CN102212576B - Method for synthesizing vitamin E linoleate by catalysis of yeast display lipase - Google Patents
Method for synthesizing vitamin E linoleate by catalysis of yeast display lipase Download PDFInfo
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- CN102212576B CN102212576B CN2011101071250A CN201110107125A CN102212576B CN 102212576 B CN102212576 B CN 102212576B CN 2011101071250 A CN2011101071250 A CN 2011101071250A CN 201110107125 A CN201110107125 A CN 201110107125A CN 102212576 B CN102212576 B CN 102212576B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention discloses a method for synthesizing vitamin E linoleate by catalysis of yeast display lipase. The method comprises the following steps of: dissolving vitamin E and linoleic acid into an organic solvent, adding the yeast display lipase, reacting for 10 to 14 hours in the absence of oxygen at the temperature of between 50 and 60 DEG C, and performing separation and purification to obtain the vitamin E linoleate, wherein the yeast display lipase is prepared by the following steps of: transferring linearly treated recombinant plasmids to pichia pastoris GS115, inoculating the obtained transformant to a buffered methanol complex (BMMY) medium, performing induced culture for 72 to 144 hours, performing centrifugal collection on bacteria, and performing flushing, biological imprinting and freeze drying on the bacteria to obtain the yeast display lipase. The lipase is displayed outside cells, and the vitamin E linoleate is synthesized by catalysis of the enzyme preparation, so that the transformation efficiency is improved, the reaction time is shortened and the production cost is reduced.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to the method that a primary yeast display lipase catalyzes and synthesizes linoleic acid ester of vitamin e.
Background technology
Natural VE (tocopherol), be important liposoluble vitamin and the natural antioxidants that has different physiological roles in the human body, it have significantly anti-oxidant, eliminate interior free yl, eliminate sexual dysfunction, stimulate circulation, prevent early ageing, preventing cancer takes place, improve functions such as immunity of organisms, is a kind of extremely important medicine and health material.Yet natural VE is easy to oxidized, and the vitamin-E product on the market is the stable derivatives through over-churning mostly.Linoleic acid ester of vitamin e (vitamin E linoleate) is a kind of derivative of vitamin-E, it except have vitamin-E significantly anti-oxidant, eliminate interior free yl, eliminate sexual dysfunction, stimulate circulation, prevent functions such as early ageing, preventing cancer, raising immunity of organisms; Also having functions such as linolic acid treatment hyperlipoproteinemia and atherosclerosis, is a kind of important medicine and health material, is widely used in fields such as medicine, food, makeup.The synthesising complex E linoleate has chemical method and biological enzyme at present, because chemical method exists conversion condition harshness (strong acid, base catalysis, High Temperature High Pressure), no specificity, product complexity, drawbacks such as by product is many, separation is difficult, poor stability, biological enzyme is more and more favored.Lipase be present VITAMIN ester synthetic in most popular enzyme, but production cost height, numerous and diverse time-consuming its commercial applications of having limited to greatly of immobilization process.
Summary of the invention
The invention provides the method that a primary yeast display lipase catalyzes and synthesizes linoleic acid ester of vitamin e, can significantly reduce the linoleic acid ester of vitamin e production cost, reach higher esterification efficient and productive rate simultaneously.
One primary yeast display lipase catalyzes and synthesizes the method for linoleic acid ester of vitamin e, comprising:
Vitamin-E and linolic acid are dissolved in the organic solvent, add the yeast display lipase, in 50～60 ℃ of reactions 10～14 hours, separation, purifying made linoleic acid ester of vitamin e under the oxygen free condition.
Preferably, described organic solvent is normal hexane.
Preferably, the add-on of vitamin-E, linolic acid and yeast display lipase is respectively 20～100g, 100～300g, 50～100g in every liter of organic solvent.
Stir speed (S.S.) is 200～250 rev/mins.
Described separation, purifying are: reaction solution is centrifugal, get supernatant liquor, and rotary evaporation is removed organic solvent, is dissolved in normal hexane, recrystallization after the washing.
Described yeast display lipase prepares by the following method:
To change pichia spp (Pichia pastoris) GS115 through the recombinant plasmid of linearization process over to, the gained transformant is inoculated in the BMMY substratum, inducing culture is centrifugal collection thalline after 72～144 hours, and thalline makes the yeast display lipase through flushing, biological trace and lyophilize;
Described recombinant plasmid is by initial carrier pPIC9K and insert the lipase gene in MF α 1 signal peptide downstream among the initial carrier pPIC9K successively and the cell walls α agglutinin gene of pichia spp GS115 is formed.
Described lipase gene can be selected for use and be the sequence of AF229435 Genbank number, and pichia spp (Pichia pastoris) GS115 is commercially produced product, can buy from Invitrogen company.Genbank number of its cell walls α agglutinin gene sequence is M28164.
Carrier pPIC9K is commercially produced product (as Invitrogen company), and there is MF α 1 signal peptide sequence in it, and (Genbank number: M17301), there is the AOX1 promotor in the signal peptide sequence upstream (Genbank number: Z46233) in this carrier simultaneously.The purpose of recombinant plasmid linearization process be for born of the same parents in genome generation homologous recombination, improve expression stability.
Preferably, the used part of described biological trace is oleic acid, and it can the inducible enzyme structural modification, improves transformation efficiency.
The present invention is by importing pichia spp cell GS115 with lipase gene and cell walls α agglutinin gene, and after the pichia spp cell induction was cultivated, lipase was expressed justacrine outside born of the same parents, utilizes cell walls α lectin that this lipase is fixed on cell surface simultaneously.Utilize this yeast display lipase that esterification is carried out catalysis, can effectively improve operational stability, thermotolerance and repeatability, because this enzyme of specificity of enzyme reaction can suppress the generation of side reaction significantly, whole transformation efficiency (in vitamin-E) is more than 78%.
Embodiment
Embodiment 1 preparation yeast display lipase
Method by synthetic, the lipase gene of synthetic Rhizopus oryzae (Rhizopus oryzae) (Genbank number: AF229435) and the cell walls α agglutinin gene (Genbank number is M28164) of pichia spp GS115, add connection peptides sequence GSSGGSGGSGGSGGSGS (linker) at lipase gene C end simultaneously, obtain nucleotide sequence pro-ROL-linker-α-agglutinin after the connection, add EcoR I and Not I restriction enzyme site simultaneously at the sequence two ends, wherein pro-ROL is lipase gene, and α-agglutinin is cell walls α agglutinin gene.
Be template with above-mentioned artificial synthesized sequence, utilize following primer right, carry out pcr amplification,
Upstream primer: 5 '-AAGGAAAAAAGAATTCGTTCCAGTTTCTGG-3 ';
Downstream primer: 5 '-TTTTCCTTTTGCGGCCGCTAATGAAACG-3 '
The PCR reaction system is: template DNA is 1 μ l, high-fidelity DNA polymerase 0.5 μ l, and dNTP (50mM) 0.4 μ l, each 0.5 μ l of upstream and downstream primer, 10 * PCR damping fluid, 5 μ l add water to 50 μ l.
The PCR operational conditions is: 94 ℃ 3 minutes, 35 circulations (94 ℃ 30 seconds, 60 ℃ 1 minute, 72 ℃ 30 seconds), 72 ℃ 10 minutes.
With EocR I and Not I simultaneously enzyme cut PCR product and pPIC9K plasmid, and under the effect of T4 ligase enzyme, spend the night and be connected to form the pPIC9K-ROL plasmid, by the electrophoresis check and reclaim plasmid.For making goal gene and pichia spp GS115 that His 4 unit points displacement reorganization take place, carry out linearization for enzyme restriction with the pPIC9K-ROL plasmid of Sal I and handle.The about 15 μ l of goal gene that linearization for enzyme restriction is handled well join in the previously prepd pichia spp GS115 competent cell, change in the electric revolving cup ice bath 15min over to, then at 1500V, 400 Ω, the 10ms that shocks by electricity under the 25uF condition, and the sorbyl alcohol of the about 1ml precooling of adding.The electricity of the above-mentioned mixing about 400 μ l of thing that change the line of production are applied on the MD flat board, and the screening positive transformant is applied to positive transformant on the G418 flat board of different concns then, the resistance screening of G418 is gone out the positive recombinant bacterial strain of multiple copied of Mut phenotype according to positive transformant.
The positive recombinant bacterial strain of multiple copied is seeded in fermentation culture 30h in the BMGY substratum, centrifugal collecting cell; Again cell is placed the BMMY substratum inducing culture 144h that contains 0.5% (volume percent) methyl alcohol, centrifugal collecting cell, after the water flushing, be seeded to 30 ℃ of cultivation 120h in the YGC substratum, 3000g collected thalline in centrifugal minute then, wash with 50mM pH7.0 phosphoric acid buffer again behind the distilled water wash, mix with 2 times of volume oleic acid then, after-80 ℃ of following pre-freezes again through the dry 24h of German Christ vacuum freeze drier, remove oleic acid with hexane wash, carry out vacuum-drying more again and remove hexane, namely obtain the yeast display lipase of handling through biological trace.
Embodiment 2 yeast display lipases catalyze and synthesize linoleic acid ester of vitamin e
Example 1 is got vitamin-E 0.472g, linolic acid 1.400g, adds the ground triangular flask that contains the 10mL normal hexane, and mixing, preheating 10min add yeast display lipase 0.5g then, fill N
2Sealing, place 85-1 type magnetic stirring apparatus to stir and begin reaction, rotating speed is 250 rev/mins, temperature of reaction remains on 45 ℃, reaction stops behind the 12h stirring, and the yeast display lipase is removed in centrifugation, gets supernatant liquor and is rotated evaporation and removes organic solvent, add normal hexane after washing 3 times and get the linoleic acid ester of vitamin e product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.
Example 2 is got vitamin-E 0.944g, linolic acid 2.800g, adds the ground triangular flask that contains the 10mL normal hexane, and mixing, preheating 10min add yeast display lipase 1g then, fill N
2Sealing, place 85-1 type magnetic stirring apparatus to stir and begin reaction, rotating speed is 200 rev/mins, temperature of reaction remains on 50 ℃, reaction stops behind the 12h stirring, and the yeast display lipase is removed in centrifugation, gets supernatant liquor and is rotated evaporation and removes organic solvent, add normal hexane after washing 3 times and get the linoleic acid ester of vitamin e product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.
Embodiment 3 adopts traditional chemical method synthesising complex E linoleate
Get vitamin-E 0.472g, linolic acid 1.400g, add the ground triangular flask that contains the 10mL normal hexane, mixing, preheating 10min fill N
2Sealing, place 85-1 type magnetic stirring apparatus to stir and begin reaction, rotating speed is 250 rev/mins, temperature of reaction remains on 45 ℃, stop to stir behind the reaction 12h, get supernatant liquor and be rotated evaporation and remove organic solvent, add normal hexane after wash 3 times to get the linoleic acid ester of vitamin e product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.
Embodiment 4 measures conversion yield and transformation efficiency
Get the unsegregated reaction product of 1mL, centrifugal removal catalyzer, the rotary evaporation solvent accurately is settled to 10mL with 100% methyl alcohol, and this solution is measured each concentration of component of solution with high performance liquid chromatography (HPLC) behind the organic membrane filtration of 0.22 μ m.The HPLC condition is as follows: Agilent Technologies 1200 series of high efficiency liquid chromatography liquid, chromatographic column: Grace Prevail Organic Acid 5u150mm * 4.6mm, column temperature: 40 ℃, moving phase: methanol/phosphoric acid (85/15/0.1), flow velocity: 1mL/min, sample size: 10 μ L, detect wavelength: 280nm.
The transformation efficiency calculation formula is as follows:
Transformation efficiency=C vitamin E-C ester/C vitamin E * 100%.
In the formula: C ester is the concentration of linoleic acid ester of vitamin e in the HPLC working sample, and C vitaminE is the concentration of vitamin-E in the preceding sample of reaction.
By the aforesaid method detection computations, among the embodiment 2, the transformation efficiency of example 1 synthesising complex E linoleate reaches 78%, and the transformation efficiency of example 2 synthesising complex E linoleate reaches 79%.And the transformation efficiency of embodiment 3 employing traditional chemical method synthesising complex E linoleate only is 57%.
Claims (1)
1. a primary yeast display lipase catalyzes and synthesizes the method for linoleic acid ester of vitamin e, comprising:
The lipase gene of synthetic Rhizopus oryzae (Rhizopus oryzae) and the cell walls α agglutinin gene of pichia spp GS115, then add the gene fragment of coding connection peptides sequence GSSGGSGGSGGSGGSGS at lipase gene C end, connect and obtain nucleotide sequence pro-ROL-linker-α-agglutinin, pro-ROL represents lipase gene, be for Genbank number: AF229435, α-agglutinin represents cell walls α agglutinin gene, be for Genbank number: M28164, the gene fragment of linker representative coding connection peptides;
Be template with the artificial synthesized sequence, utilize following primer right, carry out pcr amplification;
Upstream primer: 5 '-AAGGAAAAAAGAATTCGTTCCAGTTTCTGG-3 ';
Downstream primer: 5 '-TTTTCCTTTTGCGGCCGCTAATGAAACG-3 ';
The PCR reaction system is: template DNA is 1 μ l, high-fidelity DNA polymerase 0.5 μ l, and the dNTP0.4 μ l of 50mM, each 0.5 μ l of upstream and downstream primer, 10 * PCR damping fluid, 5 μ l add water to 50 μ l;
The PCR operational conditions is: 94 ℃ 3 minutes; 35 circulations, each circulation is: 94 ℃ 30 seconds, 60 ℃ 1 minute, 72 ℃ 30 seconds; 72 ℃ 10 minutes;
Cut PCR product and pPIC9K plasmid with EocR I and Not I while enzyme, and under the effect of T4 ligase enzyme, spend the night and be connected to form the pPIC9K-ROL plasmid, carrying out linearization for enzyme restriction with the pPIC9K-ROL plasmid of Sal I handles, the goal gene 15 μ l that linearization for enzyme restriction is handled well join in the pichia spp GS115 competent cell, change in the electric revolving cup ice bath 15min over to, then at 1500V, 400 Ω, the 10ms that shocks by electricity under the 25uF condition, and the sorbyl alcohol of adding 1mL precooling;
The electricity of the mixing thing 400 μ l that change the line of production are applied on the MD flat board, and the screening positive transformant is applied to positive transformant on the G418 flat board of different concns then, the resistance screening of G418 is gone out the positive recombinant bacterial strain of multiple copied of Mut phenotype according to positive transformant;
The positive recombinant bacterial strain of multiple copied is seeded in fermentation culture 30h in the BMGY substratum, centrifugal collecting cell; Again cell is placed the BMMY substratum inducing culture 144h that contains volume percent 0.5% methyl alcohol, centrifugal collecting cell, after the water flushing, be seeded to 30 ℃ of cultivation 120h in the YGC substratum, 3000g collected thalline in centrifugal minute then, wash with 50mM pH7.0 phosphoric acid buffer again behind the distilled water wash, mix with 2 times of volume oleic acid then, after-80 ℃ of following pre-freezes again through the dry 24h of freeze drier, remove oleic acid with hexane wash, carry out vacuum-drying again and remove hexane, namely obtain the yeast display lipase of handling through biological trace;
Get vitamin-E 0.944g, linolic acid 2.800g, add the ground triangular flask that contains the 10mL normal hexane, mixing, preheating 10min add yeast display lipase 1g then, fill N
2Sealing, place 85-1 type magnetic stirring apparatus to stir and begin reaction, rotating speed is 200 rev/mins, temperature of reaction remains on 50 ℃, reaction stops behind the 12h stirring, and the yeast display lipase is removed in centrifugation, gets supernatant liquor and is rotated evaporation and removes organic solvent, add normal hexane after washing 3 times and get the linoleic acid ester of vitamin e product in 4 ℃ of crystallizations, oven dry, pulverizing.
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| CN2011101071250A CN102212576B (en) | 2011-04-28 | 2011-04-28 | Method for synthesizing vitamin E linoleate by catalysis of yeast display lipase |
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| CN1800173A (en) * | 2005-10-14 | 2006-07-12 | 广西工学院 | Preparation of vitamin E linolenin and vitamin E oleate |
| MY162051A (en) * | 2007-05-24 | 2017-05-31 | Loders Croklaan Bv | Process for producing compositions comprising tocopherols and tocotrienols |
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