WO2007062543A1 - Monooxygenase d'alcanes a chaine longue thermophiles et son gene codant, et leurs utilisations - Google Patents

Monooxygenase d'alcanes a chaine longue thermophiles et son gene codant, et leurs utilisations Download PDF

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WO2007062543A1
WO2007062543A1 PCT/CN2005/001974 CN2005001974W WO2007062543A1 WO 2007062543 A1 WO2007062543 A1 WO 2007062543A1 CN 2005001974 W CN2005001974 W CN 2005001974W WO 2007062543 A1 WO2007062543 A1 WO 2007062543A1
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monooxygenase
chain
recombinant
thermophilic
long
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PCT/CN2005/001974
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French (fr)
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Lu Feng
Lei Wang
Wei Wang
Yun Tang
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Nankai University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic

Definitions

  • the present invention relates to an enzyme and its encoding gene and application, and more particularly to a thermophilic long-chain alkane monooxygenase and its encoding gene and application.
  • Rhodococcus (Hamamura, Appl. Environ. Microbiol, 67: 4992-4998, 2001), Rhodococcus
  • thermophilic intracellular soluble protein of the monooxygenase capable of oxidizing long-chain anthracene have not been reported.
  • SUMMARY OF THE INVENTION It is an object of the present invention to provide a gene which encodes a thermophilic oxidizable long chain anthracene Monooxygenase.
  • Another object of the present invention is to provide a thermophilic long-chain alkane monooxygenase which oxidizes the terminal methyl group of a series of long chain indole hydrocarbons of C15 to C36 to the corresponding monohydric alcohol under high temperature conditions.
  • Another object of the present invention is to provide a recombinant plasmid capable of expressing a thermophilic long-chain alkane monooxygenase.
  • a further object of the present invention is to provide a recombinant strain capable of producing a thermophilic long-chain alkane monooxygenase. It is still another object of the present invention to provide a method of using the above-described thermophilic long-chain hydrocarbon monooxygenase in the treatment of petroleum contaminants and petroleum waste water or in improving the yield of microbial petroleum.
  • the present invention proposes a gene encoding a thermophilic long-chain alkane monooxygenase having a nucleotide sequence selected from the following a), b) or c) :
  • nucleotide sequence shown in SEQ ID NO: 1; b) due to the degeneracy of the genetic code, different from SEQ ID NO: 1, but the encoded amino acid sequence is identical to the amino acid sequence encoded by SEQ ID NO: 1.
  • thermophilic long-chain hydrocarbon monooxygenase has the nucleotide sequence shown in SEQ ID NO: 1.
  • the present invention provides a thermophilic long chain hydrocarbyl monooxygenase having an amino acid sequence selected from the group consisting of d), e) or f):
  • thermophilic long-chain alkane monooxygenase has the SEQ ID NO: The amino acid sequence shown in 2.
  • the present invention provides a recombinant plasmid expressing a thermophilic long-chain hydrocarbon monooxygenase comprising at least the above gene encoding a thermophilic long-chain alkane monooxygenase.
  • the vector of the above recombinant plasmid is pCom8.
  • the vector of the above recombinant plasmid is pET-28a(+).
  • the present invention proposes a recombinant strain producing a thermophilic long-chain hydrocarbon monooxygenase into which the above-described gene encoding a thermophilic long-chain alkane monooxygenase is introduced.
  • the recombinant agent is, for example, Pseudomonas fluorescens or Escherichia coli, preferably Pseudomonas fluorescens ⁇ 2 ⁇ 1 strain or Escherichia coli BL21 strain.
  • the present invention also provides a method of treating petroleum contaminants and petroleum wastewater, the method comprising the step of using the thermophilic long-chain alkane monooxygenase in the treatment of petroleum contaminants and petroleum wastewater.
  • the present invention also provides a method for increasing the yield of microbial petroleum hydrazine, which comprises the step of using the above thermophilic long-chain alkane monooxygenase in petroleum reclaiming.
  • stringent hybridization conditions means in the present specification that a so-called specific hybridization is formed under such conditions without forming a non-specific hybridization.
  • the stringent hybridization conditions may be such that DNA having a homology of not less than 70% of each other can hybridize between DNAs having a lower value than the above-mentioned values, and preferably having a homology of not less than 90%. DNA can be crossed between.
  • the hybridization membrane is placed in a pre-hybridization solution (0.25 mol/L sodium phosphate buffer, pH 7.0, 7% SDS), 50 ° C Pre-hybridization for 30 min; discard the pre-hybrid solution, add the hybridization solution (0.25 mol/L sodium phosphate buffer, pH 7.0, 7% SDS, isotope-labeled nucleotide fragment), hybridize at 50 °C for 12 hr; discard the hybrid solution, add Washing solution I (2xSSC and 0.1% SDS), wash the membrane twice at 50 °C for 30 min each time; add the washing solution II (0.5xSSC and 0.1% SDS), and wash the membrane at 50 °C for 30 min.
  • thermophilic long-chain hydrocarbon monooxygenase of the present invention further comprises an amino acid sequence encoding the enzyme molecule expressed by the nucleotide sequence shown in SEQ ID NO: 1. Performing one or more amino acid substitutions, insertions or deletions and still have the The nucleotide sequence of an enzyme-active protein.
  • the protein obtained by subjecting the amino acid of the enzyme molecule expressed by the thermophilic long-chain alkane monooxygenase gene of the present invention to one or more amino acid substitutions, insertions or deletions can also attain the object of the present invention.
  • the present invention also includes a protein having at least 70% homology with the amino acid sequence shown by SEQ ID NO: 2, preferably having at least 90% homology, but having thermophilic hydrocarbon monooxygenase activity.
  • the term "plurality" as used above may be a number less than 100, preferably a number less than 10.
  • thermophilic long-chain hydrocarbon monooxygenase has the same performance as the known terpene monooxygenase, which has thermophilic long-chain alkane monooxygenase activity, pi of 6.86, optimum temperature. At about 60 ° C, the optimum pH is about 7.5, the molecular weight is about 50400 Daltons, and the terminal methyl group of the C15-C36 alkane can be efficiently oxidized to produce the corresponding monohydric alcohol.
  • the thermal stability of the long chain hydrazine monooxygenase of the present invention is the highest among the hydrazine monooxygenases found so far.
  • the enzyme can be widely used in the treatment of petroleum pollutants and petroleum wastewater as well as in increasing the yield of microbial petroleum.
  • the enzyme can degrade long-chain (heavy) alkanes at high temperature, improve the ratio of light-mass hydrocarbons to heavy alkanes, improve the fluidity of crude oil, etc., thereby facilitating the reclamation of crude oil and increasing the yield of petroleum.
  • the enzyme degrades long-chain alkanes at high temperatures, environmental oil contaminants caused by oil field reclamation, oil spills and overflows, and high-temperature petroleum wastewater from petrochemicals. Processing also has a good application prospect.
  • Fig. 1 is a schematic diagram showing the construction of a thermophilic long-chain hydrocarbon monooxygenase gene recombinant plasmid pComALK according to an embodiment of the present invention.
  • 2 is a schematic diagram showing the construction of a thermophilic long-chain hydrocarbon monooxygenase gene recombinant plasmid pETALK according to an embodiment of the present invention.
  • Figure 3 shows the degradation of different alkanes by Bacillus thermophilus NG80-2.
  • Figure 4 is a mass spectrum of the product of the recombinant thermophilic long-chain alkane monooxygenase of n-hexadecane of the present invention.
  • Fig. 5 is a mass spectrum of the product of the recombinant thermophilic long-chain alkane monooxygenase of the present invention.
  • Figure 6 shows the results of substrate specificity analysis of the recombinant thermophilic long chain hydroxane monooxygenase of the present invention.
  • Figure 7 is a graph showing the effect of temperature on the activity of the recombinant thermophilic long chain hydroxane monooxygenase of the present invention.
  • Figure 8 is a graph showing the effect of pH on the activity of the recombinant thermophilic long-chain alkane monooxygenase of the present invention.
  • Thermophilic Bacillus licheniformis 0-2 (CGMCC No. 1228) Total DNA extraction studies have shown that genomic bacteria (Geobacillus thermodenitrificans) NG80-2 is produced under the condition of crude oil or liquid paraffin as carbon source. More alkane monooxygenase, which is particularly suitable for oxidizing C15 ⁇ C36 long-chain alkanes under high temperature conditions. Therefore, in this example, B. thermophilus NG80-2 obtained from the oil well formation water separation in the 69-8 block of Dagang Oilfield, Tianjin, China (this strain has been deposited in the Chinese Microbial Culture Collection Committee for common microorganisms). Center, the accession number is CGMCC No.1228), take 3ml of fresh culture cultured overnight, collect the cells by centrifugation, and suspend the body in 250W 50mM Tris buffer.
  • genomic bacteria Gaobacillus thermodenitrificans
  • the above total DNA solution 0.5 ⁇ l (about 10 ng) was used as a template, and the following oligonucleotide sequence was used as a primer, and 20 cycles of PCR were carried out according to the PCR cycle parameters set as described below.
  • the set PCR cycle parameters are as follows:
  • Upstream primer 5 -GTGACCCGGGATGAC AAA AAAA ATCC ATATTAATGC-3, downstream primer 5, -TAGA AGCTTTTATAC ATTTGAAG AAATATTTCGAT-3 '
  • the above PCR product was digested with Smal and Hindlll, subjected to 0.8% agarose gel electrophoresis, and the gel was recovered. 1.3 kb digested product fragment.
  • plasmid pCom8 which was digested with the same restriction endonuclease and recovered, and transformed with P. fluorescens ⁇ 2 ⁇ 1 [This strain was obtained from JB Van of the Swiss Federal Institute of Technology Zurich Professor Beilen provided, see J. Bacterology, Mar.
  • the recombinant strain was separately introduced into E2 medium with different C atom number alkane (n-d-decyl-n-octadecane) as the sole carbon source (NaNH4HP0 4 -4H 2 0 3.5g per liter of E2 medium, ⁇ 2 ⁇ 0 4 ⁇ 2 0 7.5g, KH 2 P0 4 3.7g, 1ml trace element solution, 1ml 1M MgS0 4 '7H 2 0; per liter of trace element
  • the solution contains FeS0 4 .7H 2 0 2.78g, MnCl 2 -4H 2 0 1.98g, CoS0 4 -7H 2 0 2.81g, CaCl 2 -2H 2 0 1.47g, CuCl 2 -2H 2 0 0.17g, ZnS0 4 In the medium of -7H 2 0 0.29g), after 48 hours, the transformant strain Pseudomonas fluorescens K0B2A 1AL
  • the control strain, Pseudomonas ⁇ 2 ⁇ 1 could not grow in the medium containing the above carbon source, indicating that the recombinant plasmid pComALK was converted to fluorescent false. After the bacterium ⁇ 2 ⁇ 1, the activity of the hydrazine monooxygenase can be expressed.
  • thermophilic long-chain hydrocarbon monohydrogenase gene was cloned into Escherichia coli
  • telomere sequences were used as primers, and 20 cycles of PCR were carried out according to the PCR cycle parameters set as described below.
  • the set PCR cycle parameters are as follows:
  • Upstream arch I 5 '-CCAGAATTCATGACAAAAAAAATCCATATTAATGC-3 ' Downstream primer: 3,-TAGCTTTATAAAGAAGTTTACATATTGAGCTCGATC-5
  • the above PCR product was digested with EcoRI and Xhol, and subjected to 0.8% agarose gel electrophoresis, and the gel was recovered to recover 1.3 kb. Product fragment. It was ligated with the plasmid pET-28a(+) which was digested with the same restriction endonuclease and recovered, and transformed into competent E.
  • coli BL21 (this strain was purchased from Tianjin Development Zone Houpu Biotechnology Development Co., Ltd., part number 69387- 3) Thereafter, it was applied to LB solid medium containing 50 ⁇ ⁇ / ⁇ 1 Kan (caramycin). The cells were cultured at 37 ° C for 16 to 18 hours, and the monoclonal colonies were picked for identification.
  • the pET-28a (+) plasmid inserted with the DNA sequence of SEQ ID NO: 1 was used as the recombinant plasmid pETALK (see Fig. 2), and the recombinant large intestine containing the plasmid was used.
  • the strain of Bacillus is Escherichia coli BL21ALK.
  • E. coli BL21ALK monoclonal was introduced into 20 ml of LB medium containing 5 ( ⁇ g/ml Kan, cultured at 37 ° C, 180 rpm / min for 12 hours, and then the culture was 1% (V / V).
  • the inoculum was added to 200 ml of LB medium containing 5 ( ⁇ g/ml Kan (total 10 shake flasks), and at 37 ° C, 220 rpm/min, when A600 was 0.6, IPTG was added to a final concentration of 0.2 mM, 45 °C. Induction at 150 rpm/min for 4 hours.
  • the cells were collected by centrifugation, suspended in 50 mM Tris-Cl (pH 7.5) buffer, and the cells were disrupted by sonication, and the supernatant was centrifuged to obtain a crude extract of recombinant alkane monooxygenase.
  • the supernatant was purified by Chelating Sepharose nickel affinity column chromatography, and the obtained enzyme preparation showed a band on SDS-PAGE.
  • the recombinant alkane monooxygenase was determined by a known protein chemical standard method.
  • the basic characteristics of the recombinant enzyme measured by SDS-PAGE is about 50,000 Daltons, similar to the theoretically estimated molecular weight (50,400 Daltons); the isoelectric point of the recombinant enzyme measured by the isoelectric precipitation method.
  • the pi is 6.86; the optimum temperature for the recombinase reaction is about 60 ° C, the optimum pH value. It is 7.5.
  • the recombinase only acts on long-chain indole hydrocarbons of C15 ⁇ C36, and has the highest activity for 16 ⁇ (see Figure 6), which is consistent with the degradation of alkanes by thermophilic denitrific spore NG80-2. (See Figure 3.)
  • Example 3 Recombination of thermophilic terpene monooxygenase to oxidize long-chain anthracene
  • the activity of the above-mentioned recombinant alkane monooxygenase was measured in the range of 37-70 ° C, and it was found that the optimum temperature for maintaining the activity of the enzyme was about 60 ° C (see Fig. 7).

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Description

嗜热长链烷烃单加氧酶及其编码基因与应用
技术领域 本发明涉及一种酶及其编码基因与应用，特别涉及一种嗜热长链烷烃 单加氧酶及其编码基因与应用。 背景技术 在微生物代谢脂肪族垸烃途径的第一步酶反应中， 烷烃单加氧酶
(alkane 1 -monooxygenase EC 1.14.15.3 ) 首先将垸烃末端甲基氧化成相应 的一元醇， 然后该烷烃依次在其它酶的作用下氧化成醛、 脂肪酸， 再 β- 氧化成二氧化碳和水（Berthe-Corti等， Acata Biotechnologica, 22: 299-336 , 2002) 。 自从 Peterson等首次报道了恶臭假单胞菌产生垸烃单加氧酶之后
(Peterson等， J. Bol. Chem, 242: 4334-4340, 1967) ，相继在不同的微生物 中，如棒杆菌（Co ^ebacter ww sp.) (Cardini, J. Biol. Chem, 245: 2789-2796, 1970)， ^JK^ffW (Pseudomonas aeruginosa) (Matsuyama, Agric. Biol. Chem. 45: 9-14, 1981 ) ， 麦芽糖假丝酵母 (Candida maltosa) (Zimmer, Biochem. Biophys Res. Commun, 251 : 244-247, 1998) ， 诺卡氏茵 {Nocardioides sp.)
(Hamamura, Appl. Environ. Microbiol, 67: 4992-4998, 2001 ) ， 红球菌
{Rhodococcus sp.) (Van Hamme, Microbiol Mol Biol Rev, 67: 503-549, 2001 ) 等发现了垸烃单加氧酶， 而不动杆菌 ( cinetobacter sp.) (Sakai , Biosci. Biotechnol. Biochem, 58: 2128-2130, 1994) 产生的垸烃双加氧酶能 氧化长链垸烃 （C12〜C44) 。 已发现的垸烃单加氧酶均为膜蛋白， 并且 只氧化 C6-C12的中长链垸烃。 有关嗜热的能氧化长链垸烃的单加氧酶胞 内可溶性蛋白的酶学和分子生物学特性还未见报道。 发明内容 本发明的一个目的是提供一种基因，其能编码嗜热的能氧化长链垸烃 的单加氧酶。
本发明的另一目的是提供一种嗜热长链烷烃单加氧酶，其可在高温条 件下， 将 C15~C36的一系列长链垸烃的末端甲基氧化为相应的一元醇。 本发明的另一目的是提供一种能表达嗜热长链烷烃单加氧酶的重组 质粒。
本发明的再一目的是提供一种能产生嗜热长链烷烃单加氧酶的重组 菌。 本发明的又一目的是提供一种在石油污染物和石油废水处理或提高 微生物石油釆收率中使用上述嗜热长链垸烃单加氧酶的方法。
为了实现上述目的， 本发明釆用如下技术方案： 本发明提出一种编码嗜热长链烷烃单加氧酶的基因，其具有选自于下 列 a) 、 b) 或 c) 的核苷酸序列：
a) SEQ ID NO: 1所示的核苷酸序列； b) 由于遗传密码的简并性， 不同于 SEQ ID NO: 1但编码的氨基酸序 列与 SEQ ID NO: 1所编码的氨基酸序列相同的核苷酸序列；
c) 在严格杂交条件下与上述 a) 或 b) 中的序列杂交， 并且编码具有 活性的嗜热长链垸烃单加氧酶的核苷酸序列。
在本发明的一个实施例中，上述编码嗜热长链垸烃单加氧酶的基因具 有 SEQ ID NO: 1所示的核苷酸序列。 本发明提出一种嗜热长链垸烃单加氧酶， 其具有选自于下列 d) 、 e) 或 f) 的氨基酸序列：
d) 上述 a) 、 b) 或 c) 所述的核苷酸序列编码的氨基酸序列； e) SEQ ID NO: 2所示的氨基酸序列； f) 上述 e) 中缺失、 替换或插入一个或多个氨基酸后的氨基酸序列， 并且具有该序列的蛋白质有嗜热长链烷烃单加氧酶的活性。 在本发明的一个实施例中， 上述嗜热长链烷烃单加氧酶具有 SEQ ID NO: 2所示的氨基酸序列。 本发明提出一种表达嗜热长链垸烃单加氧酶的重组质粒，其至少包括 上述编码嗜热长链烷烃单加氧酶的基因。
在本发明的一个实施例中， 上述重组质粒的载体为 pCom8。
在本发明的另一实施例中， 上述重组质粒的载体为 pET-28a(+)。
本发明提出一种产生嗜热长链垸烃单加氧酶的重组菌，该菌内导入了 上述编码嗜热长链烷烃单加氧酶的基因。
在本发明的一个实施例中，上述重组茵例如为荧光假单胞菌或大肠杆 菌， 优选为荧光假单胞菌 ΚΟΒ2Δ 1菌株或大肠杆菌 BL21菌株。 本发明还提出一种石油污染物和石油废水的处理方法，该方法包括在 石油污染物和石油废水的处理过程中使用上述嗜热长链烷烃单加氧酶的 步骤。
本发明还提出一种提高微生物石油釆收率的方法，该方法包括在石油 开釆中使用上述嗜热长链烷烃单加氧酶的步骤。
应当指出的是， 上述提到的术语"严格杂交条件"在本说明书中的含义 是指在该条件下形成了所谓特异杂交而没有形成非特异的杂交。例如，该 严格杂交条件可以是， 相互之间的同源性不小于 70%的 DNA之间可以杂 交而低于上述数值的 DNA之间不能杂交， 优选的是同源性不少于 90%的 DNA之间可以杂交。 相对于 Southern杂交中普通洗涤条件而言， 可以例 如为如下的杂交条件： 将杂交膜置于预杂交液 （0.25mol/L磷酸钠缓冲液， pH7.0, 7%SDS )中， 50°C预杂交 30min；弃预杂交液，加入杂交液（0.25mol/L 磷酸钠缓冲液， PH7.0, 7%SDS , 同位素标记的核苷酸片段） ， 50°C杂交 12hr; 弃杂交液， 加入洗膜液 I (2xSSC和 0.1%SDS) ， 50°C洗膜 2次， 每次 30min; 加入洗膜液 II (0.5xSSC和 0.1%SDS ) ， 50°C洗膜 30min。 所属技术领域的技术人员应该知道，本发明的编码嗜热长链垸烃单加 氧酶的 DNA序列，还包括编码对 SEQ ID NO: 1所示核苷酸序列所表达的 酶分子的氨基酸序列进行一个或多个氨基酸替换、插入或缺失并仍具有该 酶活性的蛋白质的核苷酸序列。
另夕卜，对本发明的嗜热长链烷烃单加氧酶基因所表达的酶分子的氨基 酸进行一个或多个氨基酸替换、插入或缺失所得到的蛋白质也能达到本发 明的目的。 因而本发明还包括与 SEQ ID NO: 2所示的氨基酸序列具有至 少 70%的同源性，优选具有至少 90%的同源性，但同时具有嗜热垸烃单加 氧酶活性的蛋白质。 上面使用的术语"多个"可以是小于 100的数目， 优选 为小于 10的数目。 本发明提出的上述嗜热长链垸烃单加氧酶的性能不同于已知的垸烃 单加氧酶， 其具有嗜热的长链烷烃单加氧酶活性， pi为 6.86，最适温度约 为 60°C， 最适 pH值约为 7.5， 分子量约为 50400道尔顿， 可高效氧化 C15-C36烷烃的末端甲基产生相应的一元醇。本发明的长链垸烃单加氧酶 的热稳定性， 在迄今发现的垸烃单加氧酶中是最高的。
基于本发明的嗜热长链垸烃单加氧酶的上述特性和功能，该酶可以在 石油污染物和石油废水的处理中以及在提高微生物石油釆收率中广泛应 用。 一方面， 该酶在高温下能降解长链 （重质） 烷烃， 提高轻质垸烃与重 质烷烃的比例、 改善原油流动性等特性， 从而利于原油的开釆，提高石油 釆收率；另一方面，该酶在高温下对长链烷烃的降解特性，对由油田开釆、 输油作业的跑漏、溢流所引起的环境石油污染物以及由石油化工带来的高 温石油废水的处理也具有良好的应用前景。
为让本发明的上述和其它目的、特征和优点能更明显易懂， 下面特举 实施例， 并配合说明书附图， 作详细说明如下。 附图说明 图 1 为本发明实施例中的嗜热长链垸烃单加氧酶基因重组质粒 pComALK的构建模式图。 图 2 为本发明实施例中的嗜热长链垸烃单加氧酶基因重组质粒 pETALK的构建模式图。
图 3为嗜热脱氮芽胞杆菌 NG80-2对不同烷烃的降解结果。 图 4为本发明的重组嗜热长链烷烃单加氧酶作用正十六烷后的产物质 谱图。
图 5为本发明的重组嗜热长链烷烃单加氧酶作用正二十八垸后的产物 质谱图。
图 6为本发明的重组嗜热长链垸烃单加氧酶的底物特异性分析结果。 图 7为温度对本发明重组嗜热长链垸烃单加氧酶活性影响的曲线图。 图 8为 pH值对本发明重组嗜热长链烷烃单加氧酶活性影响的曲线图。
具体实施方式 下面通过具体实施例并结合附图对本发明作进一步的详细说明。 以下各实施例仅仅是用于说明而不是限制本发明。 实施例一
1.嗜热脱氮芽胞杆菌編 0-2 (CGMCC No. 1228) 总 DNA的提取 研究证明， 嗜热脱氮芽胞杆菌 {Geobacillus thermodenitrificans) NG80-2在以原油或液体石蜡为碳源条件下产生较多的烷烃单加氧酶， 该酶特别适于在高温条件下氧化 C15~C36的长链烷烃。 因此， 在本实 施例中， 釆用从中国天津大港油田官 69-8区块油井地层水分离获得的 嗜热脱氮芽胞杆菌 NG80-2 (该菌株已保藏在中国微生物菌种保藏委员 会普通微生物中心， 其保藏号为 CGMCC No.1228) , 取其过夜培养的 新鲜培养物 3ml， 离心收集菌体， 茵体悬于 250W 50mM Tris缓冲液中
(ρΗ8.0) , 加入 10μ1 0.4Μ ΕϋΤΑ (ρΗ8.0) , 混匀后 37°C保温 20min， 之后加入 30μ1 20mg/ml溶菌酶，混匀后 37°C再保温 20min，再加入 5μ1 20mg/ml蛋白酶 Κ， 温柔混匀后， 再加入 20μ1 10%SDS , 50°C保温至 溶液澄清， 分别用等体积酚:氯仿:异戊醇抽提两次， 氯仿:异戊醇抽提 一次， 最后一次的上清溶液， 加入 2.5 倍体积预冷的无水乙醇， 回收 DNA , 用 70%乙醇洗，沉淀溶于 ΙΟΟμΙ ΤΕ缓冲液（pH8.0， lOmMTris, ImMEDTA) , 加入 lOmg/ml RNase 2μ1, 65°C保温 30min， 分别用酚: 氯仿:异戊醇、 氯仿:异戊醇各抽提一次， 上清液加入 2.5倍体积预冷的 无水乙醇， 回收 DNA, 用 70%乙醇洗， 真空干燥， 沉淀溶于 50μ1 ΤΕ 缓冲液。 DNA溶液的紫外分光光度计测定结果为 Α260/Α280=1.95， Α260=0.73。
2. 嗜热长链垸烃单加氧酶基因的克隆和筛选
取前面所述的总 DNA溶液 0.5μ1 (约 10ng) 作为模板， 以下列寡 核苷酸序列作为引物， 并按下述设定的 PCR循环参数进行 20个循环 PCR。 设定的 PCR循环参数如下：
95 °C , 3min; 95 °C , 30s ; 50 °C , 45s ; 72 °C , 2min; 72 °C , 5min; 4°C , 2hr 引物序列如下：
上游引物 5， -GTGACCCGGGATGAC AAA A A A A ATCC ATATTAATGC-3， 下游引物 5， -TAGA AGCTTTTATAC ATTTGAAG AAATATTTCGAT-3 ' 将上述 PCR产物用 Smal和 Hindlll双酶切， 经 0.8%的琼脂糖凝 胶电泳，切胶回收 1.3kb酶切产物片段。与经同样限制型内切酶酶解并 切胶回收的质粒 pCom8连接，转化感受态荧光假单胞菌 ΚΟΒ2Δ 1 [该 菌株由瑞士联邦苏黎士高等理工学院 （Swiss Federal Institute of Technology Zurich)的 J. B. Van Beilen教授提供，参见 J. Bacterology, Mar. 2002, 1733-1742] 后， 涂于含 50μβ/ιη1 Gm (庆大霉素） 的 LB固体培 养基上。 35°C培养 12小时， 再在 30°C培养 24小时， 挑取单克隆菌落 提取质粒鉴定，插入有 SEQ ID NO: 1所示的 DNA序列的 pCom8质粒 为重组质粒 pComALK (见图 1 )， 含有该质粒的重组荧光假单胞茵 ΚΟΒ2Δ 1菌株为荧光假单胞菌 ΚΟΒ2Δ 1 ALK。将该重组菌株分别接入 以不同 C原子数烷烃 （正十二烧 -正十八烷） 作为唯一碳源的 E2培养 基 （每升 E2培养基含 NaNH4HP04-4H20 3.5g, Κ2ΗΡ04Ή20 7.5g， KH2P043.7g, 1ml微量元素溶液， 1ml 1M MgS04'7H20; 每升微量元 素溶液含 FeS04.7H20 2.78g， MnCl2-4H20 1.98g, CoS04-7H20 2.81g, CaCl2-2H20 1.47g, CuCl2-2H20 0.17g, ZnS04-7H20 0.29g) 中培养， 48 小时后， 转化子菌株荧光假单胞菌 K0B2A 1ALK能在分别以 15烷、 16垸、 18垸为唯一碳源的培养基中生长， 而不能在分别以 12烷、 13 烷、 14 垸为唯一碳源的培养基中生长， 而对照菌株荧光假单胞茵 ΚΟΒ2Δ 1 不能在含有上述碳源的培养基中生长， 从而表明重组质粒 pComALK转化到荧光假单胞菌 ΚΟΒ2Δ 1中后， 可表达出垸烃单加氧 酶的活性。
3.嗜热长链院烃单加氧薛基因克隆入大肠杆菌
以上述重组质粒 pComALK为模板， 以下列寡核苷酸序列作为引 物， 并按下述设定的 PCR循环参数进行 20个循环 PCR。
设定的 PCR循环参数如下：
95 °C , 3min; 95 °C , 30s; 50°C， 45s; 72 °C , 2min; 72 "C , 5min; 4°C， 2hr 引物序列如下：
上游弓 I物： 5 '-CCAGAATTCATGACAAAAAAAATCCATATTAATGC-3 ' 下游引物： 3，-TAGCTTTATAAAGAAGTTTACATATTGAGCTCGATC-5， 上述 PCR产物用 EcoRI和 Xhol双酶切， 经 0.8%的琼脂糖凝胶电 泳，切胶回收 1.3kb酶切产物片段。与经同样限制型内切酶酶解并切胶 回收的质粒 pET-28a(+)连接， 转化感受态大肠杆菌 BL21 (该菌株购自 天津开发区厚普生物技术开发有限公司， 货号为 69387-3)后，涂于含 50μβ/ηι1 Kan (卡拉霉素）的 LB固体培养基上。 37°C培养 16〜18小时， 挑取单克隆菌落鉴定，插入有 SEQ ID NO: 1的 DNA序列的 pET-28a(+) 质粒为重组质粒 pETALK (见图 2)， 含有该质粒的重组大肠杆菌菌株 为大肠杆菌 BL21ALK。釆用 Sanger双脱氧法对此 DNA片段进行了测 序。 测序结果显示， 该基因 DNA片段全长 1323bp， 由 ATG起始密码 开始， 到 TAA终止密码子结尾， 其核苷酸序列如 SEQ ID NO: 1所示。 该完整的 ORF编码一个由 440个氨基酸组成的蛋白质，该蛋白质属于 单加氧酶家族，最高相似性同荧光假单胞菌 (Pseudomo画 fl騰顧 Pf-5的单加氧酶基因， 同源性为 51%。 实施例二 重组嗜热垸烃单加氧酶的纯化和特性
将上述重组菌 E. Coli BL21ALK单克隆接入 20ml含 5(^g/ml Kan 的 LB培养基中， 37°C， 180rpm/min培养 12小时，然后将培养物按 1% (V/V) 接种量接入 200ml含 5(^g/ml Kan的 LB培养基 （共 10个摇 瓶）， 37°C， 220rpm/min培养 A600为 0.6时， 加入 IPTG至终浓度为 0.2mM, 45 °C， 150rpm/min 诱导 4 小时。 离心收集菌体， 悬于 50mMTris-Cl (pH7.5 ) 缓冲液中， 利用超声波破碎细胞， 离心上清液 为重组烷烃单加氧酶的粗提液。此上清液经螯合琼脂糖凝胶（Chelating Sepharose) 镍亲合柱层析纯化， 得到的酶制剂在 SDS-PAGE上显示一 条带。 利用已知的蛋白质化学标准方法测定此重组烷烃单加氧酶的基 本特性。 用 SDS-PAGE测得的重组酶的分子量约为 50000道尔顿， 与 理论上推算的分子量（50400道尔顿）相似；等电点沉淀法测得的重组 酶的等电点 pi为 6.86 ; 重组酶反应的最适温度为约为 60°C， 最适 pH 值约为 7.5。 该重组酶仅对 C15〜C36的长链垸烃有作用， 并对 16垸 的活性最高 （见图 6)， 这与嗜热脱氮芽胞杆茵 NG80-2发酵降解烷烃 的特性一致 （见图 3)。 实施例三 重组嗜热垸烃单加氧酶氧化长链垸烃
在 20ml旋盖试管中装入 3ml下列反应体系： ImM垸烃， 50mM Tris-Cl (pH7.5 ) , 0.001% (W/V) Plysurf A-210G表面活性剂， ImM NADH, ImM MgS04, 2mg实施例二中制得的重组垸烃单加氧酶的纯 化酶制剂。 60°C， 250rpm/min反应 5min。加入 3ml环己烷萃取， 萃取 的有机相经 FINNICAN Polaris Q气-质联用仪分析。结果显示该酶作用 长链烷烃的末端甲基， 其产物为相应的一元醇 （见图 4和图 5)。 重组烷烃单加氧酶活性测定 在 20ml旋盖试管中装入 3ml下列反应体系： ImM正十六垸， 50mM Tris-Cl , lmM MgS04， 0.001% (W/V) Plysurf A-210G表面活性剂， ImM NADH, 0.2〜0.3mg实施例二中制得的重组垸烃单加氧酶的纯化酶制剂， 一定温度下， 250rpm/min反应 5min。 加入 3ml环己垸萃取， 萃取的有机 相经气相色谱仪 （Agilent 6820) 测定垸烃降解量， 并计算酶活。
1 . 最适酶活温度
上述反应条件下， 在 37-70°C范围内测定上述重组烷烃单加氧酶的活 性， 结果表明保持该酶活性的最适温度约为 60°C (见图 7)。
2. 最适 pH 在上述反应条件下 （温度为 60°C)， 在 50mM Tris-Cl的不同 pH缓冲 液的条件下测定上述重组垸烃单加氧酶的活性，结果表明上述实施例一中 构建的重组菌株 Coli BL21ALK中表达的垸烃单加氧酶的最适 pH值约 为 7.5 (见图 8)。
3 . pH稳定性 将上述实施例二中制得的重组垸烃单加氧酶的纯化酶制剂与 pH6.0~8.5的 50mM Tris-Cl缓冲液混合， 30°C放置 24hr， 测定酶活， 其结 果如下表所示， 结果表明酶活在 pH7.0〜7.5范围内比较稳定。 本发明的嗜热长链烷烃单加氧酶在不同 pH条件下的稳定性
PH 酶活性 （μηιοΐ 烷烃 /mg蛋白 /min) 残留活性 (%) 负对照 （未加酶） 0 0
未处理 （正对照） 1.6374 100
6.0 0.1139 6.96
6.5 0.2305 14.08
7.0 0.8982 54.86
7.5 1.2268 74.92
8.0 0.5273 32.20
8.5 0.1259 7.69 虽然本发明已以较佳实施例披露如上，然其并非用以限定本发明，任 何所属技术领域的技术人员，在不脱离本发明的精神和范围内，可做些许 的更动与改进， 因此本发明的保护范围当视权利要求所界定者为准。

Claims

权 利 要 求
1. 一种编码嗜热长链垸烃单加氧酶的基因， 该基因具有选自于下列 a) 、 b) 或 c) 的核苷酸序列：
a) SEQ ID NO: 1所示的核苷酸序列；
b) 由于遗传密码的简并性， 不同于 SEQ ID NO: 1但编码的氨基酸序 列与 SEQ ID NO: 1所编码的氨基酸序列相同的核苷酸序列；
c) 在严格杂交条件下与上述 a) 或 b) 中的序列杂交， 并且编码具有 活性的嗜热长链院烃单加氧酶的核苷酸序列。
2. 根据权利要求 1所述的编码嗜热长链垸烃单加氧酶的基因，其特征 是该基因具有 SEQ ID NO: 1所示的核苷酸序列。
3.一种嗜热长链垸烃单加氧酶， 该酶具有选自于下列 d) 、 e) 或 f) 的氨基酸序列：
d) 权利要求 1所述的核苷酸序列编码的氨基酸序列；
e) SEQ ID NO: 2所示的氨基酸序列；
f) 上述 e) 中缺失、 替换或插入一个或多个氨基酸后的氨基酸序列， 并且具有该序列的蛋白质有嗜热长链院烃单加氧酶的活性。
4.根据权利要求 3所述的嗜热长链垸烃单加氧酶,其特征是该酶具有 SEQ ID NO: 2所示的氨基酸序列。
5.一种表达嗜热长链烷烃单加氧酶的重组质粒，其特征是该质粒至少 包括权利要求 1或 2所述的基因。
6. 根据权利要求 5 所述的重组质粒， 其特征是该重组质粒的载体为
Figure imgf000013_0001
7.根据权利要求 5 所述的重组质粒， 其特征是该重组质粒的载体为
Figure imgf000013_0002
8. 一种产生嗜热长链烷烃单加氧酶的重组菌，其特征是该重组菌内导 入了权利要求 1或 2所述的基因。
9.根据权利要求 8 所述的重组菌， 其特征是该重组菌为荧光假单胞 o
10.根据权利要求 9所述的重组菌， 其特征是上述荧光假单胞菌为荧 光假单胞菌 ΚΟΒ2Δ 1菌株。
11.根据权利要求 8所述的重组菌， 其特征是该重组菌为大肠杆菌。
12.根据权利要求 11所述的重组菌，其特征是上述大肠杆菌为大肠杆 菌 BL21菌株。
13.一种石油污染物和石油废水的处理方法， 该方法包括在石油污染 物和石油废水的处理过程中使用权利要求 3或 4所述的嗜热长链烧烃单加 氧酶的步骤。
14.一种提高微生物石油釆收率的方法， 其包括在石油开釆中使用权 利要求 3或 4所述的嗜热长链垸烃单加氧酶的步骤。
PCT/CN2005/001974 2005-10-17 2005-11-22 Monooxygenase d'alcanes a chaine longue thermophiles et son gene codant, et leurs utilisations WO2007062543A1 (fr)

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CN1469928A (zh) * 2000-10-16 2004-01-21 �����ɷ� 源于嗜热细菌的细胞色素p450单加氧酶
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CN1469928A (zh) * 2000-10-16 2004-01-21 �����ɷ� 源于嗜热细菌的细胞色素p450单加氧酶
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