WO2023279816A1 - 可卡因酯酶突变体及其应用 - Google Patents
可卡因酯酶突变体及其应用 Download PDFInfo
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- WO2023279816A1 WO2023279816A1 PCT/CN2022/089839 CN2022089839W WO2023279816A1 WO 2023279816 A1 WO2023279816 A1 WO 2023279816A1 CN 2022089839 W CN2022089839 W CN 2022089839W WO 2023279816 A1 WO2023279816 A1 WO 2023279816A1
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- Prior art keywords
- cocaine
- mutant
- bze
- cocaine esterase
- esterase
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- 102100021864 Cocaine esterase Human genes 0.000 title claims abstract description 34
- 108010064410 cocaine esterase Proteins 0.000 title claims abstract description 34
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- KZFBHCCLJSAHBQ-UHFFFAOYSA-N Benzoylecgonine Natural products CN1C2CCC1C(C(C2)OC(=C)c3ccccc3)C(=O)O KZFBHCCLJSAHBQ-UHFFFAOYSA-N 0.000 claims abstract description 11
- GVGYEFKIHJTNQZ-RFQIPJPRSA-N ecgonine benzoate Chemical compound O([C@@H]1[C@@H]([C@H]2CC[C@@H](C1)N2C)C(O)=O)C(=O)C1=CC=CC=C1 GVGYEFKIHJTNQZ-RFQIPJPRSA-N 0.000 claims abstract description 11
- 230000035772 mutation Effects 0.000 claims abstract description 11
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- 108090000623 proteins and genes Proteins 0.000 claims description 14
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- PHMBVCPLDPDESM-YWIQKCBGSA-N Ecgonine Natural products C1[C@H](O)[C@@H](C(O)=O)[C@H]2CC[C@@H]1N2C PHMBVCPLDPDESM-YWIQKCBGSA-N 0.000 claims description 5
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- PHMBVCPLDPDESM-UHFFFAOYSA-N d-Pseudoekgonin Natural products C1C(O)C(C(O)=O)C2CCC1N2C PHMBVCPLDPDESM-UHFFFAOYSA-N 0.000 claims description 5
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- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
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-
- 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)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01084—Cocaine esterase (3.1.1.84)
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Definitions
- the invention relates to the technical field of biomedicine, in particular to cocaine esterase mutants and applications thereof.
- BZE has similar physiological activities to cocaine, and due to its longer half-life in vivo, BZE is considered to be the main contributor to the long-term toxicity of cocaine. In addition, BZE is one of the main residual pollutants of addictive drugs in the environment and has an important impact on the ecosystem.
- Endogenous BChE is the only one confirmed to hydrolyze BZE into non-toxic ecgonine (ECG) and benzoic acid.
- ECG non-toxic ecgonine
- BChE ecgonine
- CocE can be expressed economically and efficiently by prokaryotic cell expression system, and the recombinant protein has good safety in human body (CocE T172R/G173Q mutant has completed the phase II clinical trial of cocaine detoxification, confirming that it is safe and effective. Nasser, A.F., et.al.J.Addict.Dis.2014,33,289–302), so CocE is an ideal candidate BZE metabolic enzyme. However, the catalytic efficiency of natural CocE to BZE is too low, and it needs to be greatly improved to meet the requirements of efficient removal of BZE toxicity.
- the present invention aims to design and obtain a brand-new high-activity CocE mutant to further improve the catalytic activity to the toxic metabolite BE of cocaine, which can be used for the clinical treatment of cocaine poisoning and meets the needs of clinical use.
- Cocaine esterase mutant obtained through mutation by wild-type cocaine esterase, the amino acid sequence of wild-type cocaine esterase is shown in SEQ ID No.1,
- Described cocaine esterase mutant is following one:
- the present invention further provides the application of the cocaine esterase mutant in the preparation of medicine for treating cocaine poisoning.
- the present invention further provides the application of the cocaine esterase mutant in the preparation of a hydrolyzing agent for hydrolyzing benzoylbenzoylecgonine into ecgonine and benzoic acid.
- the present invention further provides the application of the cocaine esterase mutant in controlling cocaine or benzoylecgonine pollution in water or soil.
- the invention also provides a medicine for treating cocaine poisoning, the active ingredient is the cocaine esterase mutant.
- the medicine also includes medically acceptable auxiliary agents.
- the invention also provides a reagent for controlling cocaine or benzoylecgonine pollution in water or soil, the active ingredient being the cocaine esterase mutant.
- the present invention also provides the gene encoding the cocaine esterase mutant.
- the invention also provides an expression vector comprising the gene.
- the present invention also provides recombinant expression cells containing the expression vector.
- the invention also provides the application of the gene in controlling cocaine or benzoylecgonine pollution in water or soil.
- the invention obtains a series of mutations through computer-aided design, constructs CocE mutants by point mutation PCR method; expresses and purifies recombinant protease; verifies enzyme activity through in vitro and in vivo enzymatic reactions, and screens highly active mutants.
- the present invention has found that the catalytic efficiency of the V116K mutant obtained after the 116th V (valine) of cocaine esterase is mutated into K (lysine) to the toxic metabolite benzoylecgonine of cocaine is higher than that of the wild Enzymes are greatly improved. On the basis of the V116K mutation, further screening was carried out, and the catalytic efficiency of the screened cocaine esterase mutant to the toxic metabolite benzoylecgonine of cocaine was further improved.
- Figure 1 shows the cocaine metabolic pathway.
- Fig. 2 is a technical roadmap of the present invention.
- Fig. 3 is a hyperactive CocE mutant accelerating the metabolism of BZE in rats: (A) the time-varying curve of BEZ concentration in blood; (B) the time-varying curve of blood metabolite BA concentration.
- CocE mutants were obtained by site-directed mutagenesis.
- the cDNA of wild-type CocE (GenBank#AF173165.1, Shanghai Jierui Biosynthesis) was constructed into the Escherichia coli expression vector pET-22b(+) (provided by Shanghai Jierui Biotechnology, and the C-terminal-6 ⁇ His target gene was inserted into NdeI and XhoI restriction sites).
- the primers of the mutant were designed and amplified by PCR (KOD One Mater Mix, Shanghai Toyobo) to obtain the PCR product of the mutant, and the DNA template in the product was removed with DpnI (Thermo Scientific, FD1703).
- the extraction kit extracts the mutant plasmid, and the mutant plasmid with the correct sequence is confirmed by DNA sequencing. For multiple mutations, the next round of mutations is performed after one mutation.
- the mutant primer design is shown in Table 1.
- Embodiment 2 protein expression and purification
- Escherichia coli cells were disrupted with a pre-cooled high-pressure cell disruptor (SCIENTZ JG-IA, Ningbo Xinzhi), centrifuged at 9000 rpm for 45 min, and the supernatant was collected. Mix by swirling for 2 hr to allow the protein with 6 ⁇ His to bind to the medium. Add the binding solution to the gravity column, flow out naturally under the action of gravity, and purify the target protein by gradient elution with different concentrations of imidazole.
- SCIENTZ JG-IA Ningbo Xinzhi
- the eluted fraction was collected into a 30K (Millipore) ultrafiltration tube, and the buffer was replaced by centrifugal concentration, and the protein was stored in solution S solution (50mM HEPES, 20% D-sorbitol, 1M glycine, pH 7.4).
- the protein concentration was determined by Bradford kit (Shenggong, C503031-1000).
- K eff refers to the catalytic efficiency of the corresponding enzyme for the substrate BZE (k cat /K M ).
- RCE refers to the ratio of the catalytic efficiency of the mutant enzyme to BZE and the catalytic efficiency of the wild-type enzyme to BZE.
- Embodiment 4 animal experiment in vivo
- mice Male SD rats ( ⁇ 200 g/rat) used in the experiment were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences, and were kept in a constant temperature and humidity environment. The feeding and experimental use were in accordance with the "Guidelines for the Feeding, Management and Use of Experimental Animals".
- Blood was collected from the rat femoral vein using a lancet and heparinized capillaries. First, 8 tubes of blood from the same rat were taken, 75 ⁇ L in each tube, and each tube of blood was added to 100 ⁇ L of 250 ⁇ M paraxon to inhibit the influence of endogenous metabolic enzymes on BZE, and stored at -80°C.
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Environmental & Geological Engineering (AREA)
- Medicinal Chemistry (AREA)
- Soil Sciences (AREA)
- Mycology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Water Supply & Treatment (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Gastroenterology & Hepatology (AREA)
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- General Chemical & Material Sciences (AREA)
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- Enzymes And Modification Thereof (AREA)
Abstract
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。
Description
本发明涉及生物医药技术领域，特别是涉及可卡因酯酶突变体及其应用。
可卡因滥用和成瘾是现代社会中严重的医学和社会问题。到目前为止，还没有药物获得批准用于可卡因解毒的治疗。可卡因滥用造成的灾难性医学和社会后果使得抗可卡因药物的开发成为当务之急。在体内，约40％的可卡因被丁酰胆碱酯酶(butyrylcholinesterase，BChE)和羧酸酯酶2(carboxyesterase-2，CE-2)快速生物转化为非活性代谢物芽子碱甲酯(EME)，而45％的可卡因在肝脏经羧酸酯酶1(carboxyesterase-1，CE-1)水解成有毒代谢物苯甲酰芽子碱(benzoylecgonine，BZE)(图1)。BZE与可卡因具有相似的生理活性，由于BZE体内半衰期较长，被认为是可卡因长期毒性的主要贡献者。此外，BZE是环境中成瘾性药物主要残留污染物之一，对生态体系有着重要影响。
可卡因滥用的理想治疗方法不仅需要快速消除可卡因本身的毒性，而且更需要去除可卡因有毒代谢产物BZE的毒性。基于计算机辅助设计和酶工程相结合的研究策略，科学家们设计和开发出了一系列高效且热稳定的可卡因代谢酶包括人丁酰胆碱酯酶(butyrylcholinesterase，BChE)突变体和细菌可卡因酯酶(cocaine esterase，CocE)突变体，能够有效消除可卡因的毒性(Larsen NA,Turner JM,Stevens J,Rosser SJ,Basran A,Lerner RA,Bruce NC,Wilson IA.Crystal structure of a bacterial cocaine esterase.Nat Struct Biol 2002,9(1):17-21.)。在此基础上，进一步降解BZE是实现可卡因彻底解毒的关键，因此开发针对BZE降解的高效代谢酶对可卡因的彻底解毒治疗具有重要意义。
内源BChE是唯一被证实能水解BZE为无毒的牙子碱(ecgonine，ECG)和苯甲酸。但由于天然BChE高度糖基化修饰，高效经济重组表达BChE一直是行业难题，后期开发难度大。因此寻找能够经济表达且具有高催化活性的BZE代谢酶具有重要的应用价值。
CocE可以用原核细胞表达系统经济高效地表达，且该重组蛋白在人体内具有良好的安全性(CocE T172R/G173Q突变体已完成可卡因解毒的临床试验II期，证实其 安全有效。Nasser,A.F.,et.al.J.Addict.Dis.2014,33,289–302)，因此CocE是非常理想的候选BZE代谢酶。但天然CocE对BZE的催化效率太低，亟需大幅度提高才能满足高效去除BZE毒性的要求。
发明内容
本发明旨在设计获得全新的高活性CocE突变体，进一步提高对可卡因有毒代谢物BE的催化活性，可用于可卡因中毒的临床治疗，满足临床使用需求。
基于酯酶对苯酯水解的催化机理和酯酶经典的催化三联体结构，我们通过Rosetta软件搜索发现可卡因酯酶可作为水解BZE的候选酶。我们首次发现CocE能够催化BZE水解生成芽子碱和苯甲酸，其水解BZE的催化参数为：k
cat＝301.2min
-1，K
M＝5153μM。
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以下一种：
(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。
本发明又提供了所述可卡因酯酶突变体在制备用于治疗可卡因中毒的药物中的应用。
本发明又提供了所述可卡因酯酶突变体在制备用于水解苯甲酰苯甲酰芽子碱为芽子碱和苯甲酸的水解剂中的应用。
本发明又提供了所述可卡因酯酶突变体在治理水体或土壤中可卡因或苯甲酰芽子碱污染中的应用。
本发明还提供了一种用于治疗可卡因中毒的药物，活性成分为所述可卡因酯酶突变体。
所述的药物，还包括医学上可接受的助剂。
本发明还提供了一种用于治理水体或土壤中可卡因或苯甲酰芽子碱污染的试剂，有效成分为所述可卡因酯酶突变体。
本发明还提供了编码所述可卡因酯酶突变体的基因。
本发明还提供了包含所述基因的表达载体。
本发明还提供了包含所述表达载体的重组表达细胞。
本发明还提供了所述基因在治理水体或土壤中可卡因或苯甲酰芽子碱污染中的应用。
本发明通过计算机辅助设计得到一系列突变，采用点突变PCR方法构建CocE突变体；表达重组蛋白酶并纯化；通过体外和体内酶促反应验证酶活性，筛选高活性突变体。
本发明研究发现，将可卡因酯酶第116的V(缬氨酸)突变成K(赖氨酸)后获得的V116K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。在V116K突变的基础上，进一步进行筛选，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更进一步获得提高。
图1为可卡因代谢途径。
图2为本发明技术路线图。
图3为高活性CocE突变体加速大鼠体内BZE的代谢：(A)血液中BEZ浓度随时间变化曲线；(B)血液中代谢产物BA浓度随时间变化曲线。其中，5M-51L表示T172R/G173Q/L196C/I301C/V116K/A51L；5M-51V表示T172R/G173Q/L196C/I301C/V116K/A51V。
本发明整体技术路线如图2所示。
实施例1：定点突变
采用定点突变的方法获得CocE的突变体。
首先将野生型CocE的cDNA(GenBank#AF173165.1，上海捷瑞生物合成)构建到大肠杆菌表达载体pET-22b(+)(上海捷瑞生物提供，含C端-6×His目的基因插入到NdeI和XhoI酶切位点)。以野生型CocE质粒为模板，设计突变体的引物通过PCR (KOD One Mater Mix，上海东洋纺)扩增获得突变体的PCR产物，用DpnⅠ(Thermo Scientific，FD1703)将产物中的DNA模板除去后，转化进入感受态细胞(DH5α)中使PCR产物环化，将转化后的感受态细胞菌液涂在含有100μg/ml氨苄青霉素的LB固体培养基上37℃，15hr培养，挑选单克隆并用质粒提取试剂盒提取突变体质粒，通过DNA测序确定获得序列正确的突变体质粒。对于有多处突变的，在一处突变后再进行下一轮突变。突变体引物设计如表1所示。
表1定点突变所用引物序列
实施例2：蛋白表达及纯化
将构建成功的突变体质粒转化进入大肠杆菌BL21感受态细胞中表达蛋白，将菌液接种至LB液体培养基(含100μg/ml氨苄青霉素)中，在37℃、250rpm摇床上扩大培养至OD
600＝0.6-0.8，将菌液冷却至15℃。加入IPTG(Sigma,367-93-1)至终浓 度为1mM，15℃，180rpm诱导蛋白表达15hr。收集菌体，用含150mM NaCl的50mM Tris-HCl(pH 7.4)缓冲液重悬。用预冷的高压细胞破碎仪(SCIENTZ JG-IA，宁波新芝)将大肠杆菌细胞破碎，9000rpm，45min离心后收集上清，将上清和钴介质(Takara,TALON Metal Affinity Resin)在4℃中旋转混合2hr，使带有6×His的蛋白与介质结合。将结合液加入重力柱中，在重力作用下自然流出，用不同浓度咪唑梯度洗脱的方法将目的蛋白纯化出来。收集洗脱组分到30K(Millipore)超滤管中，通过离心浓缩替换缓冲液，蛋白保存在solution S溶液(50mM HEPES,20％D-山梨醇，1M甘氨酸，pH 7.4)中。通过Bradford试剂盒(生工，C503031-1000)测定蛋白浓度。
实施例3：酶活性分析
首先建立HPLC检测底物BZE与产物苯甲酸BA的实验方法。BZE和BA在230nm处均具有较强的紫外吸收，HPLC分析以乙腈和0.1％甲酸为流动相，通过C18液相色谱柱分离BZE和BA，以紫外检测器检测BZE和BA在230nm波长的吸收，获得BZE和BA的线性标准曲线。CocE催化BZE反应的活性测定，反应温度为25℃，每组三个重复。通过将50μL底物BZE溶液与50μL酶溶液(0.1M磷酸盐缓冲液(pH7.4)稀释)来启动酶促反应。反应具体条件如表2所示。
表2高活性突变体酶对底物BZE体外催化反应条件
然后加入50μL 10％高氯酸终止反应，加入50μL的乙腈，将混合物以12000rpm离心5分钟，然后将上清液稀释至合适的倍数进样100μL。将BZE和BA出峰时间、峰面积和标曲曲线进行对比，计算反应样品中BZE残留浓度和BA生成浓度。通过计算不同底物浓度下酶催化生成BA的反应速率，使用GraphPad Prism 8绘制酶反应动力学曲线，进行Michaelis-Menten动力学分析，即可获得各突变体的k
cat和K
M。结果如表3所示。
表3高活性突变体酶对底物BZE催化动力学参数
备注：K
eff指相应酶对于底物BZE的催化效率(k
cat/K
M)。
RCE指突变体酶对BZE的催化效率与野生型酶对BZE的催化效率的比值。
实施例4：动物体内实验
实验用雄性SD大鼠(～200g/只)购自浙江省医学科学院实验动物中心，饲养于恒温恒湿的环境。饲养和实验使用均按照《实验动物饲养管理和使用指南》。
(1)BA和BZE血样标准曲线
使用采血针和肝素处理的毛细血管从大鼠股静脉采血。首先取8管来自于同一只 大鼠的血液，每管75μL，将每管血液加入到100μL 250μΜ对氧磷(paraxon)以抑制内源性代谢酶对BZE的影响，-80℃冻存。解冻后加入19.7μL含0、4、10、20、40、60、100、200μΜBA和BE标准品混合液，然后涡旋20s，后加入150μL乙腈，涡旋1min，再加入50μL 10％HClO
4，涡旋1min，17000rcf离心15min，转移上清再次离心，取约250μL上清液进HPLC分析，进样量为100μL，HPLC实验条件与实施例3相同。最终标准样品BZE和BA浓度分别为0、0.2、0.5、1、2、3、5、10μΜ。
(2)CocE高活性突变体加速体内BZE的代谢
每组5只大鼠。首先通过大鼠尾静脉注射0.2或1mg/kg的高活性CocE突变体或生理盐水，注射1分钟内尾静脉注射2mg/kg BZE。BZE注射后分别取0、2、5、10、30、60、90、120min的血样75μL，各加入100μL 250μΜ对氧磷，-80℃冻存。解冻后涡旋20s，后加入150μL乙腈，涡旋1min，再加入50μL 10％HClO
4，涡旋1min，17000g离心15min，转移上清再次离心，取上清液约250μL进HPLC分析，进样量为100μL。根据标准品的血样标准曲线计算各组大鼠不同时间点血液中BA和BZE的浓度，从而获得BZE在有无高活性CocE突变体存在时的代谢情况。
结果如图3所示，结果显示，2mg/kg BZE静脉注射大鼠体内，2min时血液中BZA浓度高达15.8μM，血液中BA浓度<0.5μM。注射高活性突变体时，大鼠血液中BZA浓度快速下降而血液中BA浓度迅速升高，表明大鼠体内有毒的BZE被高活性突变体快速清除，代谢成无毒的BA。
Claims (10)
- 可卡因酯酶突变体，其特征在于，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以下一种：(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。
- 如权利要求1所述可卡因酯酶突变体在制备用于治疗可卡因中毒的药物中的应用。
- 如权利要求1所述可卡因酯酶突变体在制备用于水解苯甲酰苯甲酰芽子碱为芽子碱和苯甲酸的水解剂中的应用。
- 如权利要求1所述可卡因酯酶突变体在治理水体或土壤中可卡因或苯甲酰芽子碱污染中的应用。
- 一种用于治疗可卡因中毒的药物，其特征在于，活性成分为权利要求1所述可卡因酯酶突变体。
- 一种用于治理水体或土壤中可卡因或苯甲酰芽子碱污染的试剂，其特征在于，有效成分为权利要求1所述可卡因酯酶突变体。
- 编码如权利要求1所述可卡因酯酶突变体的基因。
- 包含如权利要求7所述基因的表达载体。
- 包含如权利要求8所述表达载体的重组表达细胞。
- 如权利要求7所述基因在治理水体或土壤中可卡因或苯甲酰芽子碱污染中的应用。
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