AU2019208247A1

AU2019208247A1 - Preparation method and application of sludge petroleum degrading complex enzyme

Info

Publication number: AU2019208247A1
Application number: AU2019208247A
Authority: AU
Inventors: Guanhong CHEN; Xiaowen FU; Shuhai Guo; Lei Ji; Qi Li; Tianyuan LI; Jianing Wang; Yingna XING; Qiang Zhang
Original assignee: Ecology Inst Shandong Academy Of Sciences
Current assignee: Ecology Institute Shandong Academy Of Sciences
Priority date: 2018-03-22
Filing date: 2019-03-04
Publication date: 2019-10-10
Anticipated expiration: 2039-03-04
Also published as: WO2019179303A1; KR20190111947A; CN108486006B; CN108486006A

Abstract

The present invention relates to a preparation method and application of a sludge petroleum degrading complex enzyme. The preparation method comprises the following steps of: (1) disrupting Acinetobacter calcoaceticus cells, centrifuging, and taking supernatant to obtain a petroleum degrading enzyme solution 21"; and (2) mixing the petroleum degrading enzyme solution 21" and a formate dehydrogenase to obtain a sludge petroleum degrading complex enzyme. According to the present invention, the petroleum-degrading enzyme system derived from Acinetobacter calcoaceticus is mixed with the formate dehydrogenase for the first time, and it is found that the complex enzyme can be used for petroleum degradation and repair in treatment of high-concentration petroleum-contaminated sludge; an adsorbent such as diatomaceous earth is not required for immobilization, and the petroleum in the sludge can be reduced in a short time. The complex enzyme has an efficient petroleum pollution treatment capability and low production cost and thus has broad application prospects.

Description

The present invention relates to a preparation method and application of a sludge petroleum degrading complex enzyme. The preparation method comprises the following steps of: (1) disrupting Acinetobacter calcoaceticus cells, centrifuging, and taking supernatant to obtain a petroleum degrading enzyme solution 21^#; and (2) mixing the petroleum degrading enzyme solution 21^# and a formate dehydrogenase to obtain a sludge petroleum degrading complex enzyme. According to the present invention, the petroleum-degrading enzyme system derived from Acinetobacter calcoaceticus is mixed with the formate dehydrogenase for the first time, and it is found that the complex enzyme can be used for petroleum degradation and repair in treatment of high-concentration petroleum-contaminated sludge; an adsorbent such as diatomaceous earth is not required for immobilization, and the petroleum in the sludge can be reduced in a short time. The complex enzyme has an efficient petroleum pollution treatment capability and low production cost and thus has broad application prospects.

PREPARATION METHOD AND APPLICATION OF SLUDGE PETROLEUM

DEGRADING COMPLEX ENZYME

BACKGROUND OF THE INVENTION [0001] 1. Technical Field [0002] The present invention relates to a preparation method and application of a sludge petroleum degrading complex enzyme and belongs to the technical field of soil treatment.

[0003] 2. Description of Related Art [0004] In the process of production, storage and transportation, refining and processing of petroleum, there will be spills and discharges of petroleum hydrocarbons due to accidents, abnormal operations, maintenance, etc. Examples will be blowout accidents in oilfield development; leakage accidents of oil pipelines and oil storage tanks; leakage accidents of tank trucks and oil tankers; overhaul of oil well paraffin removing equipment and oilfield ground equipment; and overhaul of refining and petrochemical production equipment. A large amount of spilling petroleum hydrocarbons should be recovered as much as possible, but in some cases it is difficult to recover them. Even if a great effort is made to recover them, there will still be a part of them left, which causes pollution to the environment (soil, ground and groundwater). When petroleum enters the soil, it will destroy the soil structure and disperse the soil particles, which will reduce the water permeability of the soil. Reactive groups rich in the petroleum can combine with inorganic nitrogen and phosphorus and limit nitrification and dephosphorylation, thus reducing the contents of available phosphorus and nitrogen in the soil. In particular, polycyclic aromatic hydrocarbons (PAHs) in the petroleum are more harmful in the soil due to their carcinogenic, mutative, teratogenic activities and their ability to be enriched in plants and animals through the food chain.

[0005] Among these hazards, sludge treatment is the most difficult. Sludge is oily sludge produced by a mixture of a large amount of oil, mud and water containing petroleum hydrocarbons will be produced due to process equipment and manual operations in the upstream process of petroleum, such as exploration and development, oil and gas gathering and transportation, sewage treatment, tank bottom cleaning, and downstream petroleum refining process. The mixture of the produced oil, mud and water leaks in the environment to produce sludge. The oily sludge in the oilfield is a massive and wide-ranging pollution source with high oil content and high heavy oil composition.

[0006] Prior to the 1980s, the treatment of petroleum-contaminated soil is limited to physical and chemical methods, namely heat treatment and chemical leaching. The heat treatment method is to purify most of the organic pollutants in the soil by incineration or calcination. However, it also destroys the structure and composition of the soil, and it is costly and difficult to implement. Chemical leaching and rinsing may also achieve better degreasing effect. However, the secondary pollution problem of the chemical reagents used limits its application. In theory, the latest pyrolysis technology for oily sludge treatment can reduce petroleum by about 80%, and the remaining petroleum content is still high. Due to the high difficulty in collecting and processing sludge, complicated treatment process and since sludge belongs to hazardous waste, its harmless treatment has always been a worldwide problem. There are basically no examples of harmless and recycling treatment in oil fields of China. As the country pays more and more attention to environmental protection work, it is very necessary for oil companies to effectively reduce environmental pollution risks and various costs.

[0007] As early as the 1970s, in order to solve the problem of petroleum-caused soil pollution caused by oil leakage and oil spills when oil pipelines and oil storage tanks have failures, Esso Research and Engineering Company began to look for clean biological solutions and its laboratory research found an effective bacterial seeding method”, opening a precedent for bio remediation of petroleum-contaminated soil since the 1980s, bioremediation technologies for contaminated soil have attracted more and more attention.

Bioremediation technologies have also made great progress and are gradually maturing.

[0008] Today, countries around the world are beginning to use biological methods to repair petroleum pollution. Bioremediation is the process of using biological life metabolic activities to reduce the concentration of toxic and harmful substances in the soil environment and restore the contaminated soil to a healthy state. Chinese Patent Document CN103484447A (Application No. 201310456751.X) discloses a preparation method and application of a petroleum degrading enzyme preparation, and the preparation steps are as follows: a petroleum degrading microorganism is subjected to cell disruption to prepare a crude enzyme solution and then the crude enzyme solution is mixed with a carrier for adsorption, and separation and drying are carried out to prepare the petroleum degrading enzyme preparation. The petroleum degrading microorganism is acinetobacter calcoaceticus, preserved with the China General Microbiological Culture Collection Center (CGMCC) and its strain collection number is CGMCC No. 3915. According to the invention, the petroleum-contaminated soil is degraded by immobilizing, through an adsorbent, the enzyme system of the microorganism having the function of degrading petroleum, and thus the degradation efficiency is remarkably improved, the microbial degradation rate is increased by 30 to 50 times as compared with that of the microorganism, and the stability is improved by 15 to 20 times as compared with that of the crude enzyme solution. Although the above technical solution may be applied to petroleum-contaminated water bodies and soil remediation processes, since the petroleum concentration at which it is applied is up to 10 g/L, the actual concentration of polluted water bodies and soils that need to be treated is much higher than this concentration, and adsorbents are required for immobilization results in higher practical application costs and poor prospects.

BRIEF SUMMARY OF THE INVENTION [0009] Directed to the deficiencies of the prior art, the present invention provides a preparation method and application of a sludge petroleum degrading complex enzyme.

[0010] The technical solutions of the present invention are as follows.

[0011] A preparation method of a sludge petroleum degrading complex enzyme, comprising the following steps:

[0012] (1) disrupting Acinetobacter calcoaceticus cells, centrifuging, and taking the supernatant to obtain a petroleum degrading enzyme solution 21#; and [0013] (2) mixing the petroleum degrading enzyme solution 21# obtained in the step (1) and a formate dehydrogenase in a ratio of 1: (3-5) by protein mass to obtain a sludge petroleum degrading complex enzyme.

[0014] According to a preferred embodiment of the present invention, in the step (1), the strain collection number of Acinetobacter calcoaceticus is CGMCC No. 3915. This strain is a known strain and does not involve the preservation of strains.

[0015] According to a preferred embodiment of the present invention, in the step (1), the Acinetobacter calcoaceticus thalli are cultured in the following way:

[0016] a. inoculating Acinetobacter calcoaceticus to an LB medium at an inoculum size of 1% to 2% by mass, and carrying out seed culture for 14-16 hours at 28-32 °C and 150-180 rpm to obtain a seed solution;

[0017] b. inoculating the seed solution prepared in the step a to the LB medium at an inoculum size of 4% to 5% by mass, and carrying out expanding culture for 14-16 hours at 28-32 °C and 150-180 rpm to obtain bacterial liquid; and [0018] c. centrifuging the bacterial liquid prepared in the step b, and collecting precipitate, thus obtaining Acinetobacter calcoaceticus thalli.

[0019] According to a preferred method of the present invention, the cell disruption in the step (1) is carried out in the following way:

[0020] uniformly mixing Acinetobacter calcoaceticus thalli with a phosphate buffer solution ( pH 7.5) in a mass-to-volume ratio of 1: (15-25) (unit: g/mL); carrying out cell disruption for 17 min by intermittent ultrasonic treatment with 320 W ultrasonics, wherein ultrasonic disruption lasts for 2s each time, and the intermittent time is 2s.

[0021] According to a preferred embodiment of the present invention, in the step (1), the centrifugation is carried out at 5000 r/min for 2 min.

[0022] According to a preferred method of the present invention, in the step (2), the formate dehydrogenase is a formate dehydrogenase ChFDH with an amino acid sequence as shown in SEQ ID NO. 1.

[0023] According to a preferred method of the present invention, in the step (2), the preparation steps of the formate dehydrogenase C6FDH are as follows:

[0024] (i) constructing genetically engineered strain E. coli BL21-fdh;

[0025] (ii) inoculating the genetically engineered strain E. coli BL2\-fdh constructed in the step (i) to a seed medium at an inoculum size of 1% to 2% by mass, and carrying out seed culture at 28-32 °C and 150-180 rpm for 10-12 hours to prepare an E. coli seed solution; and [0026] (iii) inoculating the E. coli seed solution in the step (ii) to a fermentation medium at an inoculum size of 4% to 5% by mass, and culturing for 16-18 hours at 28-32 °C and 150-180 rpm, collecting the genetically engineered strain E. Coli BL2\-fdh thalli, carrying out cell disruption, centrifuging, and collecting supernatant, thus obtaining the formate dehydrogenase C6FDH.

[0027] According to a further preferred embodiment of the present invention, in the step (i), the genetic engineering strainE.coli BL2\-fdh is constructed as follows:

[0028] amplifying the formate dehydrogenase gene fdh derived from Candida boidinii and linking to an E. coli expression vector pET28a (+) to construct a recombinant expression vector pET28a (+)-fdh carrying the fdh gene; transforming a host strain E. coli

BL21 (DE3), picking transformants, and screening a recombinant E.coli OLlX-fdh expressing the formate dehydrogenase.

[0029] According to a further preferred embodiment of the present invention, in the step (ii), the seed medium comprises the following components:

[0030] peptone (10 g/L), yeast extract (5 g/L), NaCl (10 g/L), and ampicillin (100 pg/mL).

[0031] According to a further preferred embodiment of the present invention, in the step (iii), the fermentation medium comprises the following components:

[0032] peptone (10 g/L), yeast extract (5 g/L), Na2HPO4’12H2O 9 g/L, KH2PO4 (6.8 g/L), (NHyhSCM (3.3 g/L), glucose (0.5 g/L), lactose (2 g/L), MgSCfi-TEhO (0.5 g/L), CaCf (0.02 g/L), and glycerol (0.5 vol%).

[0033] According to a further preferred method of the present invention, in the step (iii), the cell disruption is carried out in the following way:

[0034] uniformly mixing the genetically engineered strain E.coli OLlX-fdh thalli with a phosphate buffer solution ( pH 7.5) in a mass-to-volume ratio of 1: (15-25) (unit: g/mL); carrying out cell disruption for 6 min by intermittent ultrasonic treatment with 195 W ultrasonics, wherein ultrasonic disruption lasts for 3 s each time, and the intermittent time is 5s.

[0035] According to a preferred embodiment of the present invention, in the step (iii), the centrifugation is carried out at 3000 r/min for 2 min.

[0036] According to the preferred method of the present invention, in the step (2), the petroleum degrading enzyme solution 21^# and the formate dehydrogenase are mixed in a mass ratio of 1:4.

[0037] A sludge petroleum degrading complex enzyme prepared by the above-described preparation method.

[0038] Application of the sludge petroleum degrading complex enzyme in recovering petroleum-contaminated sludge.

[0039] The above-mentioned application comprises the following steps:

[0040] (I) mixing the sludge petroleum degrading complex enzyme with a sodium formate solution (the mass ratio of protein to the sodium formate is 1: (4-6)) to obtain a sludge petroleum degrading complex enzyme treatment solution having a sodium formate concentration of 150-180 mmol/L; and [0041] (II) mixing the sludge with the sludge petroleum degrading complex enzyme treatment solution prepared in the step (I) in a mass-to-volume ratio of 1: (16-24) (unit: g/mL), and stirring at 25-35 °C for 6-24 hours.

[0042] According to a preferred embodiment of the present invention, in the step (I), the mass ratio of protein to the sodium formate is 1:5.

[0043] According to a preferred embodiment of the present invention, in the step (I) , the sodium formate concentration in the sludge petroleum degrading complex enzyme treatment solution is 167 mmol/L.

[0044] According to a preferred embodiment of the present invention, in the step (II) , the sludge and the sludge petroleum degrading complex enzyme treatment solution are mixed in a mass-to-volume ratio of 1:20.

[0045] Beneficial Effect [0046] 1. According to the present invention, the petroleum-degrading enzyme system derived from Acinetobacter calcoaceticus is mixed with the formate dehydrogenase for the first time, and it is found that the complex enzyme can be used for petroleum degradation and repair in treatment of high-concentration petroleum-contaminated sludge; an adsorbent such as diatomaceous earth is not required for immobilization, and the petroleum in the sludge can be reduced in a short time. The complex enzyme has an efficient petroleum pollution treatment capability and low production cost and thus has broad application prospects.

[0047] 2. The inventor finds that when the formate dehydrogenase is formate dehydrogenase ChFDH (its amino acid sequence is as shown in SEQ ID NO. 1), the petroleum-degrading enzyme system will further improve the repair efficiency of high-concentration petroleum-contaminated sludge, and the inventor is surprised. It is further found that when the recombinant bacteria are cultured in a specific medium to obtain the formate dehydrogenase ChFDH, the stability and other properties of the formate dehydrogenase ChFDH will be significantly improved.

DETAILED DESCRIPTION OF THE INVENTION [0048] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0049] Sources of biological materials [0050] Acinetobacter calcoaceticus is purchased from CGMCC, and its strain collection number is CGMCC No. 3915;

[0051] Candida boidinii is purchased from CGMCC, and its strain collection number is CGMCC 2.2378; and [0052] Plasmid pET28a (+) is purchased from Shandong Vaughan Biotechnology Co., Ltd.;

[0053] E. coli BL21 (DE3) is a commercially available product.

[0054] Embodiment 1 [0055] The Acinetobacter calcoaceticus thalli are cultured in the following way:

[0056] a. inoculating Acinetobacter calcoaceticus to an LB medium in a mass ratio of 2%, and carrying out seed culture for 15 hours at 30 °C and 160 rpm to obtain a seed solution;

[0057] b. inoculating the seed solution prepared in the step a to the LB medium in a mass ratio of 4% to 5%, and carrying out expanding culture for 15 hours at 30 °C and

160 rpm to obtain bacterial liquid; and [0058] c. centrifuging the bacterial liquid prepared in the step b, and collecting precipitate, thus obtaining Acinetobacter calcoaceticus thalli.

[0059] Embodiment 2 [0060] The preparation steps of the formate dehydrogenase ChFDH are as follows:

[0061] (i) constructing genetically engineered strain E. coli 'BLlX-fdh iⁿ the following way:

[0062] amplifying the formate dehydrogenase gene fdh derived from Candida boidinii, wherein the gene sequence of the formate dehydrogenase gene fdh is as shown in SEQ ID NO.2 and the sequence of a forward amplification primer is as shown in SEQ ID NO. 3; linking the amplified formate dehydrogenase gene fdh to an E. coli expression vector pET28a (+) to construct a recombinant expression vector pET28a (fffdh carrying the fdh gene; transforming a host strain E. coli BL21 (DE3), picking transformants, and screening a recombinant E.coli OLlX-fdh expressing the formate dehydrogenase (for specific conditions of the preparation steps, reference is made to the use instruction of the E. coli expression vector pET28a (+);

[0063] (ii) inoculating the genetically engineered strain E. coli BCli-fdh constructed in the step (i) to a seed medium at an inoculum size of 2% by mass, and carrying out seed culture at 30 °C and 160 rpm for 12 hours to prepare an E. coli seed solution, wherein [0064] the seed medium comprises the following components:

[0065] peptone (10 g/L), yeast extract (5 g/L), NaCl (10 g/L), and ampicillin (100 pg/mL); and [0066] (iii) inoculating the E. coli seed solution in the step (ii) to a fermentation medium at an inoculum size of 4% by mass, and culturing for 16 hours at 30 °C and 160 rpm, collecting the genetically engineered strain E. Coli BL2\-fdh thalli, carrying out cell disruption, centrifuging for 2 min at 3000 r/min, and collecting supernatant, thus obtaining the formate dehydrogenase C6FDH, wherein a test result shows that the amino acid sequence is as shown in SEQ ID NO. 1;

[0067] the fermentation medium comprises the following components:

[0068] peptone (10 g/L), yeast extract (5 g/L), Na2HPO4’12H2O (9 g/L), KH2PO4 (6.8 g/L), (NH4)2SO4 (3.3 g/L), glucose (0.5 g/L), lactose (2 g/L), MgSO4’7H2O (0.5 g/L), CaCf (0.02 g/L), and glycerol (0.5 vol%);

[0069] the cell disruption is carried out in the following way:

[0070] uniformly mixing the genetically engineered strain E.coli BL21-fdh thalli with a phosphate buffer solution ( pH 7.5) in a mass-to-volume ratio of 1:20 (unit: g/mL); carrying out cell disruption for 6 min by intermittent ultrasonic treatment with 195 W ultrasonics, wherein ultrasonic disruption lasts for 3s each time, and the intermittent time is 5s.

[0071] Embodiments [0072] A preparation method of a sludge petroleum degrading complex enzyme, comprising the following steps:

[0073] (1) disrupting Acinetobacter calcoaceticus cells prepared in Embodiment 1, centrifuging for 2 min at 5000 r/min, and taking the supernatant to obtain a petroleum degrading enzyme solution 21^#, wherein [0074] the cell disruption is carried out in the following way:

[0075] uniformly mixing Acinetobacter calcoaceticus thalli with a phosphate buffer solution ( pH 7.5) in a mass-to-volume ratio of 1:20 (unit: g/ml); carrying out cell disruption for 17 min by intermittent ultrasonic treatment with 320 W ultrasonics, wherein ultrasonic disruption lasts for 2s each time, and the intermittent time is 2s; and [0076] (2) mixing the petroleum degrading enzyme solution 21^# obtained in the step (1) and a formate dehydrogenase in a ratio of 1:4 by protein mass to obtain a sludge petroleum degrading complex enzyme.

[0077] Embodiment 4 [0078] This embodiment is similar to the preparation method of a sludge petroleum degrading complex enzyme as described in Embodiment 3, the difference is that the fermentation medium used in the preparation process of the formate dehydrogenase is prepared in the following way:

[0079] culturing an LB liquid medium (peptone (10 g/L), yeast extract (5 g/L), NaCl 10 (g/L)) for 2-3 hours, adding an IPTG solution till a final concentration of 0.5 mmol / L, and carrying out induction culture.

[0080] Embodiment 5 [0081] This embodiment is similar to the preparation method of a sludge petroleum degrading complex enzyme as described in Embodiment 3, the difference is that the petroleum degrading enzyme solution 21^# is mixed with the formate dehydrogenase in a ratio of 1:3 by protein mass.

[0082] Embodiment 6 [0083] This embodiment is similar to the preparation method of a sludge petroleum degrading complex enzyme as described in Embodiment 3, the difference is that the petroleum degrading enzyme solution 21^# is mixed with the formate dehydrogenase in a ratio of 1:5 by protein mass.

[0084] Reference Example 1 [0085] This embodiment is similar to the preparation method of a sludge petroleum degrading complex enzyme as described in Embodiment 3, the difference is that the petroleum degrading enzyme solution 21^# is mixed with the formate dehydrogenase in a ratio of 2:3 by protein mass.

[0086] Reference Example 2 [0087] This embodiment is similar to the preparation method of a sludge petroleum degrading complex enzyme as described in Embodiment 3, the difference is that the petroleum degrading enzyme solution 21^# is mixed with the formate dehydrogenase in a ratio of 1:1 by protein mass.

[0088] Reference Example 3 [0089] This embodiment is similar to the preparation method of a sludge petroleum degrading complex enzyme as described in Embodiment 3, the difference is that the petroleum degrading enzyme solution 21^# is mixed with the formate dehydrogenase in a ratio of 2:1 by protein mass.

[0090] Reference Example 4 [0091] The enzyme preparation is prepared as described in Embodiment 3 in Chinese Patent Document CN103484447A (Application No. 201310456751.X).

[0092] Experimental Example 1 Petroleum Degradation Rate Experiment [0093] 2 g sludge with a petroleum content of 10% (200 mg of petroleum) is accurately weighed and added to a dry triangular flask; 20 ml of the enzyme preparation prepared according to Embodiment 1, Embodiments 4-6 and Reference Examples 1-4 with sodium formate (the mass ratio of protein in the enzyme preparation to the sodium formate is 1:5) and dEEO is added until the solution reaches 40 mL, thus obtaining a sludge petroleum degrading complex enzyme treatment solution, and in this case, the concentrations of the sodium formate solutions are all 167 mmol/L and then marked as experimental groups 1-8 respectively, and 40 mL of dEEO is taken as a CK group.

[0094] The experimental groups 1-8 react with the CK group at 30 °C and 150 rpm for 12 hours, and dichloro methane is then added to terminate the reaction. The petroleum degradation rate of sludge is determined by gravimetric method. The specific steps are as follows.

[0095] 40 mL of dichloromethane is added to the enzyme degrading system of the sludge; the mixture is adequately mixed and then completely transferred to a separating funnel; 10-20 g of sodium chloride is added; 15 mL of dichloro methane is used to wash the enzyme degradation reaction vessel and then transferred to the separating funnel; the resulting solution is adequately shaken for 3 min and then is rested still for layering; the aqueous phase is placed in the original triangular flask, and the organic phase is transferred to a 100 mL Erlenmeyer flask. An enzyme degraded sludge sample is repeatedly extracted twice with dichloro methane, 15 mL each time, and extract solutions obtained through three extractions are combined in the Erlenmeyer flask. An appropriate amount of anhydrous sodium sulfate is added to the dichloromethane extract solution (until no agglomeration occurs); the solution is then covered and rested still for more than 0.5 h for dehydration. Filtration is carried out with a qualitative filter paper washed with dichloromethane in advance, and the filtrate is collected in a 100 mL Erlenmeyer conical flask. An enzyme degraded sludge sample is repeatedly extracted twice with dichloro methane, 15 mL each time, and extract solutions obtained through three extractions are combined in the Erlenmeyer flask. A rotary evaporator is used for distilling off the dichloromethane, the resulting solution is then dried naturally and weighed to calculate the petroleum degradation rate:

[0096] Degradation rate (%) = (petroleum weight of control group - petroleum weight of enzyme degradation test group) x 100%/petroleum weight of control group [0097] After test, the relevant experimental results of different experimental groups are shown in Table 1:

[0098] Table 1 Petroleum degradation rate of petroleum-contaminated sludge (sand) under different 21^# petroleum degrading cnzymc/C6FDH ratios

Experim

Experi

Experim

Exper

CK

ental	mental	ental	iment	iment	iment	iment	iment	iment	group
group	group 1	group 2	al	al	al	al	al	al
No.			group	group	group	group	group	group
			3	3	5	6	7	8
Petro leu m degradat ion rate (%)	35.1	35.6	32.0	39.7	29.9	26.6	10.3	10.7

[0099] Data analysis

It can be seen from the above data that the petroleum degradation rates of the experimental groups 1-4 (Embodiment 1, Embodiments 4 to 6) are significantly higher than those of the experimental group 5-7 (Reference Examples 1-3), from which it can be concluded that the proportional relationship between the petroleum degrading enzyme and the formate dehydrogenase can significantly affect the petroleum degradation rate. It can be seen from the data of the experimental groups 1-4 (Embodiment 1, Embodiments 4-6) and the experimental group 8 (Reference Example 4) that the complex enzyme preparation of the present invention has a more significant degradation effect than the existing known enzyme preparations when used for degrading sludge with a high petroleum rate.

[00100] Experimental Example 2 Stability Experiment of Enzyme Preparation [00101] The experimental procedure of the Experimental Example 2 is similar to what is described in Experimental Example 1, except that the prepared sludge petroleum degrading complex enzyme treatment solution is stored for 5 days at room temperature and then used for the subsequent sludge degradation experiments. After test, the petroleum degradation rates are shown in Table 2:

[00102] Table 2 Petroleum degradation rates after 5 days of storage of sludge petroleum degrading complex enzyme treatment solution at room temperature

Experi	Experime	Expe	Expe	Expe	Expe	Expe	Expe	Expe	CK
mental	ntal group	rimen	rimen	rimen	rimen	rimen	rimen	rimen	group
group	1	tai	tai	tai	tai	tai	tai	tai
No.		group	group	group	group	group	group	group
		2	3	4	5	6	7	8
Petro leu m degrada tion rate (%)	35.3		31.7	42.1	28.6	25.9	8.3	6.5

[00103] Data analysis [00104] It can be seen from the above data that the petroleum degradation rates of the experimental group 1, the experimental group 3, and the experimental group 4 (Embodiment 1, Embodiment 5, and Embodiment 6) do not change significantly from the corresponding experimental data in Table 1; however, for the experimental group 2 (Embodiment 4), since the media used in the preparation of the formate dehydrogenase are different, the enzyme preparation loses the degradation activity, and thus it can be seen that the fermentation medium described in the present invention takes an effect in providing stability to the formate dehydrogenase.

SEQUENCE LISTING

<110>	ECOLOGY INST OF 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
<213>	Candida boidinii

<400> 1

Met Lys lie Vai Leu Vai Leu Tyr Asp Ala Gly Lys His Ala Ala Asp

10 15

Glu Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly lie Ala Asn

25 30

Trp Leu Lys Asp Gin Gly His Glu Leu lie Thr Thr Ser Asp Lys Glu

4045

Gly Gly Asn Ser Vai Leu Asp Gin His lie Pro Asp Ala Asp lie lie

5560 lie Thr Thr Pro Phe His Pro Ala Tyr lie Thr Lys Glu Arg lieAsp

70 7580

Lys Ala Lys Lys Leu Lys Leu Vai Vai Vai Ala Gly Vai Gly SerAsp

9095

His lie Asp Leu Asp Tyr lie Asn Gin Thr Gly Lys Lys lie Ser Vai

100 105110

Leu Glu Vai Thr Gly Ser Asn Vai Vai Ser Vai Ala Glu His Vai Vai

115 120125

Met Thr Met Leu Vai Leu Vai Arg Asn Phe Vai Pro Ala His Glu Gin

130 135140 lie lie Asn His Asp Trp Glu Vai Ala Ala lie Ala Lys Asp AlaTyr

145 150 155160

Asp lie Glu Gly Lys Thr lie Ala Thr lie Gly Ala Gly Arg lieGly

165 170175

Tyr Arg Vai Leu Glu Arg Leu Vai Pro Phe Asn Pro Lys Glu Leu Leu

180 185190

Tyr Tyr Asp Tyr Gin Ala Leu Pro Lys Asp Ala Glu Glu Lys Vai Gly

195 200205

Ala Arg Arg Vai Glu Asn lie Glu Glu Leu Vai Ala Gin Ala Asp lie

210 215220

Vai Thr Vai Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu lieAsn

225 230 235240

Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala Trp Leu Vai AsnThr

245 250255

Ala Arg Gly Ala lie Cys Vai Ala Glu Asp Vai Ala Ala Ala Leu Glu

Ser Gly Gin Leu Arg Gly Tyr Gly Gly Asp Vai Trp Phe Pro Gin 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 Gin

305 310 315320

Thr Arg Tyr Ala Gin Gly Thr Lys Asn lie Leu Glu Ser Phe PheThr

325 330335

Gly Lys Phe Asp Tyr Arg Pro Gin Asp lie lie Leu Leu Asn Gly Glu

340 345350

Tyr Vai Thr Lys Ala Tyr Gly Lys His Asp Lys Lys <210> 2 <211> 1095 <212> DNA <213> Candida boidinii <400> 2 atgaagatcg ttttagtctt atatgatgct ggtaaacacg ctgccgatga agaaaaatta60 tacggttgta ctgaaaacaa attaggtatt gccaattggt tgaaagatca aggacatgaa120 ttaatcacca cgtctgataa agaaggcgga aacagtgtgt tggatcaaca tataccagat180 gccgatatta tcattacaac tcctttccat cctgcttata tcactaagga aagaatcgac240 aaggctaaaa aattgaaatt agttgttgtc gctggtgtcg gttctgatca tattgatttg300 gattatatca accaaaccgg taagaaaatc tccgttttgg aagttaccgg ttctaatgtt360 gtctctgttg cagaacacgt tgtcatgacc atgcttgtct tggttagaaa ttttgttcca420 gctcacgaac aaatcattaa ccacgattgg gaggttgctg ctatcgctaa ggatgcttac480 gatatcgaag gtaaaactat cgccaccatt ggtgccggta gaattggtta cagagtcttg540 gaaagattag tcccattcaa tcctaaagaa ttattatact acgattatca agctttacca600 aaagatgctg aagaaaaagt tggtgctaga agggttgaaa atattgaaga attggttgcc660 caagctgata tagttacagt taatgctcca ttacacgctg gtacaaaagg tttaattaac720 aaggaattat tgtctaaatt caagaaaggt gcttggttag tcaatactgc aagaggtgcc780 atttgtgttg ccgaagatgt tgctgcagct ttagaatctg gtcaattaag aggttatggt840 ggtgatgttt ggttcccaca accagctcca aaagatcacc catggagaga tatgagaaac900 aaatatggtg ctggtaacgc catgactcct cattactctg gtactacttt agatgctcaa actagatacg ctcaaggtac taaaaatatc ttggagtcat tctttactgg taagtttgat tacagaccac aagatatcat cttattaaac ggtgaatacg ttaccaaagc ttacggtaaa cacgataaga aataa

960

1020

1080

1095 <210> 3 <211> 33 <212> DNA <213> Synthesis <400> 3 ccggatccat gaagatygty ttagtyytwt atg 33 <210> 4 <211> 30 <212> DNA <213> Synthesis <400>

ccgtcgactt atttcttatc gtgtttaccg

Claims

1. A preparation method of a sludge petroleum degrading complex enzyme, comprising the following steps of:

(1) disrupting Acinetobacter calcoaceticus cells, centrifuging, and taking supernatant to obtain a petroleum degrading enzyme solution 21^#; and (2) mixing the petroleum degrading enzyme solution 21^# obtained in the step (1) and a formate dehydrogenase in a ratio of 1: (3-5) by protein mass to obtain a sludge petroleum degrading complex enzyme.

2. The preparation method according to claim 1, wherein, in the step (1), the strain collection number οιAcinetobacter calcoaceticus is CGMCC No. 3915.

3. The preparation method according to claim 1, wherein, in the step (1), the Acinetobacter calcoaceticus thalli are cultured in the following way:

a. inoculating Acinetobacter calcoaceticus to an LB medium at an inoculum size of 1% to 2% by mass, and carrying out seed culture for 14-16 hours at 28-32 °C and 150-180 rpm to obtain a seed solution;

b. inoculating the seed solution prepared in the step a to the LB medium at an inoculum size of 4% to 5% by mass, and carrying out expanding culture for 14-16 hours at 28-32 °C and 150-180 rpm to obtain bacterial liquid; and

c. centrifuging the bacterial liquid prepared in the step b, and collecting precipitate, thus obtaining Acinetobacter calcoaceticus thalli;

preferably, the cell disruption in the step (1) is carried out in the following way:

uniformly mixing Acinetobacter calcoaceticus thalli with a phosphate buffer solution ( pH 7.5) in a mass-to-volume ratio of 1: (15-25) (unit: g/ml); carrying out cell disruption for 17 min by intermittent ultrasonic treatment with 320 W ultrasonics, wherein ultrasonic disruption lasts for 2s each time, and the intermittent time is 2s; and preferably, in the step (1), the centrifugation is carried out at 5000 r/min for 2 min.

4. The preparation method according to claim 1, wherein, in the step (2), the formate dehydrogenase is a formate dehydrogenase ChFDH with an amino acid sequence as shown in SEQ ID NO. 1; and preferably, in the step (2), the preparation steps of the formate dehydrogenase ChFDH are as follows:

(i) constructing genetically engineered strain E. coli BL21-fdh;

(ii) inoculating the genetically engineered strain E. coli BL2\fdh constructed in the step (i) to a seed medium at an inoculum size of 1% to 2% by mass, and carrying out seed culture at 28-32 °C and 150-180 rpm for 10-12 hours to prepare an E. coli seed solution; and (iii) inoculating the E. coli seed solution in the step (ii) to a fermentation medium at an inoculum size of 4% to 5% by mass, and culturing for 16-18 hours at 28-32 °C and 150-180 rpm, collecting the genetically engineered strain E. Coli B>L2\-fdh thalli, carrying out cell disruption, centrifuging, and collecting supernatant, thus obtaining the formate dehydrogenase ChFDH.

5. The preparation method according to claim 4, wherein, in the step (i), the genetic engineering strain E.coli B>L2\-fdh is constructed as follows:

amplifying the formate dehydrogenase gene fdh derived from Candida boidinii and linking to an E. coli expression vector pET28a (+) to construct a recombinant expression vector pET28a (+)-fdh carrying the fdh gene; transforming a host strain E. coli BL21 (DE3), picking transformants, and screening a recombinant E.coli B>L2\-fdh expressing the formate dehydrogenase;

preferably, in the step (ii), the seed medium comprises the following components: peptone (10 g/L), yeast extract (5 g/L), NaCl (10 g/L), and ampicillin (100 pg/mL); more preferably, in the step (iii), the fermentation medium comprises the following components:

peptone (10 g/L), yeast extract (5 g/L), Na2HPO4’12H2O (9 g/L), KH2PO4 (6.8 g/L), (NELXSCU (3.3 g/L), glucose (0.5 g/L), lactose (2 g/L), MgSCU’TFhO (0.5 g/L), CaCf (0.02 g/L), and glycerol (0.5 vol%);

more preferably, in the step (iii), the cell disruption is carried out in the following way:

uniformly mixing the genetically engineered strain E.coli BL2\-fdh thalli with a phosphate buffer solution ( pH 7.5) in a mass-to-volume ratio of 1: (15-25) (unit: g/mL); carrying out cell disruption for 6 min by intermittent ultrasonic treatment with 195 W ultrasonics, wherein ultrasonic disruption lasts for 3s each time, and the intermittent time is 5s; and more preferably, in the step (iii), the centrifugation is carried out at 3000 r/min for 2 min.

6. The preparation method according to claim 1, wherein, in the step (2), the petroleum degrading enzyme solution 21^# and the formate dehydrogenase are mixed in a mass ratio of 1:4.

7. A sludge petroleum degrading complex enzyme prepared by the preparation method according to claim 1.

8. Application of the sludge petroleum degrading complex enzyme according to claim 7 in recovering petroleum-contaminated sludge.

9. The application of claim 8, comprising the following steps:

(I) mixing the sludge petroleum degrading complex enzyme with a sodium formate solution (the mass ratio of protein to the sodium formate is 1: (4-6)) to obtain a sludge petroleum degrading complex enzyme treatment solution having a sodium formate concentration of 150-180 mmol/L; and (II) mixing the sludge with the sludge petroleum degrading complex enzyme treatment solution prepared in the step (I) in a mass-to-volume ratio of 1: (16-24) (unit: g/mL), and stirring at 25-35 °C for 6-24 hours.

10. The application according to claim 9, wherein, in the step (I), the mass ratio of protein to the sodium formate is 1:5;

preferably, in the step (I), the sodium formate concentration in the sludge petroleum degrading complex enzyme treatment solution is 167 mmol/L; and preferably, in the step (II), the sludge and the sludge petroleum degrading complex enzyme treatment solution are mixed in a mass-to-volume ratio of 1:20.