CN113735282B - Old yellow enzyme OYE2 protein and application thereof in chromium pollution - Google Patents
Old yellow enzyme OYE2 protein and application thereof in chromium pollution Download PDFInfo
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- CN113735282B CN113735282B CN202111103840.7A CN202111103840A CN113735282B CN 113735282 B CN113735282 B CN 113735282B CN 202111103840 A CN202111103840 A CN 202111103840A CN 113735282 B CN113735282 B CN 113735282B
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- hexavalent chromium
- reductase
- cyc1
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- protein
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- 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
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Abstract
本发明公开了一种老黄酶OYE2蛋白及其在铬污染中的应用,属于重金属防治领域。本发明以Corynebacterium crenatum SYPA 5‑5中的老黄酶OYE2蛋白作为还原Cr(VI)的有效成分,其具有六价铬还原酶的活性,该铬还原酶对Cr(VI)具有较强的还原作用,能快速去除废水中Cr(VI)的作用。采用本发明的方法,在未添加本发明的六价铬还原酶的废水中,Cr(VI)浓度未发生明显变化,加入本发明的六价铬还原酶的废水中Cr(VI)浓度降到62mg/L,还原率达38%,能快速有效地去除废水中的六价铬。因此,利用这类酶中的一种或多种制成六价铬还原酶处理含Cr(VI)废水具有工业化应用潜力。
The invention discloses an old yellow enzyme OYE2 protein and its application in chromium pollution, belonging to the field of heavy metal prevention and control. The present invention uses the old yellow enzyme OYE2 protein in Corynebacterium crenatum SYPA 5‑5 as an active ingredient for reducing Cr(VI), which has the activity of hexavalent chromium reductase, and the chromium reductase has a strong reduction effect on Cr(VI). It can quickly remove Cr(VI) in wastewater. Adopt method of the present invention, in the waste water that does not add hexavalent chromium reductase of the present invention, Cr (VI) concentration does not change significantly, adds Cr (VI) concentration in the waste water of hexavalent chromium reductase of the present invention and drops to 62mg/L, with a reduction rate of 38%, it can quickly and effectively remove hexavalent chromium in wastewater. Therefore, using one or more of these enzymes to prepare hexavalent chromium reductase to treat Cr(VI)-containing wastewater has potential for industrial application.
Description
技术领域technical field
本发明涉及一种老黄酶OYE2蛋白及其在铬污染中的应用,属于重金属防治领域。The invention relates to an old yellow enzyme OYE2 protein and its application in chromium pollution, belonging to the field of heavy metal prevention and control.
背景技术Background technique
土壤和地下水中的铬(Cr)污染是国际上最关注的环境问题之一,其主要来源于铬盐生产、电镀、鞣革、颜料生产、采矿和冶金等行业。铬在环境中存在两种稳定的铬氧化态:六价铬和三价铬;与六价铬Cr(VI)相比,三价铬Cr(III)溶解性低、迁移能力弱和生物毒性低。因此,水中铬污染治理的有效策略之一就是将六价铬Cr(VI)还原成三价铬Cr(III)。其中微生物还原六价铬Cr(VI)被看作是一种具有很大应用潜力的方法,但微生物修复过程仍存在周期较长的缺点,且利用微生物进行原位修复时受环境因子影响大,与土著微生物存在相互竞争,造成修复效果不稳定。Chromium (Cr) pollution in soil and groundwater is one of the most concerned environmental problems in the world, and it mainly comes from industries such as chromium salt production, electroplating, leather tanning, pigment production, mining and metallurgy. Chromium exists in two stable chromium oxidation states in the environment: hexavalent chromium and trivalent chromium; compared with hexavalent chromium Cr(VI), trivalent chromium Cr(III) has low solubility, weak migration ability and low biological toxicity . Therefore, one of the effective strategies for the treatment of chromium pollution in water is to reduce hexavalent chromium Cr(VI) to trivalent chromium Cr(III). Among them, microbial reduction of hexavalent chromium Cr(VI) is regarded as a method with great application potential, but the microbial remediation process still has the disadvantage of a long cycle, and the use of microorganisms for in-situ remediation is greatly affected by environmental factors. There is mutual competition with indigenous microorganisms, resulting in unstable restoration effect.
目前有大量的研究报道利用六价铬还原酶修复土壤和治理污染地下水,如利用漆酶修复DDT污染的土壤、土壤酶修复铅污染土壤、固定化酶修复被农药污染的土壤、硝化细菌产生的酶对地下水进行脱氮处理等。对于Cr(VI)污染,有大量研究报道了Cr(VI)还原酶,如来源于Escherichia coli的NfsA和YieF酶,其最大还原速率分别为0.25和5.0μmol/min/mg;Pseudomonas putida KT2400中的ChrR其最大还原速率为8.8μmol/min/mg。这些蛋白酶均能在一定条件下实现Cr(VI)的有效还原,但是离大规模工业应用尚有较大差距。因此,急需一种高效治理水体中的铬污染的方法,以满足大规模工业应用。At present, there are a large number of research reports on the use of hexavalent chromium reductase to repair soil and control polluted groundwater, such as using laccase to repair DDT-contaminated soil, soil enzymes to repair lead-contaminated soil, immobilized enzymes to repair soil contaminated by pesticides, and nitrifying bacteria. Enzyme denitrification treatment of groundwater, etc. For Cr(VI) contamination, a large number of studies have reported Cr(VI) reductases, such as NfsA and YieF enzymes from Escherichia coli, whose maximum reduction rates are 0.25 and 5.0 μmol/min/mg, respectively; Pseudomonas putida KT2400 The maximum reduction rate of ChrR is 8.8μmol/min/mg. All these proteases can effectively reduce Cr(VI) under certain conditions, but there is still a big gap from large-scale industrial application. Therefore, there is an urgent need for a method for efficiently controlling chromium pollution in water to meet large-scale industrial applications.
发明内容Contents of the invention
为了得到一种高效治理水体中铬污染的方法,本发明提供了一种六价铬还原酶及其在治理水体铬污染中的应用。所述应用,是指向六价铬污染水体中投放所述六价铬还原酶,将高水溶性的六价铬还原成低水溶性的三价铬,通过降低其迁移性达到治理目的。In order to obtain a method for efficiently treating chromium pollution in water bodies, the invention provides a hexavalent chromium reductase and its application in treating chromium pollution in water bodies. The application refers to putting the hexavalent chromium reductase into the hexavalent chromium polluted water body to reduce the highly water-soluble hexavalent chromium to the low water-soluble trivalent chromium, and achieve the purpose of treatment by reducing its mobility.
本发明提供了一种酶法降解六价铬的方法,所述方法为,将氨基酸序列如SEQ IDNO.1所示的六价铬还原酶添加至含有六价铬的反应体系当中进行降解。The invention provides a method for enzymatically degrading hexavalent chromium. The method includes adding hexavalent chromium reductase with the amino acid sequence shown in SEQ ID NO.1 to a reaction system containing hexavalent chromium for degradation.
在本发明的一种实施方式中,编码所述六价铬还原酶的核苷酸序列如SEQ IDNO.2所示。其在GenBank中注释结果为核黄素氧化还原酶/NADH氧化酶,该蛋白属于OYE2家族成员,本发明将其命名为Cyc1。In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2. Its annotated result in GenBank is riboflavin oxidoreductase/NADH oxidase, and this protein belongs to the OYE2 family member, which is named Cyc1 in the present invention.
在本发明的一种实施方式中,所述六价铬还原酶来源于Corynebacteriumcrenatum SYPA5-5。In one embodiment of the present invention, the hexavalent chromium reductase is derived from Corynebacterium crenatum SYPA5-5.
在本发明的一种实施方式中,反应体系中,所述六价铬还原酶的添加量至少为0.1U/mg六价铬Cr(VI)。In one embodiment of the present invention, in the reaction system, the added amount of the hexavalent chromium reductase is at least 0.1 U/mg hexavalent chromium Cr(VI).
在本发明的一种实施方式中,所述六价铬还原酶的投放量至少为0.1U/mg六价铬Cr(VI)。In one embodiment of the present invention, the dosage of the hexavalent chromium reductase is at least 0.1 U/mg hexavalent chromium Cr(VI).
在本发明的一种实施方式中,当污染水体中的铬浓度≤100mg/L时,六价铬还原酶的投放量为0.1U/mg Cr(VI)。根据动力学最大速度Vmax=30.39umol Cr(VI)/min/mg酶蛋白折算。In one embodiment of the present invention, when the chromium concentration in the polluted water body is ≤100 mg/L, the dose of hexavalent chromium reductase is 0.1 U/mg Cr(VI). Calculated according to the kinetic maximum velocity Vmax=30.39umol Cr(VI)/min/mg enzyme protein.
在本发明的一种实施方式中,降解反应条件为:在pH 6~12的条件下进行。In one embodiment of the present invention, the degradation reaction condition is: carried out under the condition of pH 6-12.
在本发明的一种实施方式中,所述酶的最适作用pH值为8.5。In one embodiment of the present invention, the optimal pH value of the enzyme is 8.5.
在本发明的一种实施方式中,降解反应在25-40℃的温度下进行。In one embodiment of the present invention, the degradation reaction is carried out at a temperature of 25-40°C.
在本发明的一种实施方式中,所述六价铬还原酶的最适作用温度为30℃。In one embodiment of the present invention, the optimum action temperature of the hexavalent chromium reductase is 30°C.
本发明还提供了一种重组大肠杆菌,所述重组大肠杆菌含有上述氨基酸序列如SEQ ID NO.1所示的六价铬还原酶。The present invention also provides a recombinant Escherichia coli containing the hexavalent chromium reductase whose amino acid sequence is shown in SEQ ID NO.1.
在本发明的一种实施方式中,编码所述六价铬还原酶的核苷酸序列如SEQ IDNO.2所示。In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2.
在本发明的一种实施方式中,所述重组大肠杆菌以E.coli BL21(DE3)为表达宿主,以pXMJ-19为表达载体。In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host and pXMJ-19 as the expression vector.
本发明还提供了一种全细胞降解六价铬的方法,所述方法为,将含有氨基酸序列如SEQ ID NO.1所示的六价铬还原酶的微生物细胞添加至含有六价铬的反应体系当中进行降解。The present invention also provides a method for degrading hexavalent chromium by whole cells, the method is: adding the microbial cell containing the hexavalent chromium reductase as shown in SEQ ID NO.1 to the reaction containing hexavalent chromium degradation in the system.
在本发明的一种实施方式中,所述反应体系为污水环境。In one embodiment of the present invention, the reaction system is a sewage environment.
在本发明的一种实施方式中,编码所述六价铬还原酶的核苷酸序列如SEQ IDNO.2所示。In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2.
在本发明的一种实施方式中,所述微生物细胞是重组大肠杆菌。In one embodiment of the present invention, the microbial cell is recombinant Escherichia coli.
在本发明的一种实施方式中,所述重组大肠杆菌以E.coli BL21(DE3)为表达宿主,以pXMJ-19为表达载体。In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as the expression host and pXMJ-19 as the expression vector.
在本发明的一种实施方式中,所述重组大肠杆菌在反应体系中的添加量至少为1%(v/v)。In one embodiment of the present invention, the amount of the recombinant Escherichia coli added to the reaction system is at least 1% (v/v).
本发明还提供了一种提高大肠杆菌对六价铬的降解能力的方法,所述方法为,在大肠杆菌中表达氨基酸序列如SEQ ID NO.1所示的六价铬还原酶。The present invention also provides a method for improving the degradation ability of Escherichia coli to hexavalent chromium, the method is, expressing the hexavalent chromium reductase in Escherichia coli whose amino acid sequence is shown as SEQ ID NO.1.
在本发明的一种实施方式中,编码所述六价铬还原酶的核苷酸序列如SEQ IDNO.2所示。In one embodiment of the present invention, the nucleotide sequence encoding the hexavalent chromium reductase is shown in SEQ ID NO.2.
本发明还提供了氨基酸序列如SEQ ID NO.1所示的六价铬还原酶,或含有核苷酸序列如SEQ ID NO.2所示的六价铬还原酶的重组载体,或含有氨基酸序列如SEQ ID NO.1所示的六价铬还原酶的微生物细胞在制备降解六价铬的产品中的应用。The present invention also provides a hexavalent chromium reductase with an amino acid sequence as shown in SEQ ID NO.1, or a recombinant vector containing a hexavalent chromium reductase with a nucleotide sequence as shown in SEQ ID NO.2, or with an amino acid sequence Application of the microbial cell of hexavalent chromium reductase shown in SEQ ID NO.1 in the preparation of products degrading hexavalent chromium.
在本发明的一种实施方式中,所述重组载体是以pET-28a、pDXW-10、pXMJ-19为表达载体。In one embodiment of the present invention, the recombinant vectors use pET-28a, pDXW-10, pXMJ-19 as expression vectors.
在本发明的一种实施方式中,所述微生物细胞以细菌或真菌为宿主细胞。In one embodiment of the present invention, the microbial cells use bacteria or fungi as host cells.
在本发明的一种实施方式中,所述微生物细胞是以E.coli BL21、E.coli JM109、Corynebacterium crenatum SYPA5-5或Corynebacterium ATCC 13032为宿主细胞。In one embodiment of the present invention, the microbial cells are E. coli BL21, E. coli JM109, Corynebacterium crenatum SYPA5-5 or Corynebacterium ATCC 13032 as host cells.
在本发明的一种实施方式中,所述产品为化学品。In one embodiment of the invention, the product is a chemical.
本发明还提供一种能快速去除废水中Cr(VI)的六价铬还原酶的制备方法,将上述六价铬还原酶基因Cyc1构建到表达载体上,并利用原核表达系统制备高活性的铬还原酶Cyc1。The present invention also provides a preparation method of hexavalent chromium reductase that can quickly remove Cr(VI) in wastewater, construct the above-mentioned hexavalent chromium reductase gene Cyc1 on the expression vector, and use the prokaryotic expression system to prepare highly active chromium Reductase Cyc1.
在本发明的一种实施方式中,以Corynebacterium crenatum SYPA 5-5基因组为模板,设计如下引物,通过PCR扩增获得铬还原酶基因Cyc1的核酸序列(如SEQ ID NO.2所示),将核酸序列构建到pXMJ-19载体上,并转化BL21大肠杆感受态细胞,所得重组菌经诱导表达,分离纯化得到目的蛋白。所涉及的引物序列如下:In one embodiment of the present invention, the Corynebacterium crenatum SYPA 5-5 genome is used as a template, the following primers are designed, and the nucleic acid sequence (as shown in SEQ ID NO.2) of the chromium reductase gene Cyc1 is obtained by PCR amplification. The nucleic acid sequence is constructed on the pXMJ-19 vector, and transformed into BL21 Escherichia coli competent cells, the obtained recombinant bacteria are induced to express, and the target protein is obtained by separation and purification. The primer sequences involved are as follows:
F:5’-AGCTTAAAGGAGGACAACCGTGCACCACCACCACCACCACCTCCAAGCTGT-3’F: 5'-AGCTTAAAGGAGGACAACCGTGCACCACCACCACCACCACCTCCAAGCTGT-3'
R:5’-AGCCAAGTGAATTCTTAAAGTACGTAGTC-3’。R: 5'-AGCCAAGTGAATTCTTAAAAGTACGTAGTC-3'.
有益效果Beneficial effect
(1)本发明提供的来源于Corynebacterium crenatum SYPA5-5的铬还原酶对Cr(VI)具有较强的还原作用,能快速去除废水中的Cr(VI)。(1) The chromium reductase derived from Corynebacterium crenatum SYPA5-5 provided by the present invention has a strong reducing effect on Cr(VI), and can quickly remove Cr(VI) in wastewater.
(2)采用本发明的方法,在未添加本发明的六价铬还原酶的废水中,Cr(VI)浓度未发生明显变化,加入本发明的六价铬还原酶的废水中Cr(VI)浓度降到62mg/L,还原率达38%,能快速有效地去除废水中的六价铬。因此,利用这类酶中的一种或多种制成六价铬还原酶处理含Cr(VI)废水具有工业化应用潜力。(2) Adopt the method of the present invention, in the waste water that does not add hexavalent chromium reductase of the present invention, Cr (VI) concentration does not change obviously, add Cr (VI) in the waste water of hexavalent chromium reductase of the present invention The concentration drops to 62mg/L, and the reduction rate reaches 38%, which can quickly and effectively remove hexavalent chromium in wastewater. Therefore, using one or more of these enzymes to prepare hexavalent chromium reductase to treat Cr(VI)-containing wastewater has potential for industrial application.
附图说明Description of drawings
图1:本发明实施例1中Cr(VI)还原基因Cyc1的PCR扩增结果电泳图谱;其中,M:核酸Marker;泳道1、2、3和4:Cyc1PCR扩增产物。Figure 1: Electrophoretic pattern of PCR amplification results of Cr(VI) reduced gene Cyc1 in Example 1 of the present invention; wherein, M: nucleic acid marker;
图2:本发明实施例1中表达载体pXMJ-19双酶切结果电泳图;其中,M:核酸Marker;泳道1、2:pXMJ-19双酶切(EcoR I和Hind III)。Figure 2: Electropherogram of the result of double digestion of expression vector pXMJ-19 in Example 1 of the present invention; wherein, M: nucleic acid marker;
图3:本发明实施例2中铬还原酶Cyc1粗酶液的SDS-PAGE图;其中,M:蛋白Marker;泳道1:pXMJ19空载对照;泳道2:15℃诱导Cyc1蛋白表达;泳道3:20℃诱导Cyc2蛋白表达;泳道4:25℃诱导Cyc1蛋白表达;泳道5:30℃诱导Cyc1蛋白表达。Figure 3: SDS-PAGE image of the crude enzyme solution of chromium reductase Cyc1 in Example 2 of the present invention; wherein, M: protein marker; lane 1: pXMJ19 empty control; lane 2: Cyc1 protein expression induced at 15°C; lane 3: Cyc2 protein expression was induced at 20°C; Swimming lane 4: Cyc1 protein expression was induced at 25°C; Swimming lane 5: Cyc1 protein expression was induced at 30°C.
图4:本发明实施例2中铬还原酶Cyc1纯化后的SDS-PAGE图;其中,M:蛋白Marker;泳道1:30℃诱导Cyc1蛋白表达;泳道2:25℃诱导Cyc2蛋白表达;泳道3:20℃诱导Cyc1蛋白表达。Figure 4: SDS-PAGE image of purified chromium reductase Cyc1 in Example 2 of the present invention; among them, M: protein marker; lane 1: Cyc1 protein expression induced at 30°C; lane 2: Cyc2 protein expression induced at 25°C; lane 3 : Cyc1 protein expression was induced at 20°C.
图5:本发明实施例3中温度对六价铬还原酶活性的影响。Figure 5: Effect of temperature on the activity of hexavalent chromium reductase in Example 3 of the present invention.
图6:本发明实施例3中pH值对六价铬还原酶活性的影响。Fig. 6: Effect of pH value on activity of hexavalent chromium reductase in Example 3 of the present invention.
图7:本发明实施例3中不同金属离子对六价铬还原酶活性的影响。Figure 7: Effects of different metal ions on the activity of hexavalent chromium reductase in Example 3 of the present invention.
图8:本发明实施例3中不同浓度的Cr(VI)对六价铬还原酶活性的影响。Fig. 8: Effects of different concentrations of Cr(VI) on the activity of hexavalent chromium reductase in Example 3 of the present invention.
图9:本发明实施例3中重组菌对Cr(VI)的降解情况。Figure 9: Degradation of Cr(VI) by recombinant bacteria in Example 3 of the present invention.
图10:本发明实施例4中六价铬还原酶对含Cr(VI)废水的还原情况。Figure 10: Reduction of Cr(VI)-containing wastewater by hexavalent chromium reductase in Example 4 of the present invention.
具体实施方式Detailed ways
Corynebacterium crenatum SYPA 5-5是本实验室选育的一株L-精氨酸高产菌,钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5记载于公开号为CN1441055A的专利申请文本中,保藏编号为CGMCC NO.0890(此菌株在专利申请文本中的菌株编号为SDNN403,发明人在实验过程中将其重新编号为SYPA5-5);基于前期转录组和蛋白组数据分析,发现六价铬还原酶(Cyc1)在高溶氧相对于低溶氧条件下表达水平上调。为了进一步研究Cyc1的抗氧化胁迫功能,于是构建了Cyc1过表达菌株和敲除菌,发现Cyc1蛋白具有极强的氧化还原性质。以Cyc1为研究对象,分析Cyc1的圆二色谱结构,用SWISS-MODEL建模分析,检测其对Cr(VI)的还原能力,验证了Cyc1的结构与功能。该Cyc1蛋白对Cr(VI)具有较强的还原作用,能快速去除废水中Cr(VI)的作用,在含有Cr(VI)废水处理中具有重要的应用价值。Corynebacterium crenatum SYPA 5-5 is a strain of L-arginine high-yielding bacteria selected by our laboratory. Corynebacterium crenatum SYPA5-5 is recorded in the patent application text with publication number CN1441055A, and the preservation number is CGMCC NO.0890 (the strain number of this strain in the patent application text is SDNN403, and the inventor renumbered it as SYPA5-5 during the experiment); based on the analysis of the previous transcriptome and proteome data, it was found that hexavalent chromium reductase ( Cyc1) was up-regulated under high dissolved oxygen relative to low dissolved oxygen conditions. In order to further study the anti-oxidative stress function of Cyc1, a Cyc1 overexpression strain and a knockout strain were constructed, and it was found that the Cyc1 protein has extremely strong redox properties. Taking Cyc1 as the research object, the circular dichroism structure of Cyc1 was analyzed, and the SWISS-MODEL modeling analysis was used to detect its ability to reduce Cr(VI), and the structure and function of Cyc1 were verified. The Cyc1 protein has a strong reducing effect on Cr(VI), can quickly remove Cr(VI) in wastewater, and has important application value in the treatment of Cr(VI)-containing wastewater.
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(SambrookJ&RussellDW,MolecularCloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.
下述实施例中所涉及的培养基如下:The medium involved in the following examples is as follows:
LB液体培养基(g/L):5酵母粉、10胰蛋白胨、10氯化钠。LB liquid medium (g/L): 5 yeast powder, 10 tryptone, 10 sodium chloride.
LB固体培养基:在LB液体培养基的基础上,添加1.5~2.0%的琼脂粉。LB solid medium: on the basis of LB liquid medium, add 1.5-2.0% agar powder.
下述实施例中所涉及的检测方法如下:The detection methods involved in the following examples are as follows:
蛋白浓度的检测:Detection of protein concentration:
利用Bradford试剂盒测定蛋白浓度,以牛血清蛋白作为标准品。The protein concentration was determined using a Bradford kit, with bovine serum albumin as a standard.
Cr(VI)浓度的检测:Detection of Cr(VI) concentration:
二苯碳酰二肼法,抽取待测样品0.3ml,添加2.5ml 0.12M的浓硫酸溶液,向上述混合物中加入体积为0.2ml二苯碳酰二肼(丙酮溶解),使其最终浓度达到0.4mM,在543nm吸光度下检测。Diphenylcarbazide method, extract 0.3ml of the sample to be tested, add 2.5ml 0.12M concentrated sulfuric acid solution, add a volume of 0.2ml diphenylcarbazide (dissolved in acetone) to the above mixture, and make its final concentration reach 0.4mM, detected at 543nm absorbance.
下述实施例中所涉及的基因组DNA和相关质粒的提取方法:The extraction method of genomic DNA involved in the following examples and related plasmids:
Corynebacterium crenatum SYPA5-5提取基因组DNA时根据上海捷瑞细菌基因组DNA快速提取试剂盒说明书操作。质粒提取时将培养适当浓度的菌液离心后去除上清液,谷氨酸棒杆菌为革兰氏阳性菌,其重组菌株提取质粒时,需要添加适量的溶菌酶于37℃水浴0.5h,大肠杆菌的重组菌株提取质粒时无需加入溶菌酶,采用捷瑞小型质粒提取试剂盒提取,详细操作参照说明书。Corynebacterium crenatum SYPA5-5 was used to extract genomic DNA according to the instructions of the Shanghai Jierui Bacteria Genomic DNA Rapid Extraction Kit. When extracting the plasmid, centrifuge the cultured bacterial solution with an appropriate concentration and remove the supernatant. Corynebacterium glutamicum is a Gram-positive bacterium. When extracting the plasmid from its recombinant strain, it is necessary to add an appropriate amount of lysozyme and put it in a water bath at 37°C for 0.5h. There is no need to add lysozyme when extracting the plasmid from the recombinant strain of Bacillus, and it is extracted with the Jierui Small Plasmid Extraction Kit, and the detailed operation refers to the instruction manual.
下述实施例中所涉及的大肠杆菌感受态制备与化学转化法如下:The Escherichia coli competent preparation and chemical transformation method involved in the following examples are as follows:
制备大肠杆菌化转感受态细胞,采用TakaRa公司的感受态细胞制备试剂盒Competent Cell Preparation Kit,详细操作参照说明书。采用42℃热击化转至E.coliBL21,经过抗生素抗性平板筛选获得阳性转化子,提取质粒进行PCR验证及送至金唯智生物科技公司测序验证。To prepare Escherichia coli transfected competent cells, the Competent Cell Preparation Kit from TakaRa Company was used, and the detailed operation was referred to the instruction manual. Transformed into E.coliBL21 by heat shock at 42°C, positive transformants were obtained through antibiotic resistance plate screening, the plasmid was extracted for PCR verification and sent to Jinweizhi Biotechnology Company for sequencing verification.
实施例1:构建可表达Cr(VI)还原酶基因Cyc1的基因工程菌Embodiment 1: Construction can express the genetically engineered bacterium of Cr (VI) reductase gene Cyc1
1、Cyc1基因的克隆1. Cloning of Cyc1 gene
目的基因(核苷酸序列如SEQ ID NO.2所示)通过采用高保真酶PhantaR Max(p515)DNA polymerases进行PCR扩增,PCR体系为50μL的标准反应体系,PCR扩增程序为:95℃预变性5min;95℃变性30s;退火温度一般设置为58~60℃,30~60s;72℃延伸按照每分钟扩增1500bp基因设置时间;变性至延伸程序30个循环;72℃再延伸5min;4℃保存。PCR结束后将PCR产物进行核酸电泳,进行验证及胶回收纯化,结果如图1所示。回收后与线性化载体表达载体pXMJ-19进行双酶切,结果如图2所示;使用ClonExpress II One Step CloningKit连接后化转E.coli BL21(DE3)感受态细胞,挑取阳性转化子提取质粒进行PCR验证及测序验证。The target gene (the nucleotide sequence is shown in SEQ ID NO.2) is amplified by PCR using the high-fidelity enzyme PhantaR Max (p515) DNA polymerases. The PCR system is a 50 μL standard reaction system. The PCR amplification program is: 95°C Pre-denaturation for 5 minutes; denaturation at 95°C for 30s; annealing temperature is generally set at 58-60°C, 30-60s; extension at 72°C is set according to the amplification of 1500bp genes per minute; denaturation to extension program is 30 cycles; 72°C is extended for 5 minutes; Store at 4°C. After PCR, the PCR products were subjected to nucleic acid electrophoresis for verification and gel recovery and purification. The results are shown in Figure 1. After recovery, double enzyme digestion was performed with the linearized vector expression vector pXMJ-19, the result is shown in Figure 2; after connection with ClonExpress II One Step Cloning Kit, the E.coli BL21 (DE3) competent cells were transformed, and the positive transformants were picked and extracted Plasmids were verified by PCR and sequencing.
制备得到可表达六价铬还原酶Cyc1的基因工程菌E.coli BL21(DE3)/pXMJ-19-Cyc1。The genetically engineered strain E.coli BL21(DE3)/pXMJ-19-Cyc1 expressing hexavalent chromium reductase Cyc1 was prepared.
将空载pXMJ-19质粒按照上述方法导入大肠杆菌E.coli BL21(DE3)感受态细胞中,制备得到对照菌株E.coli BL21(DE3)/pXMJ-19。The empty pXMJ-19 plasmid was introduced into Escherichia coli E. coli BL21 (DE3) competent cells according to the above method, and the control strain E. coli BL21 (DE3)/pXMJ-19 was prepared.
实施例2:六价铬还原酶Cyc1蛋白的表达和纯化Example 2: Expression and purification of hexavalent chromium reductase Cyc1 protein
1、六价铬还原酶Cyc1蛋白的诱导表达1. Induced expression of hexavalent chromium reductase Cyc1 protein
将实施例1制备得到的基因工程菌E.coli BL21(DE3)/pXMJ-19-Cyc1、E.coliBL21(DE3)/pXMJ-19对照菌株分别划线至含有ChlR的LB固体培养基上,在37℃、12h的条件下进行活化后,挑取单菌落,分别接入10mL含有ChlR的LB液体培养基小瓶中,在30℃、180rpm培养16~24h后,分别制备得到种子液;The genetically engineered bacteria E.coli BL21(DE3)/pXMJ-19-Cyc1 and E.coliBL21(DE3)/pXMJ-19 control strains prepared in Example 1 were respectively streaked onto the LB solid medium containing Chl R , After activation at 37°C and 12h, single colonies were picked and inserted into 10mL vials of LB liquid medium containing Chl R , cultured at 30°C and 180rpm for 16-24h, and seed solutions were prepared respectively;
将制备得到的种子液按1%(v/v)接种量转接至4瓶50mL含有ChlR的LB液体培养基中,分别在30℃、180rpm培养4~5h后,添加异丙基β-D-1-硫代半乳糖苷(IPTG)至终浓度为1mmol·L-1,分别在15℃、20℃、25℃、30℃,180rpm条件下诱导表达16~18h后,制备得到不同温度诱导下的发酵液;The prepared seed solution was transferred to 4 bottles of 50mL LB liquid medium containing Chl R according to the inoculation amount of 1% (v/v). After culturing at 30°C and 180rpm for 4-5h, isopropyl β- D-1-thiogalactoside (IPTG) to a final concentration of 1mmol·L -1 , induced expression at 15°C, 20°C, 25°C, 30°C, 180rpm for 16-18h, prepared at different temperatures induced fermentation broth;
分别将制备得到的发酵液在4℃、8000rpm条件下离心10min收集菌体细胞。The prepared fermentation broth was centrifuged at 4° C. and 8000 rpm for 10 min to collect bacterial cells.
分别将收集的细胞采用0.1mol·L-1的PBS缓冲液(pH 7.4)洗涤两次后,重新悬浮于PBS缓冲液(pH 7.4)中,控制菌体浓度(菌体浓度为:OD600=6.0)一致的情况下,采用超声破碎仪破碎,破碎液4℃下离心20min,收集上清,SDS-PAGE分析蛋白表达情况,结果如图3所示,从图3可知,六价铬还原酶Cyc1蛋白在30℃诱导表达效果最佳;剩余粗酶液用于后续的酶活测定。The collected cells were washed twice with 0.1 mol·
制备得到含有不同温度诱导表达下的六价铬还原酶Cyc1的粗酶液。The crude enzyme solution containing the hexavalent chromium reductase Cyc1 induced and expressed at different temperatures was prepared.
2、六价铬还原酶Cyc1蛋白的纯化2. Purification of hexavalent chromium reductase Cyc1 protein
分别将步骤1制备得到的含有六价铬还原酶Cyc1的粗酶液采用0.45μm滤膜过滤后,采用Ni-NTA蛋白纯化柱进行纯化去除杂蛋白,重组酶Cyc1末端带有his标签能够与纯化柱上的Ni+进行鳌合,梯度洗脱,采用不同浓度的咪唑缓冲液梯度洗脱,结合于纯化柱上的纯化蛋白被洗脱下来,进而达到纯化酶。然后将Cyc1纯酶液做SDS-PAGE分析,如图4所示,由图4可知,六价铬还原酶Cyc1蛋白在30℃诱导表达效果最佳。The crude enzyme solution containing hexavalent chromium reductase Cyc1 prepared in
利用Bradford试剂盒测定30℃诱导表达下的六价铬还原酶Cyc1蛋白浓度,以牛血清蛋白作为标准品。The concentration of hexavalent chromium reductase Cyc1 protein under induction expression at 30°C was determined using Bradford kit, with bovine serum albumin as the standard.
六价铬还原酶Cyc1纯酶的比酶活数据在含有在20mM MOPS—NaOH缓冲液中其中包含100mM NAD(P)H、10mM FMN、1mM CrO3和10mM CaCl2,加入50μL Cyc1纯酶,在30℃、PH=8.5的条件下反应1min,通过二苯碳酰二肼法检测Cr(VI)含量,结果如表1所示。The specific enzyme activity data of hexavalent chromium reductase Cyc1 pure enzyme was contained in 20mM MOPS-NaOH buffer, which contained 100mM NAD(P)H, 10mM FMN, 1mM CrO 3 and 10mM CaCl 2 , added 50μL Cyc1 pure enzyme, in The reaction was carried out at 30° C. and pH=8.5 for 1 min, and the Cr(VI) content was detected by the diphenylcarbazide method. The results are shown in Table 1.
表1 Cyc1纯酶液在不同配体下对Cr(VI)还原的比酶活(U/mg)Table 1 Specific enzyme activity of Cyc1 pure enzyme solution for Cr(VI) reduction under different ligands (U/mg)
实施例3:六价铬还原酶Cyc1纯酶的活性分析Embodiment 3: Activity analysis of pure enzyme of hexavalent chromium reductase Cyc1
下述实验采用的是实施例2的步骤2得到的30℃条件下诱导菌株表达后经纯化的Cyc1纯化酶。The following experiments used the purified Cyc1 enzyme obtained in
1、温度对六价铬还原酶Cyc1纯酶的活性影响1. Effect of temperature on the activity of pure enzyme of hexavalent chromium reductase Cyc1
将六价铬还原酶Cyc1纯酶分别在25、27、30、33、35、37、40、42、45℃处理30min,并取样检测六价铬还原酶的活性。The pure enzyme of hexavalent chromium reductase Cyc1 was treated at 25, 27, 30, 33, 35, 37, 40, 42 and 45°C for 30 minutes, and samples were taken to detect the activity of hexavalent chromium reductase.
检测方法为:在20mM MOPS—NaOH缓冲液中,所述缓冲液中包含100mM NAD(P)H、10mM FMN、1mM CrO3和10mM CaCl2,加入50μL Cyc1纯酶,PH=8.5的条件反应1min,通过二苯碳酰二肼法检测Cr(VI)含量。The detection method is: in 20mM MOPS-NaOH buffer, the buffer contains 100mM NAD(P)H, 10mM FMN, 1mM CrO 3 and 10mM CaCl 2 , add 50μL of Cyc1 pure enzyme, and react for 1min under the condition of pH=8.5 , Cr(VI) content was detected by the diphenylcarbazide method.
结果如图5所示,六价铬还原酶Cyc1纯酶在30℃时具有最大的活性,27℃及33℃时有比较高的活性,分别为最大活性的83%和84%;25℃和42℃时其活性为最大活性的40%和32%;由此可知,六价铬还原酶在较宽的温度范围内均具有活性。The results are shown in Figure 5, the pure enzyme of hexavalent chromium reductase Cyc1 has the maximum activity at 30°C, and has relatively high activity at 27°C and 33°C, which are 83% and 84% of the maximum activity respectively; At 42°C, its activity is 40% and 32% of the maximum activity; thus, it can be seen that the hexavalent chromium reductase has activity in a wide temperature range.
2、pH对六价铬还原酶Cyc1纯酶的活性影响2. The effect of pH on the activity of pure enzyme of hexavalent chromium reductase Cyc1
不同pH值(6、7、8、8.5、9、10、11、12)条件下对六价铬还原酶的活性进行了检测。The activity of hexavalent chromium reductase was detected under different pH values (6, 7, 8, 8.5, 9, 10, 11, 12).
检测方法为:在20mM MOPS—NaOH缓冲液中,所述缓冲液中包含100mM NAD(P)H、10mM FMN、1mM CrO3和10mM CaCl2,加入50μL Cyc1纯酶,在30℃条件反应1min,通过二苯碳酰二肼法检测Cr(VI)含量。The detection method is: in 20mM MOPS-NaOH buffer, the buffer contains 100mM NAD(P)H, 10mM FMN, 1mM CrO 3 and 10mM CaCl 2 , add 50μL Cyc1 pure enzyme, react at 30°C for 1min, Cr(VI) content was detected by the diphenylcarbazide method.
结果如图6所示,六价铬还原酶在pH值为8.5时具有最大的活性,PH在8及9时有比较高的活性,分别为最大活性的60%和91%。PH在6和10时其活性为最大活性的10%和5%。由此可知,六价铬还原酶对PH要求是比较专一。The results are shown in Figure 6, the hexavalent chromium reductase has the maximum activity at pH 8.5, and has relatively high activity at
3、金属离子对六价铬还原酶Cyc1纯酶的活性影响3. Effect of metal ions on the activity of pure enzyme of hexavalent chromium reductase Cyc1
不同金属离子(Na+、K+、Mg2+、Mn2+、Zn2+、Ca2+、Cu2+、EDTA)条件下对六价铬还原酶Cyc1纯酶的活性进行了检测。The activity of pure enzyme of hexavalent chromium reductase Cyc1 was detected under different metal ions (Na + , K + , Mg 2+ , Mn 2+ , Zn 2+ , Ca 2+ , Cu 2+ , EDTA).
具体方法如下:10mM不同金属离子(浓度为:10mM)在20mM MOPS—NaOH缓冲液中,所述缓冲液中包含100mM NAD(P)H、10mM FMN和1mM CrO3,加入50μL Cyc1纯酶,在30℃、PH=8.5的条件反应1min,通过二苯碳酰二肼法检测Cr(VI)含量。The specific method is as follows: 10 mM different metal ions (concentration: 10 mM) in 20 mM MOPS-NaOH buffer, the buffer contains 100 mM NAD(P)H, 10 mM FMN and 1 mM CrO 3 , add 50 μL Cyc1 pure enzyme, in React for 1 min under the conditions of 30°C and PH=8.5, and detect the Cr(VI) content by the diphenylcarbazide method.
结果如图7所示,六价铬还原酶Cyc1纯酶在Ca2+条件下具有最大的活性,在Mg2+比较高的活性,为最大活性的60%。由此可知,六价铬还原酶Cyc1纯酶对离子的要求是比较专一.The results are shown in FIG. 7 , the pure enzyme of hexavalent chromium reductase Cyc1 has the maximum activity under the condition of Ca 2+ , and has a relatively high activity under the condition of Mg 2+ , which is 60% of the maximum activity. It can be seen that the pure enzyme of hexavalent chromium reductase Cyc1 has relatively specific requirements for ions.
4、不同浓度的Cr(VI)对六价铬还原酶活性的影响4. The effect of different concentrations of Cr(VI) on the activity of hexavalent chromium reductase
在不同Cr(VI)浓度条件下对六价铬还原酶Cyc1纯酶的活性进行了检测。The activity of the pure enzyme of hexavalent chromium reductase Cyc1 was detected under different Cr(VI) concentration conditions.
具体方法如下:在20mM MOPS—NaOH缓冲液中,所述缓冲液中包含100mM NAD(P)H、10mM FMN和50μL Cyc1纯酶,分别加入0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、0.9mM、1.0mM的CrO3,体系在30℃、PH=8.5的条件反应1min,通过二苯碳酰二肼法检测Cr(VI)含量。The specific method is as follows: In 20mM MOPS-NaOH buffer, which contains 100mM NAD(P)H, 10mM FMN and 50μL Cyc1 pure enzyme, add 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM , 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM CrO 3 , the system was reacted at 30°C and pH=8.5 for 1min, and the Cr(VI) content was detected by the diphenylcarbazide method.
结果如图8所示,六价铬还原酶Cyc1纯酶在Cr(VI)浓度>0.4mM时具有最大的活性,Km值为0.2mM。The results are shown in FIG. 8 , the pure enzyme of hexavalent chromium reductase Cyc1 has the maximum activity when the concentration of Cr(VI)>0.4mM, and the Km value is 0.2mM.
5、重组菌株对Cr(VI)降解作用5. Degradation of Cr(VI) by recombinant strains
将实施例2制备得到基因工程菌E.coli BL21(DE3)/pXMJ-19-Cyc1、E.coli BL21(DE3)/pXMJ-19对照菌株的发酵液,按1%(v/v)的接种量转接至已添加1.0mM CrO3的50mLLB液体培养基中,在30℃、180rpm条件下培养24小时,通过二苯碳酰二肼法每隔4个小时检测Cr(VI)的含量,结果如图9所示。Example 2 is prepared to obtain the fermented liquid of genetically engineered bacteria E.coli BL21 (DE3)/pXMJ-19-Cyc1, E.coli BL21 (DE3)/pXMJ-19 control strain, inoculated by 1% (v/v) The amount was transferred to 50mL LB liquid medium that had been added with 1.0mM CrO 3 , cultured at 30°C and 180rpm for 24 hours, and the content of Cr(VI) was detected every 4 hours by the diphenylcarbazide method. As shown in Figure 9.
结果表明,基因工程菌E.coli BL21(DE3)在导入外源Cyc1基因后,显著增强了该菌株降解Cr(VI)的能力。The results showed that the ability of the strain to degrade Cr(VI) was significantly enhanced after the introduction of exogenous Cyc1 gene into the genetically engineered strain E.coli BL21(DE3).
实施例4:六价铬还原酶Cyc1纯酶在含Cr(VI)废水处理中的应用Embodiment 4: the application of hexavalent chromium reductase Cyc1 pure enzyme in the treatment of Cr(VI)-containing wastewater
具体步骤如下:Specific steps are as follows:
(1)从无锡某处废水场取一定量的含Cr(VI)废水,通过二苯碳酰二肼方法对Cr(VI)高灵敏性定量检测,对该废水进行了理化性质检测,确定了废水中含有Cr(VI),并且该废水中的六价铬Cr(VI)的浓度,为100mg/L。(1) A certain amount of Cr(VI)-containing wastewater was taken from a wastewater field in Wuxi, and the high-sensitivity quantitative detection of Cr(VI) was carried out by the diphenylcarbazide method. The physical and chemical properties of the wastewater were tested, and the The waste water contains Cr(VI), and the concentration of hexavalent chromium Cr(VI) in the waste water is 100 mg/L.
(2)将实施例2制备的六价铬还原酶Cyc1纯酶(步骤2得到的30℃条件下诱导菌株表达后经纯化的Cyc1纯化酶)按0.1U/mg Cr(VI)的投加量(即1U)加入其中,30℃,pH为8.5的条件下处理6小时,测定其中残余Cr(VI)浓度,并设置不添加六价铬还原酶的对照组,结果如图10所示。(2) Add the hexavalent chromium reductase Cyc1 pure enzyme prepared in Example 2 (the purified Cyc1 purified enzyme expressed by the induced strain obtained in
由图10可知,在未添加六价铬还原酶的废水中,Cr(VI)浓度未发生明显变化,加入六价铬还原酶的废水中Cr(VI)浓度降到62mg/L,还原率达38%,能快速有效地去除废水中的六价铬。It can be seen from Figure 10 that in the wastewater without hexavalent chromium reductase, the concentration of Cr(VI) did not change significantly, and the concentration of Cr(VI) in the wastewater added with hexavalent chromium reductase dropped to 62mg/L, and the reduction rate reached 38%, can quickly and effectively remove hexavalent chromium in wastewater.
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 江南大学<110> Jiangnan University
<120> 一种老黄酶OYE2蛋白及其在铬污染中的应用<120> An old yellow enzyme OYE2 protein and its application in chromium pollution
<130> BAA211189A<130> BAA211189A
<160> 2<160> 2
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 360<211> 360
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<400> 1<400> 1
Val Ser Lys Leu Phe Thr Pro Ile Gln Ile Arg Asp Ile Thr Ile ProVal Ser Lys Leu Phe Thr Pro Ile Gln Ile Arg Asp Ile Thr Ile Pro
1 5 10 151 5 10 15
Asn Arg Val Trp Met Ser Pro Met Cys Thr Tyr Ser Ala Ala Thr GlyAsn Arg Val Trp Met Ser Pro Met Cys Thr Tyr Ser Ala Ala Thr Gly
20 25 30 20 25 30
Leu Gly Leu Pro Thr Asp Phe His Gln Ala His Tyr Ala Ala Arg AlaLeu Gly Leu Pro Thr Asp Phe His Gln Ala His Tyr Ala Ala Arg Ala
35 40 45 35 40 45
Ala Gly Gly Val Gly Leu Val Met Val Glu Ala Thr Gly Val Ser ProAla Gly Gly Val Gly Leu Val Met Val Glu Ala Thr Gly Val Ser Pro
50 55 60 50 55 60
Val Ala Pro Ile Ser Pro Val Asp Leu Gly Leu Trp Ser His Asp GlnVal Ala Pro Ile Ser Pro Val Asp Leu Gly Leu Trp Ser His Asp Gln
65 70 75 8065 70 75 80
Ile Glu Pro Phe Ser Arg Val Thr Ala Ala Ile Arg Ala Gly Gly AlaIle Glu Pro Phe Ser Arg Val Thr Ala Ala Ile Arg Ala Gly Gly Ala
85 90 95 85 90 95
Val Pro Ala Val Gln Leu Ala His Ala Gly Arg Lys Ala Ser Thr AspVal Pro Ala Val Gln Leu Ala His Ala Gly Arg Lys Ala Ser Thr Asp
100 105 110 100 105 110
Ala Pro Trp Asn Gly Gly Gly Tyr Val Gly Pro Glu Thr Asn Gly TrpAla Pro Trp Asn Gly Gly Gly Tyr Val Gly Pro Glu Thr Asn Gly Trp
115 120 125 115 120 125
Glu Thr Val Gly Pro Ser Pro Leu Ala Phe Pro Gly Leu Pro Ala ProGlu Thr Val Gly Pro Ser Pro Leu Ala Phe Pro Gly Leu Pro Ala Pro
130 135 140 130 135 140
Arg Glu Leu Thr Val Ser Glu Ile Gln Glu Val Val Gln Gln Phe AlaArg Glu Leu Thr Val Ser Glu Ile Gln Glu Val Val Gln Gln Phe Ala
145 150 155 160145 150 155 160
Gly Ala Ala Val Arg Ala Asp Gln Ala Gly Phe Asp Val Val Glu IleGly Ala Ala Val Arg Ala Asp Gln Ala Gly Phe Asp Val Val Glu Ile
165 170 175 165 170 175
His Ala Ala His Gly Tyr Leu Leu His Asn Phe Leu Ser Pro Ile SerHis Ala Ala His Gly Tyr Leu Leu His Asn Phe Leu Ser Pro Ile Ser
180 185 190 180 185 190
Asn Lys Arg Thr Asp Ser Tyr Gly Gly Ser Leu Glu Asn Arg Ala ArgAsn Lys Arg Thr Asp Ser Tyr Gly Gly Ser Leu Glu Asn Arg Ala Arg
195 200 205 195 200 205
Ile Val Leu Glu Val Ile Asp Ala Ile Arg Ala Val Trp Pro Glu GluIle Val Leu Glu Val Ile Asp Ala Ile Arg Ala Val Trp Pro Glu Glu
210 215 220 210 215 220
Lys Pro Val Phe Met Arg Ile Ser Thr Thr Asp Trp Val Glu Glu AsnLys Pro Val Phe Met Arg Ile Ser Thr Thr Asp Trp Val Glu Glu Asn
225 230 235 240225 230 235 240
Pro Gln Asp Asp Arg Glu Ser Trp Thr Leu Ser Gln Ser Arg Gln LeuPro Gln Asp Asp Arg Glu Ser Trp Thr Leu Ser Gln Ser Arg Gln Leu
245 250 255 245 250 255
Ala Leu Trp Ala Ser Glu Arg Gly Ile Asp Leu Ile Asp Ala Ser SerAla Leu Trp Ala Ser Glu Arg Gly Ile Asp Leu Ile Asp Ala Ser Ser
260 265 270 260 265 270
Gly Gly Leu Asp Ile Val Pro Ile Pro His Asp Arg Asp Tyr Gln ThrGly Gly Leu Asp Ile Val Pro Ile Pro His Asp Arg Asp Tyr Gln Thr
275 280 285 275 280 285
Ala Lys Ala Ala Asp Leu His Ala Ser Thr Gly Ala Thr Val Ala AlaAla Lys Ala Ala Asp Leu His Ala Ser Thr Gly Ala Thr Val Ala Ala
290 295 300 290 295 300
Val Gly Arg Ile Asp Asp Ala Gln Thr Ala His Asn Leu Val Asp SerVal Gly Arg Ile Asp Asp Ala Gln Thr Ala His Asn Leu Val Asp Ser
305 310 315 320305 310 315 320
Gly Asp Val Asn Ala Val Phe Leu Gly Arg Pro Leu Leu Lys Asp ProGly Asp Val Asn Ala Val Phe Leu Gly Arg Pro Leu Leu Lys Asp Pro
325 330 335 325 330 335
Ser Trp Ala Asn Gln Ala Ala Leu Thr Leu Gly Ala Glu Pro Arg TyrSer Trp Ala Asn Gln Ala Ala Leu Thr Leu Gly Ala Glu Pro Arg Tyr
340 345 350 340 345 350
Val Arg Gln Tyr Asp Tyr Val LeuVal Arg Gln Tyr Asp Tyr Val Leu
355 360 355 360
<210> 2<210> 2
<211> 1083<211> 1083
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<400> 2<400> 2
gtgtccaagc tgtttacccc aattcaaatc cgcgacatca ccatccccaa ccgcgtgtgg 60gtgtccaagc tgtttacccc aattcaaatc cgcgacatca ccatccccaa ccgcgtgtgg 60
atgtcaccga tgtgcaccta ctctgcagcc accggtttag gtcttcccac cgattttcac 120atgtcaccga tgtgcaccta ctctgcagcc accggtttag gtcttcccac cgattttcac 120
caggctcatt acgcagctcg cgcagcaggt ggtgtcggat tagtcatggt tgaagcaact 180caggctcatt acgcagctcg cgcagcaggt ggtgtcggat tagtcatggt tgaagcaact 180
ggagtgagcc ccgtagctcc catctcccca gtcgaccttg gactttggag ccatgaccaa 240ggagtgagcc ccgtagctcc catctcccca gtcgaccttg gactttggag ccatgaccaa 240
attgaaccat tctcccgagt gacagcagct attcgcgccg gtggggcagt accggccgtt 300attgaaccat tctcccgagt gacagcagct attcgcgccg gtggggcagt accggccgtt 300
caattagccc atgctggccg caaggcatcc accgatgctc cgtggaatgg tggcggatat 360caattagccc atgctggccg caaggcatcc accgatgctc cgtggaatgg tggcggatat 360
gttggaccag aaaccaatgg atgggagact gtcggcccca gccctctggc attcccaggt 420gttggaccag aaaccaatgg atgggagact gtcggcccca gccctctggc attcccaggt 420
ttgcctgctc cgcgcgagct gacggtttca gaaatccaag aggttgtgca gcagttcgct 480ttgcctgctc cgcgcgagct gacggtttca gaaatccaag aggttgtgca gcagttcgct 480
ggcgccgccg ttcgtgccga tcaggctggt tttgatgtcg tggaaattca cgcagcacac 540ggcgccgccg ttcgtgccga tcaggctggt tttgatgtcg tggaaattca cgcagcacac 540
ggctaccttt tgcataactt cctttctccg atctccaaca agcgcaccga ttcatacggc 600ggctaccttt tgcataactt cctttctccg atctccaaca agcgcaccga ttcatacggc 600
ggatctttag aaaaccgcgc tcgcatcgtg ctcgaagtca ttgatgcaat ccgcgcagtg 660ggatctttag aaaaccgcgc tcgcatcgtg ctcgaagtca ttgatgcaat ccgcgcagtg 660
tggccagagg aaaagcctgt attcatgcgc atttccacca ccgactgggt agaggaaaac 720tggccagagg aaaagcctgt attcatgcgc atttccacca ccgactgggt agaggaaaac 720
ccacaggatg atcgcgagtc ctggacgctg agccaaagca ggcagctggc tttgtgggca 780ccacaggatg atcgcgagtc ctggacgctg agccaaagca ggcagctggc tttgtgggca 780
tccgagcgcg gaattgattt gatcgatgcc tcttctggtg gcctcgacat cgtccccatt 840tccgagcgcg gaattgattt gatcgatgcc tcttctggtg gcctcgacat cgtccccatt 840
ccgcatgacc gcgattacca aaccgcgaag gccgcagatc ttcacgcaag taccggagcg 900ccgcatgacc gcgattacca aaccgcgaag gccgcagatc ttcacgcaag taccggagcg 900
acagtcgctg ctgtggggcg cattgatgac gcccaaactg cgcacaattt ggttgattct 960acagtcgctg ctgtggggcg cattgatgac gcccaaactg cgcacaattt ggttgattct 960
ggcgatgtta atgcagtttt cctcggccgt ccactgctca aggatccttc ctgggcaaac 1020ggcgatgtta atgcagtttt cctcggccgt ccactgctca aggatccttc ctgggcaaac 1020
caagcagccc tcacgctagg tgcggaaccc aggtatgttc gccaatacga ctacgtactt 1080caagcagccc tcacgctagg tgcggaaccc aggtatgttc gccaatacga ctacgtactt 1080
taa 1083taa 1083
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