of enzyme activity is defined as the amount of enzyme in U/mg required to release 1. Mu. Mol of p-nitrophenol (p-nitrophenol, p-NP) per minute of immobilized enzyme.
The following data in Table 1 demonstrate that the enzyme activity of ROL@aZIF-8 is greatly improved compared to ROL@ZIF-8 and ROL@aZIF-8 (1:3).
TABLE 1 comparison of enzyme Activity of ROL@aZIF-8 and ROL@ZIF-8
3. Characterization of immobilized Lipase ROL@aZIF-8 and its vector
The surface morphology and size of the immobilized lipase ROL@aZIF-8 and MOF support aZIF-8 were characterized using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The MOF carrier aZIF-8 and the immobilized lipase ROL@aZIF-8 prepared by the MOF carrier all show an irregular and disordered structure (shown as A, B in figure 1) and have a structure with a diameter of about 200-300 nm.
Scanning transmission electron microscopy-energy spectrum element surface scanning analysis (figure 3) proves that Zn and N elements are distributed in the MOF carrier aZIF-8 material, wherein yellow in figure 3 shows the distribution of Zn elements, red shows the distribution of N elements, and the scale is 250nm. As shown in Table 2 below, the immobilized lipase ROL@aZIF-8 was found to have N/Zn of 1.53 and 1.94, which demonstrated successful immobilization of ROL in aZIF-8, i.e., an average of one zinc ion coordinated with 1.5 nitrogen atoms in the amorphous aZIF-8 material, by elemental analysis and detection data of inductively coupled plasma emission spectrometry (ICP-OES).
TABLE 2 percent of elements aZIF-8 and ROL@aZIF-8
| Material | Weight (mg) | N(wt%) | Zn | N/Zn |
| ROL@aZIF-8 | 2.3440 | 16.33 | 39.06 | 1.94 |
| aZIF-8 | 2.2110 | 11.98 | 36.46 | 1.53 |
Referring to fig. 4, xrd analysis showed that the synthesized aZIF-8 nanoparticles exhibited strong peaks at 2θ=11.05 °, 12.14 °, 17.25 ° and 18.43 °, with significant differences from the simulated ZIF-8 diffraction peak distribution law, indicating significant changes in the crystal structure of the mof support aZIF-8 compared to ZIF-8, demonstrating the amorphous structure. Meanwhile, the synthesized immobilized lipase ROL@aZIF-8 nanocomposite map is consistent with the synthesized aZIF-8 map, which shows that the influence of ROL encapsulation on aZIF-8 material is negligible. Referring to FIG. 5,N 2 adsorption resolution curves, the synthesized ZIF-8 nanomaterial exhibits a typical type I adsorption isotherm with high levels of N 2 adsorption, indicating the microporous nature of the crystals. The presence of mesopores in aZIF-8 and ROL@aZIF-8 was confirmed by the data of Table 3 below.
TABLE 3 BET surface area and pore size of immobilized lipases ROL@aZIF-8 and aZIF-8
| Material | BET surface area (m 2g-1) | Pore volume (cm 3g-1) |
| ROL@aZIF-8 | 26.261 | 0.2177 |
| aZIF-8 | 83.897 | 0.189 |
It can be seen that the collapse of micropores toward mesopores results in a decrease in specific surface area and Kong Rongxia decrease, thereby decreasing nitrogen adsorption capacity. Meanwhile, referring to fig. 6, it is shown by thermogravimetric analysis that a curve from room temperature to 800 ℃ is recorded at a heating rate of 10 ℃ min -1 in air. The free lipase ROL curve shows two weightlessness at 30-150 ℃ and 250-500 ℃, corresponding to structural water removal, decomposition and calcination of the enzyme. aZIF-8 decomposition temperatures, starting around 350 ℃, show lower thermal stability. Whereas the second stage of immobilized lipase ROL@aZIF-8 starts from 250℃and ends up at around 350℃and a weight loss of about 5% by weight occurs in the second stage due to the decomposition of protein molecules. And the weight loss around 410 ℃ is small.
Furthermore, FT-IR analysis showed that both aZIF-8 and ROL@aZIF-8 exhibited specific absorption peaks at 692cm -1、758cm-1、953cm-1 and 1308cm -1. The strip at 1350-1500cm -1 corresponds to the stretching of the entire loop. The band at 900-1350cm -1 is due to the in-plane bending of the ring. The band below 800cm -1 is designated as out-of-plane bending. The peak at 421cm -1 is a typical Zn-N stretch pattern, illustrating the coordination of zinc ions to nitrogen atoms (FIG. 7). The immobilized lipase ROL@aZIF-8 showed specific absorption peaks of amide bonds of proteins at 1600-1700cm -1, and no corresponding band could be observed in the IR spectrum of MOF vector aZIF-8. Indicating that ROL has been successfully immobilized on the carrier (FIG. 7). From the water contact angle plot, it can be seen that the hydrophobicity of amorphous aZIF-8 decreases, and that the hydrophobicity of the ROL-immobilized aZIF-8 structure all appears to decrease (FIG. 8).
4. Influence of temperature on the enzyme Activity of immobilized Lipase ROL@aZIF-8
The influence of temperature on the hydrolytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8 is studied, and the temperature range is 30-70 ℃. The results show (FIG. 9) that the hydrolytic activity of the free lipase ROL and the immobilized lipase ROL@aZIF-8 increases with increasing temperature at 30-50 ℃. Subsequently, when the temperature was further increased from 50℃to 70℃the hydrolytic activity of the free lipase ROL and the immobilized lipase ROL@aZIF-8 was decreased. Thus, the optimal temperature for both the free lipase ROL and the immobilized lipase ROL@aZIF-8 was 50℃and the immobilized lipase exhibited better activity than the free lipase. And after immobilization, when the temperature is 30-70 ℃, the hydrolytic activity of the immobilized lipase ROL@aZIF-8 is still higher than that of the free lipase ROL. This may be the case for the amorphous aZIF-8 structure which has a protective effect on the lipase ROL.
5. Influence of pH value on the enzyme activity of immobilized lipase ROL@aZIF-8
The effect of pH on the hydrolytic activity of free lipase ROL and immobilized lipase ROL@aZIF-8 is shown in FIG. 10. The optimal pH values of the free lipase ROL and the immobilized lipase ROL@aZIF-8 are 9.0. Above or below the optimal pH, the hydrolytic activity of the free lipase ROL and the immobilized lipase ROL@aZIF-8 decreases. The free lipase ROL and the immobilized lipase ROL@aZIF-8 have higher hydrolytic activity under alkaline conditions. When the pH value is 7.5-10.0, the hydrolytic activity of the immobilized lipase ROL@aZIF-8 is higher than that of the free lipase. These results indicate that the enzyme stability is improved under pH changes due to the creation of a microenvironment that protects the enzyme molecule due to the formation of multipoint linkages (hydrophobic interactions, van der waals interactions and hydrogen bonds) between aZIF-8 and the ROL enzyme molecule.
6. Influence of storage time on the enzyme Activity of immobilized Lipase ROL@aZIF-8
One of the most important reasons for producing immobilized lipases is their storage time stability in industrial applications, as well as achieving high efficiency and reduced costs of industrial products. The stability of immobilized lipase was determined by examining the activity decay of free lipase ROL at 4℃in phosphate buffer with pH of 7.5 at 0.01mol/L for 25 days (FIG. 11). The results show that the residual activity of the immobilized lipase ROL@aZIF-8 is reduced to 1.49U/mg, the residual activity of the free lipase ROL is reduced to a relatively large extent, the enzyme activity of the ROL@aZIF-8 is higher than that of the free lipase ROL, and the storage stability is improved probably because aZIF-8 is wrapped around enzyme molecules to play a role in protecting against proteolysis and polymerization or weakening the twist degree of an enzyme active site by a chemical environment (buffer solution), so that the activity reduction of the lipase is slowed down, and the result emphasizes that the lipase-embedded MOF composite material has higher chemical and structural stability.
7. Influence of different enzyme addition amounts on the enzyme activity of immobilized lipase ROL@aZIF-8
The hydrolysis activity of the immobilized lipase ROL@aZIF-8 prepared under the condition of different enzyme addition concentrations is studied. As the enzyme concentration increased from 3mg/mL to 4mg/mL, the immobilized lipase ROL@aZIF-8 gradually increased in the ability to catalyze the hydrolysis of the substrate (FIG. 12). The enhancement of the activity of the immobilized lipase ROL@aZIF-8 is probably due to the fact that the enzymes encapsulated in the preparation process are increased, and the substrate is in more contact with enzyme molecules, so that the catalytic efficiency is improved. As the enzyme concentration increased from 4mg/mL to 7mg/mL, the immobilized lipase ROL@aZIF-8 gradually decreased in the ability to catalyze the hydrolysis of the substrate. The weakening of the activity of the immobilized lipase ROL@aZIF-8 is probably due to the fact that enzyme molecules are possibly aggregated along with the increase of concentration in the preparation process, and the immobilized lipase is less encapsulated by a carrier, so that the immobilization rate of the enzyme in the immobilized lipase is reduced, and the catalytic efficiency is reduced. The results of these studies confirm that the enzyme addition has an important regulatory effect on the catalytic activity of the immobilized lipase ROL@aZIF-8.
The results of the invention are combined to show that an amorphous ZIF-8 structure is prepared by a certain concentration ratio of metal ions and organic ligands, and the immobilized lipase ROL@aZIF-8 is prepared by immobilizing the lipase ROL in the MOF carrier aZIF-8 by adopting an in-situ self-encapsulation method. The results show that compared with free lipase ROL, the enzyme activity of the immobilized lipase ROL@aZIF-8 is improved, and the immobilized lipase ROL@aZIF-8 has higher heat stability, pH tolerance and storage time stability. Meanwhile, when the concentration of the added enzyme is 4mg/mL, the immobilized lipase ROL@aZIF-8 shows the maximum enzyme activity of 5.69U/mg. The scheme of the invention is helpful to provide a potential approach for immobilizing lipase, and deserves expansion in the catalytic application and industrial biocatalysis of different MOFs and lipases.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (6)
1. The controllable preparation method of the immobilized rhizopus oryzae lipase is characterized by comprising the steps of adding a zinc acetate solution into a 2-methylimidazole solution, adding a solution containing rhizopus oryzae lipase, stirring and reacting for 20-40 min, washing, collecting precipitate, and performing vacuum freeze-drying treatment to obtain the immobilized rhizopus oryzae lipase, namely immobilized lipase ROL@aZIF-8;
The concentration of the zinc acetate solution is 20 mmol/L, the concentration of the 2-methylimidazole solution is 33.8 mmol/L, the concentration of the solution containing rhizopus oryzae lipase is 5mg/mL, and the volume ratio of the zinc acetate solution, the 2-methylimidazole solution and the solution containing rhizopus oryzae lipase is 1:1:0.08;
the collected precipitate was lyophilized at-80 ℃ in vacuo to dryness 12 h.
2. The controllable preparation method of the immobilized rhizopus oryzae lipase according to claim 1, wherein the stirring reaction is performed at a rotation speed of 300-500 r/min.
3. The controllable preparation method of the immobilized rhizopus oryzae lipase according to claim 1, wherein the washing is carried out 3-5 times by adopting deionized water.
4. The immobilized rhizopus oryzae lipase prepared by the controllable preparation method of any one of claims 1-3, wherein the maximum enzyme activity reaches 5.69U/mg when the concentration of the added enzyme is 4 mg/mL.
5. The immobilized rhizopus oryzae lipase according to claim 4, wherein the immobilized rhizopus oryzae lipase has an enzyme activity higher than that of the free enzyme at a temperature ranging from 30 ° to 70 ° C, pH to 7.5.0.
6. The use of an immobilized rhizopus oryzae lipase according to any one of claims 4-5 in the preparation of an industrial biocatalyst.
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