CN109797141A - A kind of mould lipase of camphor tree suede branch and its encoding gene and application - Google Patents

Abstract

The invention discloses a kind of mould lipase of camphor tree suede branch and its encoding gene and applications.Protein provided by the invention, is named as McLipB, is following protein 1) or 2) or 3): 1) protein that amino acid sequence shown in sequence 1 forms in sequence table；2) in sequence table the amino acid sequence shown in N-terminal the 22nd to 565 of sequence 1 composition protein；1) or 2) 3) protein with lipase active for obtaining the protein described in by the substitution and/or deletion and/or addition of one or several amino acid residues.Present invention discover that a kind of lipase and its encoding gene mould from camphor tree suede branch, are conducted into Pichia pastoris, recombinant yeast pichia pastoris is obtained, recombinant yeast pichia pastoris should be after the fermentation of 5L fermentor middle-high density, and the fat enzyme activity of fermented supernatant fluid reaches 4304U/mL.

Description

A kind of mould lipase of camphor tree suede branch and its encoding gene and application

Technical field

The present invention relates to field of biotechnology, the mould lipase of specifically a kind of camphor tree suede branch and its encoding gene and application.

Background technique

It is raw can be catalyzed triacylglycerol ester hydrolysis as a kind of important ester linkage hydrolyzing enzyme for lipase (EC 3.1.1.3) At glycerol and free fatty acid.In addition, lipase can also a variety of reactions such as catalytic esterification, transesterification, alcoholysis, acidolysis and ammonolysis, And there is substrate specificity, location specific and stereospecificity.Therefore, lipase is widely used in food, medicine, washing The fields such as agent and cosmetics (Sarmah et al.Recent advances on sources and industrial applications of lipases.Biotechnology Progress.2018,34:5-28；Kumar et al.Synthesis of macromolecular systems via lipase catalyzed biocatalytic reactions:Applications and future perspectives.Chemical Society Reviews.2016, 45:6855-6887.)。

With the increase to lipase demand, the producing enzyme level for improving lipase is had become a hot topic of research.It is existing at present Multiple-microorganism lipase gene realizes clonal expression, such as from head mold, Mucor, root Mucor, aspergillus, mould, geotrichum candidum, Lipase (the Singh et al.Overview of fungal of Candida, bacillus, pseudomonad, Burkholderia etc. lipase:A review.Applied Biochemistry and Biotechnology.2012,166:486-520； Treichel et al.A review on microbial lipases production.Food and Bioprocess Technology.2010,3:182-196).Compared to mesophile lipase, the lipase report in thermophilic fungal source is less, as taken Xi Xinsatuo bacterium (Neosartorya fischeri) (Sun et al.Heterologous production of an acidic thermostable lipase with broad-range pH activity from thermophilic fungus Neosartorya fischeri P1.Journal of Bioscience and Bioengineering.2016, 122:539-544), thermophilic basket bacterium (Talaromyces thermophilus) (Zhang et al.Heterologous expression of an alkali and thermotolerant lipase from Talaromyces thermophilus in Trichoderma reesei.Applied Biochemistry and Biotechnology.2015,176:1722-1735), mould (Malbranchea cinnamomea) (the Tong et of camphor tree suede branch al.Characterization of a new sn-1,3-regioselective triacylglycerol lipase from Malbranchea cinnamomea.Biotechnology and Applied Biochemistry.2016,63: 471-478), thermophilic ankle section bacterium (Talaromyces thermophilus) (number of patent application: 201710071285.1) and thermophilic Hot sac fungus (Thermoascus aurantiacus) (number of patent application: 201210556924.0) etc..Industrial application is most Thermomyces lanuginosus lipase derives from rhizomucor miehei (Rhizomucor miehei) and the thermophilic hyphomycete of thin cotton like (Thermomyces lanuginosus).The enzyme in thermophilic fungal source usually has good thermal stability and preferably industry Applicability.Therefore, Thermomyces lanuginosus lipase is excavated to be of great significance for industrial production and application.

The lipase of Candida rugosa lipase like superfamily has important industrial application value, such as applies In enhancing cheese flavor, oil prodution industry, synthesizing chiral compound and degradation phthalic acid ester etc. (Houde et al.Lipases and their industrial applications:An overview.Applied Biochemistry and Biotechnology.2004,118:155-170；Fernández-Lorente et al.Specificity enhancement towards hydrophobic substrates by immobilization of lipases by interfacial activation on hydrophobic supports.Enzyme and Microbial Technology.2007,41:565-569).The lipase of the family is mainly derived from candida rugosa, geotrichum candidum (Geotrichumcandidum) and (the Gupta et al.Molecular such as geotrichum fermentans (Geotrichumfermenfans) and functional diversity of yeast and fungal lipases:Their role in biotechnology and cellular physiology.Progress in Lipid Research.2015,57:40- 54) it, there are no the report from thermophilic fungal.

Summary of the invention

The purpose of the present invention is to provide a kind of lipase, derive from the mould S168 of camphor tree suede branch.Bacterial strain was on April 19th, 2012 It is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Chaoyang District, Beijing City The institute 3 of North Star West Road 1), deposit number is CGMCC NO.6022, and classification naming is the mould (Malbranchea of camphor tree suede branch cinnamomea)。

Protein provided by the invention, is named as McLipB, is following 1) -4) in it is any:

1) protein that amino acid sequence shown in sequence 1 forms in sequence table；

2) in sequence table the amino acid sequence shown in N-terminal the 22nd to 565 of sequence 1 composition protein；

3) in N-terminal or/and the obtained fused protein of C-terminal connection label 1) or 2)；

4) by 1) -3) in it is any shown in protein by one or several amino acid residues substitution and/or missing and/or Add the obtained protein with lipase active.

Wherein, sequence 1 is made of 565 amino acid residues in sequence table.

1) or 2) or 3) in order to make the protein in convenient for purifying, can in sequence table amino acid sequence shown in sequence 1 Amino terminal or carboxyl terminal connect the sequence of upper label as shown in Table 1.

The sequence of 1 label of table

It is above-mentioned 4) in protein, the substitutions of one or several amino acid residues and/or deletion and/or addition is not More than the substitution and/or deletion and/or addition of 10 amino acid residues.

It is above-mentioned 4) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.

It is above-mentioned 4) in the encoding gene of protein can be by the way that one will be lacked in DNA sequence dna shown in sequence 2 in sequence table The codon of a or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.

The nucleic acid molecules of code for said proteins also belong to protection scope of the present invention.

The nucleic acid molecules of code for said proteins can be DNA molecular shown in any in following (a1)-(a4):

(a1) code area includes the DNA molecular of sequence 2 in sequence table；

(a2) in sequence table sequence 2 from the 64-1698 nucleotide in 5 ' ends form DNA molecular；

(a3) nucleotide sequence limited with (a1) or (a2) has 75% or 75% or more identity, and encodes with rouge The DNA molecular of the protein of fat enzymatic activity；

(a4) gene limited under strict conditions with (a1) or (a2) is with 90% or more homology and coding has rouge The DNA molecular of the protein of fat enzymatic activity；

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.

Wherein, sequence 2 is made of 1698 nucleotide in sequence table, the nucleotide coding sequence table of sequence 2 in sequence table Amino acid sequence shown in middle sequence 1.

Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA It hands over and hybridizes at 65 DEG C in experiment and wash film.

Expression cassette, recombinant vector, recombinant microorganism or the transgenic cell of nucleic acid molecules containing code for said proteins System also belongs to protection scope of the present invention.

The recombinant vector can be the DNA shown in the sequence 2 of the multiple cloning sites insetion sequence table of carrier pPIC9K points The recombinant plasmid pPIC9K-McLipB that son obtains.

The recombinant vector concretely uses the SnaBI of the replacement of DNA molecular shown in sequence 2 carrier pPIC9K in sequence table (carrier pPIC9K is cut into a sheet by restriction endonuclease SnaBI and AvrII to segment between AvrII identification sequence Section and a small fragment, the DNA are the small fragment), obtain recombinant plasmid pPIC9K-McLipB.

The recombinant bacterium can be by obtaining recombinant vector importing host microorganism.

The host microorganism can be yeast, bacterium, algae or fungi.The yeast is pichia pastoris yeast (Pichia pastoris)GS115。

Above-mentioned protein as in lipase application also in the scope of protection of the invention.

Or above-mentioned nucleic acid molecules, or, containing the expression cassette of above-mentioned nucleic acid molecules, recombinant vector, recombinant microorganism or turning base Because of cell line, the application in lipase is being prepared also in the scope of protection of the invention.

It is another object of the present invention to provide a kind of methods for preparing lipase.

The above method includes the following steps: the above-mentioned recombinant microorganism that ferments, and obtains lipase.

The above method, the fermented and cultured includes the following steps: to cultivate in recombinant microorganism fermentation medium, to glycerol Consumption completely, starts to carry out glycerol feeding, the flow acceleration of glycerol is controlled, to ensure the dissolved oxygen amount in fermentation system in 20%- Between 70%.Stop glycerol feeding when thallus weight in wet base reaches 180-220g/L；Nature enemy 0.5h again starts stream plus volume hundred Divide 100% methanol of content to carry out Fiber differentiation, obtains lipase.

Specific fermented and cultured successively passes through following steps:

1) seed liquor culture: above-mentioned recombinant bacterium being inoculated into 150mL YPD culture medium, 30 DEG C, 200rpm oscillation training It supports to OD₆₀₀Reach 2-6, obtains seed liquor；

2) seed liquor of step 1) basis culture: is inoculated in the hair of the 5L containing 1.5L basal medium with 10% inoculum concentration It is cultivated in fermentation tank, the cultivation temperature is 30 DEG C, pH 4.0, revolving speed 600rpm, completely to glycerol consumption, into glycerol stream Add the stage；

3) the glycerol feeding stage: flowing the glycerol concentration added is 50% (w/v), and temperature is 30 DEG C, pH 5.0, by adjusting The flow acceleration control dissolved oxygen of glycerol flows 4-6h between the added-time in 20%-70%；Reach 180-220g/L to thallus weight in wet base, stops Flow glycerol adding；

4) the methanol feeding stage: after stopping stream glycerol adding, hungry 0.5h starts 100% methanol induction, cultivation temperature 30 DEG C, pH 6.0, revolving speed 800rpm, dissolved oxygen 20%-70%.

Application of the above-mentioned protein in hydrolysis p-nitrophenyl ester or triglyceride also belongs to protection scope of the present invention；

Or above-mentioned protein prepares the application in milk-taste essence in hydrolysis butter and also belongs to protection scope of the present invention；

Or application of the above-mentioned protein in degradation phthalate also belongs to protection scope of the present invention.

The present invention also provides a kind of method for preparing milk-taste essence, include the following steps: to be hydrolyzed with above-mentioned protein yellow Oil obtains milk-taste essence；

Or the present invention also provides a kind of method of hydrolysis p-nitrophenyl ester or triglyceride, include the following steps: to use Above-mentioned protein hydrolysis p-nitrophenyl ester or triglyceride；

Or, including the following steps: the present invention also provides a kind of method of phthalate of degrading with above-mentioned albumen Matter hydrolyzes phthalate.

Among the above, the p-nitrophenyl ester be p-nitrophenyl butyrate, p-nitrophenyl capronate, p-nitrophenyl caprylate, P-nitrophenyl decylate, p-nitrophenyl laurate, p-nitrophenyl myristinate and/or p-nitrophenyl palmitate；

Or, the triglyceride is glyceryl triacetate, tributyrin, tricaproin, three sad glycerol Ester, decanoin, trilauryl acid glyceride, myristin, tripalmitin and/or olive oil；

Or the phthalic acid ester be dipropyl phthalate, dibutyl phthalate, phthalic acid two oneself Bis- (2- ethylhexyl) esters of ester, repefral, diethyl phthalate, phthalic acid and/or O-phthalic Sour diisobutyl ester.

Among the above, the reaction pH range of the hydrolysis is 3.0-9.0；

And/or the range of reaction temperature of the hydrolysis is 30-40 DEG C.

Present invention discover that a kind of lipase and its encoding gene mould from camphor tree suede branch, are conducted into Pichia pastoris, Recombinant yeast pichia pastoris is obtained, recombinant yeast pichia pastoris should be after the fermentation of 5L fermentor middle-high density, the fat enzyme enzyme enzyme of fermented supernatant fluid Vigor reaches 4304U/mL.Purifying obtains the pure enzyme of recombinant lipase, enzymatic activity recovery 78.2%, purification 1.2, pure enzyme Specific enzyme activity power be 544.6U/mg.The optimal reaction pH and temperature of the enzyme are respectively 7.5 and 40 DEG C, in pH 3.0-9.0 and 45 It keeps stablizing at DEG C.Recombinant lipase centering, short carbon chain triglycerides hydrolysing activity with higher, and it is special without position Property.Lipase hydrolysis butter of the invention mainly generates butyric acid, caproic acid and a small amount of octanoic acid and capric acid, is preparing milk-taste essence Aspect has application value.Can degrade a variety of phthalic acid esters of the enzyme generate corresponding phthalic monoester, and to neighbour The degradation rate of phthalic acid dipropyl, dibutyl phthalate and dihexyl phthalate is up to 90% or more.

Detailed description of the invention

Fig. 1 be recombinant yeast pichia pastoris 5L fermentation cylinder for fermentation producing enzyme course figure (▲: enzyme activity；■: thallus weight in wet base).

Fig. 2 is SDS-PAGE (M: low molecular weight standard of the recombinant yeast pichia pastoris in 5L fermentor middle-high density fermentation process Albumen；1-8: methanol induction 0h, 12h, 36h, 60h, 84h, 108h, 132h and 156h).

Fig. 3 is the purifying figure (M: low molecular weight standard protein of lipase McLipB；1: crude enzyme liquid；2: pure enzyme solution).

Fig. 4 be lipase McLipB optimal pH (▲: citrate buffer, pH 3.0-6.0；◆: phosphate-buffered Liquid, pH 6.0-8.0；■: Tris-HCl, pH 7.0-9.0；*: HEPES, pH 7.0-8.0；●: CHES, pH 8.0-10.0).

Fig. 5 be lipase McLipB pH stability (▲: citrate buffer, pH 3.0-6.0；◆: phosphate is slow Fliud flushing, pH 6.0-8.0；■: Tris-HCl, pH 7.0-9.0；*: HEPES, pH 7.0-8.0；●: CHES, pH 8.0- 10.0；△: CAPS, pH 10.0-11.0).

Fig. 6 is the optimum temperature of lipase McLipB.

Fig. 7 is the temperature stability of lipase McLipB.

Fig. 8 is the TLC analysis (S that lipase McLipB hydrolyzes olein₁-S₄: glyceryl monooleate, two oleic acid are sweet Grease, oleic acid and olein mark product；S: hydrolysate).

Fig. 9 is that the TLC of lipase McLipB degradation phthalic acid ester analyzes (S₁: O-phthalic acidity scale product；S₂: it is corresponding Phthalic monoester mark product；1: blank control；2: enzyme digestion reaction is for 24 hours).

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Following embodiment is only limited to illustrate technical solution of the present invention.

The mould S168 of camphor tree suede branch in following example.Bacterial strain is stored in Chinese microorganism strain guarantor on April 19th, 2012 It hides administration committee's common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), preservation Number be CGMCC NO.6022, classification naming be camphor tree suede branch it is mould (Malbranchea cinnamomea).

The cloning and expression of embodiment 1, the mould lipase of camphor tree suede branch

One, the amplification of gene M cLipB

RNA, the reverse transcription cDNA for extracting the mould S168 of camphor tree suede branch, using cDNA as template, with following primer amplification:

Upstream primer McLipSnaBIF:5 '-CCATGTACGTA(underscore indicates SnaBI to GCCCCGGAGAAACGC-3 ' Restriction enzyme site) and downstream primer McLipAvrIIR:5 '-CCGCCTAGG(lower stroke of TCAGTAATAGAACTTTTTGATGTTC-3 ' Line indicates AvrII restriction enzyme site).

PCR amplification condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 90s, circulation 35 times；Last 72 DEG C of extensions 5min.

Amplification, which obtains PCR product, has nucleotide shown in sequence 2 in sequence table, and unnamed gene shown in the nucleotide is Gene M cLipB, wherein sequence 2 64-1698 are gene M cLipB coded sequence, and 1-63 are signal DNA encoding peptide Sequence；The albumen of gene coding is named as albumen McLipB, and the amino acid sequence of the albumen is sequence 1 in sequence table, wherein Sequence 1 1-21 are signal peptide sequence, the 22nd to 565 sequence for albumen McLipB.

Two, the building of recombinant vector

Recombinant vector pPIC9K-McLipB is that gene M cLipB shown in sequence 2 is replaced Yeast expression carrier pPIC9K DNA molecular between SnaBI the and AvrII double enzyme site of (U.S. Invitrogen, article No. V17520), carrier expression recombination Lipase McLipB.

By recombinant vector pPIC9K-McLipB with electrotransformation pichia pastoris yeast after restriction enzyme SacI linearisation GS115 (U.S. Invitrogen, article No. C18100) constitutes recombinant bacterium GS115/pPIC9K-McLipB.

Empty carrier pPIC9K is imported in pichia pastoris yeast GS115, control recombinant bacterium GS115/pPIC9K is obtained.

Three, the lipase of recombinant bacterium expression

By above-mentioned two obtain recombinant bacterium GS115/pPIC9K-McLipB coating MD plate (2% glucose, 1.34%YNB, 4 ×10^-5% biotin), the His obtained from MD plate⁺100 μ L are taken to be coated on containing difference after the sterilized water scraping of transformant (1% yeast extract, 2% tryptone, 2% glucose, G418 concentration are respectively 1,2 and 4mg/ to the YPD plate of G418 concentration mL).After cultivating 3-5d at 30 DEG C, picking transformant is in BMGY culture medium (1% yeast extract, 2% tryptone, 100mMpH 6.0 phosphate buffer, 1.34%YNB, 4 × 10^-5% biotin, 1% glycerol) in cultivate 16-18h, 3000rpm centrifugation 5min collects thallus, with BMMY culture medium (1% yeast extract, 2% tryptone, the phosphate-buffered of 100mMpH 6.0 Liquid, 1.34%YNB, 4 × 10^-5% biotin, 0.5% methanol) thallus is resuspended to OD₆₀₀It is 1.0 or so, induces target protein table It reaches.Every adding 100% methanol for 24 hours to final concentration of 0.5% successive induction.After methanol induction 3d, take culture solution and in 10000rpm is centrifuged 10min, collects supernatant and detects enzyme activity.

The above condition of culture be 100mL triangular flask loading amount 20mL culture medium, 30 DEG C of temperature, revolving speed 200rpm.

Enzyme activity determination method: the suitably diluted enzyme solution (supernatant) of 50 μ L is added to 400 μ L 50mM pH7.5's In Tris-HCl buffer, 2min is preheated in 40 DEG C of water-baths, and the aqueous isopropanol of 50 μ L 10mM p-nitrophenyl laurates is added After react 10min.500 μ L terminators are added after completion of the reaction, 10000rpm is centrifuged 3min, and light absorption value is measured at 410nm.

Enzyme activity unit (U) is defined as in the above conditions, and reaction generates required for 1 μm of ol p-nitrophenol per minute Enzyme amount.

The preparation of terminate liquid: being the CuCl of 28g/L (w/v) to 20mL concentration₂·H₂It is slowly added to while stirring in O The Na of 40mL68.5g/L (w/v)₃PO₄·12H₂O is centrifuged 5min under the conditions of 4000rpm.Supernatant is discarded, 19.1g/L is used (w/v) Na₂B₄O₇·H₂O (solution X) rinses precipitating twice.Finally by precipitating be suspended in 100mL containing 5%SDS (w/v) and The solution X of 0.6% (w/v) NaCl.

Determining the protein quantity uses Lowry method, using bovine serum albumin as standard protein (Lowryet al.Protein measurement with the Folin phenol reagent.Journal of Biological Chemistry.1951,193:265-275)。

As a result contain lipase in the supernatant of culture solution, and the enzyme activity of lipase is 42.8U/mL.

To compare recombinant bacterium GS115/pPIC9K as control, lipase is not as a result detected.

Therefore, albumen McLipB is lipase.

Four, recombinant yeast pichia pastoris high density fermentation prepares lipase

The recombinant bacterium GS115/pPIC9K-McLipB that above-mentioned two obtain is subjected to high density fermentation in 5L fermentor.Hair Fermenting process and culture medium (fermentation basal medium BSM, PTM₁Trace salt) preparation referring to Pichia pastoris fermentation manual (Version B, 053002, Invitrogen).Entire fermentation process is by following several stages.

1) seed culture: recombinant bacterium GS115/pPIC9K-McLipB is inoculated in 150mL YPD culture medium, and 30 DEG C, 200rpm is cultivated to OD₆₀₀For 2-6 or so, as seed liquor.

2) seed liquor basis culture: is seeded to the 5L equipped with 1.5L fermentation basal medium BSM with 10% inoculum concentration Fermentor adjusts pH to 4.0 with 28% concentrated ammonia liquor, PTM is added₁4.35mL/L is to starting fermentation liquid, revolving speed 600rpm, temperature 30℃.When to the rebound of glycerol depletion dissolved oxygen (dissolved oxygen rising, this value is exactly a feature of glycerol depletion when rising), start sweet Oil stream adds.

3) glycerol feeding culture: the glycerol of stream plus 50% (w/v, g/mL) controls 30 DEG C of temperature, and pH 5.0 is monitored always Dissolved oxygen keeps dissolved oxygen in 20%-70% by adjusting flow acceleration.4-6h between the stream added-time, reaches 180-220g/ to thallus weight in wet base Stop stream after L to add.

4) 100% methanol induction culture: after stopping stream glycerol adding, hungry half an hour, stream adds 100% (volumn concentration) Methanol induction, revolving speed 800rpm, monitoring dissolved oxygen control 30 DEG C of temperature, pH 6.0 in 20%-70%.

Fermentation liquid is periodically taken in Induction Process, and precipitating and supernatant is collected by centrifugation.

Detect the thallus weight in wet base of precipitating；

Detect the enzyme activity of supernatant.

It as a result is 4304U/mL supernatant as shown in Figure 1, reaching highest through methanol induction 156h post-fermentation supernatant enzyme activity Liquid.

Electrophoresis detection supernatant, as a result as shown in fig. 2, it can be seen that the molecular weight for obtaining destination protein in supernatant is big Small is 59.1kDa, consistent with predicted molecular weight 59.9kDa.

Embodiment 2, the purifying of recombinant lipase (McLipB) and zymologic property

One, the purifying of recombinant lipase McLipB

By the citric acid-of fermented supernatant fluid (crude enzyme liquid) the 20mM pH 6.0 of methanol induction 156h in the four of embodiment 1 After sodium citrate buffer solution (buffer solution A, Citric Acid Mono and citrate dihydrate trisodium) dialysis 16h, through SP Sepharose Fast Flow (GE Healthcare, 17-0729-10, be purchased from Sigma company) strong cation exchange chromatography (1 × 10cm) into Row isolates and purifies, and loading flow velocity is 0.5mL/min.Eluent OD is eluted to buffer solution A after loading₂₈₀< 0.05 (flow velocity is 1.0mL/min), then enzyme solution is collected, is purified with 10 column volumes of buffer solution A linear elution of the NaCl containing 0-300mM Recombinant lipase McLipB (pure enzyme solution) afterwards detects purity with SDS-PAGE.

As a result as shown in figure 3, recombinant lipase McLipB is the pure enzyme of electrophoresis grade.

The specific enzyme activity power for detecting recombinant lipase McLipB, is increased to 544.6U/mg, purification by 456.9U/mg It is 1.2, the rate of recovery is 78.2% (table 2)

The purifying table of 2 lipase of table (McLipB)

A: purification refers to the ratio of the specific enzyme activity of pure enzyme and the specific enzyme activity of thick enzyme.

B: the rate of recovery refers to that the total enzyme activity power of pure enzyme accounts for the percentage composition of crude enzyme liquid total enzyme activity power.

Two, the zymologic property of recombinant lipase McLipB

1, optimal reaction pH and pH stability

Measurement lipase activity power is used to determine optimal reaction pH under 40 DEG C, 50mM difference buffer conditions Buffer is citric acid-sodium citrate buffer solution (pH 3.0-6.0, Citric Acid Mono and citrate dihydrate trisodium), phosphoric acid hydrogen two Sodium-phosphate sodium dihydrogen buffer solution (pH 6.0-8.0, disodium hydrogen phosphate and sodium dihydrogen phosphate dihydrate), Tris-HCl (pH 7.0-9.0), HEPES-NaOH (pH 7.0-8.0) and CHES-NaOH (pH 8.0-10.0).

Citric acid-trisodium citrate pH 3.0-6.0: it is molten that appropriate 50mM sodium citrate is added into 50mM citric acid solution Liquid adjusts pH respectively to 3.0,3.5,4.0,4.5,5.0,5.5 and 6.0；

Sodium dihydrogen phosphate-disodium hydrogen phosphate pH 6.0-8.0: appropriate 50mM phosphorus is added into 50mM sodium dihydrogen phosphate Sour disodium hydrogen solution adjusts pH respectively to 6.0,6.5,7.0,7.5 and 8.0；

Tris-HCl pH 7.0-9.0: be added into 50mM Tris solution appropriate 6M HCl adjust pH respectively to 7.0, 7.5,8.0,8.5 and 9.0；

HEPES-NaOH (pH 7.0-8.0) formula is that N-2- hydroxyethyl piperazine-N'-2- ethanesulfonic acid mixes adjusting with NaOH To appropriate pH.

CHES-NaOH (pH 8.0-10.0) formula 2- (ring amine) -1- ethanesulfonic acid is mixed with NaOH is adjusted to appropriate pH.

The recombinant lipase McLipB for diluting an above-mentioned preparation respectively with above-mentioned different pH value buffer, by what is diluted Enzyme solution is placed in 35 DEG C of water-baths keep the temperature 30min respectively after, be placed in ice water cooling 30min, then measured according to the method for front Enzyme activity.Using untreated enzyme solution as control, the percentage that residual enzyme activity accounts for untreated control enzyme activity is calculated.

As a result as shown in figure 4, showing that the optimal reaction pH of recombinant lipase McLipB is 7.5 (Fig. 4).

30min is handled in pH 3.0-9.0, residual enzyme activity is in 80% or more (Fig. 5).

2) optimal reactive temperature and temperature stability

The enzyme solution Tris-HCl buffer of 50mM pH 7.5 is diluted into suitable multiple, respectively the front at 25-55 DEG C Method measures enzyme activity.The measurement of temperature stability is that enzyme solution is handled to 30min at different temperature respectively, then in ice water Cooling 30min, measures residual enzyme activity in bath.

As a result as shown in fig. 6, the optimal reactive temperature of recombinant lipase McLipB is 40 DEG C.

After 45 DEG C or less heat preservation 30min, residual enzyme activity is higher than 80% (Fig. 7).

3) substrate specificity

Recombinant lipase McLipB is measured to the substrate specificity of p-nitrophenyl ester according to the method for front；With triglycerides When measuring its substrate specificity for substrate, concentration of substrate 10mM, reaction condition is the 50mM pH 7.5Tris-HCl at 40 DEG C 10min is reacted in buffer, finally with the free fatty acid generated in 10mM NaOH titration hydrolytic process.Enzyme activity unit is fixed Justice is reaction generates enzyme amount required for 1 μm of ol fatty acid per minute under the above conditions.

Recombinant lipase McLipB p-nitrophenyl capronate and p-nitrophenyl lauric acid ester hydrolysis vigor highest, specific enzyme activity Power is respectively 535.7U/mg and 531.9U/mg (table 3).

In triglycerides series substrate, the active highest of the enzyme hydrolysis tributyrin, specific enzyme activity 809.8U/ Mg hydrolyzes vigor decline (table 3) with the increase of carbon atom number in the fatty acid chain of triglycerides.

In addition, the enzyme also has hydrolysing activity (table 3) to olive oil.

The substrate specificity of 3 lipase of table (McLipB)

4) location specific

The location specific of recombinant lipase McLipB is investigated by hydrolysis olein.Contain in 1mL reaction system 100mM olein, 100U recombinant lipase McLipB and 50mM pH 7.5Tris-HCl buffer.It is placed in 35 DEG C, 12h is reacted in 180rpm shaking bath.TLC analysis is carried out after the isometric n-hexane extraction of reaction product.

TLC analysis condition: hydrolyzation sample point sample is soaked 180 after drying completely in opening up layer three times on silica gel plate with developing solution DEG C baking colour developing.Developing agent is petroleum ether/ethyl ether/acetic acid=80/20/0.5 (v/v/v), and the methanolic that color developing agent is 5% is molten Liquid.

Experimental results are shown in figure 8, has 1,2- in the product that recombinant lipase McLipB hydrolysis olein generates Glyceryl dioleate and 1,3- glyceryl dioleate illustrate that the enzyme can both act on Sn-1, can also act on Sn-2, belong to In no location specific lipase.

Embodiment 3, recombinant lipase McLipB prepare the application in milk-taste essence in hydrolysis butter

7g butter, disodium hydrogen phosphate-sodium dihydrogen phosphate of 1.75mL 0.2M pH 7.5 are added in 50mL triangular flask Buffer and recombinant lipase McLipB 50U/g butter, in 40 DEG C, 180rpm reacts 6h.After reaction, it is kept the temperature in 85 DEG C 15min is to terminate reaction.With the acid value of 0.1M KOH titration determination hydrolysate.Using solid phase microextraction-gas chromatography-mass spectrum Method (SPME-GC-MS) measures the volatile fatty acid in hydrolysate.

SPME-GC-MS condition: in 20mL extraction flask be added 5g sample, in 55 DEG C of water-baths balance 30min after, with 50 μm/ GC injection port is inserted directly into after 30 μm of DVB/CAR/PDMS absorption 30min to be detected.Chromatographic column be DB-WAX (30m × 0.25mm × 0.25 μm), carrier gas (He) flow velocity 1.0mL/min, Splitless injecting samples；250 DEG C of injector temperature, ion source temperature 230℃.Temperature program: initial temperature is 40 DEG C, keeps 3min, 170 DEG C is warming up to 5 DEG C/min, then with 10 DEG C/min liter Temperature keeps 3min to 200 DEG C, then is warming up to 230 DEG C with 5 DEG C/min, keeps 4min.

The results are shown in Table 4, recombinant lipase McLipB hydrolysis butter mainly generates butyric acid and caproic acid, and a small amount of octanoic acid and Capric acid shows that it can hydrolyze butter and prepare milk-taste essence.

4 lipase McLipB of table hydrolyzes the volatile fatty acid in butter product

The application of embodiment 4, recombinant lipase McLipB in degradation phthalic acid ester

Reaction condition: the Tris-HCl buffer of pH containing 50mM 7.5 in 1mL reaction system, 10 μm of ol phthalic acids Ester and 220U recombinant lipase McLipB are added 1M HCl and terminate and react and use same volume after 35 DEG C, 180rpm reaction for 24 hours Long-pending ethyl acetate extracts, and carries out TLC analysis.Extracting solution is placed in be dried at room temperature, uses methanol after ethyl acetate volatilization completely Dissolution, and carry out HPLC analysis.With not enzyme for blank control.

TLC analysis: developing agent is petrol ether/ethyl acetate/acetic acid (10/1/0.5, v/v/v), under 254nm ultraviolet lamp Observation.

HPLC condition: chromatographic column isC-18(4.6×250mm)；Column temperature is 35 DEG C；Mobile phase is methanol- Water-formic acid (95/5/0.1, v/v/v), flow velocity 0.8mL/min；Using variable-wavelenght detector (VWD), wavelength 240nm； Applied sample amount is 20 μ L.

It the results are shown in Table 5, recombinant lipase McLipB being capable of efficient degradation dipropyl phthalate (DPrP), O-phthalic Dibutyl phthalate (DBP) and dihexyl phthalate (DHP) generate corresponding phthalic monoester, and to the neighbour of more short carbon chain Rutgers (DMP) and diethyl phthalate (DEP), and bis- (the 2- ethyls of the phthalic acid of more Long carbon chain Hexyl) ester (DEHP) hydrolysis ability it is weaker (Fig. 9).Compared with DBP, water of the enzyme to diisobutyl phthalate (DiBP) Solution ability is decreased obviously, and illustrates that the enzyme has greater activity to straight chain substrate, and weaker to the degradation of substrates ability containing branch.

Quantitative analysis is carried out using HPLC, when reacting for 24 hours, drop of the recombinant lipase McLipB to DPrP, DBP and DHP Solution rate difference 96.0%, 94.7% and 98.9%, and to the degradation rate of DMP, DEP and DEHP lower (table 5).It, should compared with DBP Enzyme is only capable of the DiBP of degradation 50%.

The unique hydrolysis characteristic of recombinant lipase McLipB makes it have important application value in terms of environmental protection.

Degradation rate of the 5 lipase McLipB of table to phthalic acid ester

Sequence table

<110>China Agricultural University

<120>a kind of mould lipase of camphor tree suede branch and its encoding gene and application

<160>2

<170> PatentIn version 3.5

<210>1

<211>565

<212>PRT

<213>camphor tree suede branch is mould (Malbranchea cinnamomea)

<400>1

Met Lys Ser Trp Thr Arg Ala Ile Ala Thr Leu Val Ala Leu Ser Pro

1 5 10 15

Leu Thr Val Tyr Ala Ala Pro Glu Lys Arg Ala Ala Ser Pro Val Val

20 25 30

Thr Ile Ala His Pro Glu Ala Thr Ile Ile Gly Ser Ser Ala Leu Ser

35 40 45

Val Glu Thr Phe Asn Asp Ile Pro Phe Ala Ala Pro Pro Thr Gly Pro

50 55 60

Leu Arg Leu Lys Pro Pro Lys Pro Leu Asp Gly Ala Leu Gly Thr Val

65 70 75 80

Asp Ala Thr Thr Leu Ile Pro Lys Ser Cys Pro Gln Phe Tyr Phe Ser

85 90 95

Ile Asp Gln Gly Ala Ile Pro Gly Asp Ile Leu Gly Asp Leu Met Asn

100 105 110

His Pro Leu Leu Gln Lys Val Thr Asn Ala Gly Glu Asp Cys Leu Tyr

115 120 125

Leu Asn Val Gln Arg Pro Arg Gly Thr Lys Ala Gly Asp Lys Leu Pro

130 135 140

Val Leu Phe Trp Ile Tyr Gly Gly Gly Phe Gln Leu Gly Ser Thr Gln

145 150 155 160

Leu Tyr Asn Gly Ala Ser Leu Val Gln Glu Ser Met Arg Gln Gly Lys

165 170 175

Pro Ile Ile Phe Val Ala Val Asn Tyr Arg Val Gly Gly Phe Gly Phe

180 185 190

Leu Pro Gly Ala Glu Val Leu Ala Asp Gly Ser Ala Asn Leu Gly Leu

195 200 205

Leu Asp Gln Arg Leu Gly Leu Gln Trp Val Ala Asp Asn Ile Glu Ala

210 215 220

Phe Gly Gly Asp Pro Glu Lys Val Thr Ile Trp Gly Glu Ser Ala Gly

225 230 235 240

Ala Ile Ser Val Ala Ser His Met Thr Met Tyr Asp Gly Asp His Thr

245 250 255

Tyr Lys Gly Lys Pro Leu Phe Arg Gly Ala Ile Met Asn Ser Gly Ser

260 265 270

Gly Ile Pro Thr Asp Pro Val Asp Cys Pro Lys Ala Gln Ala Val Tyr

275 280 285

Asp Ser Val Val Lys Tyr Ala Gly Cys Asp Thr Ala Ser Asp Thr Leu

290 295 300

Glu Cys Leu Arg Gly Leu Asp Tyr Glu Glu Phe Leu Asp Ala Ala Asn

305 310 315 320

Ala Val Pro Gly Ile Leu Ser Tyr Ser Ser Val Ala Leu Ser Tyr Leu

325 330 335

Pro Arg Pro Asp Gly Thr Ala Phe Thr Glu Ser Pro Asp Leu Leu Ile

340 345 350

Glu Lys Gly Lys Tyr Ala Lys Val Pro Phe Ile Ile Gly Asp Gln Glu

355 360 365

Asp Glu Gly Thr Leu Phe Ala Leu Phe Gln Pro Asn Ile Thr Thr Arg

370 375 380

Gly Gln Ile Val Asp Tyr Leu Ser Glu Arg Phe Phe His His Ala Asp

385 390 395 400

Lys Ala Thr Ile Lys Gly Leu Val Asp Thr Tyr Gln Thr Ile Ser Ile

405 410 415

Asp Gly Ser Pro Phe Arg Thr Gly Leu Leu Asn Asn Trp Tyr Pro Gln

420 425 430

Phe Lys Arg Leu Ala Ala Ile Leu Gly Asp Leu Thr Phe Thr Ile Thr

435 440 445

Arg Arg Val Val Leu Asp Ile Ile Asn Arg Val Ser Pro Glu Val Pro

450 455 460

Ser Trp Ser Tyr Leu Ala Thr Tyr Asp Tyr Gly Thr Pro Ile Met Gly

465 470 475 480

Thr Phe His Gly Ser Asp Ile Leu Gln Val Phe His Gly Ile Trp Pro

485 490 495

Asn Tyr Ala Ala Arg Thr Ile Arg Gly Tyr Tyr Phe Asn Phe Val Tyr

500 505 510

Asn Leu Asp Pro Asn Asp Gly Lys Leu Pro His Trp Pro Arg Trp Ser

515 520 525

Glu Lys Arg Gln Leu Ala Glu Phe Gln Ala His Arg Val Arg Leu Leu

530 535 540

Ala Asp Asn Phe Arg Ser Asp Thr Tyr Asp Tyr Leu Val Lys Asn Ile

545 550 555 560

Lys Lys Phe Tyr Tyr

565

<210>2

<211>1698

<212> DNA

<213>camphor tree suede branch is mould (Malbranchea cinnamomea)

<400>2

atgaagtctt ggactcgagc catcgcgacg ttggtcgccc tgtctcctct gacggtttac 60

gccgccccgg agaaacgcgc tgcctctccc gtcgtgacga ttgcccaccc cgaggcaacg 120

atcatcggtt catcggcctt gtcggttgag accttcaatg acatcccctt tgcggcgcct 180

cccacggggc cgcttcgctt gaagccaccc aagccactcg atggcgccct tggcaccgtc 240

gacgcaacga ctctgatccc caagtcatgc ccccagttct attttagcat cgaccagggt 300

gccattccgg gagacatcct gggtgacctg atgaaccacc ccctgctcca gaaggtgacc 360

aacgccggcg aggattgcct gtacctcaac gtccagaggc ccagaggaac gaaagcaggg 420

gacaaactcc ctgtcctgtt ctggatctac ggcggtggct tccagctggg ctcgacacag 480

ctgtacaatg gggcgagtct ggtccaggag tccatgaggc agggcaaacc gatcatcttc 540

gtcgccgtca attaccgggt gggtggcttt ggattcttgc ccggcgcgga ggtcctggcg 600

gatggctccg ccaacctcgg cctgctcgac cagcgactgg gcctgcagtg ggtggccgac 660

aacatcgagg cctttggagg cgatccggaa aaggtcacca tctggggcga atccgccggt 720

gccatctcgg ttgcttccca catgaccatg tatgacggcg accacaccta caagggcaaa 780

cccttgttcc gcggcgcgat catgaactcg ggctccggca tccccaccga tcccgtcgat 840

tgtcccaagg cccaggccgt ctacgacagc gtggtcaagt atgccggctg tgacaccgcc 900

agtgacactc tcgagtgtct gcgagggctg gactatgagg aattcttgga cgccgcgaac 960

gccgtccccg gaatcctcag ctacagctcg gtcgctctct cctatctgcc gcggccggac 1020

ggcaccgcct tcaccgagtc gcccgatctc ttgatcgaga agggcaagta tgccaaggtt 1080

ccgttcatca tcggcgacca agaggacgaa ggcacgctct ttgctctctt ccaacccaat 1140

atcaccaccc ggggacagat cgtcgactac ctgtccgaac gcttcttcca ccacgccgac 1200

aaggctacca tcaaaggatt ggtcgacacc taccagacca tctccatcga cgggtctccc 1260

ttccgcactg gcctcctcaa caactggtac cctcagttca agcgccttgc cgctatcctg 1320

ggtgatctga ccttcaccat cacccgccgc gtggtgctcg acatcatcaa ccgggtcagt 1380

cccgaggtcc cgtcgtggtc ctatctcgcc acgtacgact acggcacacc catcatgggc 1440

accttccacg gcagcgacat cctccaggtc ttccacggca tctggcccaa ctatgccgcg 1500

cgcacgatcc gcggatacta cttcaacttc gtctacaatc tcgacccgaa cgacgggaag 1560

ctgcctcact ggcctcggtg gtctgagaag cgtcagctcg ctgagttcca ggcgcacagg 1620

gtgaggctgt tggccgacaa tttccgttct gacacctatg actacctggt caagaacatc 1680

aaaaagttct attactga 1698

Claims (10)

1. Be following 1) -4 1. protein) in it is any:

   1) amino acid sequence is protein shown in sequence 1 in sequence table；

   2) in sequence table the amino acid sequence shown in N-terminal the 22nd to 565 of sequence 1 composition protein；

   3) in N-terminal or/and the obtained fused protein of C-terminal connection label 1) or 2)；

   4) by 1) -3) in it is any shown in protein by one or several amino acid residues substitution and/or missing and/or add Protein adding and with the same function.
2. 2. encoding the nucleic acid molecules of protein described in claim 1.
3. 3. nucleic acid molecules as claimed in claim 2, it is characterised in that: the nucleic acid molecules are any in following (a1)-(a4) DNA molecular shown in kind:

   (a1) code area includes the DNA molecular of sequence 2 in sequence table；

   (a2) in sequence table sequence 2 from the 64-1698 nucleotide in 5 ' ends form DNA molecular；

   (a3) nucleotide sequence limited with (a1) or (a2) has 75% or 75% or more identity, and encodes claim 1 The DNA molecular of the protein；

   (a4) nucleotide sequence hybridization limited under strict conditions with (a1) or (a2), and encode albumen described in claim 1 The DNA molecular of matter.
4. 4. expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing nucleic acid molecules described in Claims 2 or 3.
5. 5. protein described in claim 1 is as the application in lipase；

   Or nucleic acid molecules described in Claims 2 or 3, or, expression cassette, recombinant vector containing nucleic acid molecules described in claim 4, Recombinant microorganism or transgenic cell line are preparing the application in lipase.
6. 6. a kind of method for preparing lipase includes the following steps: the recombinant microorganism described in claim 5 that ferments, obtains rouge Fat enzyme.
7. 7. according to the method described in claim 6, it is characterized by:

   The fermented and cultured includes the following steps: to cultivate in recombinant microorganism fermentation medium, and completely to glycerol consumption, stream adds Glycerol guarantees that the dissolved oxygen amount in fermentation system in 20%-70%, stops glycerol stream when thallus weight in wet base reaches 180-220g/L Add；Nature enemy 0.5h again, starts stream plus 100% methanol of volumn concentration carries out Fiber differentiation, obtains lipase.
8. 8. application of the protein described in claim 1 in hydrolysis p-nitrophenyl ester or triglyceride；

   Or protein described in claim 1 prepares the application in milk-taste essence in hydrolysis butter；

   Or application of the protein described in claim 1 in degradation phthalate.
9. 9. a kind of method for preparing milk-taste essence, the protein described in claim 1 hydrolyzes butter, obtains milk-taste essence；

   Or a kind of method of hydrolysis p-nitrophenyl ester or triglyceride, the protein described in claim 1 hydrolyze p-nitrophenyl Ester or triglyceride；

   Or, a kind of method for phthalate of degrading, the protein described in claim 1 hydrolyze phthalate.
10. 10. application according to claim 8 or method as claimed in claim 9, it is characterised in that:

    The reaction pH range of the hydrolysis is 3.0-9.0；

    And/or the range of reaction temperature of the hydrolysis is 30-40 DEG C.