Search

CN-121975765-A - Recombinant esterase, engineering bacterium and application thereof in degradation of carbaryl

CN121975765ACN 121975765 ACN121975765 ACN 121975765ACN-121975765-A

Abstract

The invention discloses recombinant esterase, engineering bacteria and application thereof in degrading carbaryl, wherein the coding gene of the recombinant esterase is cloned and expressed in escherichia coli, engineering bacteria E.coli BL21 (DE 3) -pET-28a (+) -GE003489 is constructed, and the expressed recombinant esterase is induced to have better hydrolysis capability on carbaryl. The appropriate amount of recombinant esterase is used for catalyzing and hydrolyzing the carbaryl, the concentration of the substrate is 0.1 g/L, the catalysis time is 1h, and the substrate conversion rate is 90.5%. Can be used for removing the carbaryl pollution in the environment and has potential application value.

Inventors

  • ZHANG CHAOHUI
  • Di Hengjun

Assignees

  • 浙江工业大学

Dates

Publication Date
20260505
Application Date
20260202

Claims (10)

  1. 1. A recombinant esterase derived from Pseudomonas sp, characterized in that the amino acid sequence of the recombinant esterase is shown in SEQ ID No. 2.
  2. 2. A recombinant genetically engineered bacterium comprising the recombinant esterase encoding gene of claim 1.
  3. 3. Use of the recombinant esterase of claim 1 for hydrolyzing carbaryl.
  4. 4. The method according to claim 3, wherein the method comprises the step of carrying out hydrolysis reaction at 20-60 ℃ and 120-240 rpm by using wet bacterial body crushed and extracted purified enzyme liquid obtained by induced fermentation of engineering bacteria containing recombinant esterase coding genes as a catalyst, carbaryl as a substrate and a buffer solution with pH of 7.0-9.0 as a reaction medium.
  5. 5. The use according to claim 4, wherein the reaction medium is a phosphate buffer solution having a pH of 7.4 and a pH of 50 mM.
  6. 6. The process according to claim 4, wherein the substrate is added in an amount of 0.1 to 1g/L based on the total volume of the reaction system and the catalyst is added in an amount of 80 to 200 mg/L based on the protein content.
  7. 7. The method according to claim 4, wherein the catalyst is prepared by (1) resuspension of wet cells with pre-chilled phosphate buffer at pH 7.0, 50 mM to obtain a bacterial suspension, sonication of the bacterial suspension at pH7.4, 350-450W to 10-15 min, working 2 s during sonication, intermittent 3 s, centrifugation of the resulting eluate at pH 4, 5500-8000 rpm for 30min, collecting supernatant, (2) equilibration of Ni-NTA affinity column with PB buffer at pH7.4,50 mM containing 10-20 mM imidazole, loading the supernatant obtained in step (1) at a rate of 1 ml/min, washing the column with PB buffer at pH7.4,50 mM containing 20-mM imidazole until UV absorption reaches a stable baseline, eluting protein with PB buffer at pH7.4,50 mM containing 250-300mM imidazole, collecting the protein eluate in one step, and concentrating the eluate to obtain the recombinant enzyme for the purpose.
  8. 8. The method for dialysis and concentration according to claim 7, wherein the method comprises the steps of removing salt ions in the eluent by dialysis in a buffer solution of pH7.4 and 50mM PB by using a dialysis bag with 3500 molecular weight cut-off, concentrating the cut-off solution to 1/10 of the original volume by using polyethylene glycol, and obtaining the concentrate, namely the purified enzyme solution of the recombinant esterase.
  9. 9. The use according to claim 7, wherein the concentration of wet cells in the bacterial suspension is 0.5-35 g/L and the ultrasonication conditions are 4 ℃, 400W, 15 min.
  10. 10. The method according to claim 4, wherein the wet bacterial cells are obtained by inoculating an engineering bacterium containing a recombinant esterase encoding gene into LB liquid medium containing 50. Mu.g/mL kanamycin, culturing 12-16 h in a thermostatic shaker at 37 ℃ and 200rpm to obtain a seed liquid, transferring the seed liquid into LB liquid medium containing 50. Mu.g/mL kanamycin at an inoculum size of 1%, culturing the seed liquid in a thermostatic shaker at 37 ℃ and 200rpm until OD 600 is 0.6-0.8, adding IPTG to a final concentration of 0.1 mM, placing the seed liquid in a thermostatic shaker at 24 ℃ and 180-200 rpm to induce 14 h, and centrifuging the seed liquid at 4 ℃ and 5500-8000rpm for 15min after fermentation to obtain the wet bacterial cells.

Description

Recombinant esterase, engineering bacterium and application thereof in degradation of carbaryl Field of the art The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant esterase derived from Pseudomonas sp, engineering bacteria and application thereof in degradation of carbaryl. (II) background art Carbaryl (Carbaryl) is also known as carbaryl, the chemical name 1-naphthyl-N-methyl carbamate, and is a widely used carbamate pesticide. The main insecticidal mechanism of carbaryl is the inhibition of acetylcholinesterase in insects. The carbaryl has the functions of contact killing, stomach poisoning and weak fumigation, which means that the insects can die in a poisoning way only by contacting with the liquid medicine or eating the plants sprayed with the liquid medicine. Carbaryl has been widely used in agriculture-for controlling lepidoptera pests (e.g., leaf rollers), coleoptera pests (e.g., scarab), hemiptera pests (e.g., aphids), etc. on various crops such as rice, cotton, fruit trees, and the like. Forestry-is used to control forest pests. Public health-used for preventing and controlling mosquito, fly and other sanitary pests, etc. However, carbaryl has certain disadvantages of high toxicity to bees, death of bees during flowering phase, threat to pollinated insects and ecological balance, and high toxicity to aquatic organisms, and risk of water source pollution. Has a certain threat to human health, and typical carbamate pesticide poisoning symptoms such as lacrimation, pupil constriction, muscle tremor, nausea, vomiting and the like can appear after a large amount of human body is exposed. Compared with the traditional physical and chemical treatment method, the microbial degradation method is a novel pesticide degradation way, has the advantages of low investment, no toxicity, no residue and no secondary pollution, has relatively obvious treatment effect, and is a method for removing pollutants with low cost and environmental friendliness. The microorganism can use the pesticide as the only carbon source or nitrogen source to maintain the growth of the microorganism, and the pesticide is hydrolyzed and oxidized by generating degrading enzyme, and the main mode of the microorganism degrading the pesticide is enzymatic degradation, namely that the compound enters the bacteria body in a certain mode, and then the pesticide is finally partially or completely degraded through a series of physiological and biochemical reactions under the action of various enzymes. At present, ：mcbA（from Pseudomonas sp.XWY-1）;carH （from Agrobacterium sp. XWY-2）,cehA（from Rhizobium sp. AC100）;pchA （from Pseudomonas sp. PS21）;cahA（from Arthrobacter sp. RC100）, hydrolysis products of the carbaryl hydrolase gene reported at home and abroad are alpha-naphthol. However, these enzymes have a long amino acid sequence, which is not conducive to artificial synthesis and application. (III) summary of the invention The invention aims to provide a recombinant esterase, engineering bacteria and application thereof in the hydrolysis of carbaryl, wherein the esterase has a short amino acid sequence, is easy to synthesize and express artificially, has high hydrolysis reaction activity, and can be applied to the enhanced degradation of residual carbaryl in the environment. The technical scheme adopted by the invention is as follows: The invention provides a recombinant esterase (recorded as XQest) derived from Pseudomonas sp, and the amino acid sequence of the esterase is shown as SEQ ID No. 2. The invention relates to a coding gene of the recombinant esterase, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 1. The invention also provides a recombinant plasmid (preferably pET-28a (+) -GE 003489) containing the recombinant esterase coding gene and recombinant genetic engineering bacteria (preferably E.coli BL21 (DE 3) -pET-28a (+) -GE 003489) obtained by transforming the recombinant plasmid. In addition, the invention also provides an application of the recombinant esterase in the hydrolysis of carbaryl, and in particular relates to an application of the recombinant esterase in the hydrolysis of carbaryl, wherein a purified enzyme solution obtained by carrying out induced fermentation on engineering bacteria containing recombinant esterase coding genes is used as a catalyst, carbaryl is used as a substrate, a buffer solution with pH of 7.0-9.0 is used as a reaction medium, and hydrolysis reaction is carried out under the conditions of 20-60 ℃ and 120-240 rpm (preferably 37 ℃ and 200 rpm). Preferably, the reaction medium is Phosphate (PB) buffer solution with pH of 7.4 and 50 mM, the substrate is added in an amount of 0.1-1g/L, preferably 0.1g/L, based on the total volume of the reaction system, and the catalyst is added in an amount of 80-200 mg/L, preferably 100 mg/L, based on the protein content. Further, the catalyst is prepared by (1) resuspension of wet bacterial