CN113637657B

CN113637657B - Carboxylesterase CarCB2 and whole-cell catalyst and application thereof

Info

Publication number: CN113637657B
Application number: CN202110898712.XA
Authority: CN
Inventors: 丁俊美; 刘艳; 黄遵锡; 周杨; 许波; 周峻沛
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2024-01-30
Anticipated expiration: 2041-08-05
Also published as: CN113637657A

Abstract

The invention discloses carboxylesterase CarCB2, a whole-cell catalyst and application thereof, wherein the amino acid sequence of the carboxylesterase CarCB2 is shown as SEQ ID NO.1, and the gene sequence is shown as SEQ ID NO. 2. The carboxylesterase CarCB2 is displayed on the outer membrane of the escherichia coli cell through a surface display technology to obtain the whole cell catalyst, and the cyhalothrin is degraded by using the whole cell catalyst, so that the cyhalothrin has a good degradation effect, and the whole cell catalyst has good application potential in solving the problem of pollution of pyrethroid pesticides in the environment.

Description

Carboxylesterase CarCB2 and whole-cell catalyst and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to carboxylesterase CarCB2, a whole cell catalyst thereof and application thereof.

Background

Pyrethroid pesticides are a class of synthetic pesticides based on natural pyrethrins found in pyrethrum, a third broad class of pesticides in widespread use, cyhalothrin being an important member of the pyrethroid family of pesticides and being widely used for the control of crop pests. The residue of pyrethroid pesticides in the environment is toxic to salmonids, fish, zooplankton and the like, and can destroy biological behaviors and biological processes, so that natural enemies die and the balance of food chains is destroyed. In addition, metabolites of pyrethroids have been detected in the human population and may result from food contamination due to excessive use in crop production. Pyrethroid intake can cause symptoms such as nausea, vomiting, abdominal pain, dysphagia, etc., and can be life threatening when severe. Thus, pyrethroid pesticide contamination is not only an environmental issue, but also a public health issue.

There are three methods for contaminant removal from the environment: physical, chemical and biological degradation. Because physical and chemical methods are expensive, inefficient and secondary to the environment, bioremediation, especially the mining and utilization of microbial resources of degradable pollutants, is currently the primary means of pollutant removal in the environment. Carboxylesterase is the key to microbial metabolism of pyrethroid pesticides and is responsible for ester bond cleavage. However, the use of pure enzymes in practical production and life is limited due to high cost, complicated purification, poor stability and other factors. The whole cell catalyst is prepared through gene engineering to display peptide or protein on the surface of microbial cell. In the use process, purification is not needed, the economic cost and the complexity of the process are greatly reduced, and the method has been successfully applied to the fields of recombinant vaccines, biosensors, bioremediation and the like. However, there is no report on degradation of pyrethroid pesticides using whole cell catalysts.

Disclosure of Invention

The invention aims to provide carboxylesterase CarCB2 and a whole-cell catalyst and application thereof. The whole-cell catalyst can be used for degrading the cyhalothrin pesticide without cell wall breaking and protein purification, and is a novel catalyst which is economical, effective and environment-friendly and can solve the problem of pyrethroid pesticide pollution in the environment.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

carboxylesterase CarCB2, wherein the amino acid sequence of the carboxylesterase CarCB2 is shown as SEQ ID NO. 1.

The invention also provides a gene of the carboxylesterase CarCB2, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

In another aspect of the invention, a whole cell catalyst is provided, which is a cell displaying carboxylesterase CarCB2 on the outer membrane of E.coli cells by surface display technology.

Specifically, the recombinant plasmid pET-28a (+)/CarCB 2/INPN formed by connecting carboxylesterase gene CarCB2 and ice nucleoprotein gene INPN with expression vector pET-28a (+) is transformed into E.coliBL21 (DE 3) competent cells to obtain the recombinant plasmid pET-28a (+)/CarCB 2/INPN.

Wherein, the ice nucleoprotein gene INPN is shown as SEQ ID NO. 3.

In another aspect of the invention, the whole cell catalyst described above is prepared by the following method:

connecting the CarCB2 gene and the ice nucleoprotein INPN gene with an expression vector pET-28a (+) to obtain a recombinant vector; transforming the recombinant vector into host competent cells to obtain recombinant bacteria; culturing the recombinant strain and inducing whole cell catalyst expression.

In another aspect of the present invention, the recombinant vector containing carboxylesterase gene CarCB2 and ice nucleoprotein gene INPN is also within the scope of the present invention.

Preferably, the expression vector is the vector pET-28a (+).

In another aspect of the present invention, there is provided a recombinant bacterium comprising carboxylesterase gene CarCB2 and ice nucleoprotein gene INPN.

Preferably, the host cells used for the recombinant bacteria are: coli BL21 (DE 3).

In another aspect of the invention, there is provided the use of the carboxylesterase CarCB2 and the whole cell catalyst in the degradation of cyhalothrin.

The whole-cell catalyst can be used for degrading cyhalothrin under natural conditions, and cell wall breaking and protein purification are not needed.

The beneficial effects of the invention are as follows:

compared with purified recombinant carboxylesterase CarCB2, the whole cell catalyst provided by the invention has the following advantages: the purified recombinant carboxylesterase CarCB2 retains 49% of its relative activity after 120 hours of treatment at 4deg.C and has substantially lost activity after 120 or 72 hours of treatment at 30deg.C or 37deg.C. The whole cell catalyst still maintains more than 100% of relative activity after being treated for 35 days at 4 ℃ and maintains 60.9% and 51.9% of relative activity after being treated for 35 days at 30 ℃ and 37 ℃, which shows that the whole cell catalyst has better stability than purified carboxylesterase CarCB2 and can be stably applied to pyrethroid degradation in the environment for a longer period of time; in addition, the whole cell catalyst and purified recombinant carboxylesterase CarCB2 had degradation efficiencies of 85% and 79.7% for 30mg/L cyhalothrin, respectively, at 150 min. Therefore, the whole-cell catalyst can be economically and efficiently used for solving the problem of pyrethroid pollution in the environment (such as farmlands).

Drawings

FIG. 1 is a SDS-PA GE analysis of the recombinant carboxylesterase CarCB2 of the present invention and a whole cell catalyst; wherein lanes 1 and 2 in a: e.coli BL21 (DE 3) cells containing pET-28a (+)/CarCB 2, pre-and post-induction supernatants; lane 3: purified CarCB2; lane 1 in B: e.coli BL21 (DE 3) cell disruption supernatant containing pET-28a (+); lane 2: purified CarCB2; lanes 3,4,5: e.coli BL21 (DE 3) cell-induced supernatant containing pET-28a (+)/CarCB 2/INPN, pET-28a (+)/CarCB 2/GFP and pET-28a (+)/CarCB 2/INPN/GFP, respectively; m is protein Marker;

FIG. 2 is a substrate specificity of the recombinant carboxylesterase CarCB2 and whole cell catalyst of the present invention;

FIG. 3 is an optimum reaction pH for the recombinant carboxylesterase CarCB2 of the present invention and a whole cell catalyst;

FIG. 4 is an optimum reaction temperature for the recombinant carboxylesterase CarCB2 of the present invention and a whole cell catalyst;

FIG. 5 is the temperature stability of the purified recombinant carboxylesterase CarCB2 of the present invention;

FIG. 6 temperature stability of whole cell catalysts of the present invention;

FIG. 7 shows the effect of the recombinant carboxylesterase CarCB2 and whole cell catalyst of the present invention on the degradation of cyhalothrin.

Detailed Description

The following description of the present invention will be made more complete and clear in view of the detailed description of the invention, which is to be taken in conjunction with the accompanying drawings that illustrate only some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental materials and reagents in the experimental example of the invention are as follows:

1. strains and vectors: coli E.coli BL21 (DE 3) and E.coli DH 5. Alpha., plasmids pMD18-T/inak (containing the ice nucleoprotein gene INPN) and pEGFP-N3 (containing the green fluorescent protein gene GFP) were all supplied by the present laboratory, and expression vector pET-28a (+) was purchased from Invitrogen, england Biolabs, U.S.A.; bacillus sp is supplied by the university of yunnan.

2. Enzymes and other biochemical reagents: p-nitrophenol esters (p-NPC 2 to p-NPC 12) with different carbon chain lengths were purchased from Sigma in the united states; cyhalothrin was purchased from Shanghai Ala Di Co; restriction enzymes NcoI, bamHI, hindIII, ecoRI, xhoI were purchased from baori doctor materials technology (beijing) limited; nickel-NTAAgarose is purchased from Qiagen (Germany); isopropyl- β -D-thiogalactopyranose (IPTG) and proteinase K are purchased from biofrox company, germany; the others are all domestic reagents (all available from general biochemistry reagent company).

3. Culture medium:

LB medium: 1% peptone, 1% NaCl,0.5% yeast powder, pH naturally (about 7), on the basis of which 2% (w/v) agar was added to the solid medium.

Description: the molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.

EXAMPLE 1 construction of recombinant engineering bacteria

Using Bacillus genome as template, using primers P1 and P2 to amplify CarCB2 gene, and connecting with pET-28a (+) vector (plasmid is digested with BamHI and NcoI), to obtain recombinant plasmid pET-28a (+)/CarCB 2; amplifying INPN by using the primers P3 and P4, and connecting the INPN with pET-28a (+)/CarCB 2 (the plasmid is subjected to EcoRI and HindIII double digestion) to obtain recombinant plasmid pET-28a (+)/CarCB 2/INPN; the GFP gene is amplified by using the primers P5 and P6 and is connected with pET-28a (+)/CarCB 2 (plasmid is subjected to EcoRI and HindIII double enzyme digestion) to obtain recombinant plasmid pET-28a (+)/CarCB 2/GFP; GFP was amplified using primers P7 and P8 and ligated with pET-28a (+)/CarCB 2/INPN (plasmid was digested with HindIII and XhoI) to obtain recombinant pET-28a (+)/CarCB 2/INPN/GFP.

The primer sequences are as follows (both 5 '-3'):

P1：TAAGAAGGAGATATACCATGAAAATTGTGACACCAAAACCA；

P2：ACGGAGCTCGAATTCGATCCCCAATCGAGTTTCTCTAAAAATTCG；

P3：GAAACTCGATTGGGGATCGAATATGACTCTCGACAAGGCGTTG；

P4：CTCGAGTGCGGCCGCAAGCTTGGTCTGCAAATTCTGCGGC；

P5：GAAACTCGATTGGGGATCGAATATGGTGAGCAAGGGCGAGG；

P6：GCTCGAGTGCGGCCGCAAGCTTCTTGTACAGCTCGTCCATGCC；

P7：CAGAATTTGCAGACCAAGCTTATGGTGAGCAAGGGCGAGG；

P8：GTGGTGGTGGTGGTGCTCGAGCTTGTACAGCTCGTCCATGC。

all recombinant plasmids were transformed into E.coli DH 5. Alpha. Competent cells, and all recombinant plasmids were confirmed to have been successfully constructed by colony PCR and sequencing, and the recombinant plasmids were transformed into E.coli BL21 (DE 3) competent cells, respectively.

EXAMPLE 2 inducible expression of recombinant carboxylesterase CarCB2 and Whole cell catalyst

E.coli BL21 (DE 3) strain containing recombinant plasmids of pET-28a (+)/CarCB 2 and pET-28a (+)/CarCB 2/INPN, respectively, was inoculated at an inoculum size of 1% into 5mL of liquid medium (containing 50. Mu.g/mL kanamycin) and cultured with shaking at 37℃for 12-16 hours. Then inoculating the cultured bacterial liquid into 200mL fresh culture medium again at 1% inoculum size, and shake culturing at 37deg.C for 2-3h to OD ₆₀₀ IPTG was added at a final concentration of 0.7mM after =0.5 and cultured with shaking at 15 ℃ for 20h. And centrifuging the induced bacterial liquid at 6500rpm and at 4 ℃ for 10min, and collecting bacterial bodies. E.coli BL21 (DE 3) [ pET-28a (+)/CarCB 2]The bacterial cells are suspended in ddH ₂ And (3) performing ultrasonic disruption on the thalli by using a low-temperature high-pressure cytobreaker under ice bath condition, centrifuging at 12000rpm for 40min at 4 ℃, and collecting the supernatant. Whole cell bacterial suspension in citric acid-disodium hydrogen phosphate (citric acid-Na) ₂ HPO ₄ ) (pH 7.5) a refrigerator with the temperature of 4 ℃ is placed in the reactor for standby.

Loading the supernatant after wall breaking and centrifugation to a Ni-NTA affinity chromatography column, washing the column with 20mM Tris-HCl (pH 8.0), 0-80mM imidazole and 500mM NaCl to remove the impurity protein; next, the recombinant carboxylesterase CarCB2 was eluted with 20mM Tris-HCl (pH 8.0), 500mM imidazole, 500mM NaCl and collected.

SDS-PAGE results showed that: the recombinant enzyme CarCB2 was purified and the product was a single band (. About.29 kDa) as shown in FIG. 1, lane A3; suspending in citric acid-Na ₂ HPO ₄ The whole cell catalyst in (pH 7.5) was boiled for 10min and SDS-PAGE results showed that: whole cell catalyst (CarCB 2-INPN) was expressed and has a molecular weight of about 49kDa, consistent with the theoretical molecular size, as shown in lane B3 of FIG. 1.

EXAMPLE 3 Biochemical characterization of purified CarCB2 and Whole cell catalysts

The activity of esterase CarCB2 and the activity of the whole cell catalyst are measured by adopting a p-nitrophenol method, and the method is specifically as follows: the p-NP was dissolved in acetonitrile to a concentration of 10mM; the reaction system is as follows: 420. Mu.L of buffer, 30. Mu.L of substrate with final concentration of 0.6mM, preheating for 2min at reaction temperature, adding 50. Mu.L of proper enzyme solution, reacting for 5min, adding 50. Mu.L of 1M Na ₂ CO ₃ The reaction was terminated and the absorbance value of the released p-nitrophenol was measured at a wavelength range of 405 nm. One unit of esterase activity (U) is defined as the amount of enzyme required to break down the substrate to produce 1. Mu. M p-NP per minute.

1. Recombinant carboxylesterase CarCB2 and whole cell catalyst substrate specificity

P-nitrophenol esters (p-NPC 2 to p-NPC 12) with different carbon chain lengths are used as substrates, enzymatic reaction is carried out at the pH value of 7.0/7.5 and the temperature of 30 ℃, and the utilization capacities of recombinant carboxylesterase CarCB2 and a whole cell catalyst on different substrates are measured.

FIG. 2 shows that the recombinant carboxylesterase CarCB2 and the whole cell catalyst are active on p-NPC2 to p-NPC12 and both exhibit the highest enzymatic activity on p-NPC 4.

2. Determination of the pH Activity and temperature Activity of recombinant carboxylesterase CarCB2 and Whole cell catalyst

1) Determination of optimum pH: enzyme solutions were placed in different buffers (50 mM): citric acid-Na ₂ HPO ₄ (pH 3.0–7.5)、Tris-HCl(pH 7.5–9.5)The relative residual enzyme activities of recombinant carboxylesterase CarCB2 and whole cell catalyst were determined by enzymatic reaction at 37℃with p-NPC4 as substrate, boric acid-NaOH (pH 9.5-12.0).

The results of fig. 3 show that: the recombinant carboxylesterase CarCB2 and the whole cell catalyst have activity between pH4.0 and 11.0, the enzyme activity of the whole cell catalyst is higher than that of the recombinant carboxylesterase CarCB2 between pH 7.5 and 11.0, and the recombinant carboxylesterase CarCB2 and the whole cell catalyst have the highest enzyme activity at pH 7.0 and pH 7.5 respectively.

2) Determination of optimum temperature: acid-Na at pH 7.0/7.5 ₂ HPO ₄ The relative residual enzymatic activities of recombinant carboxylesterase CarCB2 and whole cell catalyst were determined by enzymatic reaction in a buffer at 0-90℃to determine the optimum temperature. The optimum temperature and the optimum pH were measured at a maximum of 100% relative to the remaining enzyme activity.

The results of fig. 4 show: the optimal reaction temperature for both the recombinant carboxylesterase CarCB2 and the whole cell catalyst is 30 ℃.

3. Determination of the temperature stability of recombinant carboxylesterase CarCB2 and Whole cell catalysts

Temperature stability determination: recombinant carboxylesterase CarCB2 and whole cell catalyst (lyophilized powder) of the same enzyme activity unit are respectively placed at 4 ℃,30 ℃ and 37 ℃ for one month, and respectively subjected to enzymatic reaction at pH 7.0/7.5 and 30 ℃, and untreated enzyme solution is used as a control.

The results of fig. 5 show that: the recombinant carboxylesterase CarCB2 still maintains 49% of the relative activity after 120 hours of treatment at 4℃and loses the enzymatic activity after 120 hours and 72 hours of treatment at 30℃and 37 ℃. The results of fig. 6 show: the whole cell catalyst remained more than 100% relative activity after 35 days of treatment at 4 ℃ and remained 60.9% and 51.9% relative activity after 35 days of treatment at 30 ℃ and 37 ℃. The above results illustrate: compared with recombinant carboxylesterase CarCB2, the whole cell catalyst has better temperature stability.

EXAMPLE 4 degradation of cyhalothrin by recombinant carboxylesterase CarCB2 and Whole cell catalyst

At an optimum reaction pH (7.0/7.5) and temperature (30 ℃) to contain 10U of recombinase and 30mg/L of cyhalothrin (in acetoneThe degradation of cyhalothrin by recombinant carboxylesterase CarCB2 and whole cell catalyst was determined by placing in a reaction system at 10mg/mL mother liquor), sampling at time intervals with the same treatment as that of the non-emphasized carboxylesterase CarCB2 or whole cell catalyst as a control group, terminating the reaction with 10% dilute HCl, extracting the reaction product with an equal volume of ethyl acetate, and redissolving in an equal volume of methanol solution after the ethyl acetate has evaporated completely. The residual cyhalothrin was analyzed on a gas chromatography-mass spectrometry system (GC-MS-QP 2020). The gas chromatography capillary column used was DB-5MS (0.25 mm. Times.0.25 μm. Times.30 m). The initial temperature of the oven is set to 70 ℃ and then 15 ℃ for min ^-1 Heating to 200deg.C (holding for 1 min), and then heating to 5deg.C for min ^-1 Heating to 220deg.C (8 min), and cooling to 6deg.C for min ^-1 Heating to 240 deg.C (holding for 5 min), and heating to 40deg.C for min ^-1 Raise to 280 ℃ (hold 13 min) for a total time of 44min. The injector temperature was set at 300℃and helium was introduced as a carrier gas at a flow rate of 0.95 mL/min. mu.L of each reaction extract was injected in a non-split mode. All assays were repeated three times.

The results show in fig. 7: the recombinant carboxylesterase CarCB2 and the whole cell catalyst degrade 85% and 79.7% of the lambda-cyhalothrin within 150min respectively, and the lambda-cyhalothrin in the control group is not degraded. The whole-cell catalyst and the recombinant carboxylesterase CarCB2 have high-efficiency cyhalothrin degradation capability.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> university of Yunnan teachers and students

<120> carboxylesterase CarCB2 and whole cell catalyst and use thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> PRT

<213> carboxylesterase (CarCB 2)

<400> 1

Met Lys Ile Val Thr Pro Lys Pro Phe Thr Phe Lys Gly Gly Asp Lys

1 5 10 15

Ala Val Leu Leu Leu His Gly Phe Thr Gly Asn Thr Ala Asp Val Arg

20 25 30

Met Leu Gly Arg Tyr Leu Asn Glu Arg Gly Tyr Thr Cys His Ala Pro

35 40 45

Gln Tyr Lys Gly His Gly Val Pro Pro Glu Glu Leu Val His Thr Gly

50 55 60

Pro Glu Asp Trp Trp Lys Asp Val Met Asp Gly Tyr Glu Tyr Leu Gln

65 70 75 80

Ser Glu Gly Tyr Glu Asn Ile Ala Ala Cys Gly Leu Ser Leu Gly Gly

85 90 95

Val Phe Ser Leu Lys Leu Gly Tyr Thr Val Pro Ile Lys Gly Ile Val

100 105 110

Pro Met Cys Ala Pro Met His Ile Lys Ser Glu Glu Val Met Tyr Glu

115 120 125

Gly Val Leu Ser Tyr Ala Arg Asn Tyr Lys Lys Phe Glu Gly Lys Gln

130 135 140

Pro Glu Gln Ile Glu Lys Glu Met Lys Glu Phe Glu Lys Thr Pro Met

145 150 155 160

Asn Thr Leu Lys Ser Leu Gln Glu Leu Ile Ala Asp Val Arg Lys Asn

165 170 175

Val Asp Met Ile Tyr Ser Pro Thr Phe Val Val Gln Ala Arg His Asp

180 185 190

His Met Ile Asn Thr Asp Ser Ala Asp Ile Ile Tyr Asn Glu Val Glu

195 200 205

Thr Asp Asp Lys Gln Leu Lys Trp Tyr Glu Glu Ser Gly His Val Ile

210 215 220

Thr Leu Asp Lys Glu Arg Asp Leu Val His Gln Asp Val Tyr Glu Phe

225 230 235 240

Leu Glu Lys Leu Asp Trp

245

<210> 2

<211> 738

<212> DNA

<213> carboxylesterase Gene (CarCB 2)

<400> 2

atgaaaattg tgacaccaaa accatttaca tttaaaggcg gagacaaggc ggtgcttctg 60

ctgcacgggt ttacgggaaa tacggcagac gtcagaatgc tcggccgcta cttaaatgag 120

cgcggctaca cttgtcacgc gcctcaatat aaagggcacg gcgttccgcc ggaagagctg 180

gtacatacgg gacctgaaga ttggtggaaa gacgtcatgg acgggtatga atatttacaa 240

tctgaaggct atgaaaacat tgcggcctgc ggactgtcgc ttggcggggt tttttcattg 300

aaattgggtt acactgtacc cataaaggga attgttccta tgtgtgctcc gatgcacatt 360

aaaagtgaag aggttatgta cgaaggagtt ctctcctacg cgcgcaatta taaaaaattc 420

gaaggaaaac agccggagca gattgaaaag gaaatgaagg agtttgaaaa aacgccgatg 480

aatacgctca aatcgcttca ggagctgatt gccgatgtgc gcaaaaacgt cgacatgatc 540

tattccccga catttgtggt tcaggcacgg catgaccata tgattaacac agacagtgca 600

gacatcattt acaacgaggt ggaaacggac gacaaacagc tgaaatggta tgaagagtca 660

ggacatgtca ttacgctgga caaagaacgg gacctcgttc atcaggatgt gtacgaattt 720

ttagagaaac tcgattgg 738

<210> 3

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<212> DNA

<213> glacier protein Gene (INPN)

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atgactctcg acaaggcgtt ggtgctgcgt acctgtgcaa ataacatggc cgatcactgc 60

ggccttatat ggcccgcgtc cggcacggtg gaatccagat actggcagtc aaccaggcgg 120

catgagaatg gtctggtcgg tttactgtgg ggcgctggaa ccagcgcttt tctaagcgtg 180

catgccgatg ctcgatggat tgtctgtgaa gttgccgttg cagacatcat cagtctggaa 240

gagccgggaa tggtcaagtt tccgcgggcc gaggtggttc atgtcggcga caggatcagc 300

gcgtcacact tcatttcggc acgtcaggcc gaccctgcgt caacgtcaac gtcaacgtca 360

acgtcaacgt taacgccaat gcctacggcc atacccacgc ccatgcctgc ggtagcaagt 420

gtcacgttac cggtggccga acaggcccgt catgaagtgt tcgatgtcgc gtcggtcagc 480

gcggctgccg ccccagtaaa caccctgccg gtgacgacgc cgcagaattt gcagacc 537

<210> 4

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

taagaaggag atataccatg aaaattgtga caccaaaacc a 41

<210> 5

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

acggagctcg aattcgatcc ccaatcgagt ttctctaaaa attcg 45

<210> 6

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gaaactcgat tggggatcga atatgactct cgacaaggcg ttg 43

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ctcgagtgcg gccgcaagct tggtctgcaa attctgcggc 40

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gaaactcgat tggggatcga atatggtgag caagggcgag g 41

<210> 9

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gctcgagtgc ggccgcaagc ttcttgtaca gctcgtccat gcc 43

<210> 10

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cagaatttgc agaccaagct tatggtgagc aagggcgagg 40

<210> 11

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gtggtggtgg tggtgctcga gcttgtacag ctcgtccatg c 41

Claims

1. The application of the whole-cell catalyst in the degradation of cyhalothrin is characterized in that the whole-cell catalyst is prepared by combining a carboxylesterase CarCB2 coding gene and a binuclear protein geneINPNThe recombinant plasmid pET-28a (+)/CarCB 2/INPN formed by connecting the recombinant plasmid pET-28a (+) with an expression vector pET-28a (+) is transformed intoE. coliBL21 (DE 3) competent cells; the amino acid sequence of carboxylesterase CarCB2 is shown as SEQ ID NO. 1; the ice nucleoprotein geneINPNThe nucleotide sequence of (2) is shown as SEQ ID NO. 3.