CN102212602B - Method for synthesizing vitamin A lactate by catalysis of yeast display lipase - Google Patents
Method for synthesizing vitamin A lactate by catalysis of yeast display lipase Download PDFInfo
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- lipase
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 39
- 102000004882 Lipase Human genes 0.000 title claims abstract description 30
- 239000004367 Lipase Substances 0.000 title claims abstract description 30
- 235000019421 lipase Nutrition 0.000 title claims abstract description 30
- 235000019155 vitamin A Nutrition 0.000 title claims abstract description 30
- 239000011719 vitamin A Substances 0.000 title claims abstract description 30
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 title claims abstract description 26
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- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 title claims abstract description 25
- 238000002818 protein evolution Methods 0.000 title claims abstract description 25
- 229940045997 vitamin a Drugs 0.000 title claims abstract description 25
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000006555 catalytic reaction Methods 0.000 title abstract description 4
- 230000002194 synthesizing effect Effects 0.000 title abstract 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 15
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- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 6
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 4
- 244000285963 Kluyveromyces fragilis Species 0.000 claims description 4
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- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 claims description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
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- QGNJRVVDBSJHIZ-QHLGVNSISA-N retinyl acetate Chemical compound CC(=O)OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C QGNJRVVDBSJHIZ-QHLGVNSISA-N 0.000 abstract description 5
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- 241000235058 Komagataella pastoris Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
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- 125000000946 retinyl group Chemical group [H]C([*])([H])/C([H])=C(C([H])([H])[H])/C([H])=C([H])/C([H])=C(C([H])([H])[H])/C([H])=C([H])/C1=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C1(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
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- 235000019169 all-trans-retinol Nutrition 0.000 description 1
- 239000011717 all-trans-retinol Substances 0.000 description 1
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- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- Y02P20/00—Technologies relating to chemical industry
- 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
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for synthesizing vitamin A lactate by catalysis of yeast display lipase. The method comprises the following steps of: dissolving vitamin A acetate and lactic 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 A lactate, 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 A lactate 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 the vitamin A lactate.
Background technology
Vitamin A (Vogan-Neu) is a kind of natural liposoluble vitamin, have the normal vision of keeping reaction, skeleton development, epithelium function, safeguard the skin cells function, promote to grow and remove effects such as free radical, raising immunological competence, so vitamin A has been widely used in industries such as makeup, protective foods.The most frequently used in the vitamin A series is vitamin A and, but they are very easily oxidized under the condition of light, air, oxygenant and high temperature, have now had a large amount of research to concentrate on and have developed the stable vitamin A derivatives of synthesising property such as vitamin A fatty acid ester (VITAMIN palmitinic acid, oleic acid ester).Alpha hydroxy acid (modal oxyacetic acid and lactic acid) is improving skin, is promoting stratum corneum regeneration etc. to prevent that effect has been proved aspect the skin aging, but since these acid can rapid permeability to the epidermis depths, concentration was above 5% o'clock, will the grievous injury skin histology, the Alpha-hydroxy acid esters of vitamin A can effectively overcome above shortcoming, and keep higher vitamin A activity, compare with non-esterified form and have more obvious skin care effect.Vitamin A lactate (retinyl lactylate) is to make with chemosynthesis in the market, yet the synthetic toxic substance that not only exists chemical catalyst to bring of chemical method, and be easy to take place side reaction under the high temperature, condition of high voltage, produce the product of toxic side effect, also have the energy consumption height simultaneously, equipment and instrument are required problems such as high, that environmental pollution is serious, enzyme process is synthetic can avoid these problems.Have the report that utilizes lipase synthesise vitamins A lactate at present, but owing to lipase (resolvase or immobilization) making complexity, the high commercialization that limits this biocatalysis process of production cost, mass-producing are used.
Summary of the invention
The invention provides the method that a primary yeast display lipase catalyzes and synthesizes the vitamin A lactate, can significantly reduce vitamin A lactate production cost, reach higher esterification efficient and productive rate simultaneously.
One primary yeast display lipase catalyzes and synthesizes the method for vitamin A lactate, comprising:
And lactic acid are dissolved in the organic solvent, add the yeast display lipase, in 50～60 ℃ of reactions 10～14 hours, separation, purifying made the vitamin A lactate under the oxygen free condition.
Preferably, described organic solvent is normal hexane.
Preferably, the add-on of, lactic acid and yeast display lipase is respectively 20～100g, 30～100g, 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 A) is more than 87%.
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 the vitamin A lactate
Example 1 is got 0.328g, lactic acid 0.35g, 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 vitamin A lactate product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.
Example 2 is got 0.656g, lactic acid 0.7g, 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 vitamin A lactate product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.
Embodiment 3 adopts traditional chemical method synthesise vitamins A lactate
Example 1 is got 0.328g, lactic acid 0.35g, adds the ground triangular flask that contains the 10mL normal hexane, and 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 vitamin A lactate 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 retinyl acetate-C ester/C retinyl acetate * 100%.
In the formula: C ester is the concentration of vitamin A lactate in the HPLC working sample, and C retinylacetate is the concentration of in the preceding sample of reaction.
By the aforesaid method detection computations, among the embodiment 2, the transformation efficiency of example 1 synthesise vitamins A lactate reaches 87%, and the transformation efficiency of example 2 synthesise vitamins A lactates reaches 85%.And the transformation efficiency of embodiment 3 utilization employing traditional chemical method synthesise vitamins A lactates only is 38%.
Claims (1)
1. a primary yeast display lipase catalyzes and synthesizes the method for vitamin A lactate, 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 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, archaeal 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;
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; 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, 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 and contain the BMMY substratum inducing culture 144h that volume percent is 0.5% methyl alcohol, centrifugal collecting cell, after the water flushing, be seeded to 30 ℃ of cultivation 120h in the YGC substratum, the centrifugal collection thalline of 3000g 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;
Get 0.328g, lactic acid 0.35g, add the ground triangular flask that contains the 10mL normal hexane, 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 vitamin A lactate product in 4 ℃ of crystallizations, oven dry, pulverizing.
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| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
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