Cocaine esterase mutant and application thereof

文档序号：81187 发布日期：2021-10-08 浏览：31次中文

阅读说明：本技术 可卡因酯酶突变体及其应用 (Cocaine esterase mutant and application thereof ) 是由陈侠斌姚建庄侯书荣邓荇予张云童骏森于 2021-07-05 设计创作，主要内容包括：本发明公开了可卡因酯酶突变体及其应用。所述可卡因酯酶突变体由野生型可卡因酯酶经过突变所得,野生型可卡因酯酶的氨基酸序列如SEQ ID No.1所示,所述可卡因酯酶突变体为T172R/G173Q/L196C/I301C,或者再增加V116K点突变,或者再增加A51位点突变,A51位点突变为L、Y、V、F或W。本发明筛选到的可卡因酯酶突变体对可卡因有毒代谢物苯甲酰芽子碱的催化效率相对于野生型酶大大提高。(The invention discloses a cocaine esterase mutant and application thereof. The cocaine esterase mutant is obtained by mutating wild cocaine esterase, the amino acid sequence of the wild cocaine esterase is shown in SEQ ID No.1, the cocaine esterase mutant is T172R/G173Q/L196C/I301C, or V116K point mutation is added, or A51 point mutation is added, and A51 point mutation is L, Y, V, F or W. The catalytic efficiency of the cocaine esterase mutant screened by the invention on the toxic metabolite benzoylecgonine of cocaine is greatly improved compared with that of a wild enzyme.)

1. The cocaine esterase mutant is characterized in that the cocaine esterase mutant is obtained by mutating wild cocaine esterase, the amino acid sequence of the wild cocaine esterase is shown as SEQ ID No.1,

the cocaine esterase mutant is one of the following:

(1)V116K，

(2)T172R/G173Q/V116K，

(3)T172R/G173Q/L196C/I301C/V116K，

(4)T172R/G173Q/L196C/I301C/V116K/A51L，

(5)T172R/G173Q/L196C/I301C/V116K/A51Y，

(6)T172R/G173Q/L196C/I301C/V116K/A51V，

(7)T172R/G173Q/L196C/I301C/V116K/A51F，

(8)T172R/G173Q/L196C/I301C/V116K/A51W。

2. use of a cocaine esterase mutant according to claim 1 in the preparation of a medicament for treating cocaine poisoning.

3. Use of a cocaine esterase mutant according to claim 1 for preparing a hydrolysis agent for hydrolyzing benzoylbenzoylecgonine to ecgonine and benzoic acid.

4. The use of a cocaine esterase mutant according to claim 1 in the treatment of cocaine or benzoylecgonine contamination in water or soil.

5. A medicament for the treatment of cocaine intoxication wherein the active ingredient is a cocaine esterase mutant according to claim 1.

6. An agent for treating cocaine or benzoylecgonine contamination in water or soil, wherein the active ingredient is the cocaine esterase mutant according to claim 1.

7. A gene encoding a cocaine esterase mutant according to claim 1.

8. An expression vector comprising the gene of claim 7.

9. A recombinant expression cell comprising the expression vector of claim 8.

10. The use of the gene of claim 7 in the treatment of cocaine or benzoylecgonine contamination in water or soil.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a cocaine esterase mutant and application thereof.

Background

Cocaine abuse and addiction are serious medical and social problems in modern society. To date, no drug has been approved for cocaine detoxification therapy. The catastrophic medical and social consequences of cocaine abuse have made the development of anti-cocaine drugs a paramount concern. In vivo, about 40% of cocaine is rapidly biotransformed into the inactive metabolite Ecgonine Methyl Ester (EME) by butyrylcholinesterase (BChE) and carboxylesterase 2 (carboxyestererase-2, CE-2), while 45% of cocaine is hydrolyzed in the liver into the toxic metabolite Benzoylecgonine (BZE) by carboxylesterase 1 (carboxyestererase-1, CE-1) (fig. 1). BZE have similar physiological activity to cocaine and are considered to be a major contributor to long-term toxicity of cocaine due to its long half-life in BZE. BZE is one of the major residual pollutants of environmental addictive drugs and has a major impact on the ecosystem.

The ideal treatment for cocaine abuse requires not only a rapid elimination of the toxicity of cocaine itself, but also a further elimination of the toxicity of the toxic metabolite BZE of cocaine. Based on a research strategy combining computer aided design and enzyme engineering, scientists design and develop a series of highly efficient and thermostable cocaine metabolizing enzymes including human butyrylcholinesterase (BChE) mutant and bacterial cocaine esterase (CocE) mutant, which can effectively eliminate the toxicity of cocaine (Larsen NA, Turner JM, Stevens J, Rosser SJ, Basran A, Lerner RA, Bruce NC, Wilson IA. Crystal structure of a bacterial cocaine esterase. Nat Structure Biol 2002,9(1): 17-21.). On the basis, the further degradation BZE is the key to realize the thorough detoxification of cocaine, so the development of high-efficiency metabolic enzymes aiming at BZE degradation has important significance for the thorough detoxification treatment of cocaine.

Endogenous BChE is the only dentine (ECG) and benzoic acid that has been shown to hydrolyze BZE to be non-toxic. However, because of high glycosylation modification of natural BChE, efficient and economic recombinant expression of BChE is always an industrial problem, and the difficulty of later development is large. Therefore, the BZE metabolic enzyme which can be economically expressed and has high catalytic activity is important in application value.

CocE can be economically and efficiently expressed by a prokaryotic cell expression system, and the recombinant protein has good safety in human bodies (the CocE T172R/G173Q mutant has completed the clinical test II phase of cocaine detoxification, which proves that the recombinant protein is safe and effective, Nasser, A.F., et al.J.Addit.Dis.2014, 33,289 and 302), so the CocE is a very ideal candidate BZE metabolic enzyme. However, the catalytic efficiency of natural CocE to BZE is too low, and needs to be greatly improved to meet the requirement of efficiently removing BZE toxicity.

Disclosure of Invention

The invention aims to design and obtain a brand-new high-activity CocE mutant, further improve the catalytic activity of cocaine toxic metabolite BE, can BE used for clinical treatment of cocaine poisoning, and meets the clinical use requirement.

Based on the catalytic mechanism of phenyl ester hydrolysis by esterase and the classical catalytic triad structure of esterase, we found cocaine esterase to be a candidate enzyme for hydrolysis BZE by Rosetta software search. We found for the first time that CocE can catalyze BZE hydrolysis to give ecgonine and benzoic acid, with catalytic parameters for hydrolysis BZE: k is a radical of_cat＝301.2min^-1，K_M＝5153μM。

The cocaine esterase mutant is obtained by mutating wild cocaine esterase, the amino acid sequence of the wild cocaine esterase is shown in SEQ ID No.1,

the cocaine esterase mutant is one of the following:

(1)V116K，

(2)T172R/G173Q/V116K，

(3)T172R/G173Q/L196C/I301C/V116K，

(4)T172R/G173Q/L196C/I301C/V116K/A51L，

(5)T172R/G173Q/L196C/I301C/V116K/A51Y，

(6)T172R/G173Q/L196C/I301C/V116K/A51V，

(7)T172R/G173Q/L196C/I301C/V116K/A51F，

(8)T172R/G173Q/L196C/I301C/V116K/A51W。

the invention also provides application of the cocaine esterase mutant in preparing a medicine for treating cocaine poisoning.

The invention also provides application of the cocaine esterase mutant in preparing a hydrolyzing agent for hydrolyzing benzoyl ecgonine into ecgonine and benzoic acid.

The invention also provides application of the cocaine esterase mutant in treating cocaine or benzoylecgonine pollution in water or soil.

The invention also provides a medicine for treating cocaine poisoning, and the active ingredient is the cocaine esterase mutant.

The medicine also comprises medically acceptable auxiliary agents.

The invention also provides a reagent for treating cocaine or benzoylecgonine pollution in water or soil, and the active ingredient of the reagent is the cocaine esterase mutant.

The invention also provides a gene for coding the cocaine esterase mutant.

The invention also provides an expression vector containing the gene.

The invention also provides a recombinant expression cell containing the expression vector.

The invention also provides application of the gene in treating cocaine or benzoylecgonine pollution in water or soil.

The invention obtains a series of mutations through computer-aided design, and constructs a CocE mutant by adopting a point mutation PCR method; expressing the recombinant protease and purifying; the enzyme activity was verified by in vitro and in vivo enzymatic reactions, and high activity mutants were screened.

The research of the invention finds that the catalytic efficiency of the V116K mutant obtained after the V (valine) of the 116 th cocaine esterase is mutated into K (lysine) on the toxic metabolite benzoyl ecgonine of cocaine is greatly improved compared with that of the wild enzyme. And (3) further screening on the basis of the V116K mutation, and further improving the catalytic efficiency of the screened cocaine esterase mutant on a cocaine toxic metabolite, namely benzoylecgonine.

Drawings

FIG. 1 is a cocaine metabolic pathway.

FIG. 2 is a technical scheme of the present invention.

FIG. 3 shows that the highly active CocE mutant accelerates BZE metabolism in rats: (A) curve of the BEZ concentration in blood over time; (B) time-dependent profile of metabolite BA concentration in blood. Wherein, 5M-51L represents T172R/G173Q/L196C/I301C/V116K/A51L; 5M-51V denotes T172R/G173Q/L196C/I301C/V116K/A51V.

Detailed Description

The overall technical route of the invention is shown in figure 2.

Example 1: site-directed mutagenesis

The mutant of CocE is obtained by adopting a site-directed mutagenesis method.

Firstly, cDNA (GenBank # AF173165.1, Shanghai Czert biosynthesis) of wild type CocE is constructed into an Escherichia coli expression vector pET-22b (+) (provided by Shanghai Czert organism, a target gene containing C end-6 × His is inserted into NdeI and XhoI enzyme cutting sites). Using wild CocE plasmid as template, designing mutant primer, amplifying by PCR (KOD One Mater Mix, Shanghai Toyo Yan Fang) to obtain mutant PCR product, removing DNA template in the product by Dpn I (Thermo Scientific, FD1703), transforming into competent cell (DH5 alpha) to cyclize PCR product, coating transformed competent cell bacterial liquid on LB solid culture medium containing 100 ug/ml ampicillin, culturing at 37 deg.C for 15hr, selecting monoclone, extracting mutant plasmid with plasmid extraction kit, and determining to obtain mutant plasmid with correct sequence by DNA sequencing. For those with multiple mutations, one mutation is followed by the next round of mutation. Mutant primer designs are shown in table 1.

TABLE 1 primer sequences for site-directed mutagenesis

Example 2: protein expression and purification

Successfully constructed mutant plasmids are transformed into escherichia coli BL21 competent cells to express proteins, the bacterial liquid is inoculated into LB liquid culture medium (containing 100 mu g/ml ampicillin), and the mixture is expanded and cultured on a shaker at 37 ℃ and 250rpm until the OD is reached₆₀₀The bacterial solution was cooled to 15 ℃ under 0.6-0.8 ℃. IPTG (Sigma,367-93-1) was added to a final concentration of 1mM, 15 ℃ and protein expression was induced at 180rpm for 15 hr. The cells were collected and resuspended in 50mM Tris-HCl (pH7.4) buffer containing 150mM NaCl. Coli cells were disrupted with a precooled high pressure cell disrupter (SCIENTZ JG-IA, Ningbo New Ganoderma), centrifuged at 9000rpm for 45min and the supernatant was collected, and the supernatant was mixed with a cobalt medium (Takara, TALON Metal Affinity Resin) by rotation at 4 ℃ for 2hr to bind the 6 XHis-carrying protein to the medium. And adding the binding solution into a gravity column, naturally flowing out under the action of gravity, and purifying the target protein by using a gradient elution method of imidazole with different concentrations. The eluted fractions were collected into 30K (Millipore) ultrafiltration tubes, the buffer was replaced by concentration by centrifugation, and the protein was stored in solution S (50mM HEPES, 20% D-sorbitol, 1M glycine, pH 7.4). Protein concentration was determined by the Bradford kit (Bio-Industrial, C503031-1000).

Example 3: enzyme Activity assay

An experimental method for detecting the substrate BZE and the product benzoic acid BA by HPLC is firstly established. BZE and BA have strong ultraviolet absorption at 230nm, HPLC analysis uses acetonitrile and 0.1% formic acid as mobile phase, BZE and BA are separated by C18 liquid chromatographic column, BZE and BA absorption at 230nm wavelength are detected by ultraviolet detector, and linear standard curve of BZE and BA is obtained. Activity assay for CocE catalyzed BZE reaction, reaction temperature was 25 ℃, triplicate for each group. The enzymatic reaction was initiated by diluting 50. mu.L of substrate BZE solution with 50. mu.L of enzyme solution (0.1M phosphate buffer, pH 7.4). The reaction conditions are shown in Table 2.

TABLE 2 in vitro catalysis of the reaction conditions of the highly active mutant enzyme on substrate BZE

Then, 50. mu.L of 10% perchloric acid was added to terminate the reaction, 50. mu.L of acetonitrile was added, the mixture was centrifuged at 12000rpm for 5 minutes, and then the supernatant was diluted to an appropriate volume and 100. mu.L was injected. BZE and BA peak-out time, peak area and calibration curve were compared to calculate BZE residual concentration and BA formation concentration in the reaction sample. By calculating the reaction rate of BA generated by enzyme catalysis under different substrate concentrations, drawing an enzyme reaction kinetic curve by using GraphPad Prism 8, and carrying out Michaelis-Menten kinetic analysis, the k of each mutant can be obtained_catAnd K_M. The results are shown in Table 3.

TABLE 3 catalytic kinetic parameters of highly active mutant enzymes on substrate BZE

Remarking: k_effRefers to the catalytic efficiency (k) of the corresponding enzyme with respect to substrate BZE_cat/K_M)。

RCE refers to the ratio of the catalytic efficiency of the mutant enzyme pair BZE to the catalytic efficiency of the wild-type enzyme pair BZE.

Example 4: in vivo experiments in animals

Male SD rats (200 g/rat) for experiments were purchased from the center of laboratory animals of the institute of medical sciences, Zhejiang province and were bred in a constant temperature and humidity environment. The feeding and experimental use are according to the instruction of Experimental animal feeding management and use.

(1) Standard curve for BA and BZE blood samples

Blood was collected from the femoral vein of rats using a blood collection needle and heparin-treated capillary blood vessels. First, 8 tubes of blood from the same rat, 75. mu.L per tube, were taken and each tube was added to 10mu.L 250. mu.M paraoxon (paraxon) to inhibit the effect of endogenous metabolic enzymes on BZE, frozen at-80 ℃. Thawing, adding 19.7 μ L mixture containing BA and BE standard substance of 0, 4, 10, 20, 40, 60, 100, 200 μm, vortexing for 20s, adding 150 μ L acetonitrile, vortexing for 1min, and adding 50 μ L10% HClO₄Vortex for 1min, centrifuge at 17000rcf for 15min, transfer supernatant again, take about 250 μ L supernatant for HPLC analysis, sample size is 100 μ L, HPLC experimental conditions are the same as example 3. The final standard BZE and BA concentrations were 0, 0.2, 0.5, 1, 2, 3, 5, 10 μm, respectively.

(2) CocE high activity mutants accelerate BZE metabolism in vivo

Each group had 5 rats. First, 0.2 or 1mg/kg of high-activity CocE mutant or physiological saline is injected into the tail vein of the rat, and 2mg/kg of BZE is injected into the tail vein within 1 minute. BZE blood samples of 0min, 2min, 5min, 10 min, 30 min, 60 min, 90 min, and 120min are taken respectively, 100 μ L of paraoxon 250 μm is added respectively, and frozen at-80 deg.C. After thawing, vortex for 20s, add 150. mu.L acetonitrile, vortex for 1min, add 50. mu.L 10% HClO₄Vortex for 1min, centrifuge at 17000g for 15min, transfer supernatant again, take about 250 μ L supernatant for HPLC analysis, sample size is 100 μ L. The concentrations of BA and BZE in the blood were calculated at different time points for each group of rats according to the standard curve of the blood sample of the standard, thereby obtaining BZE metabolic profile in the absence of highly active CocE mutant.

The results are shown in FIG. 3, which shows that 2mg/kg BZE was injected intravenously into rats, the BZA concentration in blood was as high as 15.8. mu.M at 2min, and the BA concentration in blood was < 0.5. mu.M. When the high-activity mutant is injected, the BZA concentration in the blood of the rat is rapidly reduced, and the BA concentration in the blood is rapidly increased, which shows that toxic BZE in the body of the rat is rapidly eliminated by the high-activity mutant and metabolized into nontoxic BA.

Sequence listing

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<120> cocaine esterase mutant and application thereof

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