CN105567651B - The thermal stability catechol 1,2- dioxygenase of the macro genomic source of animal wastes, its encoding gene and preparation method thereof - Google Patents
The thermal stability catechol 1,2- dioxygenase of the macro genomic source of animal wastes, its encoding gene and preparation method thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
- C12Y113/11001—Catechol 1,2-dioxygenase (1.13.11.1)
Abstract
This application discloses a kind of thermal stability catechol 1 of the macro genomic source of animal wastes, 2- dioxygenase, amino acid sequence is as shown in SEQ ID NO.2, totally 283 amino acid, theoretical molecular weight 31.91kDa；Its encoding gene nucleotide sequence is as shown in SEQ ID NO.1, gene size 852bp.The thermal stability catechol 1, the most suitable action pH of 2- dioxygenase is 7.0, after handling 1h in 7.0~10.0 range of pH, 90% or more enzyme activity residue；Optimum temperature is 40 DEG C, and 210h is resistant under the conditions of 25 DEG C and 40 DEG C on enzyme activity substantially without influence, is typical thermal stability enzyme, can be used for suitable, the degradation of the enzymatic clarification and polycyclic aromatic hydrocarbon of cis- muconic acid.
Description
Technical field
The application belongs to microorganism and genetic engineering field, specifically, being related to a kind of macro genomic source of animal wastes
Thermal stability catechol 1,2- dioxygenase, its encoding gene and preparation method thereof.
Background technique
Aromatic compound is the second major class organic carbon source that carbohydrate is only second on the earth, in nature extensively
In the presence of.Catechol is the intermediate product of microbial metabolism phenols and most of polycyclc aromatic compounds, catechol into
The cracking of one step plays an important role to whether polycyclic aromatic hydrocarbon can thoroughly degrade.Catechol 1,2- dioxygenase (EC
It 1.13.11.1) is the key that catalysis catechol ortho position oxidation open loop aromatic ring oxygenase, which cracks to be formed
Intermediary --- suitable, cis- muconic acid, and it is further degraded to succinic acid and acetyl coenzyme A in subsequent enzymatic reaction,
And enter tricarboxylic acid cycle, finally it is degraded to H2O and CO2, therefore catechol 1,2- dioxygenase are a variety of in microbial degradation
Important role during aromatic compound.In addition, to crack the intermediate product to be formed suitable for the enzymatic catechol,
Cis- muconic acid is a kind of fine chemical material, can be used for producing engineering plastics, resin, nylon of property etc., and
Synthetic antibiotic, the agent of resistance amine, emulsifier etc..Currently, suitable, cis- muconic acid is commercially produced mainly with aromatic series
Compound sets out, and is obtained by organic chemical synthesis, will cause influence to environment.Therefore, exploitation is suitable for suitable, it is cis- oneself two
The catechol 1 of enedioic acid enzymatic clarification, 2- dioxygenase are of great significance.
In recent years, Acinetobacter [Lin et al.Protein J, 2015,34 (6): 421-433],
Arthrobacter[Eck et al.Gen,1993,123(1):87-92]、 Corynebacterium[Shen et
al.Biotechnol Lett,2004,26(7):575-580]、 Pseudomonas[Kim et al.J Basic
Microbiol,2015,55(3):354-362；Kaneko et al.Chem Lett,2011,40:381-383],
Rhodocococcus [Strachan et al.Biochem J,1998,333:741-747,Murakami et al.Gene,
1997,185 (1): 49-54] and Streptomyces [An et al.FEMS Microbiol Lett, 2001,195 (1):
17-22] etc. various bacterias catechol 1,2- dioxygenase gene is cloned in succession, expresses and identify.However as one kind
The thermal instability of biocatalyst, enzyme limits its application, Li Qin etc. application enzymatic clarification it is suitable, cis- muconic acid it is new
In technique, in order to enhance catechol 1, the stability of 2- dioxygenase has carried out immobilization, the adjacent benzene of gained immobilization to enzyme
3-5 days [patent " enzymatic clarification is suitable, cis- muconic acid new process ", disclosures also can be only used continuously in diphenol 1,2- dioxygenase
Numbers 93108610].The catechol 1 of thermal stability, 2- dioxygenase can keep for a long time higher under the conditions of certain temperature
Enzyme activity is accordingly used in the degradation of polycyclic aromatic hydrocarbon and suitable, and the Production by Enzymes of cis- muconic acid has its distinctive application advantage.So
And the catechol 1 obtained currently with conventional microbiological cultivation from above-mentioned bacterial clone, 2- dioxygenase thermal stability is not
It reaches.Such as the catechol 1 in the source Acinetobacter, 2- dioxygenase are resistant to 2h enzyme activity just at 37 DEG C of optimum temperature
Decline 10% [Lin et al. Protein J, 2015,34 (6): 421-433].
For animal especially phytophagous animal due to the various plants that ingest extensively, gastrointestinal tract is lignin and its related phenols
The environment of mass degradation, while by the metabolism of environment interaction to environmental pollutants, so that by suitable during long-term evolution
Should and natural selection, there may be the functional genes for participating in aromatic compound metabolism in gastrointestinal tract.However, traditional micro- life
Object pure culture technigne makes the uncultured microorganisms for accounting for 99% or more microbe species that can not separate acquisition, therefore passes through separation
Microorganism is cultivated to screen the popularity and validity that the conventional method of novel enzyme greatly limits screening.Metagenomics are avoided
The problem of microorganism is separately cultured, greatly extend microbial resources utilizes space, to find and find new function
Gene and biocatalyst --- enzyme provide new research strategy.Virtue is obtained from environment using technique of metagenome in recent years
The research of fragrant compounds of group metabolic enzymes is concentrated mainly on the environmental samples such as soil, activated sludge and sewage, to animal gastrointestinal tract
The research of environmental sample deficient [Beloqui et al.J Biol Chem, 2006,281 (32): 22933-22942；Fang
et al.PLoS One,2012,7(11):e50312]。
Summary of the invention
In view of this, the technical problem to be solved by the application is to provide a kind of heat of the macro genomic source of animal wastes
Stability catechol 1,2- dioxygenase, its encoding gene and preparation method thereof, thermal stability neighbour benzene two provided by the invention
The temperature stability of phenol 1,2- dioxygenase is preferable.
In order to solve the above-mentioned technical problem, this application discloses a kind of thermal stability of the macro genomic source of animal wastes neighbours
Benzenediol 1,2- dioxygenase, amino acid sequence is as shown in SEQ ID NO. 2, and totally 283 amino acid, theoretical molecular weight are
31.91kDa。
Disclosed herein as well is thermal stability catechol 1 described in a kind of coding above-mentioned technical proposal, 2- dioxygenases
Gene, nucleotide sequence is as shown in SEQ ID NO.1, gene size 852bp.
Disclosed herein as well is a kind of recombinant expression carriers comprising gene described in above-mentioned technical proposal, preferably
pEasy-E2-catPLCgl。
Disclosed herein as well is a kind of using obtained by the conversion host cell of recombinant expression carrier described in above-mentioned technical proposal
Recombinant bacterial strain, the bacterial strain includes but is not limited to Escherichia coli, saccharomycete, bacillus or Bacillus acidi lactici, is preferably recombinated
Bacterial strain BL21 (DE3)/catPLCgl.
Disclosed herein as well is thermal stability catechol 1 described in a kind of above-mentioned technical proposal, 2- dioxygenase is being closed
Cheng Shun, the application in cis- muconic acid.
Disclosed herein as well is thermostabilization catechol 1 described in a kind of above-mentioned technical proposal, 2- dioxygenase is being degraded
Application in polycyclic aromatic hydrocarbon.
Disclosed herein as well is thermal stability catechol 1 described in a kind of above-mentioned technical proposal, the systems of 2- dioxygenase
Preparation Method, comprising the following steps:
The recombinant expression carrier conversion host cell of catechol 1,2- dioxygenase gene is obtained into recombinant bacterial strain, is cultivated
Recombinant bacterial strain, induction recombination catechol 1, the expression of 2- dioxygenase；
It recycles and purifies expressed catechol 1,2- dioxygenase obtains thermal stability catechol 1, the bis- oxygenations of 2-
Enzyme.
Further, catechol 1,2- dioxygenase gene obtain in accordance with the following methods:
Fosmid mixing plasmid is extracted from macro genomic library；
Using fosmid mixing Plasmid DNA as template, with primer WFCgl-C shown in SEQ ID NO.312OF and SEQ
Primer WFCgl-C shown in ID NO.412OR carries out PCR amplification, obtains catechol 1,2- dioxygenase gene；
Specifically, the catechol 1,2- dioxygenase gene obtain in accordance with the following methods:
Microbe genome DNA is extracted from Japan's bee monkey excrement, it is " a kind of from animal wastes that extracting method can refer to patent
The middle method for extracting high molecular weight genome ", publication number 102586234A；
Size will be recycled through pulsed field gel electrophoresis separation, agarose gel electrophoresis after mentioned microorganism genomic DNA fragment
The DNA fragmentation of about 40kb, the DNA fragmentation of recycling is connect with fosmid carrier pCC1FOS and transfecting host bacterium E.coli
EPI300 is coated on the LB plate containing 12.5 μ g/mL chloramphenicol, and 37 DEG C of overnight incubations obtain transformant, and building obtains macro base
Because of a group library.
Using kit QIAGEN Large-Construct Kit, constructed by kit specification step extraction macro
The fosmid mixing plasmid of genomic library.Instrument Biorupter is interrupted with ultrasound to interrupt the fosmid mixing Plasmid DNA of 5 μ g
For the segment of 400-600bp, the DNA fragmentation interrupted is carried out with Genomic DNA Clean&Concentration kit
Purifying carries out the end-filling of DNA segment, 3 ' ends with TureseqTM DNA Sample Preparation Kit after purification
Add the PCR amplification of A base and adjunction head and DNA fragmentation (operation is carried out by kit specification).With HiSeq2000 genome
Sequenator (Illumima company) carries out gene order-checking to the above-mentioned library prepared.The data warp that gene order-checking obtains
Reading frame prediction and Local BLAST compare, and obtain catechol 1,2- dioxygenase gene catPLCgl, the gene order is such as
Shown in SEQ ID NO.1.
Using the macro genomic library fosmid mixing Plasmid DNA as template, with primer shown in SEQ ID NO.3
WFCgl-C12Primer WFCgl-C shown in OF and SEQ ID NO.412OR carries out PCR amplification.PCR response parameter are as follows: 94 DEG C pre-
It is denaturalized 5min；94 DEG C of denaturation 30sec, 63 DEG C of annealing 30sec, 72 DEG C of extension 1min, after 20 recycle, 94 DEG C are denaturalized 30sec again,
53 DEG C of annealing 30sec, 72 DEG C of extension 1min, 72 DEG C of heat preservation 7min after 10 circulations, wherein DEG C each circulation warm from 63 DEG C to 53
0.5 DEG C of degree decline.PCR result obtains target gene catPLCgl.
It obtains catechol 1, after 2- dioxygenase gene catPLCgl, it is connect with plasmid pEasy-E2, can be obtained
To recombinant expression carrier pEasy-E2-catPLCgl.PEasy-E2-catPLCgl is converted into e. coli bl21 (DE3), is obtained
Obtain recombinant escherichia coli strain BL21 (DE3)/catPLCgl.
After obtaining the recombinant strain containing recombinant expression carrier, it is cultivated, induction recombination catechol 1,2- is bis-
Oxygenation expression of enzymes recycles and purifies expressed catechol 1, and thermal stability catechol 1 can be obtained in 2- dioxygenase,
2- dioxygenase.Specifically, the method is as follows: recombinant bacterial strain is inoculated in LB (containing 100 μ g/mL with 0.1% inoculum concentration
Amp) in culture solution, 37 DEG C of quick oscillation 16h.Then this bacterium solution activated fresh LB is inoculated into 1% inoculum concentration (to contain
100 μ g/mL Amp) in culture solution, quick oscillation culture about 2~3h (OD600Reach 0.6~1.0) after, final concentration is added
The IPTG of 0.7mmol/L is induced, in 20 DEG C of continuation shaken cultivation about 20h.9500rpm is centrifuged 5min, collects thallus.With appropriate
PH7.0 Tris-HCl buffer suspension thalline after, the ultrasonic disruption thalline under ice bath.The first enzyme solution of the above concentration intracellular
Through 4 DEG C, after 12000rpm is centrifuged 10min, draws supernatant and purify destination protein with Nickel-NTA Agarose.SDS-
PAGE the result shows that, recombinate catechol 1,2- dioxygenase is expressed in Escherichia coli, through Nickel-NTA
Agarose is single band after purification.
Compared with prior art, the application can be obtained including following technical effect:
1) thermal stability catechol 1 provided by the invention, the optimal pH of 2- dioxygenase are 7.0, pH 6.0~
58% or more enzyme activity can be kept between 9.0；After the buffer of pH7.0~10.0 handles 1h, 90% or more enzyme activity residue；Most
Thermophilic degree is 40 DEG C, and about 12% and 20% enzyme activity is respectively provided at 0 DEG C and 10 DEG C；It is resistant to 1h under the conditions of 30 DEG C and 40 DEG C,
Still keep 100% enzyme activity；10min, remaining enzyme activity about 46% are resistant under the conditions of 50 DEG C；210h is resistant under the conditions of 25 DEG C and 40 DEG C
On enzyme activity substantially without influence.Under the conditions of pH 7.0 and 40 DEG C of temperature, the K of the enzymem、VmaxAnd KcatRespectively 24.9mg mL-1、
8.3μmol min-1 mg-1And 4.7s-1；SDS and Ag+Completely inhibit the enzymatic activity of 1,2 dioxygenase of catechol, Fe2+、Hg2+、
Cu2+, Triton 100 have very strong inhibiting effect to the enzyme；Catechol 1 is recombinated, 2- dioxygenase is to 3- methyl neighbour benzene two
Phenol, 4- methyl pyrocatechol opposite enzyme activity be respectively 93.5 ± 0.1% and 36.2 ± 0.3%, and to 4- chloro catechol
With hydroquinone without enzyme activity.
2) the above property shows thermal stability catechol 1 prepared by the present invention, and 2- dioxygenase is in suitable, cis- hexadiene
There is potential application prospect in terms of the degradation of the enzymatic clarification and polycyclic aromatic hydrocarbon of diacid.
Certainly, any product for implementing the application must be not necessarily required to reach all the above technical effect simultaneously.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present application, constitutes part of this application, this Shen
Illustrative embodiments and their description please are not constituted an undue limitation on the present application for explaining the application.In the accompanying drawings:
Fig. 1 is the recombination catechol 1 provided in an embodiment of the present invention in expression in escherichia coli, 2- dioxygenase
SDS-PAGE analysis, wherein M: low molecular weight protein Marker；1: the recombination catechol 1 of purifying, 2- dioxygenase；2:
Contain recombination catechol 1, the E. coli culture supernatant liquid of 2- dioxygenase；
Fig. 2 is recombination catechol 1 provided in an embodiment of the present invention, the optimal pH of 2- dioxygenase；
Fig. 3 is recombination catechol 1 provided in an embodiment of the present invention, the pH stability of 2- dioxygenase；
Fig. 4 is recombination catechol 1 provided in an embodiment of the present invention, the optimum temperature of 2- dioxygenase；
Fig. 5 is recombination catechol 1 provided in an embodiment of the present invention, the thermal stability of 2- dioxygenase；
Fig. 6 is recombination catechol 1 provided in an embodiment of the present invention, the tolerance of 2- dioxygenase temperature.
Specific embodiment
Presently filed embodiment is described in detail below in conjunction with accompanying drawings and embodiments, how the application is applied whereby
Technological means solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Test material and reagent
1, bacterial strain and carrier: bacterial strain Escherichia coli EPI300 and carrier pCC1FOS are purchased from EPICENTRE public affairs
Department, E.coli BL21 (DE3) are purchased from Novagen company, and coli expression carrier pEasy-E2 is purchased from the full formula gold biology in Beijing
Technology Co., Ltd..
2, genetic engineering operation enzyme, kit and other biochemical reagents: restriction enzyme, archaeal dna polymerase, connection
Enzyme and dNTP are purchased from TaKaRa company, and Large-Construct Kit is purchased from QIAGEN company；It is other all (equal for domestic reagent
It can be commercially available from common biochemical Reagent Company).
3, culture medium:
LB culture medium: Peptone 10g, Yeast extract 5g, NaCl 10g adds distilled water to 1000mL, and pH is certainly
So (about 7).Solid medium adds 2.0% (w/v) agar on this basis.
Illustrate: not making the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning: A Laboratory
Guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and product description
It carries out.
1 catechol 1 of embodiment, the acquisition of 2- dioxygenase gene catPLCgl
1, Japan bee monkey fecal microorganism Metagenomic library construction
Microbe genome DNA is extracted from Japan's bee monkey excrement, and (extracting method is detailed in patent, and " one kind is mentioned from animal wastes
The method for taking high molecular weight genome ", publication number: CN102586234A, the applying date: 2012-03-12), through arteries and veins after fragmentation
Rush the DNA fragmentation of field electrophoretic separation, agarose gel electrophoresis recycling size about 40kb, the DNA fragmentation and fosmid carrier of recycling
PCC1FOS connection and transfecting host bacterium E.coli EPI300, are coated on the LB plate containing 12.5 μ g/mL chloramphenicol, 37 DEG C of trainings
It supports and obtains transformant overnight, this clone library is stored in 96 orifice plates, -80 DEG C of preservations.
2, the extraction and sequencing of fosmid plasmid
Structure is extracted using kit QIAGEN Large-Construct Kit (operation is carried out by kit specification)
The fosmid mixing plasmid for the macro genomic library built.Fosmid mixing plasmid of the instrument Biorupter by 5 μ g is interrupted with ultrasound
The segment that DNA interrupts as 400-600bp, with Genomic DNA Clean&Concentration kit to the DNA interrupted
Segment is purified, and is mended after purification with the end that TureseqTM DNA Sample Preparation Kit carries out DNA fragmentation
The PCR amplification of flat, 3 ' ends plus A base and adjunction head and DNA fragmentation (operation is carried out by kit specification).Use HiSeq2000
Gene order-checking instrument (Illumima company) carries out gene order-checking to the above-mentioned library prepared.The number that gene order-checking obtains
It is compared according to through reading frame prediction and Local BLAST, obtains catechol 1,2- dioxygenase gene catPLCgl, the gene sequence
Column are as shown in SEQ ID NO.1.
3, catechol 1, the clone of 2- dioxygenase gene catPLCgl
With WFCgl-C12OF 5 '-ATGACTACGGGTACAGACAATC-3 ' and WFCgl-C12OR 5’-
GTCCTCCTTGTCCAGTGCGAAG-3 ' is primer pair, is that template carries out with macro genomic library fosmid mixing Plasmid DNA
PCR amplification.PCR response parameter are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 63 DEG C of annealing 30sec, 72 DEG C extend
1min, after 20 circulations, 94 DEG C are denaturalized 30sec again, 53 DEG C of annealing 30sec, 72 DEG C of extensions 1min, 10 recycle after 72 DEG C protect
Warm 7min, wherein DEG C each circulating temperature declines 0.5 DEG C from 63 DEG C to 53.PCR result obtains target gene catPLCgl.
2 thermal stability catechol 1 of embodiment, the preparation of 2- dioxygenase CatPLCgl
Catechol 1 prepared by embodiment 1,2- dioxygenase gene catPLCgl are connect with plasmid pEasy-E2
To recombinant expression carrier pEasy-E2-catPLCgl, then converts e. coli bl21 (DE3) and obtain recombinant escherichia coli strain
BL21(DE3)/catPLCgl.Take the coli strain BL21 (DE3) containing recombinant expression carrier pEasy-E2-catPLCgl
/ catPLCgl is inoculated in LB (containing 100 μ g/mL Amp) culture solution, 37 DEG C of quick oscillation 16h with 0.1% inoculum concentration.So
The bacterium solution of this activation is inoculated into fresh LB (containing 100 μ g/mL Amp) culture solution with 1% inoculum concentration afterwards, quick oscillation training
Support about 2-3h (OD600Reach 0.6-1.0) after, the IPTG induction of final concentration 0.7mmol/L is added, in 20 DEG C of continuation shaken cultivations
About 20h.9500rpm is centrifuged 5min, collects thallus.After suitable pH7.0 Tris-HCl buffer suspension thalline, in ice bath
Under the conditions of ultrasonic disruption thalline.The first enzyme solution of the above concentration intracellular draws supernatant after 12,000rpm, 4 DEG C of centrifugation 10min
And purify destination protein with Nickel-NTA Agarose, obtain thermal stability catechol 1,2- dioxygenase CatPLCgl.
SDS-PAGE analysis is carried out to the purifying destination protein, is as a result that the embodiment of the present invention provides referring to Fig. 1, Fig. 1
The recombination catechol 1 in expression in escherichia coli, 2- dioxygenase SDS-PAGE analysis, wherein M: low molecular weight egg
White matter Marker；1: the recombination catechol 1 of purifying, 2- dioxygenase；2: contain recombination catechol 1,2- dioxygenase
E. coli culture supernatant liquid.As shown in Figure 1, catechol 1 is recombinated, 2- dioxygenase is expressed in Escherichia coli,
It is after purification single band through Nickel-NTA Agarose.
3 thermal stability catechol 1 of embodiment, the property measurement of 2- dioxygenase CatPLCgl
1, thermal stability catechol 1, the activity analysis of 2- dioxygenase CatPLCgl
Activity determination method using spectrophotometry [Gou et al.Biotechnol Lett, 2012,34 (1):
117-123]: take 10 μ l 150mM catechol substrate solutions (final concentration of 0.5mM) and 2.94mL 50mM buffer anti-
3min is preheated at a temperature of answering, and the suitably diluted enzyme solution of 50 μ l is added and reacts 5min, 5min internal absorbance is measured at respective wavelength
Value added.Suitable as the catalysate of substrate using catechol, molar extinction coefficient of the cis- muconic acid at 260nm is
16 800/M cm.1 enzyme-activity unit (U) is defined as catalysis substrate per minute under given conditions and generates the corresponding product of 1 μm of ol
Required enzyme amount.
2, thermal stability catechol 1, the optimal pH of 2- dioxygenase and the measurement of pH stability:
The optimal pH of enzyme measures: the thermal stability catechol 1 that embodiment 2 is purified, 2- dioxygenase at 30 DEG C and
Enzymatic reaction is carried out in the buffer of 2.2~pH of pH 10.0.The pH Stability Determination of enzyme: the enzyme solution of purifying is placed in pH 2.2
In the buffer of~pH 10.0,1h is handled at 30 DEG C, carries out enzymatic reaction, at pH7.0 and 40 DEG C then with untreated
Enzyme solution is as control.Buffer are as follows: 50mM citrate-phosphate disodium hydrogen buffer (2.2~pH of pH 8.0)； 50mM Tris-
HCl (8.0~pH of pH 9.0)；50mM glycine-NaOH (9.0~pH of pH 10.0).Using catechol as substrate, reaction
5min measures the thermal stability catechol 1 of purifying, the zymologic property of 2- dioxygenase.As a result referring to figs. 2 and 3, Fig. 2 is
Thermal stability catechol 1 provided in an embodiment of the present invention, the optimal pH of 2- dioxygenase, Fig. 3 provide for the embodiment of the present invention
Thermal stability catechol 1, the pH stability of 2- dioxygenase.By Fig. 2 and Fig. 3 it is found that thermal stability provided by the invention
Catechol 1, the optimal pH of 2- dioxygenase are 7.0, and 58% or more enzyme activity can be kept between pH 6.0~9.0；Through
After the buffer processing 1h of pH7.0~10.0,90% or more enzyme activity residue.
3, thermal stability catechol 1, the optimum temperature and thermal stability determination of 2- dioxygenase:
The optimum temperature of enzyme measures: in the buffer of pH7.0, enzymatic reaction is carried out at 0~60 DEG C.The thermostabilization of enzyme
Property measurement: the enzyme solution of same enzyme amount is placed in the temperature (30 DEG C, 40 DEG C, 50 DEG C or 60 DEG C) of setting and handles 1h, or is placed in and sets
In fixed temperature (25 DEG C or 40 DEG C) after processing 210h, enzymatic reaction is carried out at pH7.0 and 40 DEG C, with untreated enzyme solution work
For control.As a result referring to fig. 4, Fig. 5 and Fig. 6, Fig. 4 be thermal stability catechol 1 provided in an embodiment of the present invention, the bis- oxygenations of 2-
The optimum temperature of enzyme, Fig. 5 are thermal stability catechol 1 provided in an embodiment of the present invention, the thermal stability of 2- dioxygenase, figure
6 be thermal stability catechol 1 provided in an embodiment of the present invention, the temperature tolerance of 2- dioxygenase.The result shows that: thermostabilization
Property catechol 1, the optimum temperature of 2- dioxygenase is 40 DEG C, and about 12% and 20% enzyme activity is respectively provided at 0 DEG C and 10 DEG C；
It is resistant to 1h under the conditions of 30 DEG C and 40 DEG C, still keeps 100% enzyme activity；10min, remaining enzyme activity about 46% are resistant under the conditions of 50 DEG C；
210h is resistant under the conditions of 25 DEG C and 40 DEG C on enzyme activity substantially without influence.
4, catechol 1 is recombinated, the Determination of Kinetic Parameters of 2- dioxygenase:
Kinetic parameter pH 7.0,40 DEG C of temperature and under the first order reaction time using the catechol of various concentration as substrate
(5-50 μM) is measured, and calculates K according to Lineweaver-Burk methodm、VmaxAnd KcatValue.After measured, in pH 7.0 and temperature
The K of the enzyme under the conditions of 40 DEG C of degreem、VmaxAnd KcatRespectively 24.9mg mL-1、8.3μmol min-1 mg-1And 4.7s-1。
5, to recombination catechol 1, the effect of vigor of 2- dioxygenase is measured for different metal ions and chemical reagent:
Metal ion and chemical reagent (final concentration of 1mM) are added in enzymatic reaction system, studies its shadow to enzyme activity
It rings.Under the conditions of 40 DEG C and 7.0 pH, measurement enzyme activity (does not add the enzymatic reaction of metal ion and chemical reagent under similarity condition
As control), as a result referring to table 1.Table 1 shows SDS and Ag+The enzymatic activity of 1,2 dioxygenase of catechol is completely inhibited,
Fe2+、Hg2+、Cu2+, Triton 100 have very strong inhibiting effect to the enzyme, remaining metal ion and chemical reagent are to the enzyme
Enzymatic activity influences smaller.
1 metal ion of table and chemical reagent are to recombination catechol 1, the effect of vigor of 2- dioxygenase
6, catechol 1 is recombinated, measurement of the 2- dioxygenase to different degradation of substrates:
Under the conditions of 40 DEG C and 7.0 pH, the different substrates of same concentrations, measurement are added in above-mentioned enzyme assay system
Enzyme activity (using catechol as the enzyme activity of substrate as control), as a result referring to table 2.Catechol 1 is recombinated, 2- dioxygenase is to 3-
Methyl pyrocatechol, 4- methyl pyrocatechol enzymatic activity be respectively 93.5 ± 0.1% and 36.2 ± 0.3%, and to 4- chloro
Catechol and hydroquinone are without enzyme activity.
The different substrates of table 2 are to recombination catechol 1, the effect of vigor of 2- dioxygenase
The above property shows thermal stability catechol 1 prepared by the present invention, and 2- dioxygenase is in suitable, cis- hexadiene two
There is potential application prospect in terms of the degradation of the enzymatic clarification and polycyclic aromatic hydrocarbon of acid.
Several preferred embodiments of the invention have shown and described in above description, but as previously described, it should be understood that the present invention
Be not limited to forms disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations,
Modification and environment, and the above teachings or related fields of technology or knowledge can be passed through within that scope of the inventive concept describe herein
It is modified.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be in this hair
In the protection scope of bright appended claims.
Claims (9)
1. a kind of thermal stability catechol 1 of the macro genomic source of animal wastes, 2- dioxygenase, which is characterized in that its ammonia
Base acid sequence is as shown in SEQ ID NO.2.
2. a kind of coding thermal stability catechol 1 described in claim 1, the gene of 2- dioxygenase.
3. gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.1.
4. a kind of recombinant expression carrier comprising gene as claimed in claim 2.
5. a kind of convert the resulting recombinant bacterial strain of host cell using recombinant expression carrier as claimed in claim 4.
6. thermal stability catechol 1 described in claim 1,2- dioxygenase is synthesizing suitable, answering in cis- muconic acid
With.
7. thermal stability catechol 1 described in claim 1, application of the 2- dioxygenase in degrading polycyclic aromatic hydrocarbons.
8. a kind of thermal stability catechol 1 as described in claim 1, the preparation method of 2- dioxygenase, which is characterized in that
The following steps are included:
The recombinant expression carrier conversion host cell of catechol 1,2- dioxygenase gene is obtained into recombinant bacterial strain, culture recombination
Bacterial strain, induction recombination catechol 1, the expression of 2- dioxygenase；
It recycles and purifies expressed catechol 1,2- dioxygenase obtains thermal stability catechol 1,2- dioxygenase.
9. thermal stability catechol 1 according to claim 8, the preparation method of 2- dioxygenase, which is characterized in that institute
Catechol 1 is stated, 2- dioxygenase gene obtains in accordance with the following methods:
Fosmid mixing plasmid is extracted from macro genomic library；
Using fosmid mixing Plasmid DNA as template, with primer WFCgl-C12OF and SEQ ID shown in SEQ ID NO.3
Primer WFCgl-C12OR shown in NO.4 carries out PCR amplification, obtains catechol 1,2- dioxygenase gene.
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| CN114854700A (en) * | 2021-02-04 | 2022-08-05 | 轻工业环境保护研究所 | Catechol 1, 2-dioxygenase and its coding radical, preparation method and application |
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