CN112010948A - Mutant protein applied to low-temperature degradation of hydroquinone wastewater and construction method - Google Patents

Mutant protein applied to low-temperature degradation of hydroquinone wastewater and construction method Download PDF

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CN112010948A
CN112010948A CN201910454531.0A CN201910454531A CN112010948A CN 112010948 A CN112010948 A CN 112010948A CN 201910454531 A CN201910454531 A CN 201910454531A CN 112010948 A CN112010948 A CN 112010948A
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孙海丽
高天鹏
常国华
刘涛
台喜生
陈映全
张威
陈熙明
刘光琇
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Abstract

本发明属于基因工程领域,具体涉及一种应用于对苯二酚废水低温降解的突变蛋白及构建方法。所述应用于对苯二酚废水低温降解的突变蛋白的氨基酸序列如SEQ ID NO.1所示,包含突变蛋白hqdAmu和突变蛋白hqdBmu,氨基酸序列如SEQ ID NO.2所示和SEQ ID NO.3所示。所述突变蛋白hqdAmu是将蛋白hqdA的第17位的甘氨酸、第88位的亮氨酸和第133位的缬氨酸分别突变为精氨酸、天冬氨酸和组氨酸;所述突变蛋白hqdBmu是将蛋白hqdB的第39位的甲硫氨酸和第148位的谷氨酸分别突变为缬氨酸和赖氨酸。本发明制备的突变蛋白,具有低温下高效降解含酚废水的特点,可在降解低温含酚废水中应用。

Figure 201910454531

The invention belongs to the field of genetic engineering, and in particular relates to a mutant protein and a construction method for low-temperature degradation of hydroquinone wastewater. The amino acid sequence of the mutant protein applied to the low-temperature degradation of hydroquinone wastewater is shown in SEQ ID NO.1, including the mutant protein hqdA mu and the mutant protein hqdB mu , and the amino acid sequence is shown in SEQ ID NO.2 and SEQ ID NO. NO.3 shows. The mutant protein hqdA mu is to mutate glycine at position 17, leucine at position 88 and valine at position 133 of protein hqdA into arginine, aspartic acid and histidine, respectively; the The mutant protein hqdB mu is the 39-position methionine and the 148-position glutamic acid of the protein hqdB are mutated to valine and lysine, respectively. The mutant protein prepared by the invention has the characteristics of efficiently degrading phenol-containing wastewater at low temperature, and can be used in degrading low-temperature phenol-containing wastewater.

Figure 201910454531

Description

一种应用于对苯二酚废水低温降解的突变蛋白及构建方法A kind of mutant protein applied to low temperature degradation of hydroquinone wastewater and construction method

技术领域technical field

本发明属于基因工程领域,具体涉及一种应用于对苯二酚废水低温降解的突变蛋白及构建方法。The invention belongs to the field of genetic engineering, and in particular relates to a mutant protein and a construction method for low-temperature degradation of hydroquinone wastewater.

背景技术Background technique

化学工业排放的废水中含酚废水是一类难处理的废水,由于低含量的酚类物质就可以有效杀灭微生物。因此化学工业产生的含酚废水无法直接排放至污水处理池中。现在主要应用于含酚废水中酚类物质降解的手段为铁碳降解法:铁碳可以在酸性水中形成微电极,这些微电极可以将酚类物质电离分解。经过铁碳反应处理后的废水中酚含量大幅度降低,不会对污水降解微生物产生杀灭作用。此时该废水方可排放至污水处理池中。由于中国北方大部分地区在秋冬季温度相对较低,铁碳反应无法有效的降解废水中的酚类物质,因此很难达到排放至污水处理池微生物处理的标准。Phenol-containing wastewater in the wastewater discharged from the chemical industry is a type of wastewater that is difficult to treat. Due to the low content of phenolic substances, it can effectively kill microorganisms. Therefore, the phenol-containing wastewater produced by the chemical industry cannot be directly discharged into the sewage treatment tank. At present, the method mainly used for the degradation of phenolic substances in phenol-containing wastewater is the iron-carbon degradation method: iron-carbon can form micro-electrodes in acidic water, and these micro-electrodes can ionize and decompose phenolic substances. The phenolic content in the wastewater treated by the iron-carbon reaction is greatly reduced, and it will not kill the microorganisms that degrade the wastewater. At this time, the waste water can be discharged into the sewage treatment tank. Due to the relatively low temperature in autumn and winter in most parts of northern China, the iron-carbon reaction cannot effectively degrade phenolic substances in wastewater, so it is difficult to meet the standard of microbial treatment in sewage treatment pools.

目前已报道用于对苯二酚降解的基因是hqdA与hqdB,它们均来自Sphingomonassp.strain TTNP3。在菌体内hqdA与hqdB共表达的蛋白可以形成二聚体共同催化降解对苯二酚。通过对HqdA与HqdB二聚体蛋白纯化后研究其催化动力学发现,该蛋白在常温下催化降解对苯二酚的效率为1.4mol对苯二酚/mol蛋白,其催化对苯二酚降解的Km=2.2μM。然而该酶却在4℃下降解对苯二酚的能力相对较弱,其催化效率仅为常温下催化效率的1/35,无法在低温状态下对对苯二酚降解。The genes that have been reported for hydroquinone degradation are hqdA and hqdB, both of which are derived from Sphiningomonassp.strain TTNP3. The proteins co-expressed by hqdA and hqdB in bacteria can form dimers to catalyze the degradation of hydroquinone. By studying the catalytic kinetics of the HqdA and HqdB dimer protein after purification, it was found that the efficiency of the protein to catalyze the degradation of hydroquinone at room temperature was 1.4mol hydroquinone/mol protein. Km=2.2 μM. However, the enzyme's ability to degrade hydroquinone at 4 °C is relatively weak, and its catalytic efficiency is only 1/35 of the catalytic efficiency at room temperature, and it cannot degrade hydroquinone at low temperature.

本发明具体提供了一种利用易错PCR技术对hqdA和hqdB进行定向进化,对突变体进行筛选后选出突变蛋白HqdAmu和HqdBmu,应用大肠杆菌对HqdAmu和HqdBmu进行重组共表达,对pETduet-1-hqdABmu2进行培养后进行高密度发酵和蛋白表达诱导得到诱导发酵液,然后从发酵液中分离出大肠杆菌进行破碎后即得到突变蛋白的大肠杆菌裂解液。本发明制备的突变蛋白,具有低温下高效降解含酚废水的特点。The invention specifically provides a kind of directed evolution of hqdA and hqdB by using error-prone PCR technology, selecting mutant proteins HqdA mu and HqdB mu after screening the mutants, and using Escherichia coli to recombine and co-express HqdA mu and HqdB mu , After culturing pETduet-1-hqdAB mu 2, high-density fermentation and protein expression induction were performed to obtain an induced fermentation broth, and then E. coli was isolated from the fermentation broth and disrupted to obtain an E. coli lysate of the mutant protein. The mutant protein prepared by the invention has the characteristics of efficiently degrading phenol-containing wastewater at low temperature.

发明内容SUMMARY OF THE INVENTION

本发明的目的是提供一种应用于对苯二酚废水低温降解的突变蛋白,所述的突变蛋白能够在低温状态下高效降解含酚废水。The purpose of the present invention is to provide a mutant protein for low-temperature degradation of hydroquinone wastewater, and the mutant protein can efficiently degrade phenol-containing wastewater at low temperature.

本发明的另一目的是提供上述应用于对苯二酚废水低温降解的突变蛋白的构建方法和应用。Another object of the present invention is to provide the above-mentioned construction method and application of the mutant protein for low-temperature degradation of hydroquinone wastewater.

为了达到上述目的,本发明采用的技术方案为:In order to achieve the above object, the technical scheme adopted in the present invention is:

一种突变蛋白,所述突变蛋白的氨基酸序列如SEQ ID NO.1所示。A mutant protein whose amino acid sequence is shown in SEQ ID NO.1.

所述突变蛋白包含突变蛋白hqdAmu和突变蛋白hqdBmu,所述突变蛋白hqdAmu的氨基酸序列如SEQ ID NO.2所示,突变蛋白hqdBmu的氨基酸序列如SEQ ID NO.3所示。The mutein comprises a mutein hqdA mu and a mutein hqdB mu , the amino acid sequence of the mutein hqdA mu is shown in SEQ ID NO.2, and the amino acid sequence of the mutein hqdB mu is shown in SEQ ID NO.3.

所述突变蛋白hqdAmu是将蛋白hqdA的第17位的甘氨酸、第88位的亮氨酸和第133位的缬氨酸分别突变为精氨酸、天冬氨酸和组氨酸;所述突变蛋白hqdBmu是将蛋白hqdB的第39位的甲硫氨酸和第148位的谷氨酸分别突变位缬氨酸和赖氨酸。The mutant protein hqdA mu is to mutate glycine at position 17, leucine at position 88 and valine at position 133 of protein hqdA into arginine, aspartic acid and histidine, respectively; the The mutant protein hqdB mu is the 39-position methionine and the 148-position glutamic acid of the protein hqdB are mutated to valine and lysine, respectively.

一种应用于对苯二酚废水低温降解的突变蛋白的构建方法,包括如下步骤:A method for constructing a mutant protein for low-temperature degradation of hydroquinone wastewater, comprising the following steps:

步骤一、分别以鞘氨醇单胞菌(Sphingomonas sp.strain)基因组为PCR扩增模板,利用PCR扩增引物P1、P2扩增hqdA,利用PCR扩增引物P3、P4扩增hqdB,其中P1、P2、P3、P4的核苷酸序列如下:Step 1. Take the genome of Sphingomonas sp.strain as a PCR amplification template, use PCR amplification primers P1 and P2 to amplify hqdA, and use PCR amplification primers P3 and P4 to amplify hqdB, wherein P1 , the nucleotide sequences of P2, P3, and P4 are as follows:

P1:AAGGATCCGATGGCCGACGTGGTCACC;P1:AAGGATCCGATGGCCGACGTGGTCACC;

P2:GCAAGCTTTCAGGCCGCCTCGGTCTGG;P2:GCAAGCTTTCAGGCCGCCTCGGTCTGG;

P3:AACATATGATGGCTATGTCCGAAGCACTTG;P3: AACATATGATGGCTATGTCCGAAGCACTTG;

P4:AACTCGAGTCAGAAAGTGACCGGGACCAC;P4: AACTCGAGTCAGAAAGTGACCGGGACCAC;

分别对扩增产物进行纯化和酶切,将酶切产物分别连接至大肠杆菌共表达载体pETduet-1中构建得到pETduet-1-hqdA和pETduet-1-hqdB;The amplified products were purified and digested respectively, and the digested products were respectively connected to the E. coli co-expression vector pETduet-1 to construct pETduet-1-hqdA and pETduet-1-hqdB;

步骤二、利用PCR扩增引物P3、P4以及易错PCR试剂盒扩增hqdB,利用PCR扩增引物P1、P2以及易错PCR试剂盒扩增hqdA,分别对扩增产物进行纯化和酶切,将酶切产物分别连接至步骤一得到的pETduet-1-hqdA和pETduet-1-hqdB中构建得到pETduet-1-hqdABmu和pETduet-1-hqdBAmuStep 2: Amplify hqdB with PCR amplification primers P3, P4 and error-prone PCR kit, amplify hqdA with PCR amplification primers P1, P2 and error-prone PCR kit, and purify and digest the amplified product respectively, The enzyme cleavage products are respectively connected to the pETduet-1-hqdA and pETduet-1-hqdB obtained in step 1 to construct pETduet-1-hqdAB mu and pETduet-1-hqdBA mu ;

步骤三、将步骤二得到的pETduet-1-hqdABmu和pETduet-1-hqdAmuB分别转化入大肠杆菌BL21中进行菌落培养,得到的菌落转入TB培养基进行菌株培养;Step 3, transform the pETduet-1-hqdAB mu and pETduet-1-hqdA mu B obtained in step 2 into Escherichia coli BL21 respectively for colony culture, and the obtained colony is transferred into TB medium for bacterial culture;

步骤四、分别以大肠杆菌最优密码子进行序列优化并全基因合成hqdAmu和hqdBmu后将其分别构建至大肠杆菌共表达载体pETduet-11中,构建得到pETduet-1-hqdABmu2;Step 4. Perform sequence optimization with the optimal codons of E. coli and synthesize hqdA mu and hqdB mu from the whole gene, respectively construct them into the co-expression vector pETduet-11 of E. coli, and construct pETduet-1-hqdAB mu 2;

步骤五、将pETduet-1-hqdABmu2转化至大肠杆菌BL21中培养后接种至培养基进行高密度发酵,发酵16-20h后,流加5-13L补料液,流加时间为4-10h,流加完毕后加入灭菌的酵母提取物至其终浓度为1-4%,加入浓度为0.1-0.8mM的IPTG于20-28℃下诱导过夜得到发酵液,诱导过程中流加5-13L诱导补料液,流加时间为12-24h,流加完毕后从发酵液中分离出大肠杆菌制备得到突变蛋白。Step 5. Transform pETduet-1-hqdAB mu 2 into Escherichia coli BL21 and inoculate it into the medium for high-density fermentation. After 16-20 hours of fermentation, add 5-13 L of feed solution, and the feeding time is 4-10 hours. , add sterilized yeast extract to a final concentration of 1-4% after the flow addition, add IPTG with a concentration of 0.1-0.8mM to induce overnight at 20-28°C to obtain a fermentation broth, and flow 5-13L during the induction process The feeding liquid is induced, and the feeding time is 12-24 h. After the feeding is completed, Escherichia coli is separated from the fermentation broth to prepare the mutant protein.

优选的,所述的步骤三中大肠杆菌BL21在含有0.005-0.015g/L的对苯二酚、0.1-0.8mMIPTG的LB固体培养基中在14℃-20℃培养48-60h。由于大肠杆菌对对苯二酚极其敏感,在含有0.005-0.015g/L的对对苯二酚培养基中就会死亡,因此需要加入IPTG。Preferably, in the third step, Escherichia coli BL21 is cultured in LB solid medium containing 0.005-0.015g/L hydroquinone and 0.1-0.8mM IPTG at 14°C-20°C for 48-60 hours. Since Escherichia coli is extremely sensitive to hydroquinone, it will die in the hydroquinone medium containing 0.005-0.015g/L, so IPTG needs to be added.

优选的,所述的步骤五中接种中采用大肠杆菌高密度发酵培养基,接种比例为1:50-1:100。Preferably, in the inoculation in the fifth step, a high-density fermentation medium of Escherichia coli is used, and the inoculation ratio is 1:50-1:100.

优选的,所述的步骤五中补料液由葡萄糖、硫酸镁、维生素和微量元素组成;Preferably, in the step 5, the feed solution is composed of glucose, magnesium sulfate, vitamins and trace elements;

优选的,所述的步骤五中诱导补料液由甘油、硫酸镁、维生素B1和微量元素组成。Preferably, the induction feeding solution in the fifth step is composed of glycerol, magnesium sulfate, vitamin B1 and trace elements.

突变蛋白在含酚废水低温降解中的应用。Application of mutant proteins in low-temperature degradation of phenol-containing wastewater.

本发明的有益效果为:The beneficial effects of the present invention are:

本发明所提供的突变蛋白,与野生型蛋白相比,在20-25℃下降解对苯二酚的能力获得了提高,但无显著性区别;在低温2-4℃下降解对苯二酚的能力为野生型蛋白的27.3倍,酶活显著性提高,其催化活性为6.4μmol/min/mg。Compared with the wild-type protein, the mutant protein provided by the present invention has an improved ability to degrade hydroquinone at 20-25°C, but there is no significant difference; it degrades hydroquinone at a low temperature of 2-4°C The capacity of the enzyme is 27.3 times that of the wild-type protein, and the enzyme activity is significantly improved, and its catalytic activity is 6.4 μmol/min/mg.

将大肠杆菌裂解液按不同比例稀释添加至含有1g/L对苯二酚的废水中,检测发现14-24g的大肠杆菌裂解液在4℃下对1吨含有0.5-2g/L对苯二酚废水处理8-16h后对苯二酚含量仅为20-80ppm。对苯二酚的含量显著性下降,即本发明所制备的突变蛋白能够在低温环境下降解对苯二酚,且降解的活性极高。The Escherichia coli lysate was diluted in different proportions and added to the wastewater containing 1g/L hydroquinone, and it was found that 14-24g of Escherichia coli lysate contained 0.5-2g/L hydroquinone for 1 ton at 4°C. After 8-16h of wastewater treatment, the hydroquinone content is only 20-80ppm. The content of hydroquinone is significantly reduced, that is, the mutant protein prepared by the present invention can degrade hydroquinone in a low temperature environment, and the degradation activity is extremely high.

附图说明Description of drawings

图1 pETduet-1-hqdABmu2的构建过程Figure 1 Construction process of pETduet-1-hqdABmu2

图2在25℃、4℃下野生型蛋白与突变蛋白酶活催化效率的柱状对比图Figure 2 Column comparison of the catalytic efficiency of wild-type protein and mutant protease activity at 25°C and 4°C

其中Wt 25摄氏度为野生型蛋白在25℃下的酶活催化效率,Wt 4摄氏度为野生型蛋白在4℃下的酶活催化效率,Mu 25摄氏度为突变型蛋白在25℃下的酶活催化效率,Mu 4摄氏度为突变型蛋白在4℃下的酶活催化效率。Among them, Wt 25°C is the enzymatic catalytic efficiency of the wild-type protein at 25°C, Wt 4°C is the enzymatic catalytic efficiency of the wild-type protein at 4°C, and Mu 25°C is the enzymatic catalytic efficiency of the mutant protein at 25°C Efficiency, Mu 4°C is the catalytic efficiency of the mutant protein at 4°C.

图3在低温下(4℃)利用高密度表达突变蛋白的大肠杆菌发酵液(14g/t)处理500ppm的对苯二酚废水后,废水中对苯二酚浓度随时间的变化趋势图,横坐标是处理时间,纵坐标是废水中对苯二酚浓度。Figure 3 shows the variation trend of hydroquinone concentration in wastewater with time after treating 500 ppm hydroquinone wastewater with Escherichia coli fermentation broth (14g/t) expressing mutant protein at high density at low temperature (4°C). The coordinate is the treatment time, and the ordinate is the hydroquinone concentration in the wastewater.

图4在低温下(4℃)利用高密度表达突变蛋白的大肠杆菌发酵液(24g/t)处理2000ppm对苯二酚废水后,废水中对苯二酚浓度随时间的变化趋势图,横坐标是处理时间,纵坐标是废水中对苯二酚浓度。Fig. 4 After treating 2000 ppm hydroquinone wastewater with Escherichia coli fermentation broth (24g/t) expressing the mutant protein at high density at low temperature (4°C), the change trend of hydroquinone concentration in wastewater with time, abscissa is the treatment time, and the ordinate is the hydroquinone concentration in the wastewater.

具体实施方式Detailed ways

下面结合附图和具体实施方式对本发明做进一步说明,但是本发明的保护范围不受实施例的限制。The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited by the embodiments.

下列实施例中的材料为:The materials in the following examples are:

所使用鞘氨醇单胞菌(Sphingomonas sp.strain)从甘肃极端环境资源与工程重点实验室菌种保藏中心获得。Sphingomonas sp. strain used was obtained from the Culture Collection Center of Gansu Key Laboratory of Extreme Environmental Resources and Engineering.

使用工具酶、DNA分子量Marker、胶回收试剂盒购、His-tag纯化试剂盒、易错PCR试剂盒、蛋白定量试剂盒等相关试剂均购自生工生物工程(上海)股份有限公司,具体的反应条件和使用的方法均参考商品说明书。Use tool enzymes, DNA molecular weight markers, gel recovery kits, His-tag purification kits, error-prone PCR kits, protein quantification kits and other related reagents were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. The specific reaction For conditions and methods of use, refer to the product manual.

下面的质粒和大肠杆菌用于DNA文库构建和基因克隆;The following plasmids and E. coli were used for DNA library construction and gene cloning;

大肠杆菌BL21感受态细胞购自索莱宝(solarbio)生物科技有限公司,pETduet-1质粒购自于湖南丰晖生物科技有限公司。Escherichia coli BL21 competent cells were purchased from Solarbio Biotechnology Co., Ltd., and the pETduet-1 plasmid was purchased from Hunan Fenghui Biotechnology Co., Ltd.

实施例1一种应用于对苯二酚废水低温降解的突变蛋白的构建和活性测定Example 1 Construction and activity assay of a mutant protein applied to hydroquinone wastewater low-temperature degradation

1.引物扩增1. Primer Amplification

设计的引物P1、P2、P3、P4序列如下:The sequences of the designed primers P1, P2, P3 and P4 are as follows:

P1:5’—AAGGATCCGATGGCCGACGTGGTCACC—3’;P1:5'-AAGGATCCGATGGCCGACGTGGTCACC-3';

P2:5’—GCAAGCTTTCAGGCCGCCTCGGTCTGG—3’;P2:5'-GCAAGCTTTCAGGCCGCCTCGGTCTGG-3';

P3:5’—AACATATGATGGCTATGTCCGAAGCACTTG—3’;P3:5'-AACATATGATGGCTATGTCCGAAGCACTTG-3';

P4:5’—AACTCGAGTCAGAAAGTGACCGGGACCAC—3’;P4:5'-AACTCGAGTCAGAAAGTGACCGGGACCAC-3';

将鞘氨醇单胞菌(Sphingomonas sp.strain)在LB培养基(蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L)于28℃下培养4天。当OD 600=1.0-1.5时,取80μl菌液加入20μl 2g/L的溶菌酶,于37℃下溶菌破壁10h,95℃加热破裂细菌5min待用。Sphingomonas sp. strain was cultured in LB medium (peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) for 4 days at 28°C. When OD 600=1.0-1.5, take 80 μl of bacterial solution and add 20 μl of 2g/L lysozyme, lyse the bacteria at 37°C for 10 hours, and heat at 95°C for 5 minutes to break the bacteria.

向PCR管中加入25μl 2X PCR Mix,2μl p1,2μl p2,2μl加热破裂的菌液为模板,加水补至50μl。向PCR管中加入25μl 2X PCR Mix,2μl p3,2μl p4,2μl加热破裂的菌液为模板,加水补至50μl。Add 25 μl of 2X PCR Mix, 2 μl of p1, 2 μl of p2, and 2 μl of heat-disrupted bacterial solution to the PCR tube as a template, and add water to make up to 50 μl. Add 25 μl of 2X PCR Mix, 2 μl of p3, 2 μl of p4, and 2 μl of heat-disrupted bacterial solution to the PCR tube as a template, and add water to make up to 50 μl.

PCR程序:95℃5min预变性,95℃40S,55℃50S,72℃90S(30个循环)。扩增完毕后利用琼脂糖凝胶电泳对扩增产物分离,切胶回收扩增出的hqdA与hqdB。PCR program: pre-denaturation at 95°C for 5 min, 40S at 95°C, 50S at 55°C, and 90S at 72°C (30 cycles). After the amplification, the amplified products were separated by agarose gel electrophoresis, and the amplified hqdA and hqdB were recovered by cutting the gel.

2.酶切2. Enzyme digestion

①利用NcoI+HindIII对hqdA进行酶切①Enzyme cleavage of hqdA with NcoI+HindIII

酶切体系:3μl buffer,NcoI+HindIII酶各1μl,25μl扩增hqdA;Enzyme digestion system: 3 μl buffer, 1 μl each of NcoI+HindIII enzymes, and 25 μl amplifying hqdA;

酶切条件:37℃下酶切2h。Digestion conditions: Digestion at 37°C for 2h.

②利用NedI+XhoI对hqdB进行酶切②Enzymatic cleavage of hqdB with NedI+XhoI

酶切体系:3μl buffer,NedI+XhoI酶各1μl,25μl扩增hqdB;Enzyme digestion system: 3μl buffer, 1μl NedI+XhoI enzyme each, 25μl amplification hqdB;

酶切条件:37℃下酶切2h。Digestion conditions: Digestion at 37°C for 2h.

利用琼脂糖凝胶电泳对扩增产物分离,切胶回收酶切好的hqdA与hqdB。The amplified products were separated by agarose gel electrophoresis, and the digested hqdA and hqdB were recovered by cutting the gel.

③用NcoI+HindIII对pETduet-1进行酶切③Enzymatic digestion of pETduet-1 with NcoI+HindIII

酶切体系:2μl buffer,酶各1μl,16μl pETduet-1。Enzyme digestion system: 2 μl buffer, 1 μl enzyme, 16 μl pETduet-1.

酶切条件:37℃下酶切12h。Digestion conditions: Digestion at 37°C for 12h.

利用琼脂糖凝胶电泳对扩增产物分离,切胶回收酶切好的pETduet-1。The amplified products were separated by agarose gel electrophoresis, and the digested pETduet-1 was recovered by cutting the gel.

3.连接3. Connect

①利用T4DNA连接酶对pETduet-1与hqdA连接① Use T4 DNA ligase to ligate pETduet-1 and hqdA

连接体系:2μl buffer,酶1μl,3μl pETduet-1,14μl hqdA,16℃下连接过夜。将连接产物转化至大肠杆菌感受态中,在含有100μg/ml氨苄青霉素的LB培养基中培养,筛选;对长出的单菌落分别用引物P1与P2对其扩增,通过检测是否能扩出hqdA来确定阳性克隆。利用测序验证克隆的正确性,构建完成pETduet-1-hqdA。对连接正确的克隆,利用质粒提取试剂盒提取质粒,利用NedI+XhoI对质粒进行酶切,酶切体系:2μl buffer,酶各1μl,16μlpETduet-1-hqdA。酶切条件:37℃下酶切12h。用琼脂糖凝胶电泳对扩增产物分离,切胶回收酶切好的质粒。Ligation system: 2μl buffer, 1μl enzyme, 3μl pETduet-1, 14μl hqdA, ligation overnight at 16°C. The ligation product was transformed into E. coli competent, cultured in LB medium containing 100 μg/ml ampicillin, and screened; the grown single colonies were amplified with primers P1 and P2, respectively, and it was detected by testing whether they could expand. hqdA to identify positive clones. The correctness of the clone was verified by sequencing, and pETduet-1-hqdA was constructed. For the clones with correct ligation, the plasmid was extracted with a plasmid extraction kit, and the plasmid was digested with NedI+XhoI. The digestion system was: 2 μl buffer, 1 μl enzyme each, and 16 μl pETduet-1-hqdA. Digestion conditions: Digestion at 37°C for 12h. The amplified products were separated by agarose gel electrophoresis, and the digested plasmid was recovered by cutting the gel.

②利用T4DNA连接酶对酶切好的质粒与hqdB连接②Use T4 DNA ligase to connect the digested plasmid to hqdB

连接体系:2μl buffer,酶1μl,3μl质粒,14μl hqdB,16℃下连接过夜。将连接产物转化至大肠杆菌感受态中,在含有100μg/ml氨苄青霉素的LB培养基中培养,筛选。对长出的单菌落分别用引物P3与P4对其扩增,通过检测是否能扩出hqdB来确定阳性克隆;利用测序验证克隆的正确性,构建完成pETduet-1-hqdAB。Ligation system: 2μl buffer, 1μl enzyme, 3μl plasmid, 14μl hqdB, ligation overnight at 16°C. The ligated product was transformed into competent E. coli, cultured in LB medium containing 100 μg/ml ampicillin, and screened. The grown single colonies were amplified with primers P3 and P4 respectively, and positive clones were determined by detecting whether hqdB could be amplified; sequencing was used to verify the correctness of clones, and pETduet-1-hqdAB was constructed.

4.蛋白表达与纯化4. Protein expression and purification

将pETduet-1-hqdAB转化入大肠杆菌BL21感受态中,利用大肠杆菌蛋白表达培养基TB对其进行培养。在37℃下将菌株培养至浓度为OD600=0.5-0.8时,加入中浓度为0.5mMIPTG,20℃下诱导过夜。离心收集菌体,按照1g菌体用6ml PBS的比例重悬菌体。利用超声波对其进行破碎,利用His-tag纯化试剂盒对其蛋白进行纯化,利用蛋白定量试剂盒对纯化好的蛋白进行定量分析。The pETduet-1-hqdAB was transformed into E. coli BL21 competent cells and cultured in E. coli protein expression medium TB. The strain was cultured at 37°C to a concentration of OD600 = 0.5-0.8, and 0.5 mM IPTG was added to induce overnight induction at 20°C. The cells were collected by centrifugation, and the cells were resuspended in 6 ml of PBS according to the ratio of 1 g of cells. It was crushed by ultrasonic wave, the protein was purified by His-tag purification kit, and the purified protein was quantitatively analyzed by protein quantification kit.

5.易错PCR5. Error-prone PCR

利用易错PCR试剂盒以上述扩增获得的hqdA与hqdB为模板,进行易错PCR扩增。向PCR管中加入25μl 2X易错PCR Mix,2μl p1,2μl p2,2μl模板,加水补至50μl。向PCR管中加入25μl 2X易错PCR Mix,2μl p3,2μl p4,2μl模板,加水补至50μl。PCR程序:95℃5min预变性,95℃40S,55℃50S,72℃90S(30个循环)。扩增完毕后利用琼脂糖凝胶电泳对扩增产物分离,切胶回收扩增出的hqdAmu与hqdBmu。Error-prone PCR amplification was performed using an error-prone PCR kit with the hqdA and hqdB obtained by the above amplification as templates. Add 25 μl 2X Error-Prone PCR Mix, 2 μl p1, 2 μl p2, 2 μl template to the PCR tube, and make up to 50 μl with water. Add 25 μl of 2X error-prone PCR Mix, 2 μl p3, 2 μl p4, 2 μl template to the PCR tube, and make up to 50 μl with water. PCR program: pre-denaturation at 95°C for 5 min, 40S at 95°C, 50S at 55°C, and 90S at 72°C (30 cycles). After the amplification, the amplified products were separated by agarose gel electrophoresis, and the amplified hqdAmu and hqdBmu were recovered by cutting the gel.

6.易错PCR文库构建6. Error-prone PCR library construction

①利用NcoI+HindIII对hqdAmu进行酶切;利用NedI+XhoI对hqdBmu进行酶切。酶切体系:3μl buffer,酶各1μl,25μl回收的DNA。酶切条件:37℃下酶切2h。利用琼脂糖凝胶电泳对扩增产物分离,切胶回收酶切好的hqdAmu与hqdBmu。① Use NcoI+HindIII to digest hqdAmu; use NedI+XhoI to digest hqdBmu. Enzyme digestion system: 3 μl buffer, 1 μl enzyme each, 25 μl recovered DNA. Digestion conditions: Digestion at 37°C for 2h. The amplified products were separated by agarose gel electrophoresis, and the digested hqdAmu and hqdBmu were recovered by cutting the gel.

②分别利用NcoI+HindIII与NedI+XhoI对pETduet-1-hqdAB进行酶切。酶切体系:3μl buffer,酶各2μl,25μlpETduet-1-hqdAB。酶切条件:37℃下酶切12h。利用琼脂糖凝胶电泳对扩增产物分离,切胶回收酶切好的载体。② pETduet-1-hqdAB was digested with NcoI+HindIII and NedI+XhoI respectively. Enzyme digestion system: 3 μl buffer, 2 μl enzyme, 25 μl pETduet-1-hqdAB. Digestion conditions: Digestion at 37°C for 12h. The amplified products were separated by agarose gel electrophoresis, and the digested vector was recovered by cutting the gel.

③利用T4DNA连接酶对酶切好的质粒分别与酶切好的hqdAmu与hqdBmu连接,连接体系:2μl buffer,酶1μl,3μl质粒,14μl hqdAmu或hqdBmu,16℃下连接过夜。将连接产物转化至大肠杆菌感受态中,在含有100μg/ml氨苄青霉素的LB培养基中培养,筛选。构建得到pETduet-1-hqdABmu和pETduet-1-hqdBAmu。③ Use T4 DNA ligase to connect the digested plasmids to the digested hqdAmu and hqdBmu respectively. The ligation system: 2 μl buffer, 1 μl enzyme, 3 μl plasmid, 14 μl hqdAmu or hqdBmu, and ligation overnight at 16°C. The ligated product was transformed into competent E. coli, cultured in LB medium containing 100 μg/ml ampicillin, and screened. The construction yielded pETduet-1-hqdABmu and pETduet-1-hqdBAmu.

7.易错PCR文库筛选及共表达突变蛋白载体构建7. Error-prone PCR library screening and co-expression mutant protein vector construction

将构建好的pETduet-1-hqdABmu和pETduet-1-hqdBAmu分别转化至大肠杆菌BL21中,在含有在0.005-0.015g/L的对苯二酚、0.1-0.8mM IPTG的LB固体培养基中在14℃-20℃培养48-60h。选取最先长出的单菌落,测序确认突变位点。利用生物信息学软件clust W对不同突变克隆中共有突变位点进行确认。利用定点突变试剂盒对pETduet-1-hqdAB上的hqdA与hqdB进行定点突变,最终构建完成pETduet-1-hqdABmu2(图1)。The constructed pETduet-1-hqdABmu and pETduet-1-hqdBAmu were transformed into E. coli BL21, respectively, in LB solid medium containing 0.005-0.015g/L hydroquinone and 0.1-0.8mM IPTG. Incubate at 14°C-20°C for 48-60h. The first single colony was selected and sequenced to confirm the mutation site. The common mutation sites in different mutant clones were confirmed by the bioinformatics software clust W. The hqdA and hqdB on pETduet-1-hqdAB were site-directed mutagenesis using a site-directed mutagenesis kit, and pETduet-1-hqdABmu2 was finally constructed (Figure 1).

8.突变蛋白表达与纯化8. Mutant protein expression and purification

将pETduet-1-hqdABmu2转化至大肠杆菌BL21感受态中培养后接种至培养基进行高密度发酵,发酵16-20h后,流加5-13L补料液,流加时间为4-10h,流加完毕后加入灭菌的酵母提取物至其终浓度为1-4%,在37℃下将菌株培养至浓度为OD600=0.5-0.8时,加入浓度为0.1-0.8mM的IPTG于20-28℃下诱导过夜得到发酵液,诱导过程中流加5-13L诱导补料液,流加时间为12-24h,流加完毕后从发酵液中分离出大肠杆菌,离心收集菌体,按照1g菌体用6ml PBS的比例重悬菌体。利用超声波对其进行破碎,利用His-tag纯化试剂盒对其蛋白进行纯化,利用蛋白定量试剂盒对纯化好的蛋白进行定量分析。The pETduet-1-hqdAB mu 2 was transformed into Escherichia coli BL21 competent medium and then inoculated into the medium for high-density fermentation. After fermentation for 16-20h, 5-13L of feed solution was added in flow for 4-10h. After the feeding is completed, add sterilized yeast extract to a final concentration of 1-4%, culture the strain at 37°C to a concentration of OD600=0.5-0.8, add IPTG with a concentration of 0.1-0.8mM to 20- Induce overnight at 28°C to obtain fermentation broth. During the induction process, 5-13 L of induction feed solution was added, and the feeding time was 12-24 h. The cells were resuspended in the ratio of 6ml PBS. It was crushed by ultrasonic wave, the protein was purified by His-tag purification kit, and the purified protein was quantitatively analyzed by protein quantification kit.

9.对苯二酚降解能力测定9. Determination of hydroquinone degradation ability

酶催化反应体系:250μl 50mM Tris pH7.0;50nM纯化的HqdAB或HqdABmu2;100μl350μM溶解于50mM Tris pH7.0的对苯二酚溶液。在不同温度下孵育不同时间,在320nm下测定吸光值的变化,以测定纯化后的HqdABmu和野生型蛋白分别在不同温度下的酶活。Enzyme-catalyzed reaction system: 250 μl 50 mM Tris pH7.0; 50 nM purified HqdAB or HqdABmu2; 100 μl 350 μM hydroquinone solution dissolved in 50 mM Tris pH7.0. Incubate at different temperatures for different times, and measure the changes in absorbance at 320 nm to determine the enzymatic activities of purified HqdAB mu and wild-type proteins at different temperatures, respectively.

将8.突变蛋白表达与纯化中得到的诱导发酵液中的大肠杆菌进行分离后称重,将大肠杆菌按照100g/L的浓度重悬于磷酸盐缓冲液中,应用高压均质机在80MPa压力下破碎1-2遍后形成大肠杆菌裂解液,将大肠杆菌裂解液按不同比例稀释添加至含有1g/L对苯二酚的废水中,检测对苯二酚的含量。8. The Escherichia coli in the induced fermentation broth obtained in the expression and purification of the mutant protein were separated and weighed, and the Escherichia coli was resuspended in phosphate buffer at a concentration of 100g/L, and a high pressure homogenizer was used at a pressure of 80MPa. After crushing for 1-2 times, an Escherichia coli lysate was formed, and the Escherichia coli lysate was diluted in different proportions and added to the wastewater containing 1 g/L hydroquinone to detect the content of hydroquinone.

10.结果分析10. Analysis of results

HqdABmu和野生型蛋白在20-25℃下降解对苯二酚的能力分别为6.8μmol/min/mg和6.1μmol/min/mg,HqdABmu在10℃和4℃下降解对苯二酚的能力比野生型蛋白强,尤其在2-4℃下降解对苯二酚的能力为野生型蛋白的27.3倍,其催化活性为6.4μmol/min/mg。实验结果如图2所示。The ability of HqdAB mu and wild-type protein to degrade hydroquinone at 20-25 °C was 6.8 μmol/min/mg and 6.1 μmol/min/mg, respectively, and HqdAB mu degraded hydroquinone at 10 °C and 4 °C, respectively. The ability is stronger than that of the wild-type protein, especially the ability to degrade hydroquinone at 2-4 °C is 27.3 times that of the wild-type protein, and its catalytic activity is 6.4 μmol/min/mg. The experimental results are shown in Figure 2.

将得到的诱导发酵液中的大肠杆菌进行分离后称重检测到大肠杆菌的湿重高达80-150g/L,添加至含有对苯二酚的废水中,检测发现14-24g的大肠杆菌裂解液在4℃下对1吨含有0.5-2g/L对苯二酚废水处理8-16h后对苯二酚含量仅为20-80ppm。实验结果如图3、图4所示。The E. coli in the obtained induced fermentation broth was separated and weighed to detect that the wet weight of E. coli was as high as 80-150g/L, added to the wastewater containing hydroquinone, and the detection found that 14-24g of E. coli lysate was The content of hydroquinone was only 20-80ppm after treating 1 ton of wastewater containing 0.5-2g/L hydroquinone at 4°C for 8-16h. The experimental results are shown in Figure 3 and Figure 4.

序列表sequence listing

<110> 兰州城市学院<110> Lanzhou City College

<120> 一种应用于对苯二酚废水低温降解的突变蛋白及构建方法<120> A mutant protein applied to low-temperature degradation of hydroquinone wastewater and its construction method

<160> 3<160> 3

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 511<211> 511

<212> PRT<212> PRT

<213> 鞘氨醇单胞菌(Sphingomonas sp. strain)<213> Sphingomonas sp. strain

<400> 1<400> 1

Met Ala Asp Val Val Thr Glu Phe Gly Ala Leu Thr Asp Tyr Arg LysMet Ala Asp Val Val Thr Glu Phe Gly Ala Leu Thr Asp Tyr Arg Lys

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Arg Gly Val Glu Ile Ile Asp Asp Asp Pro Arg Asn Tyr Val Phe SerArg Gly Val Glu Ile Ile Asp Asp Asp Pro Arg Asn Tyr Val Phe Ser

20 25 30 20 25 30

Asn Val Phe Glu Val Ala Ala Asn Ala Ala Pro Tyr Glu Arg Val AlaAsn Val Phe Glu Val Ala Ala Asn Ala Ala Pro Tyr Glu Arg Val Ala

35 40 45 35 40 45

Val Gly Lys Asn Phe Glu Tyr Val Ile Glu Ser Ala Arg Ala Glu GlyVal Gly Lys Asn Phe Glu Tyr Val Ile Glu Ser Ala Arg Ala Glu Gly

50 55 60 50 55 60

Thr Ser Gly Trp Phe Ser Cys Ala His Asp Glu Phe Val Leu Ala MetThr Ser Gly Trp Phe Ser Cys Ala His Asp Glu Phe Val Leu Ala Met

65 70 75 8065 70 75 80

Asp Gly Gln Ile Glu Val His Asp Leu Lys Leu Asp Asn Ser Asp AlaAsp Gly Gln Ile Glu Val His Asp Leu Lys Leu Asp Asn Ser Asp Ala

85 90 95 85 90 95

Tyr Val Asp Pro Asp Ser Glu Gly Ala Val Ala Ile Gly Glu Ala LeuTyr Val Asp Pro Asp Ser Glu Gly Ala Val Ala Ile Gly Glu Ala Leu

100 105 110 100 105 110

Pro Glu Gly Arg Lys Met Gly Arg Ile Val Leu Arg Arg Gly His MetPro Glu Gly Arg Lys Met Gly Arg Ile Val Leu Arg Arg Gly His Met

115 120 125 115 120 125

Ala Leu Leu Pro His Gly Ala Ala Tyr Arg Phe Tyr Ala Glu Gln ProAla Leu Leu Pro His Gly Ala Ala Tyr Arg Phe Tyr Ala Glu Gln Pro

130 135 140 130 135 140

Ala Ala Met Leu Phe Gln Ser Ile Glu Gly Ala Val Thr Val Gln LysAla Ala Met Leu Phe Gln Ser Ile Glu Gly Ala Val Thr Val Gln Lys

145 150 155 160145 150 155 160

Trp Gly Glu Ile Cys Gln Thr Glu Ala Ala Met Ala Met Ser Glu AlaTrp Gly Glu Ile Cys Gln Thr Glu Ala Ala Met Ala Met Ser Glu Ala

165 170 175 165 170 175

Leu Glu Ile Ile Asp Phe Gly Asp Ser Lys Ala Arg Thr Asp Thr GluLeu Glu Ile Ile Asp Phe Gly Asp Ser Lys Ala Arg Thr Asp Thr Glu

180 185 190 180 185 190

His Leu Ala Ile Asn Asn Glu Thr Gly Tyr Arg Ser Phe Arg Ala GlyHis Leu Ala Ile Asn Asn Glu Thr Gly Tyr Arg Ser Phe Arg Ala Gly

195 200 205 195 200 205

Gly Phe Thr Phe Thr Arg Asp Glu Tyr Phe Ala Arg Leu Thr Trp ProGly Phe Thr Phe Thr Arg Asp Glu Tyr Phe Ala Arg Leu Thr Trp Pro

210 215 220 210 215 220

Gly Gly Ser His Ile Ile Pro Ile Asp Ala Phe Leu Arg Ala Val MetGly Gly Ser His Ile Ile Pro Ile Asp Ala Phe Leu Arg Ala Val Met

225 230 235 240225 230 235 240

Arg Asp Val Ala Trp Gly Phe Phe Tyr Gly Val Val Asn Phe Asp HisArg Asp Val Ala Trp Gly Phe Phe Tyr Gly Val Val Asn Phe Asp His

245 250 255 245 250 255

Val Phe Gly Thr Ile Asn His Tyr Gly Glu Val Thr Met Phe Ala GlyVal Phe Gly Thr Ile Asn His Tyr Gly Glu Val Thr Met Phe Ala Gly

260 265 270 260 265 270

Arg Phe Asn Asp Ala Tyr Arg Asn Ala Gly Arg Asp His Glu Glu ArgArg Phe Asn Asp Ala Tyr Arg Asn Ala Gly Arg Asp His Glu Glu Arg

275 280 285 275 280 285

Phe Lys Ser Ser Ala Leu Met Ala Val Phe Lys Asp Ile Leu Ser AspPhe Lys Ser Ser Ala Leu Met Ala Val Phe Lys Asp Ile Leu Ser Asp

290 295 300 290 295 300

Trp Thr Val Glu Gly Tyr Asp Pro Phe Ala Ala Pro Met Lys Thr GlyTrp Thr Val Glu Gly Tyr Asp Pro Phe Ala Ala Pro Met Lys Thr Gly

305 310 315 320305 310 315 320

Leu Pro Trp Gly Ile Lys Asn Gly Asn Asn Asp Glu Ala Ile Ser ArgLeu Pro Trp Gly Ile Lys Asn Gly Asn Asn Asp Glu Ala Ile Ser Arg

325 330 335 325 330 335

Gln Arg Val Thr Ala Arg Arg Met Val Gly Leu Pro Gly Asp Thr ProGln Arg Val Thr Ala Arg Arg Met Val Gly Leu Pro Gly Asp Thr Pro

340 345 350 340 345 350

Val Arg Thr Asp Ala Asn Gly Phe Pro Val Asn Arg Gln Phe Ala AspVal Arg Thr Asp Ala Asn Gly Phe Pro Val Asn Arg Gln Phe Ala Asp

355 360 365 355 360 365

Val Pro Gln Glu Gln Pro Val Val Glu Ala Glu Pro Gly Phe Glu AlaVal Pro Gln Glu Gln Pro Val Val Glu Ala Glu Pro Gly Phe Glu Ala

370 375 380 370 375 380

Glu Val Ser Ala Tyr Asn Leu Phe Gly Tyr Leu Ser Arg Ser Asp ValGlu Val Ser Ala Tyr Asn Leu Phe Gly Tyr Leu Ser Arg Ser Asp Val

385 390 395 400385 390 395 400

Thr Trp Asn Pro Ser Val Cys Ser Val Val Gly Asp Ser Leu Phe CysThr Trp Asn Pro Ser Val Cys Ser Val Val Gly Asp Ser Leu Phe Cys

405 410 415 405 410 415

Pro Thr Ser Glu Glu Phe Ile Leu Pro Val Glu His Gly Asn Asp ArgPro Thr Ser Glu Glu Phe Ile Leu Pro Val Glu His Gly Asn Asp Arg

420 425 430 420 425 430

Cys Glu Trp Phe Leu Gln Leu Ser Asp Glu Ile Val Trp Asp Val LysCys Glu Trp Phe Leu Gln Leu Ser Asp Glu Ile Val Trp Asp Val Lys

435 440 445 435 440 445

Asp Lys Glu Ser Gly Lys Pro Arg Ala Arg Val Thr Ala Arg Ala GlyAsp Lys Glu Ser Gly Lys Pro Arg Ala Arg Val Thr Ala Arg Ala Gly

450 455 460 450 455 460

Asp Ile Cys Cys Met Pro Ala Asp Ile Arg His Gln Gly Tyr Ser ThrAsp Ile Cys Cys Met Pro Ala Asp Ile Arg His Gln Gly Tyr Ser Thr

465 470 475 480465 470 475 480

Lys Arg Ser Met Leu Leu Val Trp Glu Asn Gly Ser Pro Lys Ile ProLys Arg Ser Met Leu Leu Val Trp Glu Asn Gly Ser Pro Lys Ile Pro

485 490 495 485 490 495

Gln Met Ile Ala Asp Gly Thr Ala Pro Val Val Pro Val Thr PheGln Met Ile Ala Asp Gly Thr Ala Pro Val Val Pro Val Thr Phe

500 505 510 500 505 510

<210> 2<210> 2

<211> 170<211> 170

<212> PRT<212> PRT

<213> 鞘氨醇单胞菌(Sphingomonas sp. strain)<213> Sphingomonas sp. strain

<400> 2<400> 2

Met Ala Asp Val Val Thr Glu Phe Gly Ala Leu Thr Asp Tyr Arg LysMet Ala Asp Val Val Thr Glu Phe Gly Ala Leu Thr Asp Tyr Arg Lys

1 5 10 151 5 10 15

Arg Gly Val Glu Ile Ile Asp Asp Asp Pro Arg Asn Tyr Val Phe SerArg Gly Val Glu Ile Ile Asp Asp Asp Pro Arg Asn Tyr Val Phe Ser

20 25 30 20 25 30

Asn Val Phe Glu Val Ala Ala Asn Ala Ala Pro Tyr Glu Arg Val AlaAsn Val Phe Glu Val Ala Ala Asn Ala Ala Pro Tyr Glu Arg Val Ala

35 40 45 35 40 45

Val Gly Lys Asn Phe Glu Tyr Val Ile Glu Ser Ala Arg Ala Glu GlyVal Gly Lys Asn Phe Glu Tyr Val Ile Glu Ser Ala Arg Ala Glu Gly

50 55 60 50 55 60

Thr Ser Gly Trp Phe Ser Cys Ala His Asp Glu Phe Val Leu Ala MetThr Ser Gly Trp Phe Ser Cys Ala His Asp Glu Phe Val Leu Ala Met

65 70 75 8065 70 75 80

Asp Gly Gln Ile Glu Val His Asp Leu Lys Leu Asp Asn Ser Asp AlaAsp Gly Gln Ile Glu Val His Asp Leu Lys Leu Asp Asn Ser Asp Ala

85 90 95 85 90 95

Tyr Val Asp Pro Asp Ser Glu Gly Ala Val Ala Ile Gly Glu Ala LeuTyr Val Asp Pro Asp Ser Glu Gly Ala Val Ala Ile Gly Glu Ala Leu

100 105 110 100 105 110

Pro Glu Gly Arg Lys Met Gly Arg Ile Val Leu Arg Arg Gly His MetPro Glu Gly Arg Lys Met Gly Arg Ile Val Leu Arg Arg Gly His Met

115 120 125 115 120 125

Ala Leu Leu Pro His Gly Ala Ala Tyr Arg Phe Tyr Ala Glu Gln ProAla Leu Leu Pro His Gly Ala Ala Tyr Arg Phe Tyr Ala Glu Gln Pro

130 135 140 130 135 140

Ala Ala Met Leu Phe Gln Ser Ile Glu Gly Ala Val Thr Val Gln LysAla Ala Met Leu Phe Gln Ser Ile Glu Gly Ala Val Thr Val Gln Lys

145 150 155 160145 150 155 160

Trp Gly Glu Ile Cys Gln Thr Glu Ala AlaTrp Gly Glu Ile Cys Gln Thr Glu Ala Ala

165 170 165 170

<210> 3<210> 3

<211> 341<211> 341

<212> PRT<212> PRT

<213> 鞘氨醇单胞菌(Sphingomonas sp. strain)<213> Sphingomonas sp. strain

<400> 3<400> 3

Met Ala Met Ser Glu Ala Leu Glu Ile Ile Asp Phe Gly Asp Ser LysMet Ala Met Ser Glu Ala Leu Glu Ile Ile Asp Phe Gly Asp Ser Lys

1 5 10 151 5 10 15

Ala Arg Thr Asp Thr Glu His Leu Ala Ile Asn Asn Glu Thr Gly TyrAla Arg Thr Asp Thr Glu His Leu Ala Ile Asn Asn Glu Thr Gly Tyr

20 25 30 20 25 30

Arg Ser Phe Arg Ala Gly Gly Phe Thr Phe Thr Arg Asp Glu Tyr PheArg Ser Phe Arg Ala Gly Gly Phe Thr Phe Thr Arg Asp Glu Tyr Phe

35 40 45 35 40 45

Ala Arg Leu Thr Trp Pro Gly Gly Ser His Ile Ile Pro Ile Asp AlaAla Arg Leu Thr Trp Pro Gly Gly Ser His Ile Ile Pro Ile Asp Ala

50 55 60 50 55 60

Phe Leu Arg Ala Val Met Arg Asp Val Ala Trp Gly Phe Phe Tyr GlyPhe Leu Arg Ala Val Met Arg Asp Val Ala Trp Gly Phe Phe Tyr Gly

65 70 75 8065 70 75 80

Val Val Asn Phe Asp His Val Phe Gly Thr Ile Asn His Tyr Gly GluVal Val Asn Phe Asp His Val Phe Gly Thr Ile Asn His Tyr Gly Glu

85 90 95 85 90 95

Val Thr Met Phe Ala Gly Arg Phe Asn Asp Ala Tyr Arg Asn Ala GlyVal Thr Met Phe Ala Gly Arg Phe Asn Asp Ala Tyr Arg Asn Ala Gly

100 105 110 100 105 110

Arg Asp His Glu Glu Arg Phe Lys Ser Ser Ala Leu Met Ala Val PheArg Asp His Glu Glu Arg Phe Lys Ser Ser Ala Leu Met Ala Val Phe

115 120 125 115 120 125

Lys Asp Ile Leu Ser Asp Trp Thr Val Glu Gly Tyr Asp Pro Phe AlaLys Asp Ile Leu Ser Asp Trp Thr Val Glu Gly Tyr Asp Pro Phe Ala

130 135 140 130 135 140

Ala Pro Met Lys Thr Gly Leu Pro Trp Gly Ile Lys Asn Gly Asn AsnAla Pro Met Lys Thr Gly Leu Pro Trp Gly Ile Lys Asn Gly Asn Asn

145 150 155 160145 150 155 160

Asp Glu Ala Ile Ser Arg Gln Arg Val Thr Ala Arg Arg Met Val GlyAsp Glu Ala Ile Ser Arg Gln Arg Val Thr Ala Arg Arg Met Val Gly

165 170 175 165 170 175

Leu Pro Gly Asp Thr Pro Val Arg Thr Asp Ala Asn Gly Phe Pro ValLeu Pro Gly Asp Thr Pro Val Arg Thr Asp Ala Asn Gly Phe Pro Val

180 185 190 180 185 190

Asn Arg Gln Phe Ala Asp Val Pro Gln Glu Gln Pro Val Val Glu AlaAsn Arg Gln Phe Ala Asp Val Pro Gln Glu Gln Pro Val Val Glu Ala

195 200 205 195 200 205

Glu Pro Gly Phe Glu Ala Glu Val Ser Ala Tyr Asn Leu Phe Gly TyrGlu Pro Gly Phe Glu Ala Glu Val Ser Ala Tyr Asn Leu Phe Gly Tyr

210 215 220 210 215 220

Leu Ser Arg Ser Asp Val Thr Trp Asn Pro Ser Val Cys Ser Val ValLeu Ser Arg Ser Asp Val Thr Trp Asn Pro Ser Val Cys Ser Val Val

225 230 235 240225 230 235 240

Gly Asp Ser Leu Phe Cys Pro Thr Ser Glu Glu Phe Ile Leu Pro ValGly Asp Ser Leu Phe Cys Pro Thr Ser Glu Glu Phe Ile Leu Pro Val

245 250 255 245 250 255

Glu His Gly Asn Asp Arg Cys Glu Trp Phe Leu Gln Leu Ser Asp GluGlu His Gly Asn Asp Arg Cys Glu Trp Phe Leu Gln Leu Ser Asp Glu

260 265 270 260 265 270

Ile Val Trp Asp Val Lys Asp Lys Glu Ser Gly Lys Pro Arg Ala ArgIle Val Trp Asp Val Lys Asp Lys Glu Ser Gly Lys Pro Arg Ala Arg

275 280 285 275 280 285

Val Thr Ala Arg Ala Gly Asp Ile Cys Cys Met Pro Ala Asp Ile ArgVal Thr Ala Arg Ala Gly Asp Ile Cys Cys Met Pro Ala Asp Ile Arg

290 295 300 290 295 300

His Gln Gly Tyr Ser Thr Lys Arg Ser Met Leu Leu Val Trp Glu AsnHis Gln Gly Tyr Ser Thr Lys Arg Ser Met Leu Leu Val Trp Glu Asn

305 310 315 320305 310 315 320

Gly Ser Pro Lys Ile Pro Gln Met Ile Ala Asp Gly Thr Ala Pro ValGly Ser Pro Lys Ile Pro Gln Met Ile Ala Asp Gly Thr Ala Pro Val

325 330 335 325 330 335

Val Pro Val Thr PheVal Pro Val Thr Phe

340 340

Claims (9)

1. The mutein applied to the low-temperature degradation of hydroquinone wastewater is characterized in that the amino acid sequence of the mutein is shown in SEQ ID NO. 1.
2. The mutein according to claim 1, wherein the mutein comprises the mutein hqdAmuAnd the mutein hqdBmuThe mutein hqdAmuThe amino acid sequence of (A) is shown as SEQ ID NO.2, and the mutant protein hqdBmuThe amino acid sequence of (A) is shown in SEQ ID NO. 3.
3. The mutein according to claim 2, wherein the mutein hqdA is characterized in thatmuGlycine at the 17 th site, leucine at the 88 th site and valine at the 133 th site of the protein hqdA are respectively mutated into arginine, aspartic acid and histidine; the mutein hqdBmuThe methionine at the 39 th position and the glutamic acid at the 148 th position of the protein hqdB are respectively mutated into valine and lysine.
4. A construction method of mutant protein applied to low-temperature degradation of hydroquinone wastewater is characterized by comprising the following steps:
step one, taking a genome of Sphingomonas sp.strain as a PCR amplification template, amplifying hqdA by using PCR amplification primers P1 and P2, and amplifying hqdB by using PCR amplification primers P3 and P4, wherein the sequences of P1, P2, P3 and P4 are respectively as follows:
P1:AAGGATCCGATGGCCGACGTGGTCACC;
P2:GCAAGCTTTCAGGCCGCCTCGGTCTGG;
P3:AACATATGATGGCTATGTCCGAAGCACTTG;
P4:AACTCGAGTCAGAAAGTGACCGGGACCAC;
respectively purifying and enzyme-cutting the amplified products, and respectively connecting the enzyme-cut products to an escherichia coli co-expression vector pETduet-1 to construct pETduet-1-hqdA and pETduet-1-hqdB;
step two, amplifying hqdBmu by using PCR amplification primers P3 and P4 and an error-prone PCR kit, and amplifying hqdBmu by using PCR amplification primersAmplifying hqdAmu by using P1, P2 and an error-prone PCR kit, respectively purifying and enzyme-cutting the amplified products, respectively connecting the enzyme-cut products to pETduet-1-hqdA and pETduet-1-hqdB obtained in the step one to construct pETduet-1-hqdABmuAnd pETduet-1-hqdBAmu
Step three, the pETduet-1-hqdAB obtained in the step twomuAnd pETduet-1-hqdBAmuRespectively transferring the bacterial colonies into escherichia coli BL21 for colony culture, and transferring the obtained bacterial colonies into an LB culture medium for strain culture;
fourthly, carrying out site-directed mutagenesis on the hqdA and the hqdB on the pETduet-1-hqdAB by utilizing a site-directed mutagenesis kit, and finally constructing and finishing pETduet-1-hqdABmu 2;
step five, pETduet-1-hqdABmu2, transforming the strain into escherichia coli BL21 competence, culturing the strain, inoculating the strain to a culture medium for high-density fermentation, adding a feed liquid after the fermentation, adding a sterilized yeast extract until the final concentration is 1-4%, culturing the strain at 37 ℃ until the concentration is OD600 (OD 600) is 0.5-0.8, adding IPTG (isopropyl-beta-thiogalactoside) with the concentration of 0.1-0.8mM, inducing at 20-28 ℃ overnight to obtain a fermentation liquid, adding an induction feed liquid during the induction process, separating escherichia coli from the fermentation liquid after the feed is finished, centrifugally collecting the thalli, resuspending the thalli according to the proportion of 1g of the thalli with 6ml of PBS (phosphate buffer solution), crushing the thalli by using ultrasonic waves, and purifying the protein by using a His-tag purification kit to prepare the mutant protein.
5. The method for constructing the mutein of claim 4, wherein the LB solid medium in step three contains 0.005-0.015g/L hydroquinone, 0.1-0.8mM IPTG.
6. The method of claim 4, wherein the inoculation in step five is carried out using a high-density fermentation medium of Escherichia coli at an inoculation ratio of 1:50 to 1: 100.
7. The method of claim 4, wherein the feed solution in step five comprises glucose, magnesium sulfate, vitamins and trace elements.
8. The method of claim 4, wherein the inducing supplement solution of step five comprises glycerol, magnesium sulfate, vitamin B1, and trace elements.
9. The use of the mutein of claim 1 for the degradation of low temperature phenol-containing wastewater.
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