CN107699558A - Immobilised enzymes, its preparation method and its application in atrazine-contaminated soil is repaired - Google Patents
Immobilised enzymes, its preparation method and its application in atrazine-contaminated soil is repaired Download PDFInfo
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- CN107699558A CN107699558A CN201711180826.0A CN201711180826A CN107699558A CN 107699558 A CN107699558 A CN 107699558A CN 201711180826 A CN201711180826 A CN 201711180826A CN 107699558 A CN107699558 A CN 107699558A
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- Prior art keywords
- immobilised enzymes
- preparation
- atrazine
- enzyme liquid
- chitosan
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 136
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 136
- 239000002689 soil Substances 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 58
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 39
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 36
- 229920001661 Chitosan Polymers 0.000 claims abstract description 23
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 13
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 12
- 239000000661 sodium alginate Substances 0.000 claims abstract description 12
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 7
- MXWJVTOOROXGIU-UHFFFAOYSA-N atrazine Chemical compound CCNC1=NC(Cl)=NC(NC(C)C)=N1 MXWJVTOOROXGIU-UHFFFAOYSA-N 0.000 claims description 49
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 9
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- 239000011573 trace mineral Substances 0.000 claims description 9
- 235000013619 trace mineral Nutrition 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- 229910004835 Na2B4O7 Inorganic materials 0.000 claims description 5
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 claims description 5
- 239000005457 ice water Substances 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- 239000011686 zinc sulphate Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 150000008052 alkyl sulfonates Chemical class 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- IGLKELDWPZFFKF-UHFFFAOYSA-N OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P Chemical compound OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P IGLKELDWPZFFKF-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 9
- 244000005700 microbiome Species 0.000 abstract description 8
- 239000008187 granular material Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 27
- 238000006731 degradation reaction Methods 0.000 description 22
- 230000015556 catabolic process Effects 0.000 description 21
- 230000000694 effects Effects 0.000 description 19
- 238000012545 processing Methods 0.000 description 15
- 230000001954 sterilising effect Effects 0.000 description 11
- 239000002609 medium Substances 0.000 description 9
- 230000008901 benefit Effects 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 102000038379 digestive enzymes Human genes 0.000 description 6
- 108091007734 digestive enzymes Proteins 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 244000223760 Cinnamomum zeylanicum Species 0.000 description 5
- 235000017803 cinnamon Nutrition 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N CHCl3 Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 241000186063 Arthrobacter Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- -1 alkyl sulphur Chemical compound 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002170 ethers Chemical class 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011806 microball Substances 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 235000010086 Setaria viridis var. viridis Nutrition 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008953 bacterial degradation Effects 0.000 description 1
- 239000011805 ball Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 231100000507 endocrine disrupting Toxicity 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 244000230342 green foxtail Species 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000001089 mineralizing effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- FKHIFSZMMVMEQY-UHFFFAOYSA-N talc Chemical compound [Mg+2].[O-][Si]([O-])=O FKHIFSZMMVMEQY-UHFFFAOYSA-N 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- 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
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
Abstract
The invention provides a kind of immobilised enzymes, its preparation method and its application in atrazine-contaminated soil is repaired, belong to microorganism technique for fixing field.The preparation method of the immobilised enzymes comprises the following steps：The strains of HB 5 are crushed and are centrifugally separating to obtain enzyme liquid；Enzyme liquid is added in the aqueous acetic acid of chitosan and stirred, be then added dropwise in sodium hydroxide solution and stir, filter acquisition microballoon；Instilled after microballoon and sodium alginate soln are mixed in calcium chloride solution.The preparation method technique of immobilised enzymes provided by the invention is simple and convenient to operate, and can be prepared that granule size is uniform, the constitutionally stable double-deck immobilization ball for being adsorbed with enzyme.Application present invention also offers the immobilised enzymes being prepared using the above method and its in atrazine-contaminated soil is repaired.
Description
Technical field
The present invention relates to microorganism technique for fixing field, in particular to a kind of immobilised enzymes, its preparation method and its
Application in atrazine-contaminated soil is repaired.
Background technology
Atrazine (Atrazine) also known as Atrazine, the entitled chloro- 4- ethylamines -6- isopropylamines -1,3 of 2- of chemistry, 5-
S-triazine, it is that selective before seedling, after seedling closing herbicide are inhaled in one kind, its be suitable to corn, sorghum, tea place, orchard and
Annual gramineous weed is prevented and kill off in forest land etc. and broad leaved weed is handled.But with the extensive use of atrazine, it result in
Serious problem of environmental pollution, it can influence eubolism mechanism and form in organism as endocrine disruption inhibitor
Development, causes the generation of breast cancer and oophoroma, in order to solve atrazine pollution problem, it is necessary to the soil using atrazine
Handled.
Most promising with bioremediation technology in current existing atrazine processing method, it is due to high-efficiency environment friendly
The advantages of as research focus, research shows there is higher value using bacterial degradation atrazine, and microorganism can utilize
Atrazine, so that atrazine molecule takes off alkyl, de- N- alkyl and the cracking of three azo-cycles, discharges titanium dioxide as carbon source and nitrogen source
Carbon and thorough mineralizing and degrading, because the process of microbial degradation atrazine is driven by enzyme, in order to improve the effect of atrazine degraded
Fruit, it is necessary to enzyme is fixed on carrier to produce long-acting degradation effect, therefore, it is necessary to one kind can long-time stable exist with drop
Solve the immobilised enzymes product of atrazine pollutant in soil.
The content of the invention
The invention provides a kind of preparation method of immobilised enzymes, it has the advantages of technique is simple and convenient to operate.
Present invention also offers a kind of immobilised enzymes, and it has the advantages of epigranular, Stability Analysis of Structures, and can be in soil
In slowly degraded to expose the atrazine digestive enzyme of internal load.
Present invention also offers application of the above-mentioned immobilised enzymes in atrazine-contaminated soil is repaired.
What the present invention was realized in：
A kind of preparation method of immobilised enzymes, comprises the following steps：
HB-5 strains are crushed and are centrifugally separating to obtain enzyme liquid；
Enzyme liquid is added in the aqueous acetic acid of chitosan and stirred, be then added dropwise in sodium hydroxide solution and stir
Mix, filter acquisition microballoon；
Instilled after microballoon and sodium alginate soln are mixed in calcium chloride solution.
In preferred embodiments of the present invention, above-mentioned HB-5 strains are existed by 2%~4% inoculum concentration using fluid nutrient medium
More than 48h is cultivated at 28~35 DEG C, then carries out broken centrifugation operation, fluid nutrient medium includes 3~5g/L of sucrose, yeast
Medicinal extract 1~3g/L, K2HPO41.6~2.5g/L, KH2PO40.4~0.6g/L, MgSO4·7H2O0.2~0.5g/L,
NaCl0.1~0.4g/L, 2~4mL/L of trace element solution, 100~300mg/L of atrazine.
In preferred embodiments of the present invention, above-mentioned trace element solution includes EDTA2~2.5g/L, FeSO4·7H2O1
~2g/L, ZnSO4·7H2O3~5g/L, MnSO4·H2O1~2g/L, CuSO4·5H2O0.4~0.8g/L, Na2B4O7·
10H2O0.2~0.5g/L, Na2MoO4·2H2O0.25~0.5g/L.
In preferred embodiments of the present invention, above-mentioned broken centrifugation be by HB-5 strains at 4 DEG C in 8000~
9000rpm collects thalline after centrifuging 8~12min, and with 0.05~0.1mol/L phosphate buffer wash 1~3 time after
8000~9000rpm secondary centrifugings, 8~12min collects thalline, and the thalline of collection and phosphate buffer then are pressed into 1g：2~
15 are centrifuged in 9000~10000rpm at 4 DEG C after 10~20min of ice-water bath ultrasonic disruption after 3mL ratio mixing~
20min collects supernatant, and ultrasonic power is 280~350W.
In preferred embodiments of the present invention, during above-mentioned ultrasonic disruption, 5~20s is often crushed, is spaced 5~20s.
In preferred embodiments of the present invention, the mass fraction of chitosan is 2% in the aqueous acetic acid of above-mentioned chitosan
~5%, the mass ratio of enzyme liquid and chitosan is 1：30~40, the mass fraction of sodium hydroxide solution is 5%~10%.
In preferred embodiments of the present invention, the alkyl sulphur containing mass fraction 1%~2% in above-mentioned sodium hydroxide solution
Hydrochlorate.
In preferred embodiments of the present invention, the sodium alginate soln of above-mentioned microballoon and mass concentration 3%~5% presses 1：25
Instilled after~40 mass ratio mixing in the calcium chloride solution of mass fraction 4%~8%.
Present invention also offers a kind of immobilised enzymes, and it is prepared using the preparation method of above-mentioned immobilised enzymes.
Present invention also offers application of the above-mentioned immobilised enzymes in atrazine-contaminated soil is repaired.
The beneficial effects of the invention are as follows：The preparation method of immobilised enzymes provided by the invention comprises the following steps：By HB-5
Strain is broken to be centrifugally separating to obtain enzyme liquid；Enzyme liquid is added in the aqueous acetic acid of chitosan and stirred, is then added dropwise to
Stirred in sodium hydroxide solution, filter acquisition microballoon；Instilled after microballoon and sodium alginate soln are mixed in calcium chloride solution.This
Invent the preparation method technique of immobilised enzymes provided to be simple and convenient to operate, can be prepared that granule size is uniform, structure is steady
The fixed double-deck immobilization ball for being adsorbed with enzyme.Present invention also offers the immobilised enzymes being prepared using the above method, and it has
There is the advantages of epigranular, structural stability is strong, the immobilised enzymes can slowly degrade to expose internal load in soil
Atrazine digestive enzyme carries out purification operation.Present invention also offers above-mentioned immobilised enzymes answering in atrazine-contaminated soil is repaired
With.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below by embodiment it is required use it is attached
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore be not construed as pair
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is that the enzyme liquid that is prepared using embodiment 1 and immobilised enzymes are degraded the effect contrast figure of atrazine in brunisolic soil；
Fig. 2 is that the enzyme liquid that is prepared using embodiment 2 and immobilised enzymes are degraded the effect contrast figure of atrazine in brunisolic soil.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase
Product.
The embodiment of the present invention is specifically described below.
A kind of preparation method of immobilised enzymes is present embodiments provided, is comprised the following steps：
S1, it is centrifugally separating to obtain enzyme liquid by HB-5 strains are broken；Preferably, HB-5 strains are made by 2%~4% inoculum concentration
More than 48h is cultivated at 28~35 DEG C with fluid nutrient medium, then carries out broken centrifugation operation, fluid nutrient medium includes sugarcane
Sugar 3~5g/L, yeast extract 1~3g/L, K2HPO41.6~2.5g/L, KH2PO40.4~0.6g/L, MgSO4·7H2O0.2~
0.5g/L, NaCl0.1~0.4g/L, 2~4mL/L of trace element solution, 100~300mg/L of atrazine.Wherein, it is micro-
Solution preferably comprises EDTA2~2.5g/L, FeSO4·7H2O1~2g/L, ZnSO4·7H2O3~5g/L, MnSO4·H2O1~
2g/L, CuSO4·5H2O0.4~0.8g/L, Na2B4O7·10H2O0.2~0.5g/L, Na2MoO4·2H2O0.25~0.5g/
L.It is further preferred that broken centrifugation be by HB-5 strains at 4 DEG C after 8000~9000rpm centrifuges 8~12min
Thalline is collected, and 1~3 time is washed after 8000~9000rpm secondary centrifugings with 0.05~0.1mol/L phosphate buffer
8~12min collects thalline, and the thalline of collection and phosphate buffer then are pressed into 1g：Ice-water bath after 2~3mL ratio mixing
15~20min is centrifuged at 4 DEG C in 9000~10000rpm collect supernatant, ultrasonic power after 10~20min of ultrasonic disruption
For 280~350W；During ultrasonic disruption, 5~20s is preferably often crushed, is spaced 5~20s.
S2, enzyme liquid be added in the aqueous acetic acid of chitosan stirred, be added dropwise to stirring in sodium hydroxide solution,
Filtering obtains microballoon；The mass fraction of chitosan is 2%~5% in the aqueous acetic acid of preferable chitosan, and enzyme liquid and shell gather
The mass ratio of sugar is 1：30~40, the mass fraction of sodium hydroxide solution is 5%~10%；Preferably comprised in sodium hydroxide solution
The alkylsulfonate of mass fraction 1%~2%；Dry and dried at preferably 30~40 DEG C.
S3, instilled after mixing microballoon and sodium alginate soln in calcium chloride solution.Preferably, microballoon and mass concentration
3%~5% sodium alginate soln presses 1：The calcium chloride that mass fraction 4%~8% is instilled after 25~40 mass ratio mixing is molten
In liquid.
The strain that the preparation method of immobilised enzymes of the present invention chooses Arthrobacter (Arthrobacter) degradation bacteria HB-5 enters
Crushed after row culture, centrifugation extraction obtains degrading the enzyme of atrazine, then successively using chitosan and sodium alginate to enzyme
Secondary embedding balling-up is carried out, so as to obtain Stability Analysis of Structures and to temperature, humidity and the adaptable immobilised enzymes of acid-base property, to prolong
In the useful effect cycle of long digestive enzyme, ensure the repairing effect of long-time stable；Wherein, HB-5 strains are used and contains micro member
Enzyme extraction operation is carried out after the fluid nutrient medium culture of plain solution, microorganism can be promoted quickly to breed, improve the quantity of viable bacteria
And activity, so as to lift the enzyme amount and activity that extraction obtains, frozen water after then mixing the strain of culture and phosphate buffer
Ultrasonic disruption is bathed, and 5~20s is spaced after often crushing 5~20s in ultrasonic disruption, so that strain fully crushes guarantee
Activity is kept when enzyme separates out, avoids the activity of temperature rise destructive enzyme；The enzyme liquid being collected by centrifugation is added to the acetic acid of chitosan
Stirred in the aqueous solution, be added dropwise in the sodium hydroxide solution containing alkylsulfonate and stir, filter acquisition microballoon, can obtain
Obtain epigranular and embed good microballoon, the addition of alkylsulfonate can make chitosan microball fast by adjusting surface tension
Rapid-result shape, ensure that the granularity of chitosan microball is balanced, Stability Analysis of Structures；Instilled after the microballoon of preparation and sodium alginate soln are mixed
Double-deck bead is made in secondary embedding treatment in calcium chloride solution, can be further ensured that the structural stability of immobilised enzymes, extends
Its useful effect is failed.
Present invention also offers a kind of immobilised enzymes, and it is prepared using the preparation method of above-mentioned immobilised enzymes,
The immobilised enzymes has the advantages of epigranular, Stability Analysis of Structures, and the green bristlegrass that can be slowly degraded in soil and expose internal load is gone
Tianjin digestive enzyme is in order to long-acting degraded atrazine.
Present invention also offers application of the above-mentioned immobilised enzymes in atrazine-contaminated soil is repaired, the immobilised enzymes is applied
For the atrazine in effective degraded soil can be stablized in soil for a long time.
The feature and performance of the inventive method are described in further detail with reference to embodiments.
Embodiment 1
Embodiment 1 provides a kind of preparation method of immobilised enzymes, and its specific preparation process is as follows：
S101, it is centrifugally separating to obtain enzyme liquid by HB-5 strains are broken；HB-5 strains are inoculated in liquid by 2% inoculum concentration
In culture medium, thalline is collected after 8000rpm centrifuges 8min at 4 DEG C after more than 48h is cultivated at 30 DEG C, is used in combination
0.05mol/L phosphate buffer washs 3 times and collects thalline after 8000rpm secondary centrifugings 12min, then by the bacterium of collection
Body and phosphate buffer press 1g：After 3mL ratio mixing after ice-water bath ultrasonic disruption 10min at 4 DEG C in 10000rpm
Centrifugation 15min collects supernatant and obtains enzyme liquid；Ultrasonic power is 280W；During ultrasonic disruption, 5s is often crushed, is spaced 5s, wherein,
Fluid nutrient medium includes sucrose 3g/L, yeast extract 1g/L, K2HPO41.6g/L, KH2PO40.4g/L, MgSO4·7H2O0.2g/
L, NaCl0.1g/L, trace element solution 2mL/L, atrazine 100mg/L；Trace element solution preferably comprises EDTA2.5g/L,
FeSO4·7H2O1g/L, ZnSO4·7H2O5g/L, MnSO4·H2O1g/L, CuSO4·5H2O0.4g/L, Na2B4O7·
10H2O0.2g/L, Na2MoO4·2H2O0.25g/L。
S102, enzyme liquid is added in the aqueous acetic acid of chitosan stirred, shell in the aqueous acetic acid of chitosan
The mass fraction of glycan is 3%, and the mass ratio of enzyme liquid and chitosan is 1：30, then it is added dropwise to the hydrogen-oxygen that mass fraction is 5%
Change stirring in sodium solution, filtering obtains microballoon, the sodium alkyl sulfonate containing mass fraction 1.5% in sodium hydroxide solution.
S103, the calcium chloride for instilling mass fraction 4% after mixing microballoon and the sodium alginate soln of mass concentration 3% are molten
The immobilized spherule that particle diameter is 2.5mm is formed in liquid, immobilized spherule is preserved into 6h in 4 DEG C of calcium chloride solution, and store
It is standby at 4 DEG C.
Embodiment 2
Embodiment 2 provides a kind of preparation method of immobilised enzymes, and its specific preparation process is as follows：
S201, it is centrifugally separating to obtain enzyme liquid by HB-5 strains are broken；Preferably, HB-5 strains are used by 3% inoculum concentration
Thalline is collected after 9000rpm centrifuges 10min at 4 DEG C after more than 48h is cultivated at 35 DEG C of fluid nutrient medium, and uses 0.1mol/
L phosphate buffer washs 2 times and collects thalline after 9000rpm secondary centrifugings 12min, then by the thalline and phosphoric acid of collection
Salt buffer presses 1g：After 2mL ratio mixing 20min is centrifuged after ice-water bath ultrasonic disruption 15min in 9500rpm at 4 DEG C
Collect supernatant and obtain enzyme liquid；Ultrasonic power is 350W；During ultrasonic disruption, 12s is often crushed, is spaced 12s.Wherein, liquid is trained
Support base and include sucrose 5g/L, yeast extract 3g/L, K2HPO42g/L, KH2PO40.5g/L, MgSO4·7H2O0.4g/L,
NaCl0.3g/L, trace element solution 3mL/L, atrazine 250mg/L；Trace element solution includes EDTA2.5g/L,
FeSO4·7H2O2g/L, ZnSO4·7H2O4g/L, MnSO4·H2O1.5g/L, CuSO4·5H2O0.5g/L, Na2B4O7·
10H2O0.3g/L, Na2MoO4·2H2O0.4g/L。
S202, enzyme liquid is added in the aqueous acetic acid of chitosan stirred, shell in the aqueous acetic acid of chitosan
The mass fraction of glycan is 4%, and the mass ratio of enzyme liquid and chitosan is 1：35, then it is added dropwise to the hydrogen-oxygen that mass fraction is 8%
Change stirring in sodium solution, filtering obtains microballoon.
S203, the calcium chloride for instilling mass fraction 6% after mixing microballoon and the sodium alginate soln of mass concentration 4% are molten
The immobilized spherule that particle diameter is 2.5mm is formed in liquid, immobilized spherule is preserved into 5h in 4 DEG C of calcium chloride solution, and store
It is standby at 4 DEG C.
Atrazine in soil is dropped below by way of using the enzyme liquid and immobilised enzymes that are prepared in embodiment 1, embodiment 2
The effect of solution is contrasted.
The brown earth top layer plantation soil in Tai'an is chosen, removes weeds, dry branches and fallen leaves, crosses 40 mesh sieves, adjustment water content is maximum
The 60% of water-holding capacity, soil is divided into sterilization treatment group and unsterilised two groups of processing, wherein, sterilization treatment group includes control group
(BAC groups) does not add processing, addition enzyme liquid group (BAE groups) and the addition immobilised enzymes group that enzyme liquid does not add immobilised enzymes yet
(BAI groups), sterilizing group continuous sterilization 2h at 121 DEG C, unsterilised treatment group do not add enzyme liquid including control group (BNC groups)
Processing, addition enzyme liquid group (BNE groups) and the addition immobilised enzymes group (BNI groups) of immobilised enzymes are not added, and each processing sets 3 weights
It is multiple.Atrazine concentration in soil sample is adjusted to after 10mg/kg in the brown bottle loaded on 125mL respectively, per it is bottled enter soil
50g.The enzyme liquid that enzyme liquid group is prepared according to 20mL/kg amount addition embodiment 1 is added, i.e., adds enzyme in each brown bottle
Liquid 1mL；The immobilised enzymes that addition immobilised enzymes group is prepared according to 200g/kg amount addition embodiment 1, i.e., each brown bottle
Middle addition immobilised enzymes 10g；Subsequent lucifuge culture at room temperature, and 0h, 24h after culture starts, 48h, 72h, 96h,
120h and 144h is sampled from soil and is analyzed the residual quantity of atrazine, calculates degradation rate, and being fixed enzyme pollutes to two kinds
The influence of atrazine degradation rule in soil, as a result as shown in figure 1, wherein " * " represent treatment group and sterilizing control group soil sample it
Between there is significant difference, letter is different to be represented between each two treatment groups that there is significant difference, "+" to represent two kinds of soil
There is significant difference, wherein p between the respective handling group of earth<Numerical value in 0.05, Fig. 1 is parallel three times be averaged
Value, error line represent standard error.
The extraction of atrazine and analysis method are as follows in soil：25g soil samples are weighed in iodine flask with 1 percent balances
In, 70mL acetone mechanical shaking extraction, decompression suction filtration are added, iodine flask and filter residue are rinsed in three times with 30mL acetone, after filtrate merges,
It is transferred in 500mL separatory funnels, uses 50mLCHCl3Bottle,suction is washed in three times, and cleaning solution is merged into separatory funnel, is added
The sodium chloride solution of 50mL mass concentration 3%, fully vibration mix, and stand 30min, lower floor CHCl3The anhydrous sulphur of drying
Sour sodium filters the flat balloon flask into 250mL after drying.30mLCHCl is used again3Extraction is once, same to collect lower floor CHCl3, it is and upper
Secondary CHCl3Merge.10mL petroleum ethers are added into flat balloon flask, by whole extract rotary evaporators at 50 DEG C
1-2mL is concentrated into, treats column chromatography purification.The upper/lower terminal for the chromatographic column that specification is 1.5cm × 25cm is respectively added what 5g was dried
Anhydrous sodium sulfate, centre plus the 3g florisil silicas with the distilled water inactivation processing that mass fraction is 6%, chromatographic column is compacted,
Chromatographic column is eluted in advance with 10mL petroleum ethers, and then above-mentioned concentrate solution is fully transferred in chromatographic column, then is mixed with 100mL
Leacheate (petroleum ether：Ethyl acetate V：V=95：5) elute in three times, collect whole leacheates, with 50 DEG C of rotary evaporator
1-2mL is concentrated into, n-hexane constant volume to 25mL, analyzes and determines in GC-ECD.Condition determination：Chromatographic column is OV-1701 capillaries
Post (30m × 0.53mmi.d.)；Temperature：260 DEG C of injection port, 260 DEG C of detector, 230 DEG C of column temperature；Gas：Carrier gas 60kPa, tail
Blow 70kPa；Splitless injecting samples, the μ L of sample size 1, according to sample constant volume and extension rate and sample chromatogram peak and standard specimen color
The peak height of spectral peak, the concentration of atrazine in nutrient solution is calculated using external standard method.
As seen from Figure 1, with the extension of time, addition enzyme liquid, the treatment group of immobilised enzymes, atrazine are shown
Good degradation effect, during 144h, atrazine degradation rate reaches 90% or so.The treatment group and sterilization treatment of all addition enzymes
Control group compared to having significant difference, the effect that indigenous microorganism plays in atrazine degradation process, relative to enzyme liquid
It is much smaller with immobilised enzymes, and with the extension of time, immobilised enzymes finally as degradation effect of the enzyme liquid to atrazine,
This is due to that enzyme liquid and can start to be catalyzed it and hydrolyze directly with the atrazine molecule contacts in soil, therefore unsterilised
The processing degradation speed of enzyme-added liquid is most fast；And immobilised enzymes separates out from embedding medium needs the regular hour, so processing early stage
Degradation rate is less than enzyme liquid, but is remained basically stable to degradation rate both during 144h, shows that the enzyme after immobilization can more delay than enzyme liquid
Its degradation capability to atrazine of On The Drug Release, so as to applied in more long-term soil remediation.
The cinnamon soil top layer plantation soil in Shenyang is chosen, removes weeds, dry branches and fallen leaves, crosses 40 mesh sieves, adjustment water content is maximum
The 60% of water-holding capacity, soil is divided into sterilizing and unsterilised two groups of processing, wherein, sterilization treatment group includes control group (CAC groups)
Do not add processing, addition enzyme liquid group (CAE groups) and addition immobilised enzymes group (CAI groups) that enzyme liquid does not add immobilised enzymes yet,
Sterilizing group continuous sterilization 2h at 121 DEG C, unsterilised treatment group are not added enzyme liquid including control group (CNC groups) and not added yet
Processing, addition enzyme liquid group (CNE groups) and the addition immobilised enzymes group (CNI groups) of immobilised enzymes, each processing set 3 repetitions.Will
After atrazine concentration in soil sample is adjusted to 10mg/kg, it is sub-packed in 125mL brown bottle, every bottle of 50g.According to 20mL/kg's
Amount adds the enzyme liquid being prepared in embodiment 2, i.e., adds enzyme liquid 1mL in each brown bottle；Amount according to 200g/kg is added real
The immobilised enzymes being prepared in example 2 is applied, i.e., immobilised enzymes 10g is added in each brown bottle.Lucifuge culture at room temperature, and in training
The residual quantity of 0h, 24h, 48h, 72h, 96h, 120h and 144h sampling analysis atrazine after starting is supported, degradation rate is calculated, obtains
To influence of the immobilised enzymes to atrazine degradation rule in two kinds of contaminated soils of atrazine, as shown in Fig. 2 wherein " * " is represented
There is significant difference between treatment group and sterilizing control group soil sample, letter is different represent between each two treatment groups there is
Significant difference, "+" represent two kinds of soil respective handling group between there is significant difference, wherein p<In 0.05, Fig. 2
Numerical value is average value parallel three times, and error line represents standard error.
As seen from Figure 2, the principal degradation rule in cinnamon soil is similar with the degraded of brunisolic soil in Fig. 1, adds immobilization
In addition to 24h and 96h, degradation speed is most fast for two groups of processing of enzyme.In brunisolic soil, enzyme liquid is due to can directly and soil
In atrazine molecule contacts, and start to be catalyzed it and hydrolyze, therefore the processing degradation speed of unsterilised enzyme-added liquid is most fast, drop
Solution rate is maximum；But cinnamon soil, due to its own slant acidity, the optimum response pH value therefore enzyme liquid for keeping off enzyme liquid wherein can not
Play optimal degradation.And after enzyme liquid immobilization, the regular hour is needed because digestive enzyme separates out from embedding medium,
And embedded material can provide certain protective effect for enzyme molecule, therefore in each processing, at unsterilised being fixed enzyme
The degradation speed and degradation rate of reason are also higher than direct enzyme-added liquid, but when having arrived 144h, two kinds of degradeds handled to atrazine
Effect is essentially the same, and two kinds of soil compare, and almost all of processing has significant difference between different sample times
Occur, illustrate that the repairing effect of enzyme liquid and immobilised enzymes in two kinds of soil is not duplicate, show different physics and chemistry
Using same enzyme and immobilised enzymes repair the pollution of atrazine between the soil of matter, its effect is not quite similar,
And the height from block diagram is relatively upper as can be seen that the indigenous microorganism in cinnamon soil is in auxiliary enzyme liquid or immobilised enzymes degraded soil
In atrazine when, act on than obvious in brunisolic soil, because the organic matter containing high level in the cinnamon soil in Shenyang, is
The growth of indigenous microorganism therein provides preferable nutritional condition, so as to which indigenous microorganism therein can play more preferable drop
Solve the effect of atrazine.
In summary, immobilised enzymes provided in an embodiment of the present invention can effectively degrade the atrazine contained in soil,
And the structural stability of the immobilised enzymes is strong, it can slowly be degraded in soil and produce stabilization to discharge atrazine digestive enzyme
Long-acting degraded operation, will degraded compared to directly so as to adapt to different soil physical chemistry environment to carry out degradation treatment to atrazine
Enzyme, which is added in soil, has big advantage.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (10)
1. a kind of preparation method of immobilised enzymes, it is characterised in that comprise the following steps：
HB-5 strains are crushed and are centrifugally separating to obtain enzyme liquid；
The enzyme liquid is added in the aqueous acetic acid of chitosan and stirred, is then added dropwise in sodium hydroxide solution and stirs
Mix, filter acquisition microballoon；
Instilled after the microballoon and sodium alginate soln are mixed in calcium chloride solution.
2. the preparation method of immobilised enzymes according to claim 1, it is characterised in that the HB-5 strains press 2%~4%
Inoculum concentration more than 48h is cultivated at 28~35 DEG C using fluid nutrient medium, then carry out the broken centrifugation operation, institute
State fluid nutrient medium and include 3~5g/L of sucrose, yeast extract 1~3g/L, K2HPO41.6~2.5g/L, KH2PO40.4~0.6g/
L, MgSO4·7H2O0.2~0.5g/L, NaCl0.1~0.4g/L, 2~4mL/L of trace element solution, atrazine 100~
300mg/L。
3. the preparation method of immobilised enzymes according to claim 2, it is characterised in that the trace element solution includes
EDTA2~2.5g/L, FeSO4·7H2O1~2g/L, ZnSO4·7H2O3~5g/L, MnSO4·H2O1~2g/L, CuSO4·
5H2O0.4~0.8g/L, Na2B4O7·10H2O0.2~0.5g/L, Na2MoO4·2H2O0.25~0.5g/L.
4. the preparation method of immobilised enzymes according to claim 1, it is characterised in that broken centrifuge is by institute
State HB-5 strains and collect thalline after 8000~9000rpm centrifuges 8~12min at 4 DEG C, and with 0.05~0.1mol/L phosphorus
Phthalate buffer washs 1~3 time and collects thalline after 8000~9000rpm secondary centrifugings, 8~12min, then by the bacterium of collection
Body and phosphate buffer press 1g：After 2~3mL ratio mixing after 10~20min of ice-water bath ultrasonic disruption at 4 DEG C in
9000~10000rpm centrifuges 15~20min and collects supernatant, and ultrasonic power is 280~350W.
5. the preparation method of immobilised enzymes according to claim 4, it is characterised in that during the ultrasonic disruption, often break
Broken 5~20s, it is spaced 5~20s.
6. the preparation method of immobilised enzymes according to claim 1, it is characterised in that the aqueous acetic acid of the chitosan
The mass fraction of middle chitosan is 2%~5%, and the mass ratio of the enzyme liquid and the chitosan is 1：30~40, the hydrogen-oxygen
The mass fraction for changing sodium solution is 5%~10%.
7. the preparation method of immobilised enzymes according to claim 6, it is characterised in that contain in the sodium hydroxide solution
The alkylsulfonate of mass fraction 1%~2%.
8. the preparation method of immobilised enzymes according to claim 1, it is characterised in that the microballoon and mass concentration 3%
~5% sodium alginate soln presses 1：The calcium chloride solution of mass fraction 4%~8% is instilled after 25~40 mass ratio mixing
In.
9. a kind of immobilised enzymes, it is characterised in that it is the system using the immobilised enzymes as any one of claim 1-8
Preparation Method is prepared.
10. application of the immobilised enzymes as claimed in claim 9 in atrazine-contaminated soil is repaired.
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