CN102250854B - Lipase for treating oil pollutants as well as encoding gene and application thereof - Google Patents

Abstract

The invention provides a lipase for treating oil pollutants as well as an encoding gene and application thereof in the hydrolytic treatment of oil pollutants by a microbial method. The sequences of the lipase and the encoding gene thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The new lipase gene provided by the invention can be highly expressed in yeast, overcome the shortcomings of traditional pure culture techniques, can increase the expression activity of the lipase, and has important significance in promoting the application of the lipase in the treatment of oil-containing wastewater.

Description

Lipase, encoding gene and application thereof for the treatment of grease contamination

(1) technical field

The present invention relates to a kind of lipase for the treatment of grease contamination, encoding gene and the application in microbial method hydrolysis treatment grease contamination thereof.

(2) background technology

In recent years, the pollution that oil substances causes in the environment is day by day serious, has become an environmental problem that needs to be resolved hurrily.Oily(waste)water is the large and wide source of pollution of a kind of amount.Along with the raising of people's quality of life and the enhancing of environmental consciousness, the pollution problem of grease more and more receives people's concern.Grease contamination treatment process commonly used mainly contains Physical and chemical method at present, but can't thoroughly eliminate the grease in the waste water, and easily causes secondary pollution.Biological process is to process a kind of the most effective, the safest of grease contamination and method the most thoroughly.

Lipase (Lipase, GEH) is a kind of special lytic enzyme, can be hydrolyzed water-insoluble Ester, such as long chain triglycerides (fat).Outstanding feature of lipase be it can be specifically on oil-water two-phase interface, be lipid acid and glycerine with fat splitting.

In recent years, molecular biology research for lipase is deepening continuously, and has up to the present cloned a lot of lipase genes, has measured their nucleotide sequence, the albumin crystal structure of part lipase is after deliberation clear (Winkler F K, Arcy AD et al.1990 also; Brady L, 1991).

Lot of documents has been reported the expression such as the expression system that utilizes intestinal bacteria (Escherichia coli) and pichia spp (Pichia Piastoris) lipase gene, it is the lip1 gene of the codon optimization of 1647 bases that Brocca etc. have synthesized total length, realized its high efficient expression of allos in pichia spp (Pichia pastoris), yield of enzyme is up to 150U/ml (Brocca S, et al.1998).Pfeffer etc. adopt the series of optimum strategy to express Candidaantarctica lipase A (CalA) (Pfeffer J, et al.2007) in Escherichia coli Origami B.

From the yielding lipase wild mushroom that occurring in nature screens, its lipase gene generally is subjected to the rigorous regulation and control of Host Strains, and the metabolism that the lipase of expression satisfies self gets final product; And market orientation requires high efficient expression to improve lipase output.A large amount of microbial lipase gene clonings and the recombinant expressed enzyme source that can ensure that provides for the market requirement, but the expression efficiency of original lipase gene is still not high, is the bottleneck that lipase is widely applied.

Can only account in the microorganism of laboratory pure culture at present its sum less than 1%, most microorganisms can not be cultivated, and can't obtain pure bacterium colony by traditional training method of separation and purification, thereby limit the exploitation of new lipase gene.Utilize the new lipase gene of molecular biological method screening, improve the expression vigor of lipase, significant for the application that promotes lipase in oil-containing waste water is processed.

(3) summary of the invention

The invention provides a kind of new lipase gene that from environment, filters out, this gene can be in yeast cell high efficient expression, overcome the not high defective of original lipase gene expression efficiency.

A kind of lipase for the treatment of grease contamination, its aminoacid sequence is shown in SEQ ID NO:1.

The invention still further relates to the gene of the described lipase of coding claim 1.Concrete, described gene nucleotide series is shown in SEQ ID NO:2.The present invention screens the lipase gene of a new high efficient expression by making up the DNA library of active sludge.Having overcome the deficiency of traditional pure culture technigne, is a new way of seeking new lipase gene.

The lipase gene that the present invention relates to is a kind of new gene, does not have homology with isoformgene.The lipase of its coding is a kind of new lipase, compares with the lipase aminoacid sequence that other have been reported, and similarity is less than 30%.Should be pointed out that the coded aminoacid sequence of lipase gene of the present invention is carried out one or more amino acid substitutions, insertion or lack resulting functional analogue all belonging to protection scope of the present invention.In the situation that known lipase gene of the present invention, those of ordinary skill of the present invention can by this area ordinary method carrier construction, conversion, expression, obtain described lipase.

The invention still further relates to the recombinant vectors that contains described gene, the recombinant vectors that will contain described gene is converted in the Host Strains (being generally yeast), expresses, and can obtain lipase of the present invention.

The invention still further relates to the application of described gene in the preparation recombinant lipase.

The invention still further relates to the application of described lipase in microbial method hydrolysis treatment grease contamination.The optimum temperuture of lipase of the present invention is 25～35 ℃, and optimal pH is 6.5～7.5.

Beneficial effect of the present invention is mainly reflected in: the invention provides a kind of new lipase gene, can be in yeast high efficient expression, overcome the deficiency of traditional pure culture technigne, can improve the expression vigor of lipase, significant for the application that promotes lipase in oil-containing waste water is processed.

(4) description of drawings

Fig. 1 is the total DNA electrophoretogram of active sludge;

Fig. 2 is that Sau3A I enzyme is cut the DNA electrophoretogram;

Fig. 3 is the EcoR I/Not I double digestion checking electrophoretogram of restructuring plasmid pPIC9K-lipN;

Fig. 4 is the PCR checking electrophoretogram of lipN goal gene in the transformed yeast; M:Marker, A: with 5 ' AOX1 and 3 ' AOX1 as primer amplification, B: with α-factor and 3 ' AOX1 as primer amplification

Fig. 5 is the optimal pH curve of lipase;

Fig. 6 is the optimum temperuture curve of lipase.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: the structure in activated sludge DNA library

1, the extraction of activated sludge DNA

Gather mud from certain food sewage work, carry out the extraction of DNA through suitably diluting as sample.12000rmin ^-1Centrifugal 5min collects sample, and after the resuspended washing once of TE, adding N,O-Diacetylmuramidase to final concentration is 100 μ gml ^-1, 37 ℃ of water-baths 2 hours.Add 5M NaCl and adjust final concentration to 0.5M, add 40 μ l Proteinase Ks and 20%SDS (final concentration is 0.5～1%) behind the mixing, 37 ℃ are spent the night or 50 ℃ of water-bath 3h clarify to the solution thickness.After adding the saturated NaCl thermal agitation 15s of 1/3 volume, 12000rmin ^-1Centrifugal 5min collects in supernatant to the sterilization centrifuge tube, adds 0.6V Virahol mixing postprecipitation, 12000rmin ^-1Centrifugal collecting precipitation is used 70% washing with alcohol, is dissolved in after drying up in the 400 μ l sterilization deionized water, and total DNA electrophoresis as shown in Figure 1.

2, the structure in DNA library

(1) enzyme of DNA is cut

With the total DNA of the partially digested active sludge of Sau3A I, carry out enzyme with detection type reaction system first and cut, the suitableeest definite enzyme is cut concentration and time, and the type that finally is prepared is partially digested.Detection type endonuclease reaction system (10 μ L): total DNA 2 μ L, Sau3AI is an amount of, and damping fluid 1 μ L adds ddH ₂O is to final volume 10 μ L.Preparation type endonuclease reaction system (100 μ L): total DNA20 μ L, Sau3AI is an amount of, and damping fluid 10 μ L add ddH ₂O is to final volume 100 μ L.Add wherein that Sau3AI enzyme amount is cut concentration according to enzyme and the time gropes, cut the dna fragmentation that glue reclaims 2～4kb band, as the external source fragment, electrophoresis as shown in Figure 2.

(2) preparation of dephosphorization carrier

Extract the puc18 plasmid, carry out single endonuclease digestion with BamHI, the linear fragment that reclaims the 1.8kb size carries out the dephosphorylation reaction.Reaction system below in Eppendorf tube, setting up: linearizing puc18 carrier 28ul, 10 * alkaline phosphatase damping fluid, 5 μ l, CIAP (16U/ μ l TAKARA) 0.5ul adds ddH ₂O is to final volume 50 μ l.50 ℃ are reacted 30min, add the EDTA termination reaction of 1/10 volume 0.5M; Merge four effective ddH ₂O complements to 500ul; Add 400ul phenol/chloroform extracting 1 time, the sucking-off supernatant; The 3M NaAc that adds 1/10V, the ethanol of 2V precooling is at-20 degree precipitation 60min; Centrifugal collecting precipitation with drying up after 200 μ l, 70% washing with alcohol, dissolves with 20 μ lTE.Electrophoresis is quantitative, and it is in 50% the sterile glycerol that the packing of 50～100ng/ pipe is stored in final concentration, for subsequent use.

(3) transform

External source fragment and carrier are spent the night enzyme even in proportion.Get the efficient competent cell of E.coli DH5 α (every pipe is 200 μ l approximately) and melt in ice bath, every pipe adds 2 μ l enzymes and connects product, rotates gently with the mixing content, place 30min in the ice bath, centrifuge tube put into 42 ℃ circulator bath, heat shock 90s does not shake centrifuge tube.Fast centrifuge tube is moved on to make cell cooling 1～2min in the ice bath after every pipe add the LB substratum that 800 μ l are preheated to 37 ℃, behind the mixing in 37 ℃ of shaking tables, 150rmin ^-1Incubation 45min makes bacteria resuscitation, and the antibiotics resistance gene of expression plasmid coding.Conversion fluid after the coating recovery is to adding penbritin (100 μ gml ^-1) fresh LB flat board on, after bacterium liquid is completely absorbed, be inverted dull and stereotypedly in 37 ℃ of cultivations, bacterium colony appears behind 12～16h.

(4) inspection in library and preservation

150 positive colonies of picking are inoculated on the microbiotic flat board, 37 ℃ be cultured to bacterium colony and grow after, random picking Partial Conversion carries out plasmid extraction, checks that the external source fragment inserts Qing Condition, and cultured flat board is saved backup in 4 ℃.Flat board after the detection washes rear glycerol adding to final concentration 15% with sterilized water with bacterium colony, saves backup in-70 ℃.

Embodiment 2: the screening of lipase gene and sequential analysis

Place the LB liquid nutrient medium to cultivate 12h clone's in the DNA library, through suitably coating on the primary dcreening operation flat board of 2 sweet oil of surface uniform coating after the dilution, place under 37 ℃ of conditions and cultivate 24h, the bacterial strain access that is chosen at dull and stereotyped upper growth is sieved in the liquid nutrient medium again, cultivates the content of olive oil of measuring behind the 36h in every kind of fermented liquid under 37 ℃ of conditions.Choosing has the bacterial strain of strong degradation capability to sweet oil, extracts plasmid insertion sequence is checked order.Lipase gene called after lipN, carrier called after puc-lipN.

Sequencing result carries out sequence alignment at NCBI Genbank database, and the result shows there is not the sequence of discovery and this gene order homology in database, and it should be a new sequence.Through the analysis to lipase gene lipN, predict the possible encoding sequence of this enzyme, behind the removal signal peptide, comprised initiation codon and termination codon, totally 1224 Nucleotide (SEQ ID NO:2), 408 amino acid (SEQ ID NO:1) of encoding.

Above-mentioned primary dcreening operation substratum (gL ^-1): (NH ₄) ₂SO ₄2.0, KH ₂PO ₄1.0, MgSO ₄7H ₂O0.5, agar 20.0,2 sweet oil of every ware.Sieve again substratum (gL ^-1): (NH ₄) ₂SO ₄2.0, K ₂HPO ₄2.0, NaH ₂PO ₄2.0 NaCl 2.0, MgSO ₄7H ₂O 0.5, sweet oil 20.0.

Embodiment 3: the expression of lipase gene in yeast cell

1, the structure of expression vector

The design restriction enzyme site is respectively primer lipNF (ACTG AATTCATGTCCTATGTACCGTTCAG) and the lipNR (ACTGCGGCCG CCTACCAGTGGGTCGTACCAA) of EcoR I and Not I, take puc-lipN as template, amplification is with the lipase gene lipN of EcoR I/Not I restriction enzyme site, PCR product EcoR I/Not I double digestion, reclaim the 1.4kb fragment, be connected with the pPIC9K (U.S. invitrogen company) of same double digestion, Transformed E .coli DH5 α obtains recombinant plasmid pPIC9K-lipN.This recombinant plasmid is verified through EcoRI/Not I double digestion.Enzyme is cut the checking collection of illustrative plates as shown in Figure 3.

2, prepare conversion DNA

With pPIC9k-lipN Sal I or Sac I or Bgl II linearization for enzyme restriction, for yeast cell, the transformant that obtains with Sal I or Sac I linearizing is the His+Mut+ phenotype; The transformant that obtains with Bgl II is the His+Muts phenotype.Suggestion separates His+Mut+ and His+Muts yeast transformant simultaneously, is more conducive to protein expression because be difficult to the predict what structure.

3, the preparation of electric transformed competence colibacillus

In containing the 50ml centrifuge tube of 5ml YPD, the culturing yeast cell, 30 spend night; Get 30～50ul overnight culture, inoculation contains the 250ml shaking flask of 50ml fresh culture, and overnight growth is to OD600=1.3～1.5; At 4 degree, the centrifugal 5min collecting cell of 1500g is with the aqua sterilisa suspension cell of 50ml precooling; As above centrifugal, with the aqua sterilisa suspension cell of 25ml precooling; As above centrifugal, with the 1M sorbyl alcohol suspension cell of 2ml precooling; As above centrifugal, with the 1M sorbyl alcohol suspension cell of 100ul precooling.

4, electricity transforms

With 5～10ug linearizing DNA and 80uL electricity transformant mixing, transfer to the 0.1cm electricity and transform cup, ice bath 5min.Electricity Transformation Parameters: 0.75kV, 25uF, 200 Ω.The 1M sorbyl alcohol that adds immediately the 1mL precooling after the electric shock.Get on the 150ul coating MD flat board and cultivate.Hatch dull and stereotyped to clone's generation at 30 degree.

5, screening Geneticin resistance transformant

Draw 1～2ml aqua sterilisa on all HIS+ transformant flat boards; With the resuspended HIS+ transformant of sterilization sleaker, do not scratch agar; Cell suspension concentrated be transferred in the 50ml centrifuge tube of sterilization, slightly vortex (5～10S); With spectrophotometric determination concentration (1OD600=5 * 107 cells/ml); Be coated with 105 cells at every YPD flat board that contains Geneticin.(the Geneticin concentration gradient is 0,0.25,0.5,0.75,1.0,1.5,1.75,2.0,3.0 and 4.0mg/ml); Hatch flat board at 30 degree, check every day.Anti-Geneticin clone needed occur in 2～5 days.

Having studied the expression amount of phytase gene in yeast according to document is directly proportional with the copy number that it is incorporated on the karyomit(e), and its copy number is directly proportional with the ability of its anti-Geneticin, so need the transformant of the anti-G418 concentration 3.0 of screening and 4.0mg/ml, but the clone of anti-high Geneticin like this is more rare, needs the thousands of HIS+ transformant of screening to separate the clone of anti-2-4mg/ml Geneticin.

6, Geneticin resistance checking

Will from 0.4,1.0 and the transformant that obtains of the G418 flat board of 1.75mg/ml concentration line respectively on the flat board of corresponding resistant, find that colony growth is good after 5 days, and single bacterium colony occurs.The order bacterium colony in the YPD liquid nutrient medium, incubated overnight, preservation of bacteria strain.

7, PCR identifies the transformant gene

With primer 5 ' AOX 1 (GACTGGTTCCAATTGACAAGC), α-factor (TACT AT T G CCAGCATTGCTGC) and 3 ' AOX1 (GGCAAATGGCATTCTGACAT) amplifying target genes.PCR condition: 95 ℃ of 3min; 95 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 1min, 28 circulations; 72 ℃ of 5min.After PCR finishes, by 0.8% agarose electrophoresis detected magnitude.The result as shown in Figure 4, the external source linear fragment successfully is integrated into yeast chromosomal.

8, Mut+ and Muts phenotype checking

With sterilizing toothpick picking mono-clonal, on MM and MD flat board, put respectively transformant, guarantee first point on the MM flat board, each clone changes toothpick one time, and 30 degree were hatched 2 days.Two days later, observe dull and stereotyped, on the MD flat board growth fast, poor growth or the transformant of not growing are Muts on the MM flat board, the transformant that the speed of growth is the same is Mut+.Principle is that Mut+ can utilize methyl alcohol to be carbon source fast, and Muts then can not utilize methyl alcohol to be carbon source.So Mut+ can contain also Fast Growth of methyl alcohol (MM) flat board, and Muts can only contain the dull and stereotyped Fast Growth of glucose (MD).Experimental results show that according to PCR product size and the dull and stereotyped point of MD bacterium transformant is the Muts type.

9, abduction delivering

The picking mono-clonal is seeded in the 1L shaking flask that contains 100ml BMGY.28～30 degree in shaking table, 250～300rpm grows to OD600=2～6 (approximately 16～18 hours); The centrifugal 5min collecting cell of room temperature 1500-3000g is removed supernatant, with the BMMY re-suspended cell of 1/5～1/10 former culture volume (approximately 10～20ml); Place 100ml dividing plate shaking flask, cover bottleneck with 2 layers of sterile gauze or cheese cloth, put into shaking table and continue to cultivate; Every 24 hours, add methyl alcohol to final concentration and be 0.5% and induce continuing; At each following time point, get the 1ml substratum to the 1.5ml centrifuge tube.The Best Times that these samples are used for analyzing expression level and determine to induce rear collecting cell.The room temperature centrifugal 2～3min of horizontal centrifuge maximum speed of revolution.Time point: 0,6,12,24 (1 days), 36,48 (2 days), 60,72 (3 days), 84,96 (4 days); During secreting, expressing, supernatant is transferred in the single pipe, supernatant and cell precipitation are stored in-80 degree.

10, the mensuration of lipase activity

Measure lipase activity with substrate p-oil of mirbane butyric ester (p-nitrophenybutyrate).Enzyme activity unit is defined as: per minute discharges the required enzyme amount of 1umol p-nitrophenol (p-nitrophenol).Enzyme activity determination is by spending 25, and lipase hydrolysis p-oil of mirbane butyric ester is that the amount of p-nitrophenol is determined.The lipN gene is expressed in yeast cell, and lipase activity reaches the highest at 72h, is 210U/ml.

Embodiment 4: the characteristic research of lipase

1, the pH value is on the impact of lipase activity

Lipase is added in the buffer solution system of different pH values and measure enzyme activity, pH arranges from 3.0 to 10.0, gets a mensuration every 0.5.The enzyme assay reaction is carried out at 25 ℃, and the reaction times is 15min.Result such as Fig. 5 show, the optimum pH of lipase is 6.5～7.5, and in the pH8.5 scope, lipase activity can remain on more than 60% at pH5.5.

2, temperature is on the impact of lipase activity

The optimal reactive temperature of lipase is determined under the optimum pH and carries out, and the reaction times is 15min, and the temperature setting is got a mensuration from 15 ℃ to 75 ℃ every 5 ℃.Result such as Fig. 6 show, the optimum temperuture of lipase is 25～35 ℃, and the enzyme work in the time of 20～45 ℃ can keep more than 60%.

The application of embodiment 5 lipase in sewage disposal

Add lipase to carry out oil-containing waste water in certain grease company waste water treatment plant SBR technique (sequencing batch reactor) aeration tank processing by 0.1% addition.Influent quality is: grease 1200mgL-1, chemical oxygen demand (COD) (COD) 3000mgL-1.The control group oil and grease degradation rate of not using lipase is 25%, and chemical oxygen demand (COD) (COD) clearance is 70%; Oil and grease degradation rate is 95% behind the use lipase, and chemical oxygen demand (COD) (COD) clearance is 96%.Compare with control group, lipase can obviously improve the efficient that oil-containing waste water is processed.

SEQUENCE LISTING

＜110〉Zhejiang Prov Sunda Environmental Protection Co., Ltd

＜120〉for the treatment of lipase, encoding gene and the application thereof of grease contamination

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Ala Val Thr Ser Asp Glu Ser His Leu Ser Leu Gln Arg Arg Ala Ala

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Leu Ala Cys Phe Pro Asn Ile Arg Ser Asp Pro Val Asn Ala Leu Ser

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Thr Gly His Ala Leu His Ser Glu Val Glu Ala Thr Ala Pro Glu Met

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Claims (7)

1. lipase for the treatment of grease contamination, its aminoacid sequence is shown in SEQ ID NO:1.

2. the gene of coding claim 1 described lipase.

3. encoding gene as claimed in claim 2 is characterized in that described gene nucleotide series is shown in SEQ ID NO:2.

4. the recombinant vectors that contains the described gene of claim 2.

5. the application of the described gene of claim 2 in the preparation recombinant lipase.

6. the application of lipase claimed in claim 1 in the hydrolysis treatment oil-containing waste water.

7. application as claimed in claim 6 is characterized in that described hydrolysis time carries out at 25～35 ℃, pH6.5～7.5.

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