CN103387966A - A Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium - Google Patents

A Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium Download PDF

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CN103387966A
CN103387966A CN201310296638XA CN201310296638A CN103387966A CN 103387966 A CN103387966 A CN 103387966A CN 201310296638X A CN201310296638X A CN 201310296638XA CN 201310296638 A CN201310296638 A CN 201310296638A CN 103387966 A CN103387966 A CN 103387966A
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lipase
alip
penicillium cyclopium
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astaxanthin
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李颖
杨震
黄金金
关国华
陈芝
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QINHUANGDAO HUIEN BIOTECHNOLOGY CO., LTD.
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China Agricultural University
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Abstract

The present invention provides a Pichia pastoris for highly efficient heterologous expression of white Penicillium cyclopium lipase and an enzyme production medium. According to the present invention, the white Penicillium cyclopium lipase containing a self leader peptide (7 amino acids) is heterologously expressed in a genetically engineered Pichia pastoris, increasing the activity of exogenous protein lipase, and improving a part of enzymatic properties of the exogenous protein lipase. According to the enzyme production medium of the invention, through optimizing the YNB component of a BMMY medium, the medium cost is reduced by 70%. According to the invention, reliable experimental basis is provided for the industrial application of the enzyme, especially for the catalyzed hydrolysis of substrate astaxanthin ester from Haematococcus pluvialis to produce astaxanthin monomers.

Description

Pichia spp and the culture medium of the efficient white penicillium cyclopium lipase of heterogenous expression
Technical field
The present invention relates to biological technical field, the bacterial strain that relates to particularly an a kind of lipase of plant height effect heterogenous expression, the invention still further relates to a kind of method of efficient this lipase of heterogenous expression, the gene of this lipase of encoding and the application of this enzyme, and a kind of lipase heterogenous expression substratum.
Background technology
Lipase (LipaseE.C.3.1.1.3) is the lytic enzyme that a class can hydrolyzing triglyceride produces different chain length free fatty acids and glycerine.As a kind of important industrial enzymes, the series reaction such as lipase can catalytic hydrolysis, ester is synthetic, transesterify, transesterification, so it is widely used in traditional industry fields such as food, leather, feed, washing, medicine, oil and fat chemicals.
Outstanding feature of lipase is the catalysis characteristics that it is different from most lytic enzymes, it is a class nonaqueous phase enzyme, its catalyzed reaction is to carry out on water-oil interface, do not play katalysis for water soluble substrate, have the reaction conditions gentleness, by product is few and has the characteristics such as environment friendly.It is different that the lipase of different sources acts on the ester bond position of substrate triglyceride level.
Astaxanthin has another name called ASX, astaxanthin, is a kind of keto-acid carotenoid with extremely strong antioxidation, and it extensively is present in the biologies such as shrimp, crab, fish, bird, some algae and fungi.Its antioxidant property is 1000 times of natural VE, and 10 times of Lyeopene have anti-oxidant, anti-ageing, antitumor, prevention cardiovascular and cerebrovascular diseases effect, in fields such as aquaculture, medicine, foods and cosmetics, extremely wide application prospect are arranged.China, the U.S., European Union, Canadian food surveillance office etc. approved natural astaxanthin can be used as the fodder additives of aquatic animal.
Astaxanthin is except having strong anti-oxidation, the obviously immunological competence of enhancing body.Experimentation on animals shows, astaxanthin can be prevented and treated Animal diseases, and its normal growth, healthy aquaculture, raising survival rate and breeding potential are had important effect.Astaxanthin, as the fodder additives pig of feeding, can increase the seminal fluid volume of boar, improves body weight and the survival rate of porkling.The muscle function obstacle of horse, mazoitis and the microbial stomach and intestine pipeline of the helicobacter pylorus inflammation of milk cow can be prevented and treat to esterification of astaxanthin in haematococcus pluvialis powder.Add astaxanthin in poultry feed, can improve hen laying rate, promote laying hen health, reduce 50% of chick mortality ratio, and reduce vibrios to the infection of egg etc.In addition, astaxanthin is the terminal of carotenogenesis, can be not modified after entering in animal body or biochemical conversion and directly be stored in tissue, has extremely strong pigment deposition ability, can make skin and the muscle of some hydrocoles health occur and chromatic colour, make feather, skin, pin, the Xiang Jun of birds, beasts and eggs and fowl present healthy golden yellow or red.Astaxanthin Chang Zuowei tinting material is applied in the cultivation of Oncorhynchi, salmon, rainbow trout and shrimp, when improving the aquatic animal color and luster, increases its nutritive value and local flavor, improves the value of fishery products.
Astaxanthin is divided into two kinds of synthetic product and natural astaxanthins.The astaxanthin technical maturity is produced in chemosynthesis, and has realized industrialization.But chemosynthesis astaxanthin complex process, difficult control of reaction conditions, production cost is high and security is under suspicion, and U.S. FDA has forbidden that the chemosynthesis astaxanthin enters health food market.Safe, nontoxic, biological activity advantages of higher that biosynthetic natural astaxanthin has.Biological process can be divided into from fishery products by product (shell of shrimp, crab etc.) and extracting and microorganism is natural synthesizes two kinds.There are the limitations such as raw material is unstable, source is limited, content astaxanthin is low in the fishery products extraction method, can not meet the demand of industrialization.The natural synthesis method of microorganism often adopts Haematocoocus Pluvialls or phaffia rhodozyma to synthesize.Adopt at present Haematocoocus Pluvialls scale operation natural astaxanthins both at home and abroad, Haematocoocus Pluvialls algae powder is also by tinting material and the nutrition source of the approvals such as America ﹠ Canada as aquatic animal and poultry more.Though realized industrialization production, the Determination of Astaxanthin in Haematococcus Pluvialis great majority exist with the form of astaxanthin ester, complicated components, purifying and detection difficult.
Although the GB of the U.S. and Japan has been selected the method for lipase-catalyzed hydrolysis.But on technical scale, realize the lipase-catalyzed hydrolysis astaxanthin ester of application, successfully prepare the free state astaxanthin product and have no report.Research and development to application fat hydrolase catalytic hydrolysis astaxanthin ester technology have objective demand and urgency.This laboratory screens a kind of alkaline lipase that derives from white penicillium cyclopium in earlier stage, and its catalytic hydrolysis astaxanthin ester high specificity, can be for the preparation of astaxanthin monomer.
Although in early-stage Study, prove that this enzyme has specificity and attempted being applied to the technique for preparing astaxanthin monomer, because the bacterium producing multi enzyme preparation substratum is expensive, enzyme running water is flat lower, causes this enzyme high cost, has limited its industrial applications.Therefore set up the efficient secretory expression system, improve enzyme running water flat, the Optimal Medium formula, reducing costs is problem demanding prompt solution.
Summary of the invention
First purpose of the present invention is to provide the pichia pastoris phaff of the white penicillium cyclopium lipase of a kind of efficient heterogenous expression;
Second purpose of the present invention is to provide the method for utilizing the white penicillium cyclopium lipase of above-mentioned recombinant bacterium production;
The 3rd purpose of the present invention is to provide a kind of Optimal Medium of expressing this lipase.
The 4th purpose of the present invention has been to provide the purposes of above-mentioned lipase.
The present invention clones the alkaline lipase gene pro-alip that obtains comprising leading peptide from white penicillium cyclopium (Penicilliumcyclopiumvar.albus) bacterial strain, its nucleotide sequence is as shown in SEQ ID No.1, this full length gene is 795bp, the analysis showed that, GC content is 47.9%, the albumen that 264 amino acid of encoding form.By the aminoacid sequence of the white penicillium cyclopium lipase of this genes encoding as shown in SEQ ID No.2.This albumen size 28kDa, PREDICTION FOR THE ISOELECTRIC POINT is 6.16( Http:// web.expasy.org/compute_pi/), active centre is 132 Ser, 188 Asp, 241 His.Experiment shows that the lipase by this genes encoding has higher enzyme work.Measure by zymologic property, the optimal reactive temperature of this enzyme is 35 ℃, and optimal reaction pH value is 7.5, and is stable under pH7～8.5 conditions.For ease of statement, with this lipase called after Pro-ALIP.
Be to be understood that, those skilled in the art can be according to the aminoacid sequence (SEQIDNo.2) that contains the white penicillium cyclopium lipase of self leading peptide disclosed by the invention, do not affecting under its active prerequisite, replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.For example at nonactive section, (Ser) of (25) replaced with (Ala).Therefore, the white penicillium cyclopium lipase that the present invention contains leading peptide also comprises that shown in SEQ ID No.2, aminoacid sequence is substituted, replaces and/or increases one or several amino acid, have with Pro-ALIP with isoreactivity by the derivative protein that obtains of Pro-ALIP.Gene of the present invention comprises the nucleotide sequence of encoding said proteins.
In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.Thereby the white Gene Encoding Alkaline Lipase from Penicillium cyclopium that the present invention contains leading peptide also comprises by nucleotide sequence shown in SEQ ID No.1 and is substituted, lacks and/or increase one or several Nucleotide, the nucleotide sequence of the Pro-ALIP that obtains encoding.
Gene of the present invention is operably connected with expression vector, obtains expressing the recombinant expression vector of albumen of the present invention, further this recombinant expression vector is imported in appropriate host cell, obtain to express the genetic engineering bacterium of Pro-ALIP of the present invention.
In example of the present invention, be inserted on pichia pastoris phaff expression vector pPICZ α A and build and obtain recombinant expression vector pPICZ α A-pro-alip by the lipase gene pro-alip that will contain leading peptide, its plasmid construction collection of illustrative plates is as shown in Figure 2.Further, with the recombinant expression vector electricity turn enter in pichia pastoris phaff X-33 and screening obtain can high efficient expression Pro-ALIP genetic engineering bacterium, with this bacterial strain called after X33-YZ1.
The present invention also provides a kind of method for preparing above-mentioned Pro-ALIP, and it is by cultivating above-mentioned recombinant yeast pichia pastoris genetic engineering bacterium, through abduction delivering, obtains lipase Pro-ALIP.Adopting the shake-flask culture condition is 24 ℃, and initial pH is 8.0, supplements an inductor methyl alcohol in every 12 hours, to the methyl alcohol final concentration, is that 0.5%, 200rpm cultivates 120～190h.Adopt the method, shake-flask culture 164h, take sweet oil as substrate, enzyme activity is 1550U/mL.
The present invention also is provided for the culture medium of said gene recombinant, and it comprises following composition: yeast extract 8～12g/L, peptone 15～25g/L, NaH 2PO 42H 2O5.7～7.7g/L, Na 2HPO 412H 2O19.66～21.66g/L, methyl alcohol 8～12mL/L, KH 2PO 40.75～0.95g/L, K 2HPO 40.12～0.18g/L, NaCl0.08～0.12g/L, CaCl 20.08～0.12g/L, MgSO 40.4～0.6g/L.Preferably include following composition: yeast extract 10g/L, peptone 20g/L, NaH 2PO 42H 2O6.7g/L, Na 2HPO 412H 2O20.66g/L, methyl alcohol 10mL/L, KH 2PO 40.85g/L, K 2HPO 40.15g/L, NaCl0.1g/L, CaCl 20.1g/L, MgSO 40.5g/L.
The present invention has used Protocols in Molecular Biology, introduce white penicillium cyclopium lipase self leading peptide (7 amino acid), and in pichia pastoris phaff (Pichiapastoris) genetic engineering bacterium heterogenous expression, increase the activity of foreign protein lipase, improved the part zymologic property of foreign protein lipase.Experiment shows, the introducing of self leading peptide has significantly increased the secretory volume of white penicillium cyclopium lipase, has widened the catalytic reaction temperature range of white penicillium cyclopium lipase Pro-ALIP, and has improved the activity of its catalytic hydrolysis astaxanthin ester.The invention provides the recombinant bacterial strain of this enzyme of construction expression in pichia pastoris phaff (Pichiapastoris) and the method for efficient secretory expression.Adopt the method, expressing the white penicillium cyclopium lipase that contains self leading peptide can be secreted into outside born of the same parents effectively, not only reduced the separation and purification cost, and expression efficiency improves.Experiment shows: under conditions of flask fermentation, take methyl alcohol as inductor, induce, detect enzyme activity take sweet oil as substrate, enzyme activity reaches 1550U/mL.The present invention also provides its optimization BMMY substratum that uses in fermentation., by optimizing YNB component in the BMMY substratum, reduce culture medium cost and reach 70%.The present invention is the industrial applications of this enzyme, particularly, take the Astaxanthin In Haematococcus Pluvialis ester as substrate, provides reliable experimental basis in this substrate hydrolysis generation astaxanthin monomer of catalysis.
Description of drawings
Fig. 1 goal gene DNA enzyme cuts back to close electrophorogram, and swimming lane 1 is DNA standard molecular weight (kb): 4.5,3.0,2.0,1.2,0.8,0.50.2; Swimming lane 2: goal gene DNA enzyme cuts back to close 795bp fragment (arrow indication);
Fig. 2 recombinant expression vector builds schema;
Fig. 3 recombinant expression vector restriction enzyme digestion and electrophoresis figure, swimming lane 1 is the recombinant expression vector electrophoresis, and swimming lane 2 is the checking of recombinant expression vector double digestion, and swimming lane 3,4 is the checking of recombinant expression vector single endonuclease digestion, swimming lane M is DNAMarkerIII (kb): 4.5,3.0,2.0,1.2,0.8,0.5,0.2;
Fig. 4 Recombinant Pichia pastoris X33-YZ1 bacterial strain, the shake-flask culture extracellular enzyme is lived and cell density OD 600Detect;
Fig. 5 is the hydrolysis that HPLC detects the astaxanthin ester in Haematocoocus Pluvialls source, wherein, and A: before hydrolysis; B: hydrolysis 0.5h; C: hydrolysis 1h; Arrow: astaxanthin monomer peak;
The HPLC-MS/MS of Fig. 6 astaxanthin monomer identifies.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The percentage sign that relates in the present invention " % ",, if do not specify, refer to mass percent; But the per-cent of solution, except as otherwise herein provided, refer in solution 100mL contain the some grams of solute; Per-cent between liquid, refer to the ratio of capacity in the time of 20 ℃.The normal experiment operation stepss such as related enzyme is cut, connected, recovery, conversion, pcr amplification see " molecular cloning (third edition) " for details.Primer synthesizes and order-checking is completed by English fine horse (Invitrogen) biotech firm.
The preparation of embodiment 1 white penicillium cyclopium cDNA
1.1 the extraction of the white total RNA of penicillium cyclopium
(1) get appropriate white penicillium cyclopium mycelia, the filter paper suck dry moisture, liquid nitrogen grinding, add 1mLTrizol reagent (Invitrogen), vibrator vibration 1min, the standing 5min of room temperature;
(2) add the 0.2mL chloroform, vibration 15s, standing 3～5min;
（3）4℃，12000rpm，15min；
(4) draw supernatant, add the equal-volume Virahol ,-20 ℃ of precipitation 30min;
（5）4℃，12000rpm，15min；
(6) outwell supernatant, with 1mL75%DEPC-washing with alcohol precipitation, 12000rpm, 4 ℃, 5min;
(7) repeat (6) step once;
(8) outwell supernatant, 12000rpm, 4 ℃ of centrifugal 2min.With the rifle head, raffinate is drawn totally the dry 10min of spacious lid in ice; Dry 10min;
(9) add appropriate DEPC water dissolution, obtain total RNA.
1.2 the preparation of white penicillium cyclopium cDNA
Reverse transcription adopts ThermoScript II (MMLV) concrete operations of by Promega company, being produced as follows:
(1) RNA unwinds with front 70 ℃, and 5min, put 2min on ice immediately;
(2) 25 μ L reverse transcription systems:
Figure BDA00003515629000071
(3) 95 ℃ of heating 5min termination reactions, freezing preservation.
Embodiment 2 contains the design of primers of the white Gene Encoding Alkaline Lipase from Penicillium cyclopium pro-alip of leading peptide
2.1 design of primers
According to the sequence (the GenBank accession number is AF274320.1) of alip gene in GenBank, following pair of primers has been synthesized in design:
pro-alip-F（P1）：5’-CCCG GAATTCGCACCTATTTTGGAGTCGA-3’
pro-alip-R（P2）：5’-ATAAT GCGGCCGCGCTCAGATAGCCAC-3’
P1, P2 two ends are designed with respectively EcoRI and NotI restriction enzyme site (see italic in above-mentioned sequence and the part of underscore is arranged)
2.2 contain the pcr amplification of the white penicillium cyclopium lipase pro-alip of leading peptide
Adopt P1, P2 primer, with white penicillium cyclopium (ZhangHM, WuMC, GuoJ, et al.CloningandSequenceAnalysisofCompleteGeneEncodingan AlkalineLipasefromPenicilliumcyclopium.AppliedBiochemist ryand Microbiology.2011,47 (6): 586-593.) cDNA is template, and the PCR reaction system is:
Figure BDA00003515629000081
Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 40s, 54 ℃ of 40s, 72 ℃ of 1min30s, circulate 35 times; 72 ℃ of 10min; 4 ℃ of 10min.
2.3 reclaim the purpose fragment from the PCR reaction product
The employing of purifying recovery goal gene fragment is cut glue and is crossed the method for post from the PCR product, the PCR reaction product is after agarose gel electrophoresis, downcut goal gene DNA under the irradiation of ultraviolet lamp, goal gene length is 795bp(Fig. 1), the method that reclaims test kit specification sheets (available from sky root company, production code member is DP209-02) according to DNA reclaims.
2.4TA clone
PCR is reclaimed product be connected to carrier pMD18-T-Simple upper (available from TaKaRa company), the operation of the test kit that ligation provides according to TaKaRa company (CodeNo.D104A) specification sheets.
2.5 goal gene is connected to pichia pastoris phaff expression vector pPICZ α A(Invitrogen company and buys)
Respectively pMD18-T-pro-alip and pPICZ α A are carried out double digestion with restriction enzyme EcoRI and NotI, then the purpose fragment is reclaimed, with the T4 ligase enzyme, connect, obtain expression plasmid pPICZ α A-pro-alip, build schema and see accompanying drawing 2.
The detected result of expression vector pPICZ α A-pro-alip is seen accompanying drawing 3, result show external source fragment (795bp) and carrier (3.5kb) size all correct.To connect product and transform bacillus coli DH 5 alpha (available from Shanghai, giving birth to work biotechnology company limited).
Embodiment 3 contains the secreting, expressing of white Gene Encoding Alkaline Lipase from Penicillium cyclopium pro-alip in pichia pastoris phaff X-33 of leading peptide
3.1 pichia pastoris phaff X-33(Invitrogen company buys) preparation of Electroporation-competent cells and the conversion of shocking by electricity thereof
(1) single bacterium colony that picking is fresh is in the 5mLYPD liquid nutrient medium, and in 28 ℃, 200rpm cultivates 12～14h;
(2) be inoculated in the 2L triangular flask that contains the 500mLYPD substratum with 0.1% inoculum size, in 28 ℃, 200rpm cultivates 12～14h, makes its OD 600=1.3～1.5;
(3) at 4 ℃ of centrifugal 5min of lower 12000rpm, collecting cell;
(4) with twice of the sterilized water washed cell of 500～250mL ice precooling;
(5) with the 1M Sorbitol Solution USP washed cell of 20mL ice precooling once;
(6), with the 1M Sorbitol Solution USP re-suspended cell of 1mL ice precooling, about being 1.5mL to final volume, with 80 μ L, be sub-packed in little centrifuge tube.
3.2 the electric shock of pichia pastoris phaff cell transforms
(1) the 10 linearizing recombinant plasmids of μ g and 80 μ L pichia spp competent cells are mixed, place 5min on ice;
(2) competent cell that has mixed DNA is changed over to the electric revolving cup of the 0.2cm of ice precooling;
Transform under the voltage of 1.5 kilovolts;
(3) the 1M Sorbitol Solution USP that then adds the precooling of 1mL ice in the cell through transforming, mixes cell, changes the little centrifuge tube of 1.5mL over to, 30 ℃ of standing 1～2h at once;
(4) get 50～200 μ L and coat and contain 100 μ g/mLYPDS dull and stereotyped (yeast extract 1%, peptone 2%, glucose 2%, Sorbitol Powder 1M, agar 2%), in 30 ℃, cultivate 2 to 3 days observationss.
3.3 contain the screening of the recombinant bacterial strain (X33-YZ1) of pro-alip gene
3.3.1 the yeast colony PCR method is identified the correct transformant of integrating
Choose positive bacterium colony on flat board,, take 5 ' AOX1,3 ' AOX1 as primer, utilize the method for yeast colony PCR further to verify the transformant that obtains correct integration.
Primer sequence is: 5 ' AOX1:5 '-GACTGGTTCCAATTGACAAGC-3 '
3’AOX1：5′-GCAAATGGCATTCTGACATCC-3’
The treatment process of template:
(1) with aseptic suction nozzle picking colony a little, be dissolved into the D2-Buffer(1L of 50 μ L: guanidinium isothiocyanate 472.64g, 1mol/LpH8.0TrisHCl damping fluid 50mL, beta-mercaptoethanol 7mL) in mix;
(2) with mixed solution in 100 ℃ of boiling water bath 5min;
(3) 12000rpm, centrifugal 30s, abandon supernatant;
(4) with sterilized water washing precipitation 2 times;
(5) precipitation is dissolved in the ddH of 20 μ L 2O, 95 ℃ of effect 5min;
(6) obtain supernatant liquor after centrifugal and be template.
The PCR reaction system:
Figure BDA00003515629000101
Reaction conditions: 95 ℃ of 5min; 95 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2min10s, 30 circulations; 72 ℃ of 10min.
3.4 the expression of purpose lipase in pichia pastoris phaff (X33-YZ1)
3.4.1NaOH titration measuring lipase activity
(1) configuration of 0.05MNaOH: first use without CO 2The NaOH liquid storage of water configuration 5M; After accurately diluting 50 times again, take 100 ℃ of Potassium Hydrogen Phthalate 0.38g that dry to constant weight, be dissolved in 80mL without CO 2In water, calibrate its accurate concentration, then extrapolate stock concentrations; 0.05M NaOH solution with without CO 2Water liquid storage matching while using;
(2) configuration of PVA-sweet oil emulsion substrate: with sweet oil 100mL, the 300mL2%PVA-1750(polyvinyl alcohol) mix, heating is dissolved, and uses ultrasonic emulsification, power 300W, ultrasonic 3s, intermittently 5s, 99 times, circulate 2 times;
(3) get 5mL emulsion substrate, the HEPES damping fluid of 4mL0.1MpH7.5 joins in the 150mL triangular flask, is placed in 35 ℃ of water bath with thermostatic control shaking tables, 150rpm incubation 10min;
(4) get the suitable enzyme liquid that dilutes of 1mL and join in substrate and damping fluid, add 15mL dehydrated alcohol termination reaction after 35 ℃ of 150rpm reaction 10min;
(5) drip 5 phenolphthalein and make indicator, the lipid acid with 0.05MNaOH titration enzymolysis produces, stop to reaction solution is pink.
Blank operation, with above-mentioned consistent, just will join in substrate and damping fluid after fermented liquid and dehydrated alcohol hybrid reaction 10min.
Enzyme work is defined as under this condition determination, and the enzyme amount that 1min discharges 1 μ mol lipid acid is an enzyme activity unit.
3.4.2 shake flask fermentation produces enzyme
The single bacterium colony of picking pichia spp recon (X33-YZ1), be inoculated in the 25mLBMGY substratum (1% yeast powder, 2% peptone, 1% glycerine, pH6.5), in 28 ℃, the 200rpm shaking table is cultivated 16～20h to OD 600Be 6.0 left and right, by 10% inoculum size, be transferred to 50mL optimization BMMY substratum (yeast extract 10g/L, peptone 20g/L, NaH are housed 2PO 42H 2O6.7g/L, Na 2HPO 412H 2O20.66g/L, methyl alcohol 10mL/L, KH 2PO 40.85g/L, K 2HPO 40.15g/L, NaCl0.1g/L, CaCl 20.1g/L, MgSO 40.5g/L in 500mL triangular flask pH8.0), 24 ℃ are continued to cultivate, every 12 hours add 100% methyl alcohol is 0.5% in substratum to its final concentration, abduction delivering 120～190h.Often sampling at regular intervals, measure lipase activity and cell density OD 600.When no longer raising or start to reduce, enzyme work stops fermentation.
Detected result as shown in Figure 4, (initial pH is 8.0, and culture temperature is 24 ℃) shake-flask culture 164h in optimizing the BMMY substratum, the work of pichia spp recon enzyme is up to 1550U/mL.
Use above-mentioned lipase to carry out the catalytic hydrolysis of astaxanthin ester, the example of a catalytic hydrolysis astaxanthin ester below be provided:
The astaxanthin ester concentration of substrate is 200 μ g/mL, according to every microgram astaxanthin ester substrate, adds the 80U enzyme, in the 0.05M phosphate buffered saline buffer, and 30 ℃ of oscillatory reaction 1h, it is 80% that HPLC detects the astaxanthin monomer yield, the astaxanthin ester transformation efficiency can reach more than 96%.
Utilizing HPLC to analyze astaxanthin ester hydrolysis reaction front and back material changes.The lipase-catalyzed hydrolysis astaxanthin ester of Pro-ALIP effect is splendid, and after reaction 0.5h, astaxanthin ester is surplus 10% left and right only, and after reaction 1h, astaxanthin ester almost completely is hydrolyzed, and content is lower than 3%.
Rear sample carries out HPLC and HPLC-MS detects to reacting.After reaction, the maximum absorption band of main product (shown in Fig. 5 B and C arrow) is 477nm, and it is 597.5 main peak (shown in Fig. 6 arrow) that response sample has molecular weight, is the astaxanthin monomer that dissociates.
Figure BDA00003515629000131
Figure BDA00003515629000141
Figure BDA00003515629000151
Figure BDA00003515629000161
Figure BDA00003515629000171

Claims (11)

1. contain the white penicillium cyclopium lipase Pro-ALIP of self leading peptide, its aminoacid sequence is:
A) aminoacid sequence shown in SEQ ID No.2;
B) aminoacid sequence shown in SEQ ID No.2 is through replacing, lack or add the aminoacid sequence with same function of one or several amino-acid residue formation.
2. the gene of coding claim 1 described white penicillium cyclopium lipase Pro-ALIP.
3. gene as claimed in claim 2, its nucleotide sequence is as shown in SEQ ID No.1.
4. the recombinant vectors that contains claim 2 or 3 described genes.
5. recombinant vectors as claimed in claim 4, it is pPICZ α A-pro-alip.
6. the host cell that transforms of the described recombinant vectors of claim 4 or 5.
7. host cell as claimed in claim 6, it is pichia pastoris phaff X-33.
8. one kind prepares the described method that contains the white penicillium cyclopium lipase Pro-ALIP of self leading peptide of claim 1, it expresses by cultivating the described host cell of claim 6 or 7, induce the lipase gene that contains leading peptide the white penicillium cyclopium lipase Pro-ALIP that obtains to contain leading peptide.
9. method as claimed in claim 8, is characterized in that, cultivates a kind of culture medium of described host cell, and it comprises following composition: yeast extract 8～12g/L, peptone 15～25g/L, NaH 2PO 42H 2O5.7～7.7g/L, Na 2HPO 412H 2O19.66～21.66g/L, methyl alcohol 8～12mL/L, KH 2PO 40.75～0.95g/L, K 2HPO 40.12～0.18g/L, NaCl0.08～0.12g/L, CaCl 20.08～0.12g/L, MgSO 40.4～0.6g/L.
10. method as claimed in claim 8 or 9, is characterized in that, adopts the described host cell of shake-flask culture, its culture condition is 24 ℃, and initial pH is 8.0, every 12 hours supplementary inductor methyl alcohol, be that 0.5%, 200rpm cultivates 120～190h to the methyl alcohol final concentration.
Contain the application of white penicillium cyclopium lipase Pro-ALIP in the catalytic hydrolysis astaxanthin ester prepares astaxanthin monomer of self leading peptide 11. claim 1 is described.
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CN103642769A (en) * 2013-12-05 2014-03-19 青岛蔚蓝生物集团有限公司 Alkaline lipase from penicillium cyclopium
CN103667091A (en) * 2013-12-05 2014-03-26 青岛蔚蓝生物集团有限公司 Alkaline lipase production bacterial strain and application
CN103667091B (en) * 2013-12-05 2015-10-28 青岛蔚蓝生物集团有限公司 A kind of Alkaline lipase production bacterial strain and application thereof
CN103642769B (en) * 2013-12-05 2015-11-25 青岛蔚蓝生物集团有限公司 A kind of alkaline lipase deriving from penicillium cyclopium
CN106459936A (en) * 2014-05-27 2017-02-22 诺维信公司 Method for modifying a lipase
US10851356B2 (en) 2014-05-27 2020-12-01 Novozymes A/S Method for modifying characteristics of a lipase
CN107102091A (en) * 2017-06-15 2017-08-29 秦皇岛惠恩生物技术有限公司 The method for quantitatively detecting astaxanthin in haematococcus pluvialis extract using Asta E H lipase
CN107102091B (en) * 2017-06-15 2020-11-13 秦皇岛惠恩生物技术有限公司 Method for quantitatively detecting astaxanthin in haematococcus pluvialis extract by using Asta-E-H lipase
CN108192885A (en) * 2017-12-30 2018-06-22 浙江工业大学 A kind of method for improving Aspergillus oryzae lipase heterogenous expression
CN112961871A (en) * 2021-02-26 2021-06-15 源创核新(北京)新材料科技有限公司 Recombinant plasmid, genetic engineering bacterium containing recombinant plasmid and application of recombinant plasmid in preparation of dimethyl carbonate

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