CN116425316B - Application of sperm whale Mb and mutant H64D/V68I Mb in degradation of sulfonamide compounds - Google Patents
Application of sperm whale Mb and mutant H64D/V68I Mb in degradation of sulfonamide compounds Download PDFInfo
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- 102220512172 Max-interacting protein 1_H64D_mutation Human genes 0.000 title claims abstract description 29
- -1 sulfonamide compounds Chemical class 0.000 title claims abstract description 25
- 229940124530 sulfonamide Drugs 0.000 title claims abstract description 24
- 102220223348 rs1060499949 Human genes 0.000 title claims description 24
- 238000006731 degradation reaction Methods 0.000 title abstract description 18
- 230000015556 catabolic process Effects 0.000 title abstract description 15
- 241000283222 Physeter catodon Species 0.000 title abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 22
- 230000000593 degrading effect Effects 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- JNMRHUJNCSQMMB-UHFFFAOYSA-N sulfathiazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CS1 JNMRHUJNCSQMMB-UHFFFAOYSA-N 0.000 claims description 13
- 229960001544 sulfathiazole Drugs 0.000 claims description 13
- 102000036675 Myoglobin Human genes 0.000 claims description 11
- 108010062374 Myoglobin Proteins 0.000 claims description 11
- 239000002351 wastewater Substances 0.000 claims description 4
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 230000003197 catalytic effect Effects 0.000 abstract description 3
- 238000006555 catalytic reaction Methods 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 2
- 239000007800 oxidant agent Substances 0.000 abstract description 2
- 230000001590 oxidative effect Effects 0.000 abstract description 2
- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 description 10
- 229960004306 sulfadiazine Drugs 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 6
- 150000003278 haem Chemical class 0.000 description 5
- 239000008057 potassium phosphate buffer Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000010170 biological method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000000053 physical method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- 101150040714 MB gene Proteins 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- KSFOVUSSGSKXFI-GAQDCDSVSA-N CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O Chemical compound CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O KSFOVUSSGSKXFI-GAQDCDSVSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000008015 Hemeproteins Human genes 0.000 description 1
- 108010089792 Hemeproteins Proteins 0.000 description 1
- 235000000177 Indigofera tinctoria Nutrition 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229940123317 Sulfonamide antibiotic Drugs 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006735 epoxidation reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 229940097275 indigo Drugs 0.000 description 1
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000001782 photodegradation Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 229950003776 protoporphyrin Drugs 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- TZGODTCAKVHMFG-UHFFFAOYSA-N sulfanylmethoxymethanethiol Chemical compound SCOCS TZGODTCAKVHMFG-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/40—Organic compounds containing sulfur
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to application of sperm whale Mb and mutant H64D/V68IMb in degrading sulfonamide compounds, belonging to the technical field of biology, wherein the nucleotide sequence of the H64D/V68IMb is as follows: the nucleotide sequence of SEQ ID NO.1, wild type sperm whale Mb is: SEQ ID NO.2. Wild type sperm whale Mb and mutant H64D/V68IMb at H 2 O 2 The method for degrading the sulfonamide compound by biological catalysis under the condition of an oxidant, and materials used in the reaction process and products after the reaction basically cannot damage the environment or cause secondary pollution. The degradation process is very stable, the catalytic speed is high, the degradation rate can reach more than 90% in 30 minutes, and the degradation process can be accurately repeated in the follow-up process.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of sperm whale Mb and mutant H64D/V68I Mb in degrading sulfonamide compounds.
Background
The sperm whale myoglobin is a small Heme protein, and consists of Heme (Heme) and 153 amino acids, wherein the prosthetic Heme is a complex formed by (sub) iron ions and protoporphyrin (protoporphyrins), and the main biological function is oxygen storage and no catalytic function. Through protein molecular design, the methods of metal ion, hydrogen bond network, heme post-modification, domain exchange and the like are utilized, and the heme active center structure and Mb multiple biocatalysis functions including peroxidase activity can be successfully regulated and controlled. Myoglobin and its mutants are therefore also subjects of many scholars. Through gene site-directed mutagenesis, val68 valine is mutated into Ile residue, a heme-centered channel is constructed, namely His64 histidine "gate" is mutated into Asp residue, and mutant H64D/V68I Mb is obtained. Meanwhile, in recent years, heterologous expression has also attracted much attention, especially in E.coli, because of its low cost, high expression level and relatively simple isolation and purification of the expression product.
The mutant H64D/V68I Mb has been constructed in the article Molecular Engineering of Myoglobin: influence of Residue 68 on the Rate and the Enantioselectivity of Oxidation Reactions Catalyzed by H64D/V68X Myoglobin, and shows good catalytic activity on thiomethyl ether. In the Construction of biocatalysts using the myoglobin scaffold for the synthesis of indigo from indole article, it was shown that the peroxidase activity of the sulfoxylation of anisole and the epoxidation of styrene was increased.
According to the prior reports, the method for degrading the sulfonamide compound mainly comprises a physical method, a chemical method, a photodegradation method and a biological method. The physical method mainly realizes the separation of antibiotics by adsorption, ion exchange, membrane separation technology and the like. The sulfonamide compound can be removed by a physical method, and the method is not widely applied because the adsorption capacity of the adsorbent is limited. Chemical methods mainly include chlorination processes and advanced oxidation techniques. Chemical degradation of sulfonamide compounds is the complete conversion thereof to small molecules, but the high cost of catalysts and promoters limits the large-scale use of such processes. The biological method mainly adopts an aerobic biological treatment method, an anaerobic biological treatment method and an anaerobic-aerobic biological combination method. The biological method has the advantages of mild reaction conditions, low energy consumption, low treatment cost and the like, but the removal of the sulfonamide antibiotics requires a long time and depends on the environment (nitrogen source, carbon source and the like).
Therefore, the development of a method for efficiently and environmentally degrading the sulfonamide compound has profound significance for environmental protection and sustainable development.
Disclosure of Invention
The technical problem to be solved by the invention is to provide application of sperm whale Mb and mutant H64D/V68I Mb in degrading sulfonamide compounds.
The technical scheme of the invention is summarized as follows:
application of H64D/V68I Mb in degrading sulfonamide compound, mixing H64D/V68I Mb with sulfonamide compound wastewater to make sulfonamide compound concentration in reaction mixture be 0.2mM/L, myoglobin mutant concentration be 10 μm/L, adding H with final concentration of 1mM/L 2 O 2 The reaction is carried out at 37 ℃ in the dark and pH6.2。
Further, the sulfonamide compounds are sulfathiazole and sulfadiazine.
The nucleotide sequence of the H64D/V68I Mb is as follows:
atggttctgtctgaaggtgaatggcagctggttctgcatgtttgggctaaagttgaagctgacgtcgctggtcatggtcaggacatcttgattcgactgttcaaatctcatccggaaactctggaaaaattcgatcgtttcaaacatctgaaaactgaagctgaaatgaaagcttctgaagatctgaaaaaagatggtgttaccattttaactgccctaggtgctatccttaagaaaaaagggcatcatgaagctgagctcaaaccgcttgcgcaatcgcatgctactaaacataagatcccgatcaaatacctggaattcatctctgaagcgatcatccatgttctgcattctagacatccaggtaacttcggtgctgacgctcagggtgctatgaacaaagctctcgagctgttccgtaaagatatcgctgctaagtacaaagaactgggttaccagggttaatga
the invention also provides application of the sperm whale wild myoglobin in preparation of a preparation of the degradation sulfonamide compound, wherein the nucleotide sequence of the wild myoglobin is as follows:
atggttctgtctgaaggtgaatggcagctggttctgcatgtttgggctaaagttgaagctgacgtcgctggtcatggtcaggacatcttgattcgactgttcaaatctcatccggaaactctggaaaaattcgatcgtttcaaacatctgaaaactgaagctgaaatgaaagcttctgaagatctgaaaaaacatggtgttaccgtgttaactgccctaggtgctatccttaagaaaaaagggcatcatgaagctgagctcaaaccgcttgcgcaatcgcatgctactaaacataagatcccgatcaaatacctggaattcatctctgaagcgatcatccatgttctgcattctagacatccaggtaacttcggtgctgacgctcagggtgctatgaacaaagctctcgagctgttccgtaaagatatcgctgctaagtacaaagaactgggttaccagggttaatga
further, the sulfonamide compounds are sulfathiazole and sulfadiazine.
The sulfonamide compounds are of various types, and therefore, the invention selects two types with relatively high content and high attention in the environment: sulfathiazole and sulfadiazine.
The method of the invention not only establishes a method that the H is H64D/V68I Mb 2 O 2 The method for degrading the sulfonamide compound by biological catalysis under the condition of an oxidant, and materials used in the reaction process and products after the reaction basically cannot damage the environment or cause secondary pollution. The degradation process is very stable and the catalysis speed is highThe degradation rate is high, the degradation rate in 30 minutes can reach more than 90 percent, and the degradation rate can be accurately repeated in the follow-up process.
Drawings
FIG. 1 shows the purification of Wild Type (WT) sperm whale myoglobin and its mutant H64D/V68I Mb;
FIG. 2 is a bar graph of H64D/V68I Mb degradation Sulfathiazole (ST) at different concentrations;
FIG. 3 is a bar graph of Sulfadiazine (SD) degradation at different concentrations of H64D/V68I Mb.
Detailed Description
The invention is further illustrated below in connection with specific examples:
in various embodiments:
the LB medium is: tryptone (tryptone) 10g/L; yeast extract (yeast extract) 5g/L; naCl 10g/L, natural pH, water and sterilizing.
The potassium phosphate buffer solution is: monopotassium phosphate (KH) 2 PO 4 ) And dipotassium hydrogen phosphate (K) 2 HPO 4 ) The balance was water, adjusted according to concentration and pH, and filtered using a filter membrane having a pore size of 0.22 μm.
Example 1
(1) Myoglobin mutant preparation: the Mb gene of pMbt7-7 was used to express the Wild Type (WT) sperm whale Mb and H64D/V68I Mb in BL21 (DE 3) cells. The WT Mb gene is used as a template, the mutation is performed by Polymerase Chain Reaction (PCR) box type mutagenesis, after the mutation is completed, gene sequencing is performed by a biological company, comparison is performed with a wild type gene, a sequence with successful mutation is selected according to an amino acid codon comparison table, an H64D/V68IMb gene is constructed, and the gene is introduced into BL21 (DE 3) cells.
(2) Protein culture, expression, purification and concentration determination: coli containing H64D/V68I Mb was cultured in LB medium containing ampicillin (100 mg/L), harvested in late log phase, lysed and sonicated. Cell debris was removed by centrifugation and the supernatant was separated by ammonium sulfate precipitation. The pellet was dissolved in a minimum amount of potassium phosphate buffer (10 mM, pH 6.0) and then dialyzed against the same buffer. Application of dialysate to Balanced cations with the same bufferSub-column (Hiprep) TM CM FF 16/60). The cation exchange column was eluted with a linear gradient from 10mM potassium phosphate buffer (pH 6.0) to 40mM (pH 9.0). H64D/V68I Mb was further purified by eluting with potassium phosphate buffer (50 mM, pH 7.0) using gel size exclusion chromatography (HiPrep 26/60Sephacryl S-100 HR), and the concentration of Mb mutant was determined by the pyridine hemochromatograms method as shown in FIG. 1.
(3) Degrading sulfonamide compounds: mixing the sulfathiazole-containing wastewater with the H64D/V68I Mb obtained in the step (2), and mixing the sulfathiazole-containing wastewater with the WT Mb in the other group. The concentration of sulfonamide compound in the reaction mixture was 0.2mM/L, the concentrations of H64D/V68I Mb and WT Mb were 10. Mu.M/L, and 1mM/L of H was added respectively 2 O 2 . The reaction was carried out at 37℃for 30min in the absence of light at pH 6.2.
Determination of sulfathiazole and sulfadiazine concentrations using High Performance Liquid Chromatography (HPLC) analysis, use of Shimadzu LC-20A equipped with SPD-M20A UV-visible detector, use of Waters SunFire TM C18 (4.6X150 mm,5 μm) and UV-visible detectors. The mobile phases of SD and ST were ultrapure water (0.01% phosphoric acid) and acetonitrile, respectively, in a ratio of 70:30 (v/v) the UV detection wavelengths were 268nm and 270nm, respectively. The flow rate of the detection procedure was 1mL/min and the column oven was set at 25 ℃.
As shown in FIG. 2, through result analysis, H64D/V68I Mb catalyzes the sulfathiazole to degrade, and the degradation efficiency is 99.68%; WT Mb catalyzes sulfathiazole degradation with a degradation efficiency of 85.93%.
Example 2
This example replaces the sulfathiazole of example 1 with sulfadiazine, otherwise identical to example 1. Wherein H64D/V68I Mb catalyzes sulfadiazine to degrade with a degradation efficiency of 91.61%; WT Mb catalyzes sulfadiazine degradation with a degradation efficiency of 77.88%, as shown in FIG. 3.
Claims (1)
- The application of H64D/V68I Mb in degrading sulfonamide compound is characterized in that the application method is to mix H64D/V68I Mb with the sulfonamide compound wastewater to make the concentration of sulfonamide compound in the reaction mixture be 0.2mM/L and the concentration of myoglobin mutant H64D/V68I Mb be 10 mu M/L,adding H with final concentration of 1mM/L 2 O 2 The reaction is carried out at 37 ℃ in the dark and pH6.2, and the nucleotide sequence of the H64D/V68I Mb is as follows: SEQ ID NO.1, wherein the sulfonamide compound is sulfathiazole.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2185605A1 (en) * | 1996-09-16 | 1998-03-17 | Harry Tong | Myoglobin with peroxidase activity |
| CN104085975A (en) * | 2014-06-23 | 2014-10-08 | 陕西师范大学 | Method for degrading sulfadimethoxine under catalysis of chloroperoxidase |
| CN106277362A (en) * | 2016-08-31 | 2017-01-04 | 天津大学 | Method with pycnoporus samguineus degraded sulfonamides compound |
| CN108484754A (en) * | 2018-03-26 | 2018-09-04 | 南华大学 | A kind of preparation and application of the artificial metal hydrolase based on metal ion-myoglobins mutant complexes |
| CN108752469A (en) * | 2018-07-02 | 2018-11-06 | 南华大学 | A kind of biological preparation method that the dyestuff based on myoglobins mutant is indigo |
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2023
- 2023-03-10 CN CN202310229633.9A patent/CN116425316B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2185605A1 (en) * | 1996-09-16 | 1998-03-17 | Harry Tong | Myoglobin with peroxidase activity |
| CN104085975A (en) * | 2014-06-23 | 2014-10-08 | 陕西师范大学 | Method for degrading sulfadimethoxine under catalysis of chloroperoxidase |
| CN106277362A (en) * | 2016-08-31 | 2017-01-04 | 天津大学 | Method with pycnoporus samguineus degraded sulfonamides compound |
| CN108484754A (en) * | 2018-03-26 | 2018-09-04 | 南华大学 | A kind of preparation and application of the artificial metal hydrolase based on metal ion-myoglobins mutant complexes |
| CN108752469A (en) * | 2018-07-02 | 2018-11-06 | 南华大学 | A kind of biological preparation method that the dyestuff based on myoglobins mutant is indigo |
Non-Patent Citations (1)
| Title |
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| Monooxygenation of an Aromatic Ring by F43W/H64D/V68I Myoglobin Mutant and Hydrogen Peroxide;Thomas D. Pfister等;《The Journal of Biological Chemistrry》;第280卷(第13期);第12858-12866页 * |
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