CN116179508A - PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, coding gene and engineering bacteria - Google Patents
PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, coding gene and engineering bacteria Download PDFInfo
- Publication number
- CN116179508A CN116179508A CN202210875888.8A CN202210875888A CN116179508A CN 116179508 A CN116179508 A CN 116179508A CN 202210875888 A CN202210875888 A CN 202210875888A CN 116179508 A CN116179508 A CN 116179508A
- Authority
- CN
- China
- Prior art keywords
- ispetase
- csp
- pet
- mutant enzyme
- hydrolase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 101000693873 Unknown prokaryotic organism Leaf-branch compost cutinase Proteins 0.000 title claims abstract description 36
- 101000693878 Ideonella sakaiensis (strain NBRC 110686 / TISTR 2288 / 201-F6) Poly(ethylene terephthalate) hydrolase Proteins 0.000 title claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 30
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
- 241000894006 Bacteria Species 0.000 title claims abstract description 15
- 102220011290 rs312262819 Human genes 0.000 claims abstract description 41
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 229920000139 polyethylene terephthalate Polymers 0.000 claims description 48
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 48
- 239000013612 plasmid Substances 0.000 claims description 19
- -1 polyethylene terephthalate Polymers 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000007986 glycine-NaOH buffer Substances 0.000 claims description 5
- 230000003301 hydrolyzing effect Effects 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 abstract description 7
- 238000006731 degradation reaction Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 5
- 238000002844 melting Methods 0.000 abstract description 5
- 230000008018 melting Effects 0.000 abstract description 5
- 230000000593 degrading effect Effects 0.000 abstract description 4
- 229920002799 BoPET Polymers 0.000 abstract description 2
- 238000002795 fluorescence method Methods 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 229920003023 plastic Polymers 0.000 description 7
- 239000004033 plastic Substances 0.000 description 7
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 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 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- BCBHDSLDGBIFIX-UHFFFAOYSA-N 4-[(2-hydroxyethoxy)carbonyl]benzoic acid Chemical compound OCCOC(=O)C1=CC=C(C(O)=O)C=C1 BCBHDSLDGBIFIX-UHFFFAOYSA-N 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 108010005400 cutinase Proteins 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 108090000371 Esterases Proteins 0.000 description 2
- 101000989724 Ideonella sakaiensis (strain NBRC 110686 / TISTR 2288 / 201-F6) Mono(2-hydroxyethyl) terephthalate hydrolase Proteins 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- QPKOBORKPHRBPS-UHFFFAOYSA-N bis(2-hydroxyethyl) terephthalate Chemical compound OCCOC(=O)C1=CC=C(C(=O)OCCO)C=C1 QPKOBORKPHRBPS-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000012536 storage buffer Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241001509283 Ideonella Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000012206 bottled water Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 229920006351 engineering plastic Polymers 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 239000013502 plastic waste Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Images
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
- 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)
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
- C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
- C08J2367/02—Polyesters derived from dicarboxylic acids and dihydroxy compounds
-
- 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
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Sustainable Development (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, a coding gene and engineering bacteria, and a PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, named IsPETase S92P/D157A cSP the IsPETase S92P/D157A The amino acid sequence of the-cSP is shown as SEQ ID NO.1As shown, the melting temperature is greatly improved and the thermal stability is obviously enhanced as measured by a differential scanning fluorescence method. Using IsPETase S92P/D157A cSP degrading PET film and obviously improving the degradation effect.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering and biological engineering, and particularly relates to a PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, a coding gene, a plasmid containing the coding gene and engineering bacteria.
Background
Polyethylene terephthalate (PET) is the most abundant polyester plastic, consisting of terephthalic acid (TPA) and Ethylene Glycol (EG) from crude oil linked by ester bonds. PET has the characteristics of simple synthesis, low price, firmness, durability and the like, PET products comprise fiber-grade PET and non-fiber-grade PET (such as bottles, films, engineering plastics and the like), and packaging is the largest non-fiber-grade application market of PET and is also the field with the fastest speed increase. At present, most bottled water and soft drinks are filled by PET; PET is also used more widely in the fields of food, pharmaceutical, cosmetic and daily chemical packaging.
It follows that a large amount of PET is introduced into the environment by means of production or waste disposal, accumulating in the global ecosystem and thus causing serious environmental damage. PET has a high proportion of aromatic terephthalate units, which reduces chain mobility and is therefore a very difficult polyester to hydrolyze, which can exist in nature for 16-48 years. At present, the main treatment methods of PET plastics are landfill, incineration, pyrolysis and chemical degradation, secondary pollution to the environment exists, and over 4000 ten thousand tons/year of PET plastics are abandoned/landfilled in the nature, so that global pollution is extremely serious.
In recent years, biological treatment technology is gradually applied to the treatment of waste plastics due to the characteristics of high-efficiency degradation, low economic cost, environmental protection and the like. At present, good research progress has been made in the field of enzymatic degradation of PET plastics, and the possibility is provided for further realizing industrial-level recycling.
To date, many PET hydrolases in the form of esterases, lipases or cutinases have been identified as the most widely studied plastic degrading enzymes. However, these PET hydrolases are mostly required to degrade PET at a temperature of 50 ℃ or higher and exhibit extremely low activity at normal temperature, and thus cannot be used to degrade PET plastics abandoned in nature in situ.
In 2016, japanese team discovered a bacterium Ideonella sakaiensis-F6 that uses PET as the primary energy and carbon source, from which PET hydrolases IsPETase and MHETase, isPETase were subsequently identified, first hydrolyzing PET polymer to MHET, then feruloyl-like esterase MHETase further hydrolyzes MHET to TPA and EG. IsPETase favors PET substrates over the high activity of other lipases or cutinases on p-nitrophenyl esters. Compared with low crystallinity cutinase LCC, fusarium oxysporum cutinase and Thermififida fusca hydrolase, the hydrolytic activity of IsPETase on PET film is 5.5 times, 88 times and 120 times higher respectively at 30 ℃. Although IsPETase has shown the strongest PET degradation activity among enzymes tested at moderate temperatures to date, its poor thermal stability still limits its use in plastic waste treatment.
Therefore, there is a need for an enzyme that has high thermostability and can maintain activity for a long period of time, thereby improving the yield of degraded PET.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a PET hydrolase IsPETase-cSP mutant enzyme with high heat stability, which has the advantages of remarkably enhancing the heat stability and remarkably improving the yield of a reaction for degrading PET.
It is a second object of the present invention to provide a gene encoding a PET hydrolase IsPETase-cSP mutant enzyme having high thermostability.
A third object of the present invention is to provide a recombinant plasmid containing the above gene.
The fourth object of the present invention is to provide an engineering bacterium containing the recombinant plasmid.
It is a fifth object of the present invention to provide the use of the PET hydrolase IsPETase-cSP mutant enzyme having high thermostability for hydrolyzing PET.
The technical scheme of the invention is summarized as follows:
a PET hydrolase IsPETase-cSP mutant enzyme with high heat stability is named IsPETase S92P /D157A cSP the IsPETase S92P/D157A Amino acid sequence of cSP is as SEQ ID No.1 shows
A nucleotide sequence of the gene for encoding the PET hydrolase IsPETase-cSP mutant enzyme with high heat stability is shown in SEQ ID NO. 2.
Recombinant plasmid pET-22b-IsPETase containing the above gene S92P/D157A -cSP。
Engineering bacterium BL21 (DE 3)/pET-22 b-IsPETase containing recombinant plasmid S92P/D157A -cSP。
The application of the PET hydrolase IsPETase-cSP mutant enzyme with high heat stability in hydrolyzing PET; PET is a shorthand for polyethylene terephthalate.
Preferably, the above application comprises the steps of:
adding PET into 50mM glycine-NaOH buffer solution with pH value of 8.5-9.5 to make the final concentration be 60-100 mg/mL; adding PET hydrolase IsPETase-cSP mutant enzyme IsPETase S92P/D157A cSP to a final concentration of 400-600 nM; incubating for 1-3 days at 30-45 ℃ and 50-220 rpm.
The invention has the advantages that:
experiments prove that the PET hydrolase IsPETase-cSP mutant enzyme IsPETase with high heat stability S92P/D157A cSP is highly thermally stable and gives a significant increase in the yield of the reaction for degrading PET.
Drawings
FIG. 1 is a schematic representation of the product from hydrolysis of the PET hydrolase IsPETase.
FIG. 2 is a schematic representation of the structure of recombinant plasmid pET-22b-IsPETase-cSP.
FIG. 3 is a recombinant plasmid pET-22b-IsPETase S92P/D157A -cSP schematic structure.
FIG. 4 shows the PET hydrolases IsPETase-cSP and IsPETase S92P/D157A -cSP differential scanning fluorescence detection. Wherein: WT: isPETase-cSP; PA IsPETase S92P/D157A -cSP。
FIG. 5 shows the PET hydrolases IsPETase-cSP and IsPETase S92P/D157A cSP catalytic degradation of polyethylene terephthalate yield. WT: isPETase-cSP; PA IsPETase S92P/D157A -cSP；A：30℃；B：40℃；C：45℃。
Detailed Description
The technical solution of the present invention is further described in detail below by means of the accompanying drawings and specific examples, which are only illustrative, and the scope of protection of the present invention is not limited thereto.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Example 1
Contains a mutant enzyme (IsPETase) encoding a PET hydrolase IsPETase-cSP having high thermostability S92P/D157A -cSP) recombinant plasmid pET-22b-IsPETase S92P/D157A -cSP, comprising the following steps:
according to the requirements of Beijing full gold biological biotechnology Co., ltd,
the modified PET hydrolase IsPETase-cSP gene (the amino acid sequence of the modified PET hydrolase is shown as SEQ ID NO.3, and the gene is shown as SEQ ID NO. 4) is used as a template, and the primers are designed as follows:
S92P-F5'-ccacgctcgaccagccgCccagccgctcgtcg-3' (mutation at position 92) (SEQ ID NO. 5)
S92P-R5'-Gcggctggtcgagcgtggagttggtgtc-3' (mutation at position 92) (SEQ ID NO. 6)
D157A-F5'-cgcaggccccgtgggCGagctcgaccaacttc-3' (mutation at position 157) (SEQ ID NO. 7)
D157A-R5'-CGcccacggggcctgcggcgccgcggctttc-3' (mutation at position 157) (SEQ ID NO. 8)
Constructing recombinant plasmid containing mutant gene by adopting seamless connection and inverse PCR technology according to the requirements of a seamless recombination kit, and mainly comprises two steps:
(1) The recombinant plasmid pET-22b-IsPETase-cSP (see FIG. 2, construction method, see the applied patent (application number: 202110900376.8), the invention name is PET hydrolase IsPETase mutant enzyme and coding gene and engineering bacteria) is used as a template, S92P-F (SEQ ID NO. 5) and S92P-R (SEQ ID NO. 6) are used as primers for the first round of PCR amplification (95℃)2min;95℃30s,58℃30s,72℃3.5min,30 cycles; 72 ℃ for 10 min), subjecting the PCR product to DMT enzyme digestion, nucleic acid electrophoresis and gel cutting recovery to obtain purified gene fragments, connecting the fragments under the action of seamless recombination ligase, transforming the fragments into DH5 alpha competent cells, screening by ampicillin flat-plate medium, extracting plasmids and verifying by sequencing to obtain the coded IsPETase S92P Recombinant plasmid pET-22b-IsPETase of-cSP mutant enzyme S92P -cSP；
(2) Recombinant plasmid pET-22b-IsPETase S92P -cSP as template, D157A-F (SEQ ID NO. 7) and D157A-R (SEQ ID NO. 8) as primers to perform a second PCR amplification (2 min at 95 ℃,30 s at 58 ℃, 3.5min at 72 ℃,30 cycles; 10min at 72 ℃) and purifying the obtained PCR product according to the same steps as step (1), linking fragments under the action of a seamless recombination ligase, transforming into DH5 alpha competent cells, obtaining the coding IsPETase through ampicillin plate medium screening, plasmid extraction and sequencing verification S92P/D157A Recombinant plasmid pET-22b-IsPETase of-cSP mutant enzyme S92P/D157A cSP, the structure of which is shown in FIG. 3;
IsPETase S92P/D157A the amino acid sequence of cSP is shown in SEQ ID NO. 1.
Coding for a mutant enzyme IsPETase of the PET hydrolase IsPETase-cSP with high thermostability S92P/D157A The nucleotide sequence of the gene of cSP is shown in SEQ ID NO. 2.
The ampicillin plate medium comprises the following formula: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of sodium chloride, 15g/L of agar powder and 50mg/L of ampicillin.
The PCR reaction system and the PCR program refer to Beijing full gold biotechnology Co., ltd
The requirements of the FastPfu DNA Polymerase kit were as shown in the following table: />
Example 2
BL21(DE3)/pET-22b-IsPETase S92P/D157A Construction of cSP engineering bacteria, which comprises the following steps:
recombinant plasmid pET-22b-IsPETase S92P/D157A Transforming cSP into BL21 (DE 3) competent cells, and coating on ampicillin plate medium to obtain positive recombinant BL21 (DE 3)/pET-22 b-IsPETase S92P/D157A cSP the recombinant plasmid pET-22b-IsPETase is successfully constructed S92P/D157A -cSP;
control bacterium BL21 (DE 3)/pET-22 b-IsPETase-cSP was constructed as described above.
Example 3
Engineering bacterium BL21 (DE 3)/pET-22 b-IsPETase S92P/D157A Inducible expression of cSP and purification of the protein of interest
Respectively inoculating the two engineering bacteria into LB liquid culture medium, and culturing at 37 ℃ and 220r/min overnight; the overnight culture bacterial liquid is respectively inoculated into fresh LB liquid culture medium according to the inoculum size of 1 percent, when the culture is carried out at 37 ℃ and 220r/min until the OD600 is about 0.8, 0.1 percent (v/v) of IPTG is added, and the temperature is reduced to 16 ℃ for induction expression for 20 hours. And (5) centrifuging at 4000rpm for 15min to collect engineering bacteria wet cells.
And re-suspending engineering bacteria wet cells in a bacteria-destroying buffer solution, and carrying out bacteria-destroying treatment on the re-suspended wet cells by using a high-pressure cell disrupter. After the bacteria are broken, the bacterial liquid is centrifugated for 60min at 10000rpm to remove cell fragments, the supernatant fluid is adsorbed with target protein through a Ni-NTA filling column, and the non-specific adsorbed impurity protein is washed by using impurity washing buffer solution. Eluting the target protein by using an eluting buffer solution, concentrating the eluting solution by a protein concentrating tube, and further exchanging the eluting buffer solution to a preservation buffer solution by the protein concentrating tube.
The formula of the LB liquid medium is as follows: 5g/L of yeast extract, 10g/L of tryptone and 10g/L of sodium chloride;
the formula of the bacteria-destroying buffer solution is as follows: 50mM Tris-HCl,150mM NaCl,10mM Imidazole,pH =7.5;
the formula of the impurity washing buffer solution is as follows: 50mM Tris-HCl,150mM NaCl,20mM Imidazole,pH =7.5;
the formula of the elution buffer is as follows: 50mM Tris-HCl,300mM NaCl,300mM Imidazole,pH =7.5;
the formula of the storage buffer solution is as follows: 50mM Na 2 HPO 4 -HCl，100mM NaCl，pH＝7.0；
The control strain BL21 (DE 3)/pET-22 b-IsPETase-cSP was expressed by induction as described above, and the protein was purified and concentrated under the same conditions.
Example 4
PET hydrolase IsPETase-cSP mutant enzyme IsPETase S92P/D157A Determination of the melting temperature (T) by differential scanning fluorescence (cSP) m Value of
The mutant enzyme IsPETase was used S92P/D157A -cSP protein samples were added to 96-well plates, each well having the following composition: mu.l of storage buffer, 9. Mu.l of protein solution (0.4 mg/mL), 1. Mu.l of SYPRO Orange staining solution (250X). The 96-well plate was centrifuged at 2000rpm at 4.0℃for 1 minute and tested using a real-time fluorescent quantitative PCR instrument. The test conditions were: heating the sample from 30 ℃ to 90 ℃ at a rate of 0.3 ℃/s; excitation wavelength was 465nm, emission wavelength was 580nm, and fluorescence values were detected every 0.03 seconds. The lowest point of the derived curve obtained by the test is the melting temperature (T) m Values) of 70.3 c, see figure 4.
The melting temperature is defined as the temperature at which 50% of the protein is in the folded state and 50% of the protein is in the depolymerized state.
The PET hydrolase IsPETase-cSP was tested in the same manner and its melting temperature (T m Values) were 46.7℃as shown in FIG. 4.
Example 5
Use of a highly thermostable PET hydrolase IsPETase-cSP mutant enzyme IsPETase S92P/D157A -cSP catalytic degradation of polyethylene terephthalate (PET)
Polyethylene terephthalate was added to 50mM glycine-NaOH buffer pH 9.0 to a final concentration of 60mg/mL; adding IsPETase S92P/D157A cSP to a final concentration of 400nM; incubate at 30℃for 1 day at 50 rpm. The total concentration of the hydrolysis products in the reaction liquid obtained by HPLC detection is 761.2 mu M,see a in fig. 5.
The polyethylene terephthalate (PET) was catalytically degraded by the PET hydrolase IsPETase-cSP in the same manner, and the total concentration of the hydrolysis products in the reaction solution obtained by HPLC detection was 34.6. Mu.M, see A in FIG. 5.
Example 6
Use of a highly thermostable PET hydrolase IsPETase-cSP mutant enzyme IsPETase S92P/D157A -cSP catalytic degradation of polyethylene terephthalate (PET)
Polyethylene terephthalate was added to a 50mM glycine-NaOH buffer pH 8.5 to a final concentration of 80mg/mL; adding IsPETase S92P/D157A cSP to a final concentration of 500nM; incubate at 40℃for 3 days at 100 rpm. The total concentration of the hydrolysis products in the reaction solution obtained was 2485.4. Mu.M by HPLC, see B in FIG. 5.
Polyethylene terephthalate (PET) was catalytically degraded by the same method using the PET hydrolase IsPETase-cSP, and the total concentration of the hydrolysis products in the reaction solution obtained by HPLC detection was 349.2. Mu.M, see B in FIG. 5.
Example 7
Use of a highly thermostable PET hydrolase IsPETase-cSP mutant enzyme IsPETase S92P/D157A -cSP catalytic degradation of polyethylene terephthalate (PET)
Polyethylene terephthalate was added to 50mM glycine-NaOH buffer pH 9.5 to a final concentration of 100mg/mL; adding IsPETase S92P/D157A cSP to a final concentration of 600nM; incubate at 45℃for 2 days at 220 rpm. The total concentration of the hydrolysis products in the reaction solution obtained was 490.9. Mu.M by HPLC, see C in FIG. 5.
The polyethylene terephthalate (PET) was catalytically degraded by the PET hydrolase IsPETase-cSP in the same manner, and the total concentration of the hydrolysis products in the reaction solution obtained by HPLC detection was 56.8. Mu.M, see C in FIG. 5.
The total concentration of the hydrolysates was the sum of the concentrations of the three hydrolysates bis (2-hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA).
The detection conditions of the HPLC are as follows: the ultraviolet detector, characteristic absorption peak of 240nm, ZORBAX Eclipse Plus C reversed phase chromatographic column (5 μl,250mm×4.6 mm), mobile phase A is 0.1% formic acid water solution, mobile phase B is acetonitrile, mobile phase is raised from 5% acetonitrile to 70% acetonitrile in 20min, column temperature is 30deg.C, sample injection amount is 10 μl, and flow rate is 0.8ml/min.
Claims (6)
1. A PET hydrolase IsPETase-cSP mutant enzyme with high heat stability is named IsPETase S92P/D157A -cSP, characterized in that said IsPETase S92P/D157A The amino acid sequence of cSP is shown in SEQ ID NO. 1.
2. The gene for coding the PET hydrolase IsPETase-cSP mutant enzyme with high heat stability is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
3. Recombinant plasmid pET-22b-IsPETase containing the gene of claim 2 S92P/D157A -cSP。
4. Engineering bacterium BL21 (DE 3)/pET-22 b-IsPETase containing recombinant plasmid of claim 3 S92P/D157A -cSP。
5. The use of a PET hydrolase IsPETase-cSP mutant enzyme having high thermostability according to claim 1 for hydrolyzing PET; PET is a shorthand for polyethylene terephthalate.
6. The use according to claim 5, characterized by the steps of:
adding PET into 50mM glycine-NaOH buffer solution with pH value of 8.5-9.5 to make the final concentration be 60-100 mg/mL; adding IsPETase S92P/D157A cSP to a final concentration of 400-600 nM; incubating for 1-3 days at 30-45 ℃ and 50-220 rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210875888.8A CN116179508B (en) | 2022-07-25 | 2022-07-25 | PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, coding gene and engineering bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210875888.8A CN116179508B (en) | 2022-07-25 | 2022-07-25 | PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, coding gene and engineering bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116179508A true CN116179508A (en) | 2023-05-30 |
| CN116179508B CN116179508B (en) | 2024-06-11 |
Family
ID=86438941
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210875888.8A Active CN116179508B (en) | 2022-07-25 | 2022-07-25 | PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, coding gene and engineering bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116179508B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927411A (en) * | 2022-12-21 | 2023-04-07 | 天津大学 | Esterase mutant gene, protein expressed by gene and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367408A (en) * | 2016-10-20 | 2017-02-01 | 天津大学 | PET lytic enzyme mutant, coding gene and application thereof |
| CN108467857A (en) * | 2018-03-14 | 2018-08-31 | 四川大学 | PET water solution enzyme mutant and its application |
| CN113774041A (en) * | 2021-08-06 | 2021-12-10 | 天津大学 | PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium |
-
2022
- 2022-07-25 CN CN202210875888.8A patent/CN116179508B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367408A (en) * | 2016-10-20 | 2017-02-01 | 天津大学 | PET lytic enzyme mutant, coding gene and application thereof |
| CN108467857A (en) * | 2018-03-14 | 2018-08-31 | 四川大学 | PET water solution enzyme mutant and its application |
| CN113774041A (en) * | 2021-08-06 | 2021-12-10 | 天津大学 | PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927411A (en) * | 2022-12-21 | 2023-04-07 | 天津大学 | Esterase mutant gene, protein expressed by gene and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116179508B (en) | 2024-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114854713B (en) | PET hydrolase IsPETase-cSP mutant enzyme, coding gene and engineering bacteria | |
| Hyeon et al. | Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications | |
| CN102286441B (en) | Low-temperature esterase and coding gene and use thereof | |
| CN112159478B (en) | Fusion protein and application thereof in polymer degradation | |
| CN103215244B (en) | Alkaline pectinase PelN, as well as encoded gene and application thereof | |
| CN116179508B (en) | PET hydrolase IsPETase-cSP mutant enzyme with high thermal stability, coding gene and engineering bacteria | |
| CN116121223B (en) | Esterase mutant with polyester degradation activity and application thereof | |
| CN113684196A (en) | Purification method of high-temperature-resistant polyethylene terephthalate hydrolase | |
| CN105647844A (en) | Recombinant bacteria using xylose to produce glycollic acid and building method and application of recombinant bacteria | |
| CN113774041B (en) | PET hydrolase IsPETase mutant enzyme, coding gene and engineering bacterium | |
| CN116904422A (en) | Deep sea-derived PET hydrolase dsPETase01 and application thereof | |
| CN112708589A (en) | Genetically engineered bacterium, construction method thereof and application of genetically engineered bacterium in fermentation production of 5-hydroxytryptophan | |
| CN116606873B (en) | Esterase mutant gene for decomposing polyester, protein expressed by gene and application of esterase mutant gene | |
| Han et al. | The increased efficiency of porphyran hydrolysis by constructing a multifunctional enzyme complex from marine microorganisms | |
| CN118773163A (en) | PET hydrolase IsPETaseS92P/D157AMutant of-cSP and application thereof | |
| CN118222540B (en) | Heat-resistant plastic hydrolase AtPETase mutant and application thereof | |
| CN101423814B (en) | Clostridium for synthesizing glutathion and construction method and use thereof | |
| WO2024227372A1 (en) | Ispetase-8×chimera recombinase, and encoding gene, recombinant plasmid engineering bacterium and use thereof | |
| CN117305276A (en) | PET degrading enzyme IsPETase-PN mutant enzyme and encoding gene, recombinant plasmid, engineering bacteria and application thereof | |
| WO2024227370A1 (en) | Ispetase-4×chimera recombinase, and encoding gene, recombinant plasmid, engineering bacterium and use thereof | |
| CN117887686A (en) | Polyethylene terephthalate hydrolase and application thereof in degrading PET (polyethylene terephthalate) products | |
| CN114181922B (en) | Recombinant esterase, gene, recombinant bacterium and application of recombinant esterase and recombinant bacterium in degradation of phthalate | |
| CN117904076A (en) | Novel feruloyl esterase for efficiently degrading polyethylene terephthalate and application thereof | |
| CN116286567B (en) | Recombinant escherichia coli producing alpha-amino acid ester acyltransferase, and construction method and application thereof | |
| CN117448296A (en) | PET hydrolase mutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |