CN102212583B - Method for catalyzing and synthesizing octenyl succinic anhydride modified starch ester through yeast show lipase - Google Patents

Abstract

The invention discloses a method for catalyzing and synthesizing octenyl succinic anhydride modified starch ester through yeast show lipase, comprising the following steps of: dissolving grain starch in water; after imbibition, adding octenyl succinic anhydride and the yeast show lipase; stirring and reacting at 34-36 degrees centigrade for 1-1.5 h; and separating and purifying to obtain starch sodium ester sodium octenyl succinate. A method for preparing the yeast show lipase comprises the following steps of: transforming linearly treated recombinant plasmids into pichia pastoris GS115; inoculating the obtained transformant into a BMMY (buffered methanol-complex medium) culture medium; after inducing and culturing for 72-144 h, centrifugally collecting thallus; and washing, biologically imprinting, freezing and drying the thallus so as to obtain the yeast show lipase. By showing the lipase outside cells, the octenyl succinic anhydride modified starch ester is catalyzed and synthesized through the lipase preparation. By means of the method disclosed by the invention, the transformation efficiency is improved; furthermore, the reaction time is shortened; and the production cost is reduced.

Description

The yeast display lipase catalyzes and synthesizes the method for starch octenyl succinate anhydride

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 starch octenyl succinate anhydride.

Background technology

Starch octenyl succinate anhydride is at present unique by U.S. FDA and China's approval starch ester as foodstuff additive, is the most effective foodstuff additive of stabilizing food product opaque system.Starch octenyl succinate anhydride all is by octenyl succinic acid anhydride (Octenyl Succinic Anhydride at present, OSA) generate through esterification under weak subtracting property condition with starch slurry, the subject matter that exists be esterification speed inadequately fast, the reaction times is long, cause side reaction product to generate (side reaction I) and starch octenyl succinate anhydride generation hydrolysis (side reaction II), thereby influence esterification efficient, and make the side reaction product that mixes in the starch octenyl succinate anhydride finished product.

In chemical method synthesizing octene base succinic acid starch ester, biological enzyme can obtain the reaction product of high substitution value within a short period of time.With regard to the same reaction system, biological enzyme is compared chemical method and is significantly promoted reaction efficiency and productive rate, all once also is significantly increased with stability.Simultaneously, adopt biological enzyme to avoid chemical method severe reaction conditions, no specificity, product complexity, drawbacks such as by product is many, separation is difficult, poor stability.Lipase be present esterification 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 starch octenyl succinate anhydride, this method can improve starch octenyl succinate anhydride sodium synthetic esterification efficient and productive rate, reduces synthetic cost.

One primary yeast display lipase catalyzes and synthesizes the method for starch octenyl succinate anhydride, comprising:

Cereal starch is water-soluble, add octenyl succinic acid anhydride and yeast display lipase behind the imbibition, 34～36 ℃ of following stirring reactions 1～1.5 hour, separate, purifying makes starch octenyl succinate anhydride.

In weight part, reacting each proportioning raw materials can be as follows: 100～120 parts in water, 32～35 parts of cereal starchs, 1～1.2 part of octenyl succinic acid anhydride, 1～2 part of yeast display lipase.

Preferably, described separation, purification process are: reacting liquid pH value is adjusted to 7.0, and through precipitation, washing, spraying drying obtains powder.

Preferably, described stir speed (S.S.) is 200～300 rev/mins.

Described yeast display lipase prepares by the following method:

Change the recombinant plasmid of linearization process over to pichia spp (Pichia pastoris) GS115, 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 to represent lipase esterification is carried out catalysis, operational stability, thermotolerance and repeatability have not only been improved, reaction times also shortens greatly, dropped to 1～2 hour from original 12 hours, to represent the lipase production cost low for this yeast surface in addition, catalyzes and synthesizes the transformation efficiency height.

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 T4DNA 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 25 μ F conditions, 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 pH 7.0 phosphoric acid buffers 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 starch octenyl succinate anhydride

Example 1 is got 350g cereal starch water and is mixed with 35% starch milk (starch butt), makes the starch milk water-swelling in 65 ℃ of processing 15min that absorb water down, the cooling back adds the yeast display lipase of the above-mentioned preparation of 1g, adds the 10g octenyl succinic acid anhydride then in batches, places 85-1 type magnetic stirring apparatus to stir and begins reaction, rotating speed is 100 rev/mins, temperature of reaction remains on 34 ℃, stops to stir behind the reaction 1h, regulates pH value to 7.0, through precipitation, washing, spraying drying obtains powder.

Example 2 is got 400g cereal starch water and is mixed with 40% starch milk (starch butt), makes the starch milk water-swelling in 65 ℃ of processing 15min that absorb water down, the cooling back adds the yeast display lipase of the above-mentioned preparation of 2g, adds the 15g octenyl succinic acid anhydride then in batches, places 85-1 type magnetic stirring apparatus to stir and begins reaction, rotating speed is 150 rev/mins, temperature of reaction remains on 36 ℃, stops to stir behind the reaction 1.5h, regulates pH value to 7.0, through precipitation, washing, spraying drying obtains powder.

Embodiment 3 adopts traditional chemical method synthesizing octene base succinic acid starch ester

Example 1 is got 350g cereal starch water and is mixed with 35% starch milk (starch butt), makes the starch milk water-swelling in 65 ℃ of processing 15min that absorb water down, add the 10g octenyl succinic acid anhydride then in batches, place 85-1 type magnetic stirring apparatus to stir and begin reaction, rotating speed is 100 rev/mins, and temperature of reaction remains on 34 ℃, stop to stir behind the reaction 1h, regulate pH value to 7.0, through precipitation, washing, spraying drying obtains powder.

Embodiment 4 measures substitution value and phosphorus content

Accurately take by weighing the 2.0g starch octenyl succinate anhydride and place the 250mL beaker, after wetting with the pure Virahol of 10mL, add the aqueous isopropanol of 15mL 2.5mol/L hydrochloric acid, magnetic agitation 30 minutes, add 50mL 90% (preparation by weight percentage) aqueous isopropanol then, continue to stir 10 minutes.Sample is moved into B, with 90% washed with isopropyl alcohol to there not being Cl ^-(with the check of 0.1mol/L Silver Nitrate).Sample is moved in the beaker of 500mL, adding distil water is to 300mL again, and boiling water bath 20 minutes adds 2 phenolphthalein, uses 0.1mol/L NaOH titration to pink while hot.Substitution value (DS) calculation formula is as follows:

Substitution value=0.1624A/ (1-0.210A), in the formula: A is the amount of substance that every gram starch octenyl succinate anhydride consumes 0.1mol/L NaOH standardized solution, mmol.

Substitution reaction efficient=actual substitution value/theoretical substitution value * 100%

According to aforesaid method the starch octenyl succinate anhydride sample that embodiment 2 and embodiment 3 make is detected, wherein among the embodiment 2, substitution value and the reaction efficiency of example 1 product are respectively 0.0198 and 91.5%, and substitution value and the reaction efficiency of example 2 products are respectively 0.0192 and 87.1%.And the substitution value of embodiment 3 products and reaction efficiency only are 0.0175 and 75%.

Claims (1)

1. a primary yeast shows that fatty acid enzyme catalyzes and synthesizes the method for starch octenyl succinate anhydride, 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, 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 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, then at 1500V, 400 Ω, the 10ms that shocks by electricity under the 25 μ F conditions, and the sorbyl alcohol of the about 1mL precooling of adding;

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, 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, namely obtain the yeast display lipase of handling through biological trace;

Getting 350g cereal starch water is mixed with 35% starch milk (starch butt), makes the starch milk water-swelling in 65 ℃ of processing 15min that absorb water down, the cooling back adds the yeast display lipase of the above-mentioned preparation of 1g, adds the 10g octenyl succinic acid anhydride then in batches, places 85-1 type magnetic stirring apparatus to stir and begins reaction, rotating speed is 100 rev/mins, temperature of reaction remains on 34 ℃, stops to stir behind the reaction 1h, regulates pH value to 7.0, through precipitation, washing, spraying drying obtains powder.