WO2015043136A1 - 去除邻硝基苯甲酸的酶制剂及应用 - Google Patents

去除邻硝基苯甲酸的酶制剂及应用 Download PDF

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WO2015043136A1
WO2015043136A1 PCT/CN2014/072435 CN2014072435W WO2015043136A1 WO 2015043136 A1 WO2015043136 A1 WO 2015043136A1 CN 2014072435 W CN2014072435 W CN 2014072435W WO 2015043136 A1 WO2015043136 A1 WO 2015043136A1
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enzyme
enzyme preparation
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nitrobenzoic acid
leu
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虞方伯
管莉菠
骆林平
单胜道
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浙江农林大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

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  • the invention belongs to the field of enzyme genetic engineering and enzyme engineering, and relates to an enzyme preparation for removing o-nitrobenzoic acid and an application thereof.
  • Nitroaromatic compounds are widely found in nature and are mainly used in the production of dyes, insecticides, explosives, pesticides, pharmaceuticals and other chemical products. With the rapid development of industry and agriculture, the nitroaromatic compounds entering the environment are increasing, causing serious pollution to the ecological environment on which humans depend. Furthermore, due to the presence of nitro and benzene ring structures, nitroaromatic compounds are more difficult to biodegrade than other compounds. How to reduce and remove the harm of such compounds to the ecological environment has become a hot issue of common concern of the whole society.
  • nitroaromatic compounds mainly includes physical, chemical and microbial degradation methods. Although physical and chemical methods can effectively remove such substances, they are costly, require special equipment and equipment, and are easily corroded by acids and alkalis, posing certain risks. In fact, the degradation and conversion of nitroaromatic compounds in the environment is mainly accomplished by microorganisms (microorganisms are mostly metabolized by a series of enzymatic reactions). Therefore, how to make full use of functional microbial resources and remove specific pollutants has become an urgent need to repair related environments.
  • O-Nitrobenzoic acid (2-NBA) is an important intermediate for the organic synthesis and preparation of antioxidants and rust inhibitors for dyes, perfumes, greases and lubricating oils, and its industrial demand is enormous.
  • the substance is harmful to the environment, pollutes the water and the atmosphere, and easily forms acid rain in atmospheric chemistry and atmospheric physical changes.
  • 2-NBA will cause serious damage to animals and plants, and the reproduction and development of fish will be seriously affected.
  • acidification of water can cause changes in the composition of aquatic organisms, increased acid-tolerant algae and fungi, and reduced root plants, bacteria and vertebrates, and reduced decomposition rate of organic matter. How to remove 2-NBA efficiently, at low cost, and environmentally friendly is of great research significance.
  • the object of the present invention is to provide an enzyme preparation for removing o-nitrobenzoic acid against the deficiencies of the prior art.
  • Another object of the invention is to provide the use of the enzyme preparation.
  • an enzyme preparation for removing o-nitrobenzoic acid wherein the enzyme preparation is an aqueous solution or a buffer solution, and comprises an o-nitrobenzoic acid degrading enzyme having an amino acid sequence of SEQ ID No. As shown in Fig. 1, and MnCl2 having a final concentration of 0.1-0.3 mM, the pH of the reaction system is 6.0-8.0.
  • the buffer solution is generally 50-100 having a pH of 7.2-7.7. mM sodium phosphate buffer or potassium phosphate buffer.
  • the pure enzyme content of the o-nitrobenzoic acid degrading enzyme is 0.03-0.1 mg/mL.
  • the final concentration of MnCl2 was 0.2 mM.
  • a preparation method of an enzyme preparation for removing o-nitrobenzoic acid which is characterized in that: a sample of purified enzyme solution is taken 20
  • the enzyme preparation was prepared by diluting to a volume of 1 liter with a pH of 7.5 in 50 mM sodium phosphate buffer, and the final concentration of manganese chloride in the enzyme preparation was 0.1-0.3 mM.
  • the purified enzyme solution sample is prepared as follows: a strain containing a recombinant plasmid containing the nbaB gene coding fragment and incubated in LB medium at 37 ° C, and adding IPTG to a final concentration of 1 when the A600nm value is 0.6 is selected.
  • the cells were collected by centrifugation, resuspended in 50 mM sodium phosphate buffer pH 7.5, and the cells were sonicated in an ice water bath - the power was 400 W ultrasonic disruption for 5 s, pause 15 s, a total of 60 cycles; then, centrifuged at 10000 g at 4 ° C 30
  • the cell debris is removed, and the supernatant is a crude enzyme extract; the crude enzyme extract is purified using a one-stop histidine-tagged protein purification kit to obtain a purified enzyme solution sample.
  • the use of the enzyme preparation for degrading o-nitrobenzoic acid in soil is 18-36 ° C, pH It is 6.0-8.5.
  • the present invention is derived from Pseudomonas sp.
  • the gene fragment responsible for 2-NBA metabolism, nbaB, was cloned in ONBA-17.
  • the invention can provide effective biotechnology means for the degradation of 2-NBA, and is of great significance for the repair of 2-NBA polluted environment.
  • the bacterium is Pseudomonas
  • the strain ONBA-17 of sp.) was deposited on September 2, 2013 at the General Microbiology Center of the China Microbial Culture Collection Management Committee, numbered CGMCC No. 8095.
  • the address of the depositary is the Institute of Microbiology, Institute of Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.
  • the classification of the fungus is Pseudomonas sp.
  • the enzyme preparation of the present invention has a high enzyme activity and is excellent in degrading 2-NBA;
  • the enzyme preparation of the invention adopts the microbial degradation enzyme prepared by genetic engineering means, has low cost and is favorable for large-scale promotion;
  • the enzyme preparation of the present invention degrades 2-NBA by enzymatic method, and has the advantages of good effect, simple operation, safety, mild reaction conditions, and no secondary pollution.
  • Figure 1 is the total DNA of Pseudomonas sp. ONBA-17 (Genomic DNA) electrophoresis detection map.
  • Figure 2 is a graph showing the effect of pH on the activity of NbaB enzyme.
  • Figure 3 is a plot of 2-NBA content in soil samples from different treatments.
  • the total DNA of the cells was extracted by SDS high salt precipitation method. Pick a single colony on the LB plate and connect to 100 The mL LB liquid medium was cultured to a stationary phase. Collect the cells by centrifugation and add an equal volume of TEN solution [10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH). 8.0), 0.1 M NaCl] Wash the cells, collect the cells by centrifugation, and suspend them in 10 mL of TE [10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH).
  • the appropriate total DNA of the extracted cells was appropriately diluted, and the mass was detected by 0.75% (w/v%) agarose electrophoresis (as shown in Fig. 1), and it was found that the total DNA extracted was mostly concentrated at 23 About kb, there is basically no RNA present, the purity is better, and the concentration is higher.
  • nbaB-f 5'-ACGACCATATGAGTTACCAAAACTTAG-3’ (underlined as NdeI restriction site, gene sequence is SEQ ID No. 3);
  • nbaB-r 5'-CATCAGAATTCGGAAGACCAGGAGC-3’ (Underlined as the EcoRI restriction site, the gene sequence is SEQ ID No. 4).
  • 25 ⁇ L amplification system 10 ⁇ Taq DNA polymerase reaction buffer 2.5 ⁇ L; dNTP (25 mM) 2 ⁇ L; primer (25 pmol/ ⁇ L) each 0.5 ⁇ L; Mg2+ buffer (25 mM) 2.5 ⁇ L; total DNA obtained in Example 1 0.5 ⁇ L (about 100 Ng); Taq enzyme (5 U/ ⁇ L) 0.3 ⁇ L; ddH2O 16.2 ⁇ L.
  • Reaction parameters pre-denaturation at 95 ° C for 5 min, denaturation at 94 ° C for 30 sec, annealing at 52 ° C 30 Sec, extended at 72 ° C for 1 min, amplified for 25 cycles, and finally extended at 72 ° C for 5 min.
  • the amplified product was detected by 0.75% (w/v%) agarose electrophoresis and the amplified fragment was found to have the expected size (1.9). Kb or so) matches. Cut the gel containing the amplified fragment of nbaB gene into sterile 1.5 The cells were weighed in a mL centrifuge tube, and the target fragment was recovered using a BIOMIGA gel recovery and purification kit, and stored at 4 ° C until use.
  • the obtained PCR product was subjected to TA cloning and subsequent operations with reference to the "Molecular Cloning Experimental Guide", and the vector pMD was cloned.
  • 19-T and related reagents were purchased from Bao Bio Biotechnology Co., Ltd. (TaKaRa, Dalian). After picking up the plasmid and verifying the correct size of the insert by agarose gel electrophoresis, the clones were picked and sent to Shanghai for sequencing.
  • the obtained nbaB encoding gene sequence is as SEQ As shown in ID No. 2, the corresponding amino acid sequence is SEQ ID No. 1 is shown.
  • the genetic sequence alignment was first filed on the National Center for Biotechnology Information (NCBI) website and then searched by GenBank through the BLAST search engine.
  • nbaB gene amplification product of the above Example 2 was digested with NdeI and EcoRI, The fragment was then inserted into the pET21b (+) vector digested with the same restriction endonuclease as described above.
  • the nbaB gene was initiated by the T7 promoter, and the expression product had six histidine tags at the C-terminus, and ampicillin was used as a screening marker.
  • the linked fragments were verified by Shanghai Biotech sequencing to ensure PCR No mutations were introduced during the process.
  • Recombinant plasmid transferred to Escherichia coli BL21 (DE3), then picked the strain containing the recombinant plasmid (No. Y-6), and verified the introduction of the foreign gene by Shanghai Biotech.
  • Y-6 was cultured in LB medium at 37 ° C. When the A600nm value was 0.6, IPTG was added to the final concentration of 1 mM was subsequently expressed overnight at 20 °C to avoid inclusion body production. The cells were collected by centrifugation, resuspended in 50 mM sodium phosphate buffer (pH 7.5), and sonicated in an ice water bath (power 400 W) Broken cells - ultrasonic disruption for 5 s, pause for 15 s, for a total of 60 cycles.
  • One-stop histidine-tagged protein purification kit (Ni-NTA Spin) Kit, QIAGEN) purified the above crude enzyme extract to obtain a purified enzyme solution sample (in which the pure enzyme content of o-nitrobenzoic acid degrading enzyme was 2.5 mg/mL), all operations were carried out in a low temperature environment.
  • the buffer is 50 mM sodium phosphate buffer (pH 7.5) to 200 ⁇ M 2-NBA was used as a substrate, and the purified enzyme solution samples mentioned in Examples 3-5 were added, and the final concentration was 0.2.
  • mM water-soluble metal compound test metal ions include: lithium chloride, manganese chloride, silver nitrate, nickel chloride, zinc chloride, cadmium chloride, copper chloride, ferrous chloride, barium chloride And cobalt chloride).
  • test metal ions include: lithium chloride, manganese chloride, silver nitrate, nickel chloride, zinc chloride, cadmium chloride, copper chloride, ferrous chloride, barium chloride And cobalt chloride).
  • a treatment in which the above test metal compound was not added was set as a control.
  • the results are shown in Table 1.
  • Nickel, silver, lithium, copper and cobalt ions have obvious inhibitory effects on enzyme activity.
  • Test object Relative activity (%) Test object Relative activity (%) Control 100 (0.2) * NiCl 2 70 (5.2) AgNO 3 96 (1.6) ZnCl 2 100 (0.2) LiCl 79 (2.7) MnCl 2 144 (0.2) CdCl 2 101 (0.3) FeCl 2 98 (1.4) CuCl 2 84 (1.8) BaCl 2 98 (2.6) CoCl 2 76 (2.6)
  • the soil with 2-NBA pollution history around the 2-NBA manufacturer was collected and quickly brought back to the laboratory. After homogenization and other steps, the 2-NBA residue was determined. The results showed that the residual amount of 2-NBA in the soil samples was 126.8. Mg/kg. The samples were divided into 3 portions and processed in 3 portions.
  • the specific test design is as follows: No. 1 treatment. Only collect soil samples, spray equal amount of water and stir evenly, the final moisture content of soil samples is controlled at 60%; No. 2 treatment.
  • the enzyme preparation as described above was sprayed into the collected soil sample, and stirred uniformly, and the final moisture content of the soil sample was controlled at 60%;
  • the above enzyme preparation to which manganese chloride was not added was sprayed into the collected soil sample, and stirred uniformly, and the final moisture content of the soil sample was controlled at 60%.
  • Set 3 repetitions per treatment, at fixed time intervals (6 h) Sampling and analysis of 2-NBA residues in the soil.
  • the addition of the enzyme preparation treatment (treatment Nos. 2 and 3) was significantly improved in the removal rate of 2-NBA in the soil sample compared with the control treatment (treatment No. 1).
  • the 2-NBA removal rate was significantly improved.
  • Treatment of 2-NBA in soil sample No. 2 after adding enzyme preparation 30 h achieved complete removal of 2-NBA (below the detection limit), while the 2-NBA residues of treatments 1 and 3 were 116.1 and 33.3 mg/kg, respectively, with a significant difference.
  • Ggcccccccgg caaagtggt ggttaaggcc aactggaagc Cttttgcgga aaactttgta 600

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Abstract

本发明提供了一种去除邻硝基苯甲酸的酶制剂及其应用。所述酶制剂为水溶液或缓冲溶液，包含一种邻硝基苯甲酸降解酶，其氨基酸序列如SEQ ID NO.1所示，以及终浓度为0.1-0.3mM的MnCl2，反应体系pH值为6.0_8.0。酶制剂为缓冲溶液时，该缓冲溶液一般为pH值7.2-7.7的50-100mM磷酸钠缓冲液或磷酸钾缓冲液。

Description

去除邻硝基苯甲酸的酶制剂及应用 技术领域
本发明属于酶基因工程及酶工程领域，涉及一种去除邻硝基苯甲酸的酶制剂及应用。
背景技术
随着我国化学工业的快速发展，有机合成所产三废（废水、废气、废渣）产生量呈逐年递增之势。上述三废中存在大量对生物体有毒害作用的物质，对环境造成了极为严重的污染和危害，威胁到人类和其它生物的安全和健康。其中，又以硝基芳香化合物的相关污染问题最为突出。
硝基芳香化合物广泛存在于自然界中，主要应用于染料、杀虫剂、炸药、农药、医药及其它化工产品的生产。随着工农业的迅速发展，进入到环境中的硝基芳香化合物日益增多，给人类赖以生存的生态环境造成了严重的污染。此外，由于硝基和苯环结构的存在，使得硝基芳香化合物比其它化合物更难于生物降解。如何减少和去除此类化合物对生态环境的危害，已成为全社会共同关注的热点问题。
硝基芳香化合物的去除主要有物理法、化学法和微生物降解法三种。物理和化学方法虽然能够有效去除此类物质，但成本高，需要特制的仪器和装备，且酸、碱易腐蚀，具有一定的危险性。实际上，硝基芳香化合物在环境中的降解和转化主要是由微生物完成的（微生物对其的代谢多通过一系列酶促反应完成）。因此，如何充分利用好功能微生物资源，去除特异性污染物，成为了修复相关环境的迫切需要。
邻硝基苯甲酸（2-NBA）是有机合成和制备染料、香料，润滑脂和润滑油的抗氧剂和金锈剂等的重要中间体，其工业需求量十分巨大。该物质对环境有危害，会对水体和大气造成污染，易在大气化学和大气物理变化中形成酸雨。当pH值降到 5.0以下时，2-NBA会给动、植物造成严重危害，鱼的繁殖和发育会受到严重影响。此外，水体酸化还会导致水生生物的组成结构发生变化，耐酸的藻类、真菌增多，而有根植物、细菌和脊椎动物减少，有机物的分解率降低。如何高效、低成本，且环境友好的去除2-NBA颇具研究意义。
 技术问题
本发明的目的是针对现有技术的不足，提供一种去除邻硝基苯甲酸的酶制剂。
本发明的另一目的是提供该酶制剂的应用。
 技术解决方案
本发明解决其技术问题所采用的技术方案是：
一种去除邻硝基苯甲酸的酶制剂，所述的酶制剂为水溶液或缓冲溶液，包含一种邻硝基苯甲酸降解酶，其氨基酸序列如SEQ ID No. 1所示，以及终浓度为0.1-0.3 mM的MnCl2，反应体系pH值为6.0-8.0。酶制剂为缓冲溶液时，该缓冲溶液一般为pH值7.2-7.7的50-100 mM磷酸钠缓冲液或磷酸钾缓冲液。进一步的，所述的邻硝基苯甲酸降解酶的纯酶含量为0.03-0.1 mg/mL。所述的MnCl2终浓度为0.2 mM。
一种去除邻硝基苯甲酸的酶制剂的制备方法，其特征在于：取纯化酶液样本20 mL和氯化锰，用pH值7.5的50 mM磷酸钠缓冲液定容至1 L制成酶制剂，酶制剂中氯化锰的终浓度为0.1-0.3 mM。
所述纯化酶液样本的制备方法如下：挑取含有重组质粒的菌株,该重组质粒上含有nbaB基因编码片段，用LB培养基于37℃培养，待A600nm值为0.6时，加入IPTG至终浓度1 mM，随后于20℃过夜表达,离心收集细胞，重悬于50 mM pH 7.5的磷酸钠缓冲液，在冰水浴中超声破碎细胞——功率为400 W超声波破碎5 s，暂停15 s，共进行60个循环；随后，于4℃，10000 g离心30 min去除细胞碎片，上清液即为粗酶提取液；使用一站式组氨酸标签蛋白纯化试剂盒对上述粗酶提取液进行纯化，获得纯化酶液样本。
所述的酶制剂在降解土壤中邻硝基苯甲酸中的应用。应用所述酶制剂降解土壤中邻硝基苯甲酸时，体系温度为18-36℃，pH 为6.0-8.5。
本发明从Pseudomonas sp. ONBA-17中克隆得到负责2-NBA代谢的基因片段——nbaB。本发明能够为2-NBA的降解提供有效的生物技术手段，对于2-NBA污染环境的修复具有重要意义。该菌是假单胞菌属（Pseudomonas sp.）的菌株ONBA-17，于2013年9月2日在中国微生物菌种保藏管理委员会普通微生物中心保藏，编号为CGMCC No. 8095。保藏单位的地址为北京市朝阳区北辰西路1 号院中科院微生物研究所，该菌的分类命名为假单胞菌Pseudomonas sp.。
 有益效果
本发明酶制剂的有益效果主要体现在以下几个方面：
（1）通过优选添加金属化合物，使得本发明的酶制剂酶活高，降解2-NBA效果优异；
（2）本发明的酶制剂，所采用的微生物降解酶采用基因工程手段制备，成本低，有利于大规模推广；
（3）本发明的酶制剂，采用酶法降解2-NBA，不仅效果好，且操作简单、安全、所需反应条件温和，不形成二次污染。
 附图说明
图1是Pseudomonas sp. ONBA-17总DNA（Genomic DNA）电泳检测图。
图2 是pH值对NbaB酶活力的影响图。
图3 是不同处理土壤样品中2-NBA含量变化曲线。
 本发明的最佳实施方式
在本发明中，若非特指，所有的份、百分比均为重量单位，所有的设备和原料等均可从市场购得或是本行业常用的。
Pseudomonas sp. ONBA-17总DNA（Genomic DNA）的提取
1.1 Pseudomonas sp. ONBA-17的扩大培养
在无菌操作环境下，将-70℃保存的Pseudomonas sp. ONBA-17以接种针挑取小块，放置、涂布于Luria-Bertani（LB）固体培养基之上。28℃，静置培养3 d。挑选单菌落，经2次转接，观察发现平板上菌落形态一致（无杂菌）。这里所述的“平板”和Luria-Bertani（LB）固体/液体培养基等均为微生物研究/生产领域常见术语、培养基、技术和方法，具体参见《分子克隆实验指南》（J. 萨姆布鲁克，等著. 分子克隆实验指南（第三版）[M]. 黄培堂，等译. 北京：科学出版社，2008）。下文中如无特别备注或说明，均为微生物研究常用技术、方法、培养基、试剂和药品，且均在《分子克隆实验指南》中有所记录。
1.2 Pseudomonas sp. ONBA-17总DNA的提取
菌体总DNA的提取采用SDS高盐沉淀法。挑取LB平板上的单菌落，接至100 mL的LB液体培养基培养至稳定期。离心收集菌体，加等体积的TEN溶液[10 mM Tris-Cl (pH 8.0)，1 mM EDTA (pH 8.0)，0.1 M NaCl]洗涤菌体，离心收集菌体，悬浮于10 mL TE[10 mM Tris-Cl (pH 8.0)，1 mM EDTA (pH 8.0)]溶液中，加入100 μL溶菌酶（100 mg/mL），37℃水浴1 h，加入40 μL蛋白酶K（20 mg/mL），再加入300 μL 20%（w/v%）的SDS，37℃水浴过夜（约12 h）。加入1/2体积饱和NaCl剧烈振荡，12000 g离心10 min，上清用等体积酚：氯仿抽提2次，12000 g离心10 min，收集上清，加入等体积TE溶液（稀释调整NaCl浓度），0.6倍体积异丙醇沉淀，12000 g离心15 min，70%（v/v%）乙醇洗涤沉淀，乙醇挥发后溶于100 μL TE中。
将提取到的菌体总DNA适当稀释后，经0.75%（w/v%）琼脂糖电泳检测其质量（如图1所示），发现所提取的总DNA大部分集中在23 kb左右，基本上没有RNA存在，纯度较好，浓度较高。
 本发明的实施方式
实施例2-NBA降解酶编码基因nbaB的获得
经过大量的分析实验，从24对自行设计的引物中筛选出一对有效扩增引物（分别记为SEQ ID No.3和SEQ ID No.4）。
nbaB-f，5’-ACGACCATATGAGTTACCAAAACTTAG-3’ （下划线为NdeI酶切位点，基因序列为SEQ ID No.3）；
nbaB-r，5’-CATCAGAATTCGGAAGACCAGGAGC-3’ （下划线为EcoRI酶切位点，基因序列为SEQ ID No.4）。
25 µL扩增体系：10×Taq DNA聚合酶反应缓冲液2.5 µL；dNTP（25 mM）2 µL；引物（25 pmol/µL）各0.5 µL；Mg2+缓冲液（25 mM）2.5 µL；实施例1中所得总DNA 0.5 µL（约100 ng）；Taq酶（5 U/µL）0.3 µL；ddH2O 16.2 µL。反应参数：95℃预变性5 min，94℃变性30 sec，52℃退火30 sec，72℃延伸1 min，扩增25个循环，最后72℃终延伸5 min。
扩增产物经0.75%（w/v%）琼脂糖电泳检测，发现扩增片段与预期大小（1.9 kb左右）相符。切取含有nbaB基因扩增片段的凝胶，放入无菌1.5 mL离心管中称重，使用BIOMIGA胶回收纯化试剂盒进行目的片段回收，4℃保存备用。
所得PCR产物参照《分子克隆实验指南》，进行TA克隆和后续操作，克隆用载体pMD 19-T及相关试剂均购自宝生物生物技术有限公司（TaKaRa，大连）。挑取质粒经琼脂糖凝胶电泳验证插入片段大小正确后，挑取克隆子交上海生工测序。所得nbaB编码基因序列如SEQ ID No. 2所示，其相应氨基酸序列如SEQ ID No. 1所示。基因序列比对先是登录美国国家生物技术信息中心（NCBI）网站，再经BLAST搜索引擎搜寻GenBank完成。
结果显示，同nbaB编码基因序列同源的功能基因最高序列同源性仅为84%（U49504.1），且U49504.1指代基因未见其关于2-NBA降解的报道。而同NbaB氨基酸序列同源的蛋白最高序列同源性仅为82%（YP_987297.1），且YP_987297.1所指代的双加氧酶未见其关于2-NBA降解的报道。
实施例3-表达载体的构建、表达与纯化
用NdeⅠ和EcoRI消化上述实施例2中的nbaB基因扩增产物, 再将片段插入到用上述同种限制性内切酶消化过的pET21b(+)载体中。在重组质粒中，nbaB基因由T7启动子启动，表达产物C端带有6个组氨酸标签，氨苄青霉素为筛选标记。联入片段经上海生工测序验证，以确保PCR 过程中没有引入突变。
重组质粒转入Escherichia coli BL21（DE3），随后挑取含有重组质粒的菌株（编号为Y-6），经上海生工测序验证外源基因导入情况。Y-6用LB培养基于37℃培养，待A600nm值为0.6时，加入IPTG至终浓度1 mM，随后于20℃过夜表达，以避免包涵体产生。离心收集细胞，重悬于50 mM的磷酸钠缓冲液（pH 7.5），在冰水浴中超声（功率为400 W）破碎细胞——超声波破碎5 s，暂停15 s，共进行60个循环。随后，于4℃，10000 g离心30 min去除细胞碎片。上清液即为粗酶提取液，低温保存。该步骤实验进行同时，设置未转入重组质粒的E. coli BL21（DE3）作为对照，并同样进行诱导、离心、破碎和收集粗酶提取液等步骤。
使用一站式组氨酸标签蛋白纯化试剂盒（Ni-NTA Spin Kit，QIAGEN）对上述粗酶提取液进行纯化，获得纯化酶液样本（其中邻硝基苯甲酸降解酶的纯酶含量为2.5 mg/mL），所有操作均在低温环境下进行。
实施例4 NbaB 对2-NBA的作用效果
采用上述实施例3中的纯化酶液样本，向0.5 mL的反应体系（含50 mM的磷酸钠缓冲液，pH 7.5，25℃）中加入终浓度为200 µM的2-NBA作为反应底物，并测定NbaB酶活力。一个酶活力单位（U）定义为：25℃条件下，每分钟转化1 µmol 2-NBA所需的酶量。比活力表示为每毫克蛋白的酶活力单位。蛋白浓度以牛血清白蛋白作为标准参照，通过Bradford法测定。2-NBA含量测定 参照Yoshie Hasegawa等人的方法（Yoshie Hasegawa et al. 2000. A novel degradative pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fuorescens strain KU-7. FEMS Microbiology Letters，190：185-190）进行 （下同）。
结果显示，上述纯化酶液样本对2-NBA的酶活力为3446 U/mg（该值为5次实验重复的平均值，标准偏差为2.1 U/mg），对照（实施例3中提到的未转入重组质粒E. coliBL21的粗酶提取纯化物）无2-NBA降解能力，这也进一步证实了实施例2中所得基因片段的功能作用。
将纯化酶液样本在50℃水浴60 min后，再通过上述方法测定酶活力。结果显示，酶活力较为稳定，保持了82.1%（该值为4次实验重复的平均值，标准偏差为1.6%）的初始酶活力。
实施例5 反应体系pH对酶活性的影响
参照实施例4准备反应体系，缓冲液仍为50 mM的磷酸钠缓冲液（测试pH值分设5.5，6.0，6.5，7.0，7.5，8.0，8.5，9.0），以200 µM的2-NBA为底物，并添加实施例3和4中提及的纯化酶液样本，结果如图2所示。由图2可知，当反应体系pH值介于6.0和8.0之间时，酶活力保持在较高水平（较高水平指最大酶活性的75%及以上）。
实施例6 添加金属化合物对酶活性的影响
参照实施例4准备反应体系，缓冲液为50 mM的磷酸钠缓冲液（pH值7.5），以200 µM的2-NBA为底物，添加实施例3-5中提及的纯化酶液样本，以及终浓度为0.2 mM的易溶于水的金属化合物（测试金属离子包括：氯化锂、氯化锰、硝酸银、氯化镍、氯化锌、氯化镉、氯化铜、氯化亚铁、氯化钡和氯化钴）。同时，设置不添加上述测试金属化合物的处理作为对照。结果如表1所示，镍、银、锂、铜和钴离子对酶活有明显的抑制作用，锌、亚铁、钡和镉离子对酶活力影响不显著，锰离子添加可使酶活力较对照提高44%，达显著差异水平。
表1、金属离子对酶活力的影响
测试物 相对活性
（%） 		测试物 相对活性
（%）
对照 100 (0.2) * NiCl2 70 (5.2)
AgNO3 96 (1.6) ZnCl2 100 (0.2)
LiCl 79 (2.7) MnCl2 144 (0.2)
CdCl2 101 (0.3) FeCl2 98 (1.4)
CuCl2 84 (1.8) BaCl2 98 (2.6)
CoCl2 76 (2.6)
* 括号内数值表示标准差（S.D.，n= 3）
实施例7 用酶制剂去除2-NBA污染土壤中的2-NBA残留
取纯化酶液样本20 mL和适量氯化锰，用pH值7.5的50 mM磷酸钠缓冲液定容至1 L制成酶制剂，酶制剂中氯化锰的终浓度为0.2 mM。
采集2-NBA生产厂家周边有2-NBA污染史的土壤，迅速带回实验室，经匀化等步骤后，测定其中的2-NBA残留量。结果显示，所采土壤样品中2-NBA残留量为126.8 mg/kg。将所采样品，均分成3份，每份3个处理。具体试验设计如下：1号处理。仅为采集土样，喷施等量清水并搅拌均匀，土样终含水率控制在60%；2号处理。向所采集土样中喷施如上所述的酶制剂，并搅拌均匀，土样终含水率控制在60%；3号处理。向所采集土样中喷施未添加氯化锰的上述酶制剂，并搅拌均匀，土样终含水率控制在60%。每个处理设3个重复，按照固定时间间隔（6 h）采样，并分析土壤中2-NBA的残留情况。结果如图3所示，添加酶制剂处理（2和3号处理）较对照处理（1号处理）土样中2-NBA去除速率提升显著。而2号同3号处理相比，其2-NBA去除速率又显著提升。2号处理土样中的2-NBA，在添加酶制剂后30 h实现了对2-NBA的完全去除（低于检测限），而同时期1和3号处理2-NBA残留分别为116.1和33.3 mg/kg，差异达显著水平。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
 工业实用性
序列表自由内容
SEQUENCE LISTING
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<120> 去除邻硝基苯甲酸的酶制剂及应用
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Claims (7)

  1. 一种去除邻硝基苯甲酸的酶制剂，其特征在于：所述的酶制剂为水溶液或缓冲溶液，包含一种邻硝基苯甲酸降解酶，其氨基酸序列如SEQ ID No. 1所示，以及终浓度为0.1-0.3 mM的MnCl2，反应体系pH值为6.0-8.0。
  2. 根据权利要求1所述的酶制剂，其特征在于：所述的邻硝基苯甲酸降解酶的纯酶含量为0.03-0.1 mg/mL。
  3. 根据权利要求1所述的酶制剂，其特征在于：所述的MnCl2终浓度为0.2 mM。
  4. 一种去除邻硝基苯甲酸的酶制剂的制备方法，其特征在于：取纯化酶液样本20 mL和氯化锰，用pH值7.5的50 mM磷酸钠缓冲液定容至1 L制成酶制剂，酶制剂中氯化锰的终浓度为0.1-0.3 mM。
  5. 权利要求1所述的酶制剂的制备方法，其特征在于：所述纯化酶液样本的制备方法如下：挑取含有重组质粒的菌株,该重组质粒上含有nbaB基因编码片段，用LB培养基于37℃培养，待A600nm值为0.6时，加入IPTG至终浓度1 mM，随后于20℃过夜表达,离心收集细胞，重悬于50 mM pH 7.5的磷酸钠缓冲液，在冰水浴中超声破碎细胞——功率为400 W超声波破碎5 s，暂停15 s，共进行60个循环；随后，于4℃，10000 g离心30 min去除细胞碎片，上清液即为粗酶提取液；使用一站式组氨酸标签蛋白纯化试剂盒对上述粗酶提取液进行纯化，获得纯化酶液样本。
  6. 权利要求1所述的酶制剂在降解土壤中邻硝基苯甲酸中的应用。
  7. 如权利要求6 所述的应用，其特征在于：应用权利要求1 所述酶制剂降解土壤中邻硝基苯甲酸时，体系温度为18-36℃，pH 为6.0-8.5。
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