CN102212590B - Method for catalytically synthesizing sucrose palmitate with yeast display lipase - Google Patents

Abstract

The invention discloses a method for catalytically synthesizing sucrose palmitate with yeast display lipase. The method comprises the following steps: dissolving sucrose and palmitic acid in an organic solvent; adding yeast display lipase to react at 50-60 DEG C for 10-14 hours; and carrying out separation and purification to prepare the sucrose palmitate, wherein the yeast display lipase is prepared by transforming recombinant plasmid which is subjected to linearization into Pichia pastoris GS115, inoculating the obtained transformant in a BMMY culture medium, carrying out inducing culture for 72-144 hours, centrifuging to collect a thallus, and carrying out washing, bio-imprinting and freeze-drying on the thallus. By displaying the lipase outside the cell and utilizing the lipase preparation to catalytically synthesize the sucrose palmitate, the invention improves the transformation efficiency, shortens the reaction time and lowers the production cost.

Description

The yeast display lipase catalyzes and synthesizes the method for Surfhope SE Cosme C 1616

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 Surfhope SE Cosme C 1616.

Background technology

Sucrose ester is as a kind of safe surface promoting agent, its surfactivity effect and widely the scope of application be subjected to people's attention.Japan, the U.S., European Economic Community various countries, Food and Argriculture OrganizationFAO (FAO), the emulsifying agent that the equal approved sucrose ester of The World Health Organization (WHO) and China is food, makeup and medicine.Different with general tensio-active agent, sucrose ester is all energy biological degradations under aerobic and anaerobic condition, and this makes the three wastes of dairy industry product become the environmental friendliness product.China's sucrose is cheap, purity is high, and import plam oil price is lower, edible safety.Therefore, its purposes constantly is developed, and market potential is huge, has development prospect.

For a long time; the chemical preparation of sucrose ester utilizes the method for sucrose direct esterification more, because sucrose has 8 hydroxyls that reactive behavior is close, so the esterification action site is difficult to control; the single product of wishing to get specified structure is very difficult, often needs tediously long protection and de-protected step.Lipase be present sucrose 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 Surfhope SE Cosme C 1616, can significantly reduce the Surfhope SE Cosme C 1616 production cost, reach higher esterification efficient and productive rate simultaneously.

One primary yeast display lipase catalyzes and synthesizes the method for Surfhope SE Cosme C 1616, comprising:

Sucrose and palmitinic acid are dissolved in the organic solvent, add the yeast display lipase, in 50～60 ℃ of reactions 10～14 hours, separation, purifying made Surfhope SE Cosme C 1616.

Preferably, described organic solvent is the trimethyl carbinol.

Preferably, the add-on of sucrose, palmitinic acid and yeast display lipase is respectively 20～100g, 50～100g, 1～2g in every liter of organic solvent.

Described reaction places shaking bath to carry out, and the shaking bath rotating speed is 150～200 rev/mins.

Preferably, before separation and purification, add molecular sieve, continue stirring reaction 10～12h, pin moisture, promote esterification further to carry out.

Described separation, purifying are: the centrifuging and taking supernatant liquor, 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;

The initial carrier of described recombinant plasmid is carrier pPIC9K, goal gene is by the lipase gene of connection peptides sequence connection and the cell walls α agglutinin gene of pichia spp GS115, the C of MF α 1 signal peptide sequence end among the described lipase gene connection carrier pPIC9K.

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, conversion rate of esterification is more than 80%.

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 Surfhope SE Cosme C 1616

Example 1 is got sucrose 0.2g, palmitinic acid 0.5g, add the tool plug triangular flask that contains the 10mL trimethyl carbinol, mix, preheating 10min, add yeast display lipase 0.01g then, place shaking bath to begin reaction, rotating speed is 200 rev/mins, temperature of reaction remains on 50 ℃, add 0.5g molecular sieve (aperture is less than 2nm) behind the reaction 12h, after continuing reaction 12h, yeast display lipase and molecular sieve are removed in centrifugation, get supernatant liquor and are rotated evaporation and remove organic solvent, add normal hexane after washing 3 times and get the Surfhope SE Cosme C 1616 product in 4 ℃ of crystallizations, oven dry, pulverize and get final product.

Example 2 is got sucrose 1g, palmitinic acid 1g, add the tool plug triangular flask that contains the 10mL trimethyl carbinol, mixing, preheating 10min, add yeast display lipase 0.02g then, place shaking bath to begin reaction, rotating speed is 180 rev/mins, temperature of reaction remains on 55 ℃, add 0.5g molecular sieve (aperture is less than 2nm) behind the reaction 12h, after continuing reaction 12h, yeast display lipase and molecular sieve are removed in centrifugation, get supernatant liquor and are rotated evaporation and remove organic solvent, add normal hexane after washing 3 times and get the Surfhope SE Cosme C 1616 product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.

Embodiment 3 adopts traditional chemical method synthesis of sucrose cetylate

Get sucrose 0.2g, palmitinic acid 0.5g, add the tool plug triangular flask that contains the 10mL trimethyl carbinol, mixing, preheating 10min place shaking bath to begin reaction, rotating speed is 200 rev/mins, temperature of reaction remains on 50 ℃, adds 0.5g molecular sieve (aperture is less than 2nm) behind the reaction 12h, after continuing to react 12h, molecular sieve is removed in centrifugation, get supernatant liquor and be rotated evaporation and remove organic solvent, add normal hexane after wash 3 times to get the Surfhope SE Cosme C 1616 product in 4 ℃ of crystallizations, oven dry, pulverizing get final product.

Embodiment 4 measures conversion yield and transformation efficiency

Use silica gel g thin-layer plate, in 105 ℃ of activation 1h, developping agent is chloroform: methyl alcohol: (v: v), (solvent is chloroform to acetic acid=4: 4: 3: methyl alcohol=1: 1 with 5% anthrone solution, v: v) be developer, the silica-gel plate that is sprayed with developer is placed 110 ℃ of heating 10min.Sucrose ester shows bluish voilet, and sucrose is blue look (initial point).Reactant system reaction forward and backward add respectively the 20mL acetone (1: 1, v: v), make indicator with phenolphthalein, with the KOH solution titration of 0.07mol/L, average respectively as acid number, determine the transformation efficiency of esterification with the reduction value of system acid number.

The calculation of yield formula is as follows:

Productive rate=C ester * n

In the formula: C ester is the concentration of Surfhope SE Cosme C 1616 in the dropping point working sample, and n is the diluted sample multiple.

Esterification efficient: transformation efficiency (%)=(1-product acid number/palmitinic acid acid number) * 100%

By the aforesaid method detection computations, among the embodiment 2, the transformation efficiency of example 1 synthesis of sucrose cetylate reaches 80.5%, and the transformation efficiency of example 2 synthesis of sucrose cetylates reaches 82.3%.And the transformation efficiency of embodiment 3 employing traditional chemical method synthesis of sucrose cetylates only is about 50%.

Claims (1)

1. a primary yeast display lipase catalyzes and synthesizes the method for Surfhope SE Cosme C 1616, comprising:

The lipase gene of synthetic Rhizopus oryzae (Rhizopus oryzae) and the cell walls α agglutinin gene of pichia spp GS115, add the gene fragment of coding connection peptides GSSGGSGGSGGSGGSGS simultaneously at lipase gene C end, obtain nucleotide sequence pro-ROL-linker-α-agglutinin after the connection, wherein pro-ROL is lipase gene, Genbank number is AF229435, α-agglutinin is cell walls α agglutinin gene, Genbank number is M28164, and linker is the gene fragment of 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 '

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, carry out the linearization for enzyme restriction processing with the pPIC9K-ROL plasmid of Sal I; The about 15 μ l of goal gene that linearization for enzyme restriction is handled well add in the pichia spp GS115 competent cell, change in the electric revolving cup, and 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 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 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 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 sucrose 0.2g, palmitinic acid 0.5g, add the tool plug triangular flask that contains the 10mL trimethyl carbinol, mixing, preheating 10min, add yeast display lipase 0.01g then, place shaking bath to begin reaction, rotating speed is 200 rev/mins, temperature of reaction remains on 50 ℃, add the 0.5g molecular sieve behind the reaction 12h, molecular sieve bore diameter is less than 2nm, after continuing to react 12h, yeast display lipase and molecular sieve are removed in centrifugation, get supernatant liquor and be rotated evaporation and remove organic solvent, add normal hexane after wash 3 times to get the Surfhope SE Cosme C 1616 product in 4 ℃ of crystallizations, oven dry, pulverizing.