CN102286110B - Method for extracting porphyra polysaccharide through ultrasonic treatment with cooperation of yeast display type polygalacturonase - Google Patents
Method for extracting porphyra polysaccharide through ultrasonic treatment with cooperation of yeast display type polygalacturonase Download PDFInfo
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- CN102286110B CN102286110B CN2011102639586A CN201110263958A CN102286110B CN 102286110 B CN102286110 B CN 102286110B CN 2011102639586 A CN2011102639586 A CN 2011102639586A CN 201110263958 A CN201110263958 A CN 201110263958A CN 102286110 B CN102286110 B CN 102286110B
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Abstract
The invention discloses a method for extracting porphyra polysaccharide through ultrasonic treatment with cooperation of yeast display type polygalacturonase. The method comprises the following steps of: adding dry porphyra powder and yeast display type polygalacturonase to water, mixing uniformly, stirring to react at 35-37 DEG C for 30-45min, crushing at 28-35 DEG C for 1-2 hours by utilizing ultrasonic with power of 730-750W to obtain a cell disruption liquid, separating and purifying to obtain the porphyra polysaccharide. According to the method disclosed by the invention, and the polygalacturonase is displayed outside pichia pastoris cells and is cooperated with the ultrasonic to crush porphyra cells, thus the process is simple and convenient, the condition is gentle, the process is easy to be controlled and is safe and reliable, and higher-yield and higher-purity porphyra polysaccharide can be obtained.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a primary yeast displaying type polygalacturonase synergistic supersonic wave and handle the method for extracting laver amylose.
Background technology
Laver contains the abundant active polysaccharide of being made up of the polygalacturonic acid sulfuric ester, is present in the laver cell wall.Laver amylose is nutritious, the physiological function that not only has general polysaccharides and food fibre and had, but also have multiple biological activitys such as anticoagulation, reducing blood-fat, mutation, anti-ageing and anti-radiation.Owing to also contain a large amount of laver albumen in the laver, mutually combine with laver amylose, influence the separation of laver amylose, for this reason, extraction, separation, the purification process of laver amylose are significant to its biological activity of follow-up study in the research laver.
At present, from laver, extract laver amylose and adopt the chemical extraction method more, have problems such as extraction time length, solvent-oil ratio is big, the extraction yield is lower, security is not high.Publication number is that the application for a patent for invention of CN1872882A discloses a kind of method for preparing laver amylose; Laver through smash, alcohol reflux, ultrasonic disruption, water-bath extracting, concentrate, organic solvent extraction, decolouring, refrigeration, vacuum freezedrying, obtain laver amylose.This method has kept the activity of laver amylose largely, and the laver amylose yield is higher, and pigment content is low, but this method still adopts number of chemical reagent to extract laver amylose, and step is many, complex operation, and extraction conditions is complicated, and security is low.
Will from laver, obtain laver amylose fully, should destroy the laver cell structure as much as possible, the polygalacturonic acid sulfuric ester of partly degrading makes in its water soluble.Enzymolysis process is compared the chemical extraction method and is had simple to operate, mild condition, control easily, characteristics such as safe and reliable, is widely used in the extraction and the preparation of active substance at present, but adopts the Enzymatic Extraction speed of response slower merely, and the reaction times is longer.The ultrasonication smudge cells also is a method commonly used in present natural product extraction, the active substance preparation, but ultrasonication more high-power, the long period causes the inactivation of active substance easily; On the other hand; The simple ultrasonication of using; The polygalacturonic acid sulfuric ester of laver cell wall is kept perfectly basically, only produces a small amount of space, influences the laver cell crushing efficiency; And the excessive polygalacturonic acid sulfuric ester of molecular weight is water insoluble, and combines with the laver cell fragment and to be difficult to separate.Adopting the fragmentation of enzymolysis process synergistic supersonic wave from laver, to extract laver amylose also studies less.
Yeast cell surface presenting and expressing system is the proteinic eucaryon display systems of a kind of immobilization expressing heterologous; Have behind glycosylation, the protein translation folding and with the advantage of the similar mechanism of secretion of Mammals; Compare phage and Bacillus coli cells surface display technology, more help efficiently expressing the complicated albumen of biologically active.Research shows that multiple enzyme comprises that vitamin H ligase enzyme, organophosphor hydrolytic enzyme etc. all successfully are illustrated in yeast cell surface, and enzyme work is compared wild-type enzyme raising is in various degree all arranged.Pichia yeast expression system is as a kind of new display protein system, have be easy to cultivate, characteristics such as breeding is fast, adjustable expression of exogenous gene, the correlative study developed recently is rapid, becomes the host bacterium that has application prospect in the yeast surface display system.
Summary of the invention
The invention provides a primary yeast displaying type polygalacturonase synergistic supersonic wave and handle the method for extracting laver amylose; With the laver is raw material; Utilize pichia spp surface display polygalacturonase; Handle broken laver cell through the enzymolysis synergistic supersonic wave again, can obtain to high yield the laver amylose of higher degree.
One primary yeast displaying type polygalacturonase synergistic supersonic wave is handled the method for extracting laver amylose, comprising:
Laver dry powder, yeast displaying type polygalacturonase are added mixing in the entry; In 35～37 ℃ of stirring reactions 30～45 minutes; Utilizing power in 28～35 ℃ again is the ultrasonic disruption 1～2 hour of 730～750W, obtains cytoclasis liquid, and separation and purification makes laver amylose.
The particle diameter of described laver dry powder is preferably 60～100 orders.
Preferably, in every gram laver dry powder, the add-on of described yeast displaying type polygalacturonase is 0.05～0.08g; More preferably, in every premium on currency, the add-on of described laver dry powder is 20～25g, and the add-on of described yeast displaying type polygalacturonase is 1～2g.
Preferably, described stir speed (S.S.) is 100～200 rev/mins.Through stirring, can promote the interaction between yeast displaying type polygalacturonase and the laver cell, improve the enzymolysis efficiency of yeast displaying type polygalacturonase to the laver cell wall.
Fragmentation through described ultrasonication promotion laver cell more helps the Degradation of yeast displaying type polygalacturonase to the laver cell wall.UW on the one hand, can produce great pressure through cavatition as a kind of physical energy, causes cell walls even the whole organism of the thing that is broken to break and makes intracellular organic matter discharge, spread or be dissolved out; On the other hand, the UW of suitable intensity can make the conformation generation forward of enzyme molecule transform, thereby improves the activity of enzyme, quickens enzyme digestion reaction.
Because ultrasonic frequency is high, energy is big; Can produce significant heat effect in the mechanism; In order to reduce the influence that heat effect is lived to yeast displaying type polygalacturonase enzyme, described hyperacoustic treatment temp is 28～35 ℃, and hyperacoustic power is 730～750W.
Described separation and purification comprises: the broken liquid of pair cell carries out centrifugal treating, gets supernatant, and supernatant obtains laver amylose through saltouing, go post precipitation.
Described saltouing can be operated according to following steps: in supernatant, add ammonium sulfate, left standstill 11～13 hours in 3～5 ℃; Wherein, the add-on of described ammonium sulfate is counted 200～250g whenever to go up clear liquid.Adopt the salt precipitation method, can the laver albumen that be dissolved in the supernatant be removed.Ammonium sulfate has significant especially salting out, in weakly acidic solution or neutral solution, can make albumen precipitation.
Described yeast displaying type polygalacturonase can prepare through 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 yeast displaying type polygalacturonase through flushing and lyophilize;
Described recombinant plasmid is by initial carrier pPIC9K and insert the polygalacturonase gene in MF α 1 signal peptide downstream among the initial carrier pPIC9K successively, the cell walls α agglutinin gene of pichia spp GS115 is formed.
Described polygalacturonase gene can be selected for use and be the sequence of HQ446162.1 Genbank number, and pichia spp (Pichia pastoris) GS115 is a commercially produced product, can buy from Invitrogen company.Genbank number of the cell walls α agglutinin gene sequence of pichia spp GS115 is M28164.
Carrier pPIC9K is commercially produced product (like an 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 upper reaches (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.
The present invention is through importing pichia spp cell GS115 with polygalacturonase gene and cell walls α agglutinin gene; After the pichia spp cell induction is cultivated; Polygalacturonase is expressed justacrine outside born of the same parents; Utilize cell walls α lectin that this polygalacturonase is fixed on the pichia spp cell surface simultaneously, can effectively improve the activity of enzyme.The polygalacturonic acid sulfuric ester of laver cell wall is formed in the depolymerization of yeast displaying type polygalacturonase degradable; Destroy the laver cell wall construction; Combine ultrasonication through the polygalacturonase enzymolysis, then the degraded depolymerization of polygalacturonic acid sulfuric ester can further improve the cavitation effect that follow-up ultrasonication produces, and promotes laver cell broken; And the polygalacturonic acid sulfuric ester water soluble that part degraded back molecular weight diminishes, thus with the laver cell chip separation.
The present invention utilizes yeast displaying type polygalacturonase that the polygalacturonic acid sulfuric ester of forming the laver cell wall is carried out enzymolysis so that its water soluble; And then synergistic supersonic wave handles broken laver cell, can efficiently obtain to contain the laver content extracting solution of laver amylose; Again through saltout with centrifugal treating removing the laver albumen in the extracting solution, can obtain the higher laver amylose of purity in high yield ground.Compare the traditional chemical extraction method, this method has that technology is easy, mild condition, be easy to control, characteristics such as safe and reliable; Compare and adopt the wild-type enzyme enzymolysis, this method enzymolysis efficiency is higher, and the running time is short, and it is all higher to extract yield and product purity.
Embodiment
The preparation of yeast displaying type polygalacturonase
Method through synthetic; Synthetic polygalacturonase gene (Genbank number: HQ446162.1) with the cell walls α agglutinin gene of pichia spp GS115 (Genbank number: M28164); Simultaneously add connection peptides sequence GSSGGSGGSGGSGGSGS (linker) at polygalacturonase gene C end; Obtain nucleotide sequence PGA-linker-α-agglutinin after the connection; Add EcoR I and Not I restriction enzyme site simultaneously at the sequence two ends, wherein PGA is the polygalacturonase gene, and α-agglutinin is a cell walls α agglutinin gene.
With above-mentioned artificial synthesized sequence is template, utilizes following primer right, carries out pcr amplification,
Upstream primer: 5 '-ATGTTGTTATCGACACACAGTATTGTTCTG-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-PGA plasmid, through electrophoresis check and recovery plasmid.For making goal gene and pichia spp GS115 that His 4 unit points displacement reorganization take place, with Sal I the pPIC9K-PGA plasmid is carried out linearization for enzyme restriction 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 15 minutes, the 10ms that under 1500V, 400 Ω, 25uF condition, shocks by electricity then, 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 was seeded in the BMGY substratum fermentation culture 30 hours, centrifugal collecting cell; Again cell is placed the BMMY substratum inducing culture 144 hours that contains 0.5% (volume percent) methyl alcohol; Behind the centrifugal collecting cell, water flushing, be seeded in the YGC substratum 30 ℃ and cultivated 120 hours; 5000g collected thalline in centrifugal 10 minutes then; Again with 50mM pH 7.0 phosphoric acid buffers washings, again through dry 24 hours of German Christ vacuum freeze drier, obtain yeast displaying type polygalacturonase after-80 ℃ of following pre-freezes behind the distilled water wash.
The mensuration of laver amylose yield
The laver amylose Determination on content adopts the phenolsulfuric acid colourimetry, is standard substance with the D-semi-lactosi.The extraction yield (T) of laver amylose is defined as the amount of extracting the laver amylose obtain and the ratio of laver raw material dry weight.
Embodiment yeast displaying type polygalacturonase synergistic supersonic wave is handled and is extracted laver amylose
Embodiment 1 gets particle diameter 60 purpose laver dry powder 20 grams and adds in the 1000 gram water, and mixing is mixed with suspension-s; Add yeast displaying type polygalacturonase 1 gram according to method for preparing then, stirring reaction is 30 minutes under 35 ℃ of conditions, and stir speed (S.S.) is 100 rev/mins; Carry out ultrasonic disruption, ultrasonic power is 730W, and the supersound process time is 65 minutes, and the supersound process temperature is 30 ℃; Through 5000g centrifugal 10 minutes, remove deposition, obtain containing the supernatant of laver content; In supernatant, add ammonium sulfate, make that the concentration of ammonium sulfate reaches 20% (W/V) in the supernatant, left standstill 12 hours in 4 ℃; Through 5000g centrifugal 10 minutes, remove deposition, obtain containing the solution of laver amylose.
The measuring method that adopts above-mentioned laver amylose yield detects the extraction yield of laver amylose in the present embodiment, and the result shows that the extraction yield (T) of laver amylose is 38.4% in the present embodiment.
Embodiment 2 gets particle diameter 100 purpose laver dry powder 25 grams and adds in the 1000 gram water, and mixing is mixed with suspension-s; Add yeast displaying type polygalacturonase 2 grams according to method for preparing then, stirring reaction is 45 minutes under 37 ℃ of conditions, and stir speed (S.S.) is 200 rev/mins; Carry out ultrasonic disruption, ultrasonic power is 750W, and the supersound process time is 85 minutes, and the supersound process temperature is 35 ℃; Through 5000g centrifugal 10 minutes, remove deposition, obtain containing the supernatant of laver content; In supernatant, add ammonium sulfate, make that the concentration of ammonium sulfate reaches 25% (W/V) in the supernatant, left standstill 12 hours in 4 ℃; Through 5000g centrifugal 10 minutes, remove deposition, obtain containing the solution of laver amylose.
The measuring method that adopts above-mentioned laver amylose yield detects the extraction yield of laver amylose in the present embodiment, and the result shows that the extraction yield (T) of laver amylose is 39.5% in the present embodiment.
Claims (5)
1. a primary yeast displaying type polygalacturonase synergistic supersonic wave is handled the method for extracting laver amylose, comprising:
Laver dry powder, yeast displaying type polygalacturonase are added mixing in the entry; In 35 ~ 37 ℃ of stirring reactions 30 ~ 45 minutes; Utilizing power in 28 ~ 35 ℃ again is the ultrasonic disruption 1 ~ 2 hour of 730 ~ 750W, obtains cytoclasis liquid, and separation and purification makes laver amylose; In every gram laver dry powder, the add-on of described yeast displaying type polygalacturonase is 0.05 ~ 0.08g;
Described yeast displaying type polygalacturonase prepares through 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 yeast displaying type polygalacturonase through flushing and lyophilize;
Described recombinant plasmid is by initial carrier pPIC9K and insert the polygalacturonase gene in MF α 1 signal peptide downstream among the initial carrier pPIC9K successively, the cell walls α agglutinin gene of pichia spp GS115 is formed;
The Genbank of said polygalacturonase gene number is HQ446162.1;
Genbank number of said cell walls α agglutinin gene is M28164.
2. method according to claim 1 is characterized in that: the particle diameter of described laver dry powder is 60 ~ 100 orders.
3. method according to claim 1 is characterized in that: described stir speed (S.S.) is 100 ~ 200 rev/mins.
4. method according to claim 1 is characterized in that: described separation and purification comprises: the broken liquid of pair cell carries out centrifugal treating, gets supernatant, and supernatant obtains laver amylose through saltouing, go post precipitation.
5. method according to claim 4 is characterized in that: described saltouing comprises: in supernatant, add ammonium sulfate, left standstill 11 ~ 13 hours in 3 ~ 5 ℃; Wherein, the add-on of described ammonium sulfate is counted 200 ~ 250g whenever to go up clear liquid.
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