WO2009030074A1

WO2009030074A1 - Thermophilic long chain alkanol dehydrogenase and its encoding gene and uses thereof

Info

Publication number: WO2009030074A1
Application number: PCT/CN2007/002825
Authority: WO
Inventors: Lei Wang; Xueqian Liu; Lu Feng
Original assignee: Nankai University
Priority date: 2007-09-06
Filing date: 2007-09-26
Publication date: 2009-03-12
Also published as: CN101381735A

Abstract

An alcohol dehydrogenase, which is isolated from Geobacillus thermodenitrificans NG80-2, consists of 387 amino acids shown in SEQ ID NO: 2. The enzyme has ability to degrade long chain alkanol of chain length longer than C16. It can be widely act on long chain alkanol of chain length between C12 and C30, and optimum substrate is octyl alcohol. A gene encoding the enzyme, a recombinant plasmid pET-28a (+) expressing the enzyme, and a recombinant Escherichia coli BL21 producing the same are further provided.

Description

TECHNICAL FIELD The present invention relates to an enzyme and a gene encoding the same, and more particularly to an alcohol dehydrogenase capable of degrading long-chain sterols and a gene encoding the same. Background technique

Alcohol Dehydrogenase (ADH, EC 1.1.1.1) is one of the most important redox catalysts in living organisms. Many alcohol metabolisms are catalyzed by alcohol dehydrogenase. The enzyme has a wide range of applications in biocatalysis and biomedical fields. In organisms, many alcohol metabolisms are catalyzed by alcohol dehydrogenases. Alcohol dehydrogenase has been found in animals, plants, microorganisms, eukaryotes, and prokaryotic bacteria, and at least 25 EC-numbered enzymes are called alcohol dehydrogenases.

Alcohol dehydrogenases come from a wide variety of sources. The alcohol dehydrogenases in the degradable alkaloids that have been reported so far are mainly in the following three categories: Type I, Zn ²⁺ -dependent long-chain alcohol dehydrogenase, approximately 350 amino acid residues per subunit; , short-chain alcohol dehydrogenase, each subunit contains 250 amino acid residues, such as Bt-ADH; type III, Fe ²⁺ -dependent alcohol dehydrogenase, approximately 38 kDa size (Tomohisa Kato et al, Journal of Bioscience and Bioengineering, 2001. vol. 91, No.l, 100-102). Alcohol dehydrogenases can act on a wide variety of substrates, including fatty alcohols such as ethanol, aromatic alcohols, and cinnamyl alcohol.

In the study of alcohol dehydrogenase, the related species are: Thermoanaerobacter ethanolicus JW200, Thermoanaerobacter ethanolicus ATCC 31550, Thermotoga hypogea sp. Nov , Mycobacterium bovis BCG, Clostridium butyricum VPI1718); but the study is most clear Yeast. The yeast alcohol dehydrogenase is a tetramer with a single subunit peptide chain containing 347 amino acid residues and a subunit molecular weight of 35,000. Yeast alcohol dehydrogenase has strong specificity for substrates, and has high activity for ethanol and other linear primary alcohols. Only a few secondary and side alkanols have active amino acid residue sequences. 143~283 are coenzyme binding regions, amino acids. The residue sequence of 1 to 142 and 284 to 336 is a catalytically active region. When the coenzyme and the enzyme bind, the conformation of the enzyme changes. After the catalytic domain rotates by about 10°, it turns to the coenzyme binding domain, and the latter rotates by about 1.5°. A crack is formed between the two domains, the two domains and the crack form a pocket, and the substrate and the coenzyme are combined in a deep pocket formed in the middle, the side chain on one side of the pocket is hydrophobic, and the other The side chain is hydrophilic and contains catalytically active Zn ² ⁺ and its hexameric body (F. Mark Dickinson et al, Chemico-Biological Interactions 130-132 (2001) 417-423; Ueda M et al., Methods Enzymol. 1990 ; 188: 171-5). The latest patents and literature reports on alcohol dehydrogenase genes. For example, Mao Yumin et al. reported an alcohol dehydrogenase and its gene (No. 01126435.7, application date 2001.08.09); P. Hildebrandt et al. reported the alcohol dehydrogenase of Pseudomonas fluorescens DSM50106 ( Appl Microbiol Biotechnol, 59:483 - 487, 2002); Peter J. Holt et al. reported the alcohol dehydrogenase of Thermoanaerobacter ethanolicus JW200 (FEMS Microbiology Letters, 190: 57-62, 2000); Jean-Marc Wilkin et al. reported alcohol dehydrogenase in Mycobacterium bovis BCG (Eur. J. Biochem, 262: 299-307, 1999). At present, the functions and mechanisms of the degradation of terpene hydrocarbons by microorganisms are mainly concentrated in seven species, such as Pseudomonas, Acinetobacter, Prauserella, Rhodococcus. A dozen strains of the genus (P. Hildebrandt et al, Appl Microbiol Biotechnol, 2002, 59:483-487; Peter J. Holt et al, Microbiology Letters, 2000, 190: 57-62; Ce line Raynaud et al, PNAS 2003, April 29: 5010-5015; Jean-Marc Wilkinl et al, Eur. J. Biochem, 1999, 262: 299-307; Ryoko Iwamoto et al, Archives of Biochemistry and Biophysics, 2002, 398:203 - 212), alkane degradation pathway The genes in each step of the reaction are mostly dispersed in different positions in the genome, or clustered in a plasmid (such as Pseudomonas putida OCT plasmid). Although there are many reports on the study of sterol dehydrogenase, the substrate of its action is still limited to medium- and short-chain alkanols, especially ethanol and butanol. Alcohol dehydrogenases with alkanols above 16C have not yet been used. See the report (Akio Tani et al, Appl. Environ. Microbiol, Dec. 2000, p. 5231-5235; Ishige, T. et al, Appl. Environ. Microbiol. 2000, 66: 3481-348; ME Singer, J Bacteriol. 1985 December; 164(3): 1017- 1024; T Kotani et al, J Bacteriol, Dec. 2003, p: 7120-7128; Ching T. Hou et al, Applied And Environmental Microbiology, July 1983, p. 98-105; Kesen Ma et al, Journal Of Bacteriology, Feb. 1999 , p. 1163-1170).

Long-chain alkanol dehydrogenase is a key enzyme in the petroleum hydrocarbon degradation pathway. The study of this enzyme provides a more powerful mechanism for studying the mechanism of degradation of long-chain alkanes in Bacillus thermophilus NG80-2. The evidence also provides a theoretical basis for exploring the metabolic pathways of other alkane-degrading bacteria. At the same time, the thermophilicity of the enzyme and the ability to degrade long-chain alkanols have important industrial application value, and can be applied to the treatment of petroleum pollutants and petroleum wastewater and to improve microbial oil recovery. Therefore, further research on long-chain alcohol dehydrogenase is needed. Disclosure of the Invention An object of the present invention is to provide a gene which encodes a thermophilic long-chain alkanol dehydrogenase;

Another object of the present invention is to provide a thermophilic long-chain sterol alcohol dehydrogenase which can degrade long-chain sterols (above 16C);

Another object of the present invention is to provide a recombinant plasmid capable of expressing a thermophilic long-chain sterol alcohol dehydrogenase;

A further object of the present invention is to provide a recombinant bacterium capable of producing a thermophilic long-chain alkanol dehydrogenase.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention proposes a gene encoding a thermophilic long-chain sterol alcohol dehydrogenase having a nucleotide sequence selected from a), b) or c):

a) the nucleotide sequence shown in SEQ ID NO: 1;

b) a nucleotide sequence which differs from the SEQ ID NO: 1 but which encodes an amino acid sequence identical to the amino acid sequence encoded by SEQ ID NO: 1 due to the degeneracy of the genetic code; c) hybridizing under stringent hybridization conditions to the sequences in a) or b) above, and encoding the nucleotide sequence of the active alcohol dehydrogenase.

In one embodiment of the present invention, the gene encoding the thermophilic long-chain alkanol dehydrogenase has the nucleotide sequence shown in SEQ ID NO: 1.

The present invention provides a thermophilic long chain sterol alcohol dehydrogenase having an amino acid sequence selected from the group consisting of d), e) or f):

d) the amino acid sequence encoded by the nucleotide sequence described in a), b) or c) above; e) the amino acid sequence set forth in SEQ ID NO: 2; f) one or more of the above e) deleted, replaced or inserted The amino acid sequence after the amino acid, and the protein having the sequence has the activity of an alcohol dehydrogenase.

In one embodiment of the present invention, the thermophilic long-chain alkanol dehydrogenase has the amino acid sequence of SEQ ID NO: 2. The present invention proposes a recombinant plasmid expressing a thermophilic long-chain sterol alcohol dehydrogenase comprising at least the above-described gene encoding an alcohol dehydrogenase.

In one embodiment of the invention, the vector of the above recombinant plasmid is pET-28a (+). The present invention proposes a recombinant strain for producing a thermophilic long-chain sterol alcohol dehydrogenase into which the above-described gene encoding an alcohol dehydrogenase is introduced.

In one embodiment of the invention, the recombinant strain is Escherichia coli, preferably Escherichia coli BL21 strain. The invention also provides the use of a thermophilic long chain sterol alcohol dehydrogenase, characterized in that the enzyme is used in the treatment of petroleum contaminants and petroleum wastewater.

The invention also provides the use of a thermophilic long chain alkanol dehydrogenase, characterized in that the enzyme is used to enhance microbial oil recovery during petroleum production.

It should be noted that the term "stringent conditions" mentioned above means in the present specification that a so-called specific hybridization is formed under such conditions without forming a non-specific hybridization. For example, the stringent condition may be that DNA having a homology of not less than 70% of each other can hybridize between DNAs having a lower than the above value, and preferably DNA having a homology of not less than 90%. It Can be crossed. Relative to the usual washing conditions in Southern hybridization, for example, the following hybridization conditions: 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-hybridization 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 hybridization solution and 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. It will be apparent to those skilled in the art that the DNA sequence encoding an alcohol dehydrogenase of the present invention further comprises one or more amino acids encoding the amino acid sequence of the enzyme molecule expressed by the nucleotide sequence shown by SEQ ID NO: 1. A nucleotide sequence of a protein that is replaced, inserted or deleted and still has the activity of the enzyme.

Further, the protein obtained by subjecting the amino acid of the enzyme molecule expressed by the alcohol dehydrogenase gene of the present invention to one or more amino acid substitutions, insertions or deletions can also attain the object of the present invention. Thus, 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 alcohol dehydrogenase activity. The term "plurality" as used above may be a number less than 100, preferably a number less than 10.

Compared with the known alcohol dehydrogenase, the alcohol dehydrogenase proposed by the present invention is a novel alcohol dehydrogenase, and the alcohol of the present invention is compared by analyzing the amino acid sequence deduced from the alcohol dehydrogenase gene proposed by the present invention. Most of the similarities of dehydrogenases compared to the amino acid sequences of other reported enzymes are less than 45%. Alcohol dehydrogenase with similarity higher than 45% has not been functionally identified, mainly from Bacillus kaustophilus HTA426 (YP-148778.1); Bacillus licheniformis ATCC 14580 (YP-080422.1); Bacillus cereus G9241 (ZP-00239930); Bacillus thuringiensis str. Al Hakam (YP-896094); Bacillus thuringiensis serovar konkukian str. 97-27 (YP-037785.1) ); Bacillus cereus ATCC 10987 (NP-90044.1); Bacillus cereus E33L (YP-085061.1). It can be seen that, after comparing the amino acid sequence of the alcohol dehydrogenase of the present invention with the amino acid sequences of other reported alcohol dehydrogenases, the strains having a similarity of more than 45% are basically subjected to sequence alignment. In the study, no relevant literature was found for the identification of the function of the dehydrogenase gene of the corresponding strain. The alcohol dehydrogenase of the invention belongs to type III, Fe ²⁺ -dependent alcohol dehydrogenase, by amino acid sequence similarity of the enzyme.

Compared with the prior art, the beneficial effects of the above technical solution of the present invention are as follows: In addition, the alcohol dehydrogenase of the present invention is different from the reported alcohol dehydrogenase, and the enzyme activity is detected by different substrates, which proves that the enzyme has The ability to withstand high temperature degradation of long chain alkanols (greater than 16 carbons). Although the optimum substrate for the enzyme is octanol, it has a certain ability to degrade from long-chain alcohols between dodecanol and tridecyl alcohol. The optimum reaction temperature is 60 ° C, and it has high thermal stability. This is the manifestation of its thermophilicity.

The degradation of long-chain alkanes is the hydroxylation of terpene hydrocarbons to long-chain sterols by anthracene monooxygenases, followed by further dehydrogenation of alkanol dehydrogenases to long-chain alkanals, followed by dehydrogenation by dehydrogenase. The alkanoic acid is grown, and long-chain citrate coenzyme A is formed by the long-chain citrate coenzyme A ligase to enter the tricarboxylic acid cycle and finally completely oxidize to carbon dioxide and water. Among the petroleum hydrocarbon degradation pathways, the long-chain sterol dehydrogenase in Bacillus thermophilus NG80-2 is a key enzyme important in the high temperature and efficient use of the long-chain alkane of the catalytic product of alkane monooxygenase. Alcohol, which further dehydrogenates, improves the catalytic efficiency in the degradation pathway of anthraquinone; on the other hand, it has a broader range of substrates than long-chain hydrocarbon monooxygenase, which not only catalyzes long-chain sterols, but also Short-chain alcohols have high catalytic ability. Therefore, the study of long-chain sterol dehydrogenase is not only beneficial to understand the mechanism of the degradation of long-chain guanidinium by Bacillus thermophilus NG80-2, but also provides a theoretical basis for exploring the metabolic pathways of other alkane-degrading bacteria. Enzyme construction and high-efficiency expression of engineering strains and large-scale purification have broader industrial application prospects and economic value.

Based on the above-described characteristics and functions of the long-chain alkanol dehydrogenase of the present invention, the enzyme can be widely used in the treatment of petroleum pollutants and petroleum wastewater as well as in enhancing microbial oil recovery. On the one hand, the enzyme can degrade long-chain sterols at high temperatures, thereby strengthening the reaction of long-chain ruthenium through the aforementioned degradation pathway, increasing the ratio of light strontium hydrocarbons to heavy hydrazines, improving the fluidity of crude oil, etc., which is beneficial to Crude oil extraction, enhanced oil recovery; on the other hand, the enzyme's degradation characteristics of long-chain hydrocarbons at high temperatures, environmental oil pollutants caused by oil spills, oil spills, overflows and overflows The treatment of high temperature petroleum wastewater brought by petrochemical industry also has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram showing the construction of an alcohol dehydrogenase gene recombinant plasmid according to an embodiment of the present invention; FIG. 2 is a view showing an SDS-PAGE electrophoresis pattern of the alcohol dehydrogenase of the present invention;

Figure 3 shows the specific activity of the alcohol dehydrogenase of the present invention on different substrates;

Figure 4 shows the specific activity of the alcohol dehydrogenase of the present invention at different temperatures;

Figure 5 shows the specific activity of the alcohol dehydrogenase of the present invention at different pH values;

Fig. 6A and Fig. 6B are views showing the gas phase detection of the alcohol dehydrogenase activity of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be further described in detail by way of specific embodiments and with reference to the accompanying drawings. The following examples are merely illustrative and not limiting of the invention.

Embodiment 1

1. The extraction of total DNA from Bacillus thermophilus NG80-2 (CGMCC No. 1228) demonstrated that the gene encoding alcohol dehydrogenase can be isolated from the NG80-2 genome of the genus Bacillus thermophilus i GeobaciUus thermodenitrificans. Therefore, in the present embodiment, Bacillus thermophilus NG80-2 obtained from the oil well formation water separation in the 69-8 block of Dagang Oilfield, Tianjin, China (which is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee) No. 12, CGMCC No. 1228, dated October 09, 2004, has applied for domestic invention patent and obtained authorization, name of the invention: Bacillus thermophilus and its screening and application, patent number: ZL200410072759.7), take 3 ml of the fresh culture cultured overnight, the cells were collected by centrifugation, and the cells were suspended in 250 μl of 50 mM Tris buffer (pH 8.0), and 10 μl of 0.4 M EDTA (pH 8.0) was added, and the mixture was mixed for 37°. C for 20 min, then add 30 μl of 20 mg/ml lysozyme, mix for 30 min at 37 °C, add 5 μl of 20 mg/ml proteinase K, mix gently, then add 20 μl of 10% SDS. Incubate at 50 ° C until the solution is clarified, extract twice with an equal volume of phenol: chloroform: isoamyl alcohol, extract once with chloroform: isoamyl alcohol, the last supernatant solution, add 2.5 volumes of pre-cooled absolute ethanol , recover DNA, wash with 70% ethanol, dissolve the precipitate in 100 L of TE buffer (pH8.0, lOmMTris, ImMEDTA), was added 10mg / ml RNase 2 microliters, The mixture was incubated at 65 ° C for 30 min, and extracted with phenol:chloroform:isoamyl alcohol, chloroform:isoamyl alcohol, and the supernatant was added with 2.5 volumes of pre-cooled anhydrous ethanol to recover DNA, washed with 70% ethanol, vacuum. Dry and precipitate in 50 μl of TE buffer. The results of the ultraviolet spectrophotometer of the DNA solution were A260/A280=1.95 and A260=0.73.

2. Cloning and screening of the alcohol dehydrogenase gene of the present invention

0.5 μL (about 10 ng) of the total DNA solution described above 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:

95 ° C, 5 min; 95 ° C, 30 s; 50 ° C, 45 s; 72 ° C, 2 min; 72 ° C, 5 min; 4 ° C,

The 2 hr primer sequence is as follows:

Upstream primer: 5,-CGGAATTCATGCAAAATTTTACGTTTCGCA -3 '; Downstream primer: 5,-ACGCGTCGACTTATAAAGACGCACGCAAAATG -3, The above PCR product was digested with Ecom and Sail, and subjected to 0.8% agarose gel electrophoresis, and the Ukb digestion product was recovered by gel-cutting. Piece. It was ligated with the plasmid pET-28a (+) which was digested with the same restriction endonuclease and recovered, and transformed into competent E. coli DH5a, and then applied to LB containing 5 (^g/ml Kan (kanamycin)). Incubate on solid medium for 12 hours at 37 °C, pick up the monoclonal transfer to 20 ml LB medium for culture (5 (^g/ml Kan), and incubate at 37 °C for 12 hours, extract the plasmid and insert it. The pET-28a(+) plasmid of the DNA sequence of SEQ ID NO: 1 is the recombinant plasmid PLW1168 (see Figure 1), and the recombinant plasmid pLW1168 is transferred into the expression host E. coli BL21 (this strain can be developed into the development zone of the Tianjin Development Zone The recombinant strain containing the recombinant plasmid is H1454, and the recombinant plasmid PLW1168 can transfect Escherichia coli BL21 with alcohol dehydrogenase activity and kanamycin resistance.

The DNA fragment was sequenced using the Sanger method. The sequencing revealed that the insert contained a 1161 bp open reading frame encoding a protein consisting of 387 amino acids. Example Purification and Characterization of Glycol Dehydrogenase

The above recombinant H1454 monoclonal was inserted into 20 ml of LB medium containing 50 μg/ml Kan, cultured at 37 ° C, 180 rpm / min for 12 hours, and then the culture was inoculated at 1% (v/v) inoculum. 200ml LB medium containing 50 μg/ml Kan (10 shake flasks in total), 37 ° C, 220 rpm / min culture OD600 0.6, add IPTG to the final concentration of O.lmM, 45 °C, 180 rpm / min Induction for 2.5 hours. The cells were collected by centrifugation, suspended in a buffer containing 50 mM Tris-HCl (pH 8.0), and the cells were disrupted by ultrasonication, and the supernatant was centrifuged to obtain a crude extract of alcohol dehydrogenase. The supernatant was purified by Chelating Sepharose nickel affinity column chromatography, and the resulting enzyme preparation showed a band on SDS-PAGE (see Figure 2). The basic properties of this alcohol dehydrogenase are determined by known standard methods of protein chemistry. The molecular weight of the recombinase measured by SDS-PAGE was 45,000 Daltons, which is similar to the theoretically estimated molecular weight (42,800 Daltons); the isoelectric point pi of the recombinant enzyme measured by the isoelectric precipitation method was 5.18. The alcohol dehydrogenase acts on long chain alcohols of C15 to C30. Embodiment 3

1. Specific activity when determining different substrates of the alcohol dehydrogenase of the present invention

The alcohol dehydrogenase prepared in the above Example 2 was tested for different substrates by a method of preparing a 200 μl reaction system, and the final concentration of the coenzyme NAD+ was 1 m, and adding 1 mM methanol, ethanol, butanol, hexanol, respectively. Substances in octanol, decyl alcohol, decadiol, tetradecanol, cetyl alcohol, stearyl alcohol, eicosyl alcohol, tetradecanol, octacosanol, tridecyl alcohol, dehydrogenation of alcohol at a certain concentration The enzyme was supplemented to 200 μl with 50 mM Tris-HCl, pH 8.0. The mixture was mixed and reacted in a 60 ° C water bath for 10 minutes. After the reaction was completed, the reaction was terminated by ice bath for 5 minutes. It was extracted with 1:1 trichloromethane and then the light absorption was measured at 340 nm. The amount of enzyme required to produce ΙμιηοΙΝΑΟΗ per minute is defined as one enzyme activity unit. The results of the measurement are shown in Fig. 3. As can be seen from the table, the optimum substrate for the alcohol dehydrogenase is octanol.

2. Determination of the specific activity of the alcohol dehydrogenase of the present invention at different temperatures. The optimum reaction temperature of the alcohol dehydrogenase prepared in the above Example 2 is determined by: preparing a 200 μl reaction system, the substrate octanol The final concentration was 100 mM, the final concentration of coenzyme NAD+ was 1 mM, and a certain concentration of alcohol dehydrogenase was supplemented to 200 μl with 50 mM Tris-HCl pH 8.0. Mix and mix in 45° (:, 50 ° C > 55 ° C, 60 ° C, 65 ° C 70 ° C, 75 ° C water bath 10 Minutes, after the reaction was over, ice bath for 5 minutes to terminate the reaction. Extraction was carried out with 1:1 chloroform, and then light absorption was measured at 340 nm. The results of the measurement are shown in Figure 4. As can be seen from the figure, the optimum reaction temperature of the alcohol dehydrogenase is about 60 °C.

3. Determination of the specific activity of the alcohol dehydrogenase of the invention at different pH

The optimum _P H value of the alcohol dehydrogenase prepared in the above Example 2 is determined by: preparing a 200 μl reaction system, the final concentration of the substrate octanol is 100 mM, and the final concentration of the coenzyme NAD+ is lmM, which is certain The concentration of alcohol dehydrogenase was supplemented to 200 μl with a buffer of pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0 (see Table 1 for formulation). The mixture was mixed and reacted in a water bath at 60 ° C for 10 minutes. After the reaction was completed, the reaction was terminated by an ice bath for 5 minutes. Extraction was carried out with 1:1 trichloromethane, and then light absorption was measured at 340 nm. The results of the assay are shown in Figure 5. As can be seen from the figure, the optimum pH of the alcohol dehydrogenase is about 8.0.

Buffer used in alcohol dehydrogenase enzyme activity assay

4. Determination of the influence of different ions on the alcohol dehydrogenase of the present invention

The alcohol dehydrogenase prepared in the above second embodiment is subjected to different ion influence determination by the following method: preparing a 200 μl reaction system, the final concentration of the substrate octanol is 100 mM, and the final concentration of the auxiliary 醸 NAD+ is lmM, respectively FeCl ₂ , FeS0 ₄ , CuS0 ₄ , MgCl ₂ , CoCl ₂ , ZnS0 ₄ , MnCl ₂ , A1C1 ₃ , CaCl ₂ , NiCl ₂ , NaCl, KC1, EDTA to 1 mM or SDS to 0.05%, a certain concentration of alcohol dehydrogenase , supplemented to 200 μl with 50 mM Tris-HCl pH 8.0. The mixture was mixed and reacted in a water bath at 60 ° C for 10 minutes. After the reaction was completed, the reaction was terminated by an ice bath for 5 minutes. Extraction was carried out with 1:1 trichloromethane, and then light absorption was measured at 340 nm. The amount of enzyme required to produce ΙμιηοΙΝΑΟΗ per minute is defined as one unit of enzyme activity, and the results are shown in Table 2. Effect of Different Ions on the Activity of Alcohol Dehydrogenase

5. Gas phase detection of alcohol dehydrogenase activity of the present invention

The alcohol dehydrogenase prepared in the above second embodiment is subjected to gas phase determination by: preparing a reaction system of 1 ml, the final concentration of the substrate cetyl alcohol is 0.5 mM, and the w/w ratio of the surfactant to the substrate is 6%. The final concentration of coenzyme NAD+ was 1 mM, a fixed concentration of alcohol dehydrogenase, supplemented to 1 ml with 50 mM Tris-HCl pH 8.0. Mix and react at 60 ° C for a certain period of time. After the reaction was completed, the reaction was terminated by ice bathing for 5 minutes. The positive ruthenium extraction containing pentadecane as an internal standard was added, and the reduction of the reaction substrate was measured by an agilent 6820 gas chromatograph system. The results of the measurement are shown in Fig. 6A and Fig. 6B. As can be seen from the figure, as the reaction time is extended, the substrate is continuously reduced. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the invention, and any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention will fall into the present invention. The scope of protection of the present invention.

Claims

Claim

A gene encoding a thermophilic long-chain sterol alcohol dehydrogenase, characterized in that the gene has a nucleotide sequence selected from a), b) or c):

a) the nucleotide sequence shown in SEQ ID NO: 1;

b) a nucleotide sequence different from SEQ ID NO: 1 but having the same amino acid sequence as the amino acid sequence encoded by SEQ ID NO: 1;

c) hybridizing to the sequence in a) or b) above under stringent hybridization conditions and encoding the nucleotide sequence of the active alcohol dehydrogenase.

The thermophilic long-chain sterol alcohol dehydrogenase according to claim 1, which has an amino acid sequence selected from the following d), e) or f):

d) the amino acid sequence encoded by the nucleotide sequence described in a), b) or c) above; e) the amino acid sequence set forth in SEQ ID NO: 2;

f) an amino acid sequence in which one or more amino acids are deleted, replaced or inserted in the above e), and the protein having the sequence has an alcohol dehydrogenase activity.

The thermophilic long-chain sterol alcohol dehydrogenase according to claim 2, wherein the alcohol dehydrogenase has at least 70% homology with the amino acid sequence of SEQ ID NO: 2, The protein having this sequence has an alcohol dehydrogenase activity.

The thermophilic long-chain sterol alcohol dehydrogenase according to claim 3, wherein the alcohol dehydrogenase has at least 90% homology with the amino acid sequence of SEQ ID NO: 2, The protein having this sequence has an alcohol dehydrogenase activity.

The thermophilic long-chain sterol alcohol dehydrogenase according to claim 2, wherein the plurality of amino acid sequences having the amino acid sequence of SEQ ID NO: 2 are deleted, replaced, or inserted as 2 -100.

The thermophilic long-chain sterol alcohol dehydrogenase according to claim 5, wherein the amino acid sequence having the amino acid sequence of SEQ ID NO: 2 is deleted, substituted, or inserted as 2 -10.

The thermophilic long-chain alkanol dehydrogenase according to claim 2, wherein the alcohol dehydrogenase is a quinone-type, Fe ²⁺ -dependent alcohol dehydrogenase.

A recombinant plasmid expressing a thermophilic long-chain alkanol dehydrogenase, characterized in that it comprises at least the gene encoding the alcohol dehydrogenase of claim 1.

A recombinant plasmid expressing a thermophilic long-chain sterol alcohol dehydrogenase according to claim 8, wherein the vector of the recombinant plasmid is pET-28a (+).

A recombinant strain producing the thermophilic long-chain sterol alcohol dehydrogenase according to claim 2, wherein the gene encoding the alcohol dehydrogenase according to claim 1 is introduced into the recombinant bacterium.

A recombinant strain producing a thermophilic long-chain sterol alcohol dehydrogenase according to claim 10, wherein the recombinant strain is Escherichia coli.

A recombinant strain producing a thermophilic long-chain sterol alcohol dehydrogenase according to claim 11, wherein the Escherichia coli is an Escherichia coli BL21 strain.

Use of the thermophilic long-chain sterol alcohol dehydrogenase according to any one of claims 2 to 7, characterized in that the enzyme is used in the treatment of petroleum pollutants and petroleum wastewater.

Use of the thermophilic long-chain sterol alcohol dehydrogenase according to any one of claims 2 to 7, characterized in that the enzyme is used in the process of petroleum exploitation to enhance microbial oil recovery.