KR20190111947A - Method and application of sludge petroleum degrading complex enzyme - Google Patents

Abstract

The present invention relates to a method and application of the sludge petroleum decomposition complex enzyme, the production method comprises the following steps. (1) Acinetobacter calcoaceticus cells are subjected to cell disruption, centrifugation and the supernatant taken to prepare petroleum dehydrogenase solution 21 #. (2) A sludge petroleum degrading enzyme is prepared by mixing the prepared petroleum dehydrogenase solution 21 ^# with formate dehydrogenase. The present invention first found that the petroleum degrading system of the acetobacter calcoaceticus-derived bacteria can be mixed with formic acid dehydrogenase and then used to treat petroleum decomposition and recovery in high concentrations of petroleum contaminated sludge. This not only does not need to passivate the adsorbents such as diatomaceous earth, but also can reduce the oil in the sludge in a short time, has a high efficiency of petroleum contamination treatment, low production cost and broad prospects for use.

Description

Method and application of sludge petroleum degrading complex enzyme

TECHNICAL FIELD The present invention relates to the field of soil treatment, and more particularly, to a method and application of sludge petroleum degrading complex enzyme.

Accidents, abnormal operation and maintenance of oil production, storage and transportation, refining processing and use can result in the release and release of petroleum hydrocarbons. Examples include oil well ejection during oil field development, oil and oil tank leaks, oil and oil tanker leaks, oil well wax removal and oil field maintenance, oil refinery and petrochemical production equipment maintenance, etc. . Due to the large amount of petroleum hydrocarbons spilled, they should be recovered as much as possible, but in some cases recovery is difficult, so even with the greatest possible recovery, some remain and can pollute the environment (soil, ground and groundwater). When petroleum enters the soil, it destroys the soil structure and disperses the soil particles, reducing the water permeability of the soil. The rich reactor combines with inorganic nitrogen, phosphorus and limits nitrification and dephosphorylation, reducing the effective phosphorus and nitrogen content in the soil. In particular, polycyclic aromatic hydrocarbons (PAHs) are more harmful because they accumulate in plants and animals step by step and accumulate in soil through food chains and activities such as carcinogenesis, mutations, and malformations.

Of these risks, sludge treatment is the most difficult. The sludge may contain oil, mud, and water mixtures containing large amounts of petroleum hydrocarbons due to processes such as exploration and development of upstream oil, oil and gas collection transportation, sewage treatment, tank bottom cleaning and downstream petroleum refining processes. Leakage into the environment produces oily sludge. Oily sludge in oilfields is the largest and broadest source of contamination with high oil content and high heavy oil content.

Prior to the 1980s, treatment of soils contaminated with petroleum hydrocarbons was limited to physical and chemical methods, namely heat treatment and chemical leaching. The heat treatment method can purify most organic pollutants in the soil through incineration or calcination. At the same time, however, the soil structure and components are destroyed, costly and difficult to implement. Chemical leaching and water washing can also yield relatively good petroleum removal effects. However, their application is limited due to secondary contamination of the chemical reagents used. Theoretically, the latest pyrolysis technology for treating oily sludge has an oil reduction of about 80% but the residual petroleum content is still high. Since sludge is difficult to collect and treat, complex to process and belong to dangerous wastes, the harmless treatment of this is a global challenge, and there is basically no case for harmless and resource treatment in each of China's oil fields. As national interest in environmental protection increases, it is urgent for oil companies to effectively reduce environmental pollution risks and costs.

In the early 1970s, to solve the problem of soil contamination by oil spills and leaks in oil pipelines and oil storage tanks, the US Esso Research and Engineering began looking for clean biological solutions and The discovery of the “bacteria-seeding method” took the first step in the biological restoration of petroleum-contaminated soil. Since the 1980s, people's interest in bioremediation techniques for contaminated soils has increased significantly, and this technology is gradually maturing.

At present, countries around the world have started to recover from oil pollution using biological methods. Biological recovery is the process of restoring a contaminated soil to a healthy state by utilizing the biological metabolic activity of the organism to reduce the concentration of toxic substances in the soil environment. Chinese patent document CN103484447A (Application No. 201310456751.X) relates to the production method and application of petroleum dehydrogenase preparations, and the production method is as follows. In other words, the microorganisms that break down petroleum are subjected to cell disruption to prepare a crude enzyme solution, and then the adsorbed crude enzyme solution and the carrier are mixed and adsorbed. The microorganism is Acinetobacter Calcoaceticus, the species number is CGMCC No.3915, and the depositing institution is the Normal Microorganism Center of the China Microbial Species Preservation Management Committee. The present invention is to decompose soil contaminated with petroleum by immobilizing the enzyme system of the microorganism having a petroleum decomposition function with an adsorbent, the decomposition efficiency is significantly improved by 30 to 50 times the rate of microbial decomposition and the stability is 15 compared to the crude enzyme solution To 20-fold improvement. Although the above technique can be applied to petroleum contaminated water and soil recovery process, the applied petroleum concentration is up to 10g / L, and the contaminated water and soil concentration that need to be treated is much higher than the above concentration and must be immobilized with adsorbent. The actual application cost is relatively high, so the utilization prospect is not high.

An object of the present invention is to overcome the disadvantages of the prior art by providing a method and application of the sludge petroleum degrading complex enzyme.

The technical solution of the present invention is as follows, and the method for producing the sludge petroleum degrading complex enzyme comprises the following steps.

(1) Acinetobacter calcoaceticus cells are subjected to cell disruption, centrifugation and the supernatant taken to prepare petroleum dehydrogenase solution 21 ^# .

(2) The petroleum dehydrogenase 21 ^# prepared in step (1) and formic acid dehydrogenase are mixed in a ratio of protein mass ratio 1: (3 to 5) to prepare a sludge petroleum degrading complex enzyme.

Preferably, the species number of Acinetobacter calcoaceticus in step (1) is CGMCC No. 3915. The bacterium is a known strain and does not participate in the species deposit.

Preferably, in step (1), the cells of Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) is prepared by culturing according to the following method.

a. Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) is transferred to LB medium according to the inoculation amount of 1% to 2% by mass ratio and incubated for 14 to 16 hours at 28 to 32 ℃ and 150 to 180 rpm conditions to prepare a seed solution.

b. Seed solution prepared in step a is transferred to LB medium according to the inoculation amount of 4% to 5% by mass ratio, and expanded to 14 to 16 hours under conditions of 28 to 32 ℃ and 150 to 180rpm to prepare a bacterial solution.

c. Centrifugation of the microbial solution prepared in step b and the precipitates were collected to prepare Acinetobacter calcoaceticus cells.

Preferably, the bacterial disruption of step (1) comprises the following steps.

Acinetobacter calcoaceticus cells are uniformly mixed with a phosphate buffer of pH 7.5 in a ratio of mass volume ratio 1: (15 to 25) and the unit is g / ml; Cell disruption is performed for 17 minutes using intermittent sonication under 320W of ultrasonic conditions. The ultrasonic disruption time is 2s and the intermittent time is 2s each time.

Preferably, the centrifugation conditions in step (1) is centrifuged for 2 minutes at 5000 r / min.

Preferably, in step (2), the formic acid dehydrogenase is formic acid dehydrogenase (C b FDH), and the amino acid sequence is represented by SEQ ID NO.1.

Preferably, the preparation method of formic acid dehydrogenase (C b FDH) in the step (2) is as follows.

(i) Genetically modified strain E. coli (E. coli BL21- fdh ) is made.

(ii) the genetically modified strain E. coli (E. coli BL21- fdh ) prepared in step (i) was transferred to the seed medium at an inoculation amount of 1% to 2% by mass, and 10 to 28 ° C under conditions of 28 to 32 ° C and 150 to 180 rpm. E. coli seed solution is prepared by incubating the seeds for 12 hours.

(iii) the E. coli seed solution of step (ii) was transferred to the fermentation medium at an inoculation amount of 4% to 5% by mass, and incubated for 16 to 18 hours under conditions of 28 to 32 ° C and 150 to 180 rpm. E. coli BL21- fdh ) cells are collected, and supernatant is collected by cell disruption and centrifugation to prepare formate dehydrogenase (C b FDH).

Preferably, the method for constructing the genetically modified strain E. coli (E. coli BL21- fdh ) in step (i) is as follows.

The formic acid dehydrogenase gene ( fdh ) derived from Candida boidinii was expanded and linked to an E. coli expression vector (pET28a (+)) to construct a recombinant expression vector (pET28a (+)- fdh ) having the fdh gene. The host bacterium Escherichia coli (BL21 (DE3)) is transformed, and the transformant is selected to obtain a recombinant Escherichia coli (E. coli BL21- fdh ) expressing formate dehydrogenase.

More preferably, in step (ii) the seed medium component is

10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), and 100 μg / mL ampicillin.

More preferably, the fermentation medium component in step (iii),

Peptone 10g / L, yeast extract 5g / L, Na ₂ HPO _{4 and 12H 2 O 9g / L, KH} 2 PO 4 6.8g / L, (NH 4) 2 SO 4 3.3g / L, glucose 0.5g / L, lactose and 2g / L, MgSO _{4 and 7H 2 O 0.5g / L, CaCl} 2 0.02g / L, glycerol is calculated as 0.5% by volume%.

Preferably, the cell disruption step in step (iii) is as follows.

Genetically engineered strain E. coli BL21- fdh cells are uniformly mixed with a phosphate buffer of pH 7.5 in a ratio of mass volume ratio 1: (15 to 25) and the unit is g / ml; Cell disruption is performed for 6 minutes using intermittent sonication under 195W of ultrasonic conditions. The ultrasonic disruption time is 3s and the intermittent time is 5s each time.

 Preferably, the centrifugation conditions in step (iii) are centrifuged at 3000 r / min for 2 minutes.

Preferably, the mixed mass ratio of the petroleum dehydrogenase 21 ^# and formic acid dehydrogenase in step (2) is 1: 4.

Sludge petroleum degrading complex enzyme obtained by the above production method.

Application of the sludge petroleum degrading enzyme in petroleum contaminated sludge recovery.

The application includes the following steps.

(I) The sludge petroleum decomposing enzyme is mixed with sodium formate solution, and the sludge petroleum decomposing enzyme treatment liquid having a mass ratio of 1: (4 to 6) and a sodium formate concentration of 150 to 180 mmol / L for protein and sodium formate. Manufacture.

(II) The sludge petroleum degrading complex enzyme treatment liquid prepared in step (I) is mixed at a ratio of mass volume ratio 1: (16 to 24), and the unit is g / ml, and it is 6 to 24 under 25 to 35 ° C. What is necessary is just to agitate for time.

Preferably the mass ratio of protein and sodium formate in step (I) is 1: 5.

Preferably, the sodium formate concentration in the sludge petroleum decomposing complex treatment solution in step (I) is 167 mmol / L.

Preferably, in step (II), the sludge and the sludge petroleum decomposing complex enzyme treatment solution are mixed in a ratio of mass volume ratio of 1:20.

The beneficial effects of the present invention are as follows.

1. The present invention first finds that the petroleum degrading system of acetobacter calcoaceticus-derived bacteria can be mixed with formic acid dehydrogenase and then used to treat petroleum decomposition and recovery in high concentration petroleum contaminated sludge. This eliminates the need for the passivation of adsorbents such as diatomaceous earth, and can reduce the oil in the sludge in a short time, has a high efficiency of petroleum pollution treatment, low production cost, and a wide range of utilization prospects.

2. When the formate dehydrogenase is formic acid dehydrogenase (C b FDH) (amino acid sequence is SEQ ID NO.1), the present inventors further improve the recovery efficiency for high concentration petroleum contaminated sludge of petroleum dehydrogenase system, and at the same time, when a by culturing a recombinant bacterium obtained formate dehydrogenase (FDH C b), it was confirmed that the characteristics such as stability of the formate dehydrogenase (FDH C b) is remarkably improved.

Hereinafter, the technical solutions of the present invention will be described in more detail with reference to examples, which do not limit the protection scope of the present invention.

Biomaterials Source:

Acinetobacter calcoaceticus was purchased from the China Microbial Species Preservation Management Committee Ordinary Microbial Center, and the species number is CGMCC No.3915.

Candida boidinii was purchased from China's Microbial Species Preservation Control Committee Ordinary Microbial Center. The species number is CGMCC 2.2378.

Plasmid (pET28a (+)) was purchased from Shandong Biotech Co., Ltd. (山東 沃恩 生物 科技 有限公司).

Escherichia coli (BL21 (DE3)) is a commercially available product.

Example  One

Acinetobacter calcoaceticus cells are prepared by culturing according to the following method.

a. Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) is transferred to LB medium according to the inoculation amount of 2% by mass ratio and incubated for 15 hours at 30 ℃, 160rpm conditions to prepare a seed solution.

b. Seed solution prepared in step a is transferred to LB medium according to the inoculation amount of 4% to 5% by mass ratio and expanded incubation for 15 hours at 30 ℃, 160rpm conditions to prepare a bacterial solution.

c. The microbial solution prepared in step b is centrifuged to collect the precipitate to prepare Acinetobacter calcoaceticus cells.

Example  2

The production method of formic acid dehydrogenase (C b FDH) is as follows.

(i) Genetically modified strain E. coli (E. coli BL21- fdh ) is made, and the construction method is as follows.

That is, Candida seen dini (Candida boidinii) gained by expanding the formate dehydrogenase gene (fdh) of origin, the gene sequence, for example, is represented by SEQ ID NO.2, the upstream extension primer sequence contains SEQ ID NO. G. The formic acid dehydrogenase gene ( fdh ) after expansion was linked to the E. coli expression vector (pET28a (+)), and the recombinant expression vector (pET28a (+)- fdh ) having the fdh gene was constructed and obtained. Escherichia coli (BL21 (DE3)) is transformed, and transformants are selected to obtain recombinant E. coli (E. coli BL21- fdh ) expressing formate dehydrogenase. Refer to the instructions for use of the E. coli expression vector (pET28a (+)) for specific step conditions.

(ii) E. coli BL21- fdh , the genetically modified strain E. coli BL21- fdh prepared in step (i) was transferred to the seed medium at an inoculation amount of 2% by mass, and the seeds were incubated for 12 hours at 30 ° C. and 160 rpm. Prepare a liquid.

The seed medium component,

10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), and 100 μg / mL ampicillin.

(iii) transfer the E. coli seed solution of step (ii) to the fermentation medium at an inoculation amount of 4% by mass, and incubate for 16 hours at 30 ° C. and 160 rpm to obtain E. coli BL21- fdh cells. Collected, centrifuged at 3000 r / min for 2 minutes through cell disruption, and the supernatant collected to prepare formic acid dehydrogenase (C b FDH). Upon detection, the amino acid sequence is represented, for example, by SEQ ID NO.1.

The fermentation medium component,

Peptone 10g / L, yeast extract 5g / L, Na ₂ HPO _{4 and 12H 2 O 9g / L, KH} 2 PO 4 6.8g / L, (NH 4) 2 SO 4 3.3g / L, glucose 0.5g / L, lactose and 2g / L, MgSO _{4 and 7H 2 O 0.5g / L, CaCl} 2 0.02g / L, glycerol is calculated as 0.5% by volume%.

The cell disruption step is as follows.

Genetically engineered strain E. coli BL21- fdh cells are uniformly mixed with a phosphate buffer of pH 7.5 in a ratio of mass volume ratio of 1:20 and the unit is g / ml; Cell disruption is performed for 6 minutes using intermittent sonication under 195W of ultrasonic conditions. The ultrasonic disruption time is 3s and the intermittent time is 5s each time.

Example  3

The method for producing the sludge petroleum degrading complex enzyme includes the following steps.

(1) Acinetobacter calcoaceticus cells prepared in Example 1 were subjected to cell disruption, centrifuged at 5000 r / min for 2 minutes, the supernatant was taken, and petroleum dehydrogenase solution 21 ^# was prepared.

The cell disruption step is as follows.

Acinetobacter calcoaceticus cells are uniformly mixed with a phosphate buffer of pH 7.5 at a mass volume ratio of 1:20, and the unit is g / ml; Cell disruption is performed for 17 minutes using intermittent sonication under 320W of ultrasonic conditions. The ultrasonic disruption time is 2s and the intermittent time is 2s each time.

(2) The petroleum dehydrogenase 21 ^# prepared in step (1) and formic acid dehydrogenase were mixed according to the ratio of the protein by mass ratio 1: 4 to prepare a sludge petroleum degrading complex enzyme.

Example  4

The difference from the manufacturing method of the sludge petroleum decomposing complex enzyme of Example 3 is a fermentation medium employ | adopted during the process of manufacturing a formate dehydrogenase, The component is as follows.

Induction was performed by incubating LB liquid medium (10 g / L of peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride (NaCl)) for 2 to 3 hours, and then adding IPTG solution to a final concentration of 0.5 mmol / L.

Example  5

The difference from the method for producing the sludge petroleum degrading complex enzyme of Example 3 is that the mass ratio of the protein of petroleum dehydrogenase 21 ^# and formic acid dehydrogenase is 1: 3.

Example  6

The difference from the method for producing the sludge petroleum degrading complex enzyme of Example 3 is that the mass ratio of the protein of petroleum dehydrogenase 21 ^# and formic acid dehydrogenase is 1: 5.

Comparative example  One

The difference from the manufacturing method of the sludge petroleum decomposing complex enzyme of Example 3 is that the mass ratio of the protein of petroleum dehydrogenase 21 ^# and formate dehydrogenase is 2: 3.

Comparative example  2

The difference from the method for producing the sludge petroleum decomposing enzyme of Example 3 is that the mass ratio of the protein of the petroleum dehydrogenase 21 ^# and the formic acid dehydrogenase is 1: 1.

Comparative example  3

The difference from the manufacturing method of the sludge petroleum decomposing enzyme of Example 3 is that the mass ratio of the protein of petroleum dehydrogenase 21 ^# and formate dehydrogenase is 2: 1.

Comparative example  4

Enzyme preparations were prepared as described in Example 3 of Chinese Patent Document CN103484447A (Application No. 201310456751.X).

Experimental Example  1 Petroleum Degradation Rate Experiment

Accurately measure 2 g of oil 10% sludge (petroleum content 200 mg), place it in a dry Erlenmeyer flask and mix 20 ml of the enzyme preparation prepared according to Examples 1, 4-6 and Comparative Examples 1-4, respectively with sodium formate. The mass ratio of protein and sodium formate in the enzyme preparation was 1: 5, and the sludge petroleum decomposing complex treatment solution was prepared by supplementing up to 40 mL with dH ₂ O. At this time, the concentration of the sodium formate solution is all 167mmol / L and each of the experimental groups 1 to 8 and at the same time take 40mL of dH ₂ O to CK group.

The experimental groups 1 to 8 were enzymatically decomposed with the CK group for 12 hours at 30 ° C. and 150 rpm, and the reaction was terminated by adding dichloromethane. The sludge petroleum decomposition rate was measured using the gravimetric method and the specific steps are as follows.

Add 40 mL of dichloromethane to the sludge enzymatic digestion system, mix thoroughly, transfer all to a separatory funnel, add 10 to 20 g of sodium chloride, wash the enzyme digestion reaction vessel with 15 mL dichloromethane, transfer to a separatory funnel, and shake thoroughly for 3 minutes. The layers were left to stand, the water phase was placed in the original Erlenmeyer flask and the organic phase was transferred to a 100 ml conical flask. Samples of enzyme digestion sludge are extracted twice with dichloromethane, using an amount of 15 ml each time, and the extracts are combined three times in a conical flask. Appropriate amount of anhydrous sodium sulfate is added to the dichloromethane extract (until no more lumps are formed), capped and allowed to stand for at least 0.5 hours for dehydration. Filtration was carried out using qualitative filter paper previously washed with dichloromethane, and the filtrate was collected in a 100 ml conical flask. Samples of enzymatic sludge sludge extraction are repeated twice with dichloromethane, using an amount of 15 ml each time, and the extracts are combined three times in a conical flask. Dichloromethane was distilled using a rotary evaporator, naturally dried and weighed to calculate the petroleum decomposition rate.

% Degradation = (control oil weight-lyase enzyme weight) x 100% / control oil weight

The relevant experimental results of the different experimental groups that have been detected are shown in Table 1 below.

Table 1 Petroleum degradation rates of sludge under different 21 ^# petroleum dehydrogenase / Cb FDH blending conditions

Data analysis

As can be seen from the above data, since the petroleum decomposition rate of Experimental Groups 1 to 4 (Examples 1 and 4 to 6) is significantly higher than Experimental Groups 5 to 7 (Comparative Examples 1 to 3), petroleum decomposing enzymes and formic acid from the It can be seen that the proportional relationship between both dehydrogenases has a significant effect on the rate of petroleum decomposition. As can be seen from the data of Experimental Groups 1 to 4 (Example 1, Examples 4 to 6) and Experimental Group 8 (Comparative Example 4), the complex enzyme formulation of the present invention has a higher oil content than conventionally known enzyme formulations. Decomposes more pronounced decomposition effect.

Experimental Example  2: Enzyme Formulation Stability Experiment

Experimental procedure is the same as in Experimental Example 1, the difference is that after the sludge petroleum decomposition complex enzyme treatment solution is prepared and stored for 5 days at room temperature, subsequent sludge decomposition experiments are carried out. Detected petroleum decomposition rate is shown in Table 2 below.

Table 2 Petroleum decomposition rate after sludge petroleum decomposition complex enzyme treatment solution is stored at room temperature for 5 days

Data analysis

As can be seen from the above data, the petroleum decomposition rates of Experimental Group 1, Experimental Group 3 and Experimental Group 4 (Examples 1, 5 and 6) did not change significantly with the corresponding experimental data in Table 1, but Experimental Group 2 (Example 4). ) Is different from the medium used for the preparation of formic acid dehydratase, the enzyme preparation lost the degradation activity, as can be seen here, the fermentation medium described in the present invention has the effect of providing stability of formic acid dehydratase.

                         SEQUENCE LISTING <110> ECOLOGY INSTITUTE SHANDONG ACADEMY OF SCIENCES   <120> PREPARATION METHOD AND APPLICATION OF SLUDGE PETROLEUM DEGRADING COMPLEX ENZYME <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 364 <212> PRT Candida boidinii <400> 1 Met Lys Ile Val Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp 1 5 10 15 Glu Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn             20 25 30 Trp Leu Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu         35 40 45 Gly Gly Asn Ser Val Leu Asp Gln His Ile Pro Asp Ala Asp Ile Ile     50 55 60 Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg Ile Asp 65 70 75 80 Lys Ala Lys Lys Leu Lys Leu Val Val Val Ala Gly Val Gly Ser Asp                 85 90 95 His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly Lys Lys Ile Ser Val             100 105 110 Leu Glu Val Thr Gly Ser Asn Val Val Ser Val Ala Glu His Val Val         115 120 125 Met Thr Met Leu Val Leu Val Arg Asn Phe Val Pro Ala His Glu Gln     130 135 140 Ile Ile Asn His Asp Trp Glu Val Ala Ala Ile Ala Lys Asp Ala Tyr 145 150 155 160 Asp Ile Glu Gly Lys Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly                 165 170 175 Tyr Arg Val Leu Glu Arg Leu Val Pro Phe Asn Pro Lys Glu Leu Leu             180 185 190 Tyr Tyr Asp Tyr Gln Ala Leu Pro Lys Asp Ala Glu Glu Lys Val Gly         195 200 205 Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala Gln Ala Asp Ile     210 215 220 Val Thr Val Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu Ile Asn 225 230 235 240 Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala Trp Leu Val Asn Thr                 245 250 255 Ala Arg Gly Ala Ile Cys Val Ala Glu Asp Val Ala Ala Ala Leu Glu             260 265 270 Ser Gly Gln Leu Arg Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro         275 280 285 Ala Pro Lys Asp His Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala     290 295 300 Gly Asn Ala Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln 305 310 315 320 Thr Arg Tyr Ala Gln Gly Thr Lys Asn Ile Leu Glu Ser Phe Phe Thr                 325 330 335 Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile Ile Leu Leu Asn Gly Glu             340 345 350 Tyr Val Thr Lys Ala Tyr Gly Lys His Asp Lys Lys         355 360 <210> 2 <211> 1095 <212> DNA Candida boidinii <400> 2 atgaagatcg ttttagtctt atatgatgct ggtaaacacg ctgccgatga agaaaaatta 60 tacggttgta ctgaaaacaa attaggtatt gccaattggt tgaaagatca aggacatgaa 120 ttaatcacca cgtctgataa agaaggcgga aacagtgtgt tggatcaaca tataccagat 180 gccgatatta tcattacaac tcctttccat cctgcttata tcactaagga aagaatcgac 240 aaggctaaaa aattgaaatt agttgttgtc gctggtgtcg gttctgatca tattgatttg 300 gattatatca accaaaccgg taagaaaatc tccgttttgg aagttaccgg ttctaatgtt 360 gtctctgttg cagaacacgt tgtcatgacc atgcttgtct tggttagaaa ttttgttcca 420 gctcacgaac aaatcattaa ccacgattgg gaggttgctg ctatcgctaa ggatgcttac 480 gatatcgaag gtaaaactat cgccaccatt ggtgccggta gaattggtta cagagtcttg 540 gaaagattag tcccattcaa tcctaaagaa ttattatact acgattatca agctttacca 600 aaagatgctg aagaaaaagt tggtgctaga agggttgaaa atattgaaga attggttgcc 660 caagctgata tagttacagt taatgctcca ttacacgctg gtacaaaagg tttaattaac 720 aaggaattat tgtctaaatt caagaaaggt gcttggttag tcaatactgc aagaggtgcc 780 atttgtgttg ccgaagatgt tgctgcagct ttagaatctg gtcaattaag aggttatggt 840 ggtgatgttt ggttcccaca accagctcca aaagatcacc catggagaga tatgagaaac 900 aaatatggtg ctggtaacgc catgactcct cattactctg gtactacttt agatgctcaa 960 actagatacg ctcaaggtac taaaaatatc ttggagtcat tctttactgg taagtttgat 1020 tacagaccac aagatatcat cttattaaac ggtgaatacg ttaccaaagc ttacggtaaa 1080 cacgataaga aataa 1095 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <400> 3 ccggatccat gaagatygty ttagtyytwt atg 33 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <400> 4 ccgtcgactt atttcttatc gtgtttaccg 30

Claims (10)

In the manufacturing method of sludge petroleum decomposing enzyme,
Steps below:
(1) proceeding cell disruption to Acinetobacter calcoaceticus cells, centrifugation, and taking the supernatant to prepare petroleum dehydrogenase solution 21 ^# ;
(2) mixing the petroleum dehydrogenase 21 ^# prepared in step (1) with formic acid dehydrogenase according to a ratio of the protein by mass ratio 1: (3 to 5) to prepare a sludge petroleum degrading complex enzyme;
Method for producing a sludge petroleum decomposing complex comprising a. The method of claim 1,
In the step (1), the species number of Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) is CGMCC No. 3915, the method for producing a sludge petroleum degrading complex enzyme. The method of claim 1,
In the step (1), the cells of Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) is prepared by culturing according to the following method,
a. Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) was transferred to LB medium according to the inoculation amount of 1% to 2% by mass ratio and incubated for 14 to 16 hours under conditions of 28 to 32 ° C and 150 to 180 rpm to prepare a seed solution;
b. The seed solution prepared in step a was transferred to LB medium according to the inoculation amount of 4% to 5% by mass ratio and expanded incubated for 14 to 16 hours under conditions of 28 to 32 ° C. and 150 to 180 rpm;
c. Centrifuging the microbial solution prepared in step b and collecting a precipitate to prepare Acinetobacter calcoaceticus cells;
Preferably, the bacterial disruption of step (1),
Acinetobacter calcoaceticus cells are uniformly mixed with a phosphate buffer of pH 7.5 in a ratio of mass volume ratio 1: (15 to 25) and the unit is g / ml; Cell disruption is carried out for 17 minutes by adopting the intermittent ultrasonic treatment method under the ultrasonic conditions of 320W, each time the ultrasonic disruption time is 2s, and the intermittent time is 2s;
Preferably, in the step (1), the centrifugation conditions are sludge petroleum decomposing enzyme, characterized in that the centrifugation for 2 minutes at 5000r / min. The method of claim 1,
In step (2), the formic acid dehydrogenase is formic acid dehydrogenase (C b FDH) and the amino acid sequence is represented by SEQ ID NO.1;
Preferably, in the step (2), the production method of formic acid dehydrogenase (C b FDH),
(i) to create a genetically engineered strain E. coli (E. coli BL21- fdh );
(ii) the genetically modified strain E. coli (E. coli BL21- fdh ) prepared in step (i) was transferred to the seed medium at an inoculation amount of 1% to 2% by mass, and 10 to 28 ° C under conditions of 28 to 32 ° C and 150 to 180 rpm. E. coli seed solution was prepared by culturing seeds for 12 hours;
(iii) the E. coli seed solution of step (ii) was transferred to the fermentation medium at an inoculation amount of 4% to 5% by mass, and incubated for 16 to 18 hours under conditions of 28 to 32 ° C and 150 to 180 rpm. E. coli BL21- fdh ) cells are collected, and the supernatant is collected through cell disruption and centrifugation to prepare formic acid dehydrogenase (C b FDH). The method of claim 4, wherein
In the step (i), the construction method of the genetically modified strain E. coli (E. coli BL21- fdh ),
The formic acid dehydrogenase gene ( fdh ) derived from Candida boidinii was expanded and linked to an E. coli expression vector (pET28a (+)) to construct a recombinant expression vector (pET28a (+)- fdh ) having the fdh gene. Transforming the host bacterium Escherichia coli (BL21 (DE3)) and selecting the transformants to obtain recombinant E. coli (E. coli BL21- fdh ) expressing formate dehydrogenase;
More preferably, in step (ii), the seed medium component is
10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 100 μg / mL ampicillin;
More preferably, the fermentation medium component in step (iii),
Peptone 10g / L, yeast extract 5g / L, Na ₂ HPO _{4 and 12H 2 O 9g / L, KH} 2 PO 4 6.8g / L, (NH 4) 2 SO 4 3.3g / L, glucose 0.5g / L, lactose and 2g / L, MgSO _{4 and 7H 2 O 0.5g / L, CaCl} 2 0.02g / L, glycerol and 0.5% calculated in accordance with the volume%;
More preferably, in step (iii), the cell disruption step,
Genetically engineered strain E. coli BL21- fdh cells are uniformly mixed with a phosphate buffer of pH 7.5 in a ratio of mass volume ratio 1: (15 to 25) and the unit is g / ml; Cell disruption was carried out for 6 minutes using an intermittent sonication method under 195 W of ultrasonic conditions, each time an ultrasonic disruption time was 3s, and the intermittent time was 5s;
More preferably, in the step (iii), the centrifugation conditions are sludge petroleum decomposing complex enzyme, characterized in that the centrifugation for 2 minutes at 3000r / min. The method of claim 1,
In the step (2), the mass ratio of the petroleum dehydrogenase 21 ^# and formic acid dehydrogenase is 1: 4, characterized in that the sludge petroleum decomposing enzyme. Sludge petroleum degrading enzyme obtained by the method of claim 1. Application of the sludge petroleum decomposition complex enzyme of claim 7 in petroleum contaminated sludge recovery. The method of claim 8,
The application involves the following steps:
(I) The sludge petroleum decomposing enzyme is mixed with sodium formate solution, and the sludge petroleum decomposing enzyme treatment liquid having a mass ratio of 1: (4 to 6) and a sodium formate concentration of 150 to 180 mmol / L for protein and sodium formate. Manufacturing step;
(II) The sludge petroleum degrading complex enzyme treatment liquid prepared in step (I) is mixed at a ratio of mass volume ratio 1: (16 to 24), and the unit is g / ml, and it is 6 to 24 under 25 to 35 ° C. Stirring for time;
Application of petroleum contaminated sludge recovery of sludge petroleum decomposition complex enzyme comprising a. The method of claim 9,
In step (I), the mass ratio of protein and sodium formate is 1: 5;
Preferably, in the step (I), in the sludge petroleum decomposition complex enzyme treatment solution, the sodium formate concentration is 167 mmol / L;
Preferably, in the step (II), the sludge and sludge petroleum decomposing complex enzyme treatment liquid is mixed in a ratio of mass volume ratio 1:20, the application of sludge petroleum decomposing enzyme in petroleum contaminated sludge recovery.