CN106755195A

CN106755195A - A kind of method that carrageenan oligosaccharide is prepared by Asia centipede algae

Info

Publication number: CN106755195A
Application number: CN201611156183.1A
Authority: CN
Inventors: 李江
Original assignee: First Institute of Oceanography SOA
Current assignee: First Institute of Oceanography SOA
Priority date: 2016-12-14
Filing date: 2016-12-14
Publication date: 2017-05-31
Anticipated expiration: 2036-12-14
Also published as: CN106755195B

Abstract

The present invention provides a kind of method for preparing carrageenan oligosaccharide, it is after the homogenate of Asia centipede algae is first digested with cellulase and high temperature carrageenase, again by activated-charcoal column, being eluted with the ethanol solution of redistilled water and 5 25% grade different volumes fraction gradients respectively, carrageenan oligosaccharide is obtained.The amino acid sequence of its high temperature carrageenase is SEQ ID NO:1.The present invention prepares alga oligosaccharides from centipede algae first；Secondly, the present invention realizes that a step prepares the technique of alga oligosaccharides using the preparation technology of high temperature carrageenase combination complex cellulase, greatly improves the preparation efficiency of alga oligosaccharides, using the preparation technology, the yield of carrageenan oligose is up to the 8% of centipede algae dry weight.

Description

A kind of method that carrageenan oligosaccharide is prepared by Asia centipede algae

Technical field

The invention belongs to active material preparing technical field, and in particular to one kind prepares carrageenan oligosaccharide by Asia centipede algae Method.

Background technology

Low molecule glucide in marine alga all derives from natural resources, and safe, effect is special, public credibility Height, therefore used as health care, medical cosmetic addition, medical auxiliary materials, the specific use of medicine, the speed of its research and extension is fast, should It is wide with scope, therefore after succeeding in developing, once obtain one seat, its direct economic benefit in health care, medicinal cosmetic field It is huge.And by deep processing, limitation of the tangleweed as additives such as food, chemical industry will be broken through, by the sale band of product The cultivation of dynamic marine alga, and thus will indirectly drive the cultivation of algae, increase the ecological environment for cultivating income, improving aquaculture sea area.

But the preparation of alga oligosaccharides is always the bottleneck for restricting its application, the method for preparing carrageenan oligosaccharide mainly has three Kind：Biological enzyme, chemical method and Physical.Traditional chemical method and the major defect of Physical be reaction condition it is whard to control, The product degree of polymerization is very high and distributing inhomogeneity, and a large amount of waste water for producing easily pollute environment.Biological enzyme can substantially overcome these Defect, has the advantages that the gentle easy to control, Substratspezifitaet of reaction condition is good, but existing carrageenase is typically derived from normal temperature Microorganism, major part is thermally labile enzyme, when temperature is higher than 50 DEG C will fast deactivation, seriously limit it few in carragheen Application in sugared preparation technology.

The content of the invention

To solve the problems, such as alga oligosaccharides traditional preparation methods inefficiency, the present invention provides one kind and prepares carrageenan oligosaccharide Preparation technology, so as to make up the deficiencies in the prior art.

The method of the present invention is comprised the following steps：

1) Asia centipede algae is rinsed well, chopping, homogenate are made into the homogenate of 3-5% (w/v)；

2) in the homogenate of centipede algae, cellulase is added, enzymolysis 1-4h or more is carried out in 55 DEG C of effects；

Preferably, adding cellulase according to the ratio of the U/g of mass ratio 1.0-3.5 ten thousand；

3) then addition high temperature carrageenase, 55 DEG C, 1-7h is digested under the conditions of 35rpm；

Preferably, adding high temperature carrageenase according to the amount of 0.5-11.0U/g centipede algaes；

4) enzymolysis liquid weighs the enzymolysis liquid of concentration through centrifugation, concentration, is slowly added into activated-charcoal column, respectively steaming again The ethanol solution wash-out of the different volumes fraction gradient such as water and 5-25%, flow control is 3.0~4.0ml/min；Collect various Eluent is concentrated into right amount, freeze-drying, obtains carrageenan oligosaccharide.

The amino acid sequence of described high temperature carrageenase is SEQ ID NO:1；

Activated carbon in the activated-charcoal column used in the above method, is after first being soaked with watery hydrochloric acid, then to remove watery hydrochloric acid, is used Soaked in absolute ethyl alcohol, then absolute ethyl alcohol addition distilled water immersion is poured out, finally by activated carbon dry for standby.

The present invention prepares alga oligosaccharides from centipede algae first；Secondly, the present invention is combined compound using high temperature carrageenase The preparation technology of cellulase, realizes that a step prepares the technique of alga oligosaccharides, greatly improves the preparation efficiency of alga oligosaccharides, Using the preparation technology, the yield of carrageenan oligose is up to the 8% of centipede algae dry weight.

Brief description of the drawings

Fig. 1：The SDS-PAGE collection of illustrative plates of thermally-stabilised carrageenase DNA recombinant expression

Fig. 2：The optimum temperature figure of thermally-stabilised carrageenase；

Fig. 3：Digest the chromatography testing result figure of oligosaccharides；Wherein note：M is compareed for standard oligosaccharide；Be followed successively by enzyme activity enzyme control, 15min、30min、60min、90min、120min、150min、180min、360min、720min、1440min。

Fig. 4：Influence figure of the addition of cellulase to hydrolysis result；

Fig. 5：Influence figure of the action time of cellulase to hydrolysis result；

Fig. 6：Influence figure of the addition of carrageenase to hydrolysis result；

Fig. 7：Influence figure of the enzymolysis time to hydrolysis result.

Specific embodiment

The preparation of alga oligosaccharides generally uses chemical method and Physical, and the method major defect is not easily-controllable reaction condition System, poor product quality, and easily pollute environment.And being prepared using biological enzyme can substantially overcome these defects, but due to existing It is thermally labile enzyme that carrageenase is most, when temperature is higher than 50 DEG C will fast deactivation, seriously limit it in OK a karaoke club Application in glue oligosaccharides preparation technology.The invention provides a kind of thermally-stabilised carrageenase, the enzyme is degraded system under the conditions of 55 DEG C Standby carrageenan oligosaccharide, not only substantially increases the dissolubility of substrate and product, reduces the adherent effect of material and reactor, effectively Improve the preparation efficiency of carrageenan oligosaccharide.

Wherein the detection method of alga oligosaccharides the step of it is as follows：

A, capillary point sample pencil gently draw a straight line at the 2cm of silica gel plate bottom, for convenience of point sample, in point sample It is preceding to do a mark at 2cm on straight line.With 0.5mm glass point samples capillary respectively by carrageenan oligosaccharide standard items and card Draw glue oligosaccharide sample point sample in the mark on silica gel plate straight line, when with capillary point sample, capillary should be perpendicular to silica gel plate Direction point sample, and the time of capillary contact silica gel plate should be as far as possible short, and not so point sample spot diameter is difficult to control to, point sample spot Spot diameter will control 2mm, allow point of sample to spontaneously dry.

B, chromatography cylinder exhibition layer will have been put excellent silica gel plate bottom (point sample end) and be put into the chromatography cylinder for having filled developing agent, Chromatoplate must be laid flat, and developing agent liquid level must not exceed point of sample, and chromatography cylinder closing, opens up layer from bottom to top during chromatography, works as exhibition Layer forward position is reached during away from 1 centimeters of thin plate top, takes out silica gel plate, is dried in 60 DEG C of baking ovens.

C, chromogenic reagent uniformly spray on the chromatographic silica gel plate dried developer, are placed in 60 DEG C of baking ovens and add Heat, until showing chromatography spot.

The present invention is described in detail below in conjunction with specific embodiment.

Embodiment 1：The preparation of thermally-stabilised carrageenase and its sequence signature

Microbiological specimens pick up from Cali of Indonesia Anda island east coast hot spring sample (5 ° 44 ' 46 " N, 105 ° 35 ' 12 " E), identify that the determination bacterium belongs to for bacillus (Bacillus) through 16S DNA；Preliminary fermentation experiments show that the bacterial strain exists Ability with degraded carrageenan under high temperature.The bacterial strain that will be purified is added in the glycerite containing 30wt%, is stored in -80 DEG C and is surpassed In low temperature refrigerator.

(1) target DNA fragment is prepared

Bacillus bacterium (Bacillus) seed liquor is inoculated in Zobell 2216E sea water mediums, in 50 DEG C, After cultivating 24h in the shaken cultivation case of 150rpm, (Tiangen companies, DP302- are purchased from bacterial genomes extracts kit 02) chromosomal DNA is extracted, its electrophoresis DNA concentration is detected, DNA length is more than 40kb, meets requirement for construction data base.

(2) genomic DNA is partial digested

Bacillus bacterium (Bacillus) chromosomal DNA is partial digested through Sau3AI restriction endonucleases, Sau3AI endonuclease digestions After 60min, Insert Fragment is obtained.After digestion 60min, just digestion is complete for chromosomal DNA, it is adaptable to build genomic library； After the digestion time is more than 80min, excessively, each fragment is not evenly distributed for digestion, and mostly small fragment, it is unfavorable for building storehouse.Cause This selects digestion 60min as the incomplete digestion time of bacterial strain HT19 chromosomal DNAs.

(3) structure of genomic library

According to pET22b carriers (being purchased from Novagen companies) and Insert Fragment about 1:3 mole are compared to coupled reaction.Reaction In 16 DEG C of connections overnight, concentration connection product takes 1 μ l transformed competence colibacillus cell E.coli DH5 α to system respectively to 3 μ l, cultivates It is coated with afterwards and the LB plating mediums containing ampicillin (50 μ g/ml), IPTG (20 μ g/ml) and X-Gal (40 μ g/ml) On, in 37 DEG C of quiescent cultures overnight to visible colonies, according to bacterium colony indigo plant hickie reaction screening transformant.Random picking about 18000 Individual bacterium colony is preserved as the clone of genomic library, random from flat board to select 15 white single bacterium colonies, extracts plasmid, is used BcaBEST Primer M13-47 and BcaBEST Primer RV-M expand purpose segment through PCR, and primer sequence is as follows：

BcaBEST Primer M13-47：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’

BcaBEST Primer RV-M：5’-GAGCGGATAACAATTTCACACAGG-3’

PCR reaction conditions are：95 DEG C of predegeneration 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 240s, 30 Individual circulation；72 DEG C of extension 10min.

Each clone has exogenous DNA after testing, and minimum Insert Fragment is 800bp or so, and maximum Insert Fragment 6kb is left The right side, average Insert Fragment size is about 4kb or so.

Being screened from genomic library has the clone of substantially hydrolysis circle around one.Extract recombinant plasmid and be sequenced, obtain The complete ORF of heat production stabilization carrageenase.The complete length of ORF is 1125bp, and 374 amino acid, theoretical molecular are encoded altogether It is 42.5kDa.

Embodiment 2：The recombinant expression carrier of thermally-stabilised carragheen builds

By in the thermally-stabilised carragheen gene cloning of present invention acquisition to expression vector, recombinant strains are built.First Based on full length sequence, the primer of design amplification full genome：

Sense primer F-GTCGTCGTCGACCAGTAATTTTACCGGTATC；

Anti-sense primer R-CATAAATCTAGACACTCACGACTAAAGTATCT；

PCR amplifications confirm full length gene sequence.Using the method construction expression plasmid of enzyme cutting clone, i.e., with using BamH The restriction endonucleases of I/Xba I carry out double digestion to the PCR primer and expression vector pET-30 (a) of thermally-stabilised carrageenase respectively, reclaim pure Change；It is attached using T4 DNA Ligase, and converts the competent cell of TOP10.(it is purchased from using plasmid extraction kit Tiangen companies) extract positive colony plasmid, after testing, nucleotide sequence such as SEQ ID NO：Shown in 2, the gene code 400 amino acid, amino acid sequence such as SEQ ID NO：Shown in 1.

The plasmid of positive colony obtained above is transformed into e. coli bl21 (DE3) expression bacterial strain, construction expression Recombinant bacterial strain；Detailed process is as follows：

- 80 DEG C of BL21 of preservation are taken, in thawed on ice, 10 μ l recombinant plasmids is drawn with the pipette rifle point of precooling, added The BL21 for just having melted；Jog is mixed, and ice bath places 30min, is quickly transferred in 42 DEG C of water-baths, accurate to place 90 seconds；Rapidly It is transferred on ice, cools down 2min；800 μ l LB fluid nutrient mediums are subsequently adding, in 37 DEG C, 150rpm, 45min；Coating contains and blocks that The LB plating medium incubated overnights of the μ g/mL of mycin 50.The above-mentioned expression recombinant bacterial strain for building is transferred to 100ml and contains card In the LB fluid nutrient mediums of that mycin 50 μ g/mL, 37 DEG C of Amplification Cultures are until bacterium solution OD₆₀₀When value is up to 0.6,15 DEG C of coolings 30min, adds isopropyl-beta D-thio galactopyranoside (IPTG) to make its final concentration of 1.0mmol/L, 15 DEG C of shaking table cultures 24h.Low-temperature centrifugation collects supernatant, concentrated freeze-dried.Then Ni is carried out²⁺Affinity chromatography, because the recombinant protein N end expressed contains 6 × His tag, affine can be adsorbed onto in chromatographic column, after the imidazole solution gradient elution of various concentrations, collect eluent, freeze It is standby.The SDS-PAGE electrophoresis detection results of the induced expression of recombinant protein and after purification recombinant protein are shown in Fig. 1.

(5) thermally-stabilised carrageenase Activity determination and enzyme specificity analysis

Thermally-stabilised carrageenase activity obtained above is determined using DNS (3,5- dinitrosalicylic acids) method.Specific behaviour Make as follows：

Take 1mL inactivations respectively (contains 0.2wt% with the thermally-stabilised carrageenase enzyme liquid and 2mL substrate solutions of no inactivation Kappa-carrageenan) in 60 DEG C of thermostatic water-bath 30min, take 1mL product and be added in 1.5mL DNS solution, boil Water-bath 5min.Room temperature, plus distilled water are then rapidly cooled to 25mL, are mixed and OD values is surveyed under 520nm wavelength.The heat for measuring is steady It is 800U/ml to determine carrageenase activity.

In addition, thermally-stabilised carrageenase enzyme liquid obtained above is directly dripped on solid plate culture medium, contaminated with iodine solution It can be seen that obvious transparent circle, it, with carragheen degrading activity, is follow-up grinding to show that thermally-stabilised carrageenase is after color Study carefully and apply there is provided good material.

To determine the heat resistance of thermally-stabilised carrageenase, respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C Carry out the enzyme activity that enzymatic reaction 30min, DNS method determine thermally-stabilised carrageenase.It is relative its to be calculated with enzyme activity peak as 100% Enzyme activity.

After measured, thermally-stabilised carrageenase optimal reactive temperature is 60 DEG C (Fig. 2), under conditions of being 80 DEG C in temperature, should Enzyme still has 50% or so enzyme activity, and more existing carrageenase has obvious high temperature resistance characteristic.

Thermally-stabilised carrageenase after purifying is lyophilized is that can be used for the preparation of centipede algae oligosaccharides, and we have first verified that the heat is steady Determine composition analysis of the carrageenase to centipede phycocolloid catabolite, as a result see Fig. 3.

Embodiment 2

A, Asia centipede algae is rinsed well, the chopping of centipede algae, homogenate are made into the homogenate of 5% (w/v)；According to 0.5- 3.5 ten thousand U/g centipedes algaes add cellulase, 55 DEG C, digest 2h under the conditions of 35rpm after, then according to the amount of 5.0U/g centipede algaes Addition carrageenase, 55 DEG C, 6h is digested under the conditions of 35rpm, the reduced sugar growing amount and viscosity for determining each experimental group make reduced sugar The relation curve of relative amount, relative viscosity and enzyme addition, it is determined that most suitable cellulase addition.

As seen from Figure 4, different amounts of cellulase is added, the cellulose in the centipede algae of Asia is entered under the conditions of 55 DEG C Row 2h degrades.Result shows that in the range of 0.5~2.5 ten thousand U/g centipede algaes, the growing amount of reduced sugar is in cellulase addition Obvious ascendant trend, enzymolysis fluid viscosity is also in be decreased obviously trend.Under conditions of 2.5 ten thousand U/g centipede algaes, the life of reduced sugar It is no longer obvious into the variation tendency measured and digest fluid viscosity.Therefore, on the premise of cellulose degradation effect is ensured, selection is more The enzyme addition manner of saving, the most suitable addition of cellulase is defined as 2.5 ten thousand U/g centipede algaes.Collection, concentration enzymolysis liquid are standby With.

B, by 100g activated carbons first with 10% watery hydrochloric acid 500ml soaked overnights.Watery hydrochloric acid is poured out, adds 500ml anhydrous Ethanol soaks, then pours out absolute ethyl alcohol addition distilled water immersion, finally by 80 DEG C of dry for standby of activated carbon；

C, enzymolysis liquid weigh the enzymolysis liquid of concentration through centrifugation, concentration, are slowly added into activated-charcoal column (4 × 60cm), 8% The ethanol solution wash-out of volume fraction gradient, flow control is 3.0~4.0ml/min.Various eluents are collected to be concentrated into right amount, Freeze-drying, it is standby.Enzymolysis product composition, purity and yield are shown in Table 1.

Asia centipede algae oligosaccharides yield after the degraded of table 1

Embodiment 2

A, Asia centipede algae is rinsed well, the chopping of centipede algae, homogenate are made into the homogenate of 5% (w/v)；According to 2.5 Ten thousand U/g centipedes algaes add cellulase, 55 DEG C, digest 1-4h under the conditions of 35rpm after, then add according to the amount of 5.0U/g centipede algaes Plus carrageenase, 55 DEG C, 6h is digested under the conditions of 35rpm, determine the reduced sugar growing amount and viscosity of each experimental group；Make reduced sugar phase To the relation curve of content, relative viscosity and enzyme addition, it is determined that most suitable cellulase addition.

It is determined that on the basis of most suitable cellulase addition, the degradation time to cellulase is probed into, as a result It was found that, enzymolysis 2.5h or so growing amounts of reduced sugar and the viscosity of enzymolysis liquid no longer change substantially, illustrate degraded substantially completely (figure 5).Collection, concentration enzymolysis liquid are standby.

C, enzymolysis liquid weigh the enzymolysis liquid of concentration through centrifugation, concentration, are slowly added into activated-charcoal column (4 × 60cm), with The ethanol solution wash-out of 8% volume fraction gradient, flow control is 3.0~4.0ml/min.Collect various eluents be concentrated into it is suitable Amount, freeze-drying is standby.Enzymolysis product composition, purity and yield are shown in Table 2.

Asia centipede algae oligosaccharides yield after the degraded of table 2

Embodiment 3

A, Asia centipede algae is rinsed well, the chopping of centipede algae, homogenate are made into the homogenate of 5% (w/v)；According to 2.5 Ten thousand U/g centipedes algaes add cellulase, 55 DEG C, digest 2.5h under the conditions of 35rpm after, then according to 1.0-9.0U/g centipede algaes Amount addition carrageenase, determines the reduced sugar growing amount and viscosity of each experimental group, to determine by 55 DEG C, 6h is digested under the conditions of 35rpm The optimum amount of carrageenase.

As shown in fig. 6, the most suitable addition of carrageenase is about 7.0U/g centipede algaes, degraded under conditions of the addition 6h, the amount ascensional range of reduced sugar eases up, while the viscosity B coefficent of enzymolysis liquid is no longer obvious.Therefore 7.0U/g centipede algaes are selected Carrageenase addition is used as most suitable addition.Collection, concentration enzymolysis liquid are standby.

C, enzymolysis liquid weigh the enzymolysis liquid of concentration through centrifugation, concentration, are slowly added into activated-charcoal column (4 × 60cm), with The ethanol solution wash-out of 8% volume fraction gradient, flow control is 3.0~4.0ml/min.Collect various eluents be concentrated into it is suitable Amount, freeze-drying is standby.Enzymolysis product composition, purity and yield are shown in Table 3.

Asia centipede algae oligosaccharides yield after the degraded of table 3

Embodiment 4

A, Asia centipede algae is rinsed well, the chopping of centipede algae, homogenate are made into the homogenate of 5% (w/v)；According to 2.5 Ten thousand U/g centipedes algaes add cellulase, 55 DEG C, digest under the conditions of 35rpm after 2.5h and then add according to the amount of 7.0U/g centipede algaes Plus carrageenase, 55 DEG C, 1-7h is digested under the conditions of 35rpm, the reduced sugar growing amount and viscosity of each experimental group are determined, to determine most Good enzymolysis time.

As seen from Figure 7, the growing amount and viscosity of reduced sugar no longer change substantially after enzymolysis 4h, it is thus determined that carrageenase The best use of time is 4h.Collection, concentration enzymolysis liquid are standby.

C, enzymolysis liquid weigh the enzymolysis liquid of concentration through centrifugation, concentration, are slowly added into activated-charcoal column (4 × 60cm), with The ethanol solution wash-out of 8% volume fraction gradient, flow control is 3.0~4.0ml/min.Collect various eluents be concentrated into it is suitable Amount, freeze-drying is standby.Enzymolysis product composition, purity and yield are shown in Table 4.

The yield of Asia centipede algae oligosaccharides after the degraded of table 4

The present invention first with centipede algae as raw material, using the preparation technology of high temperature carrageenase combination complex cellulase, Realize that a step prepares the technique of alga oligosaccharides, greatly improve the preparation efficiency of alga oligosaccharides, using the preparation technology, OK a karaoke club The yield of oligosaccharides up to centipede algae dry weight 8%.

SEQUENCE LISTING

<110>Oceanographic Inst. No.1 of State Bureau of Oceanography

<120>A kind of method that carrageenan oligosaccharide is prepared by Asia centipede algae

<130>

<160> 2

<170> PatentIn version 3.5

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Met Ala Phe Ile Asn Ile Lys Pro Glu Leu Lys Gln Asn Met Glu Arg

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Claims

1. a kind of method for preparing carrageenan oligosaccharide, it is characterised in that described method includes the steps：

1) Asia centipede algae is rinsed well, chopping, homogenate are made into homogenate；

4) enzymolysis liquid weighs the enzymolysis liquid of concentration through centrifugation, concentration, is slowly added into activated-charcoal column, respectively with redistilled water and The ethanol solution wash-out of the different volumes fraction gradient such as 5-25%, flow control is 3.0~4.0ml/min；Collect various wash-outs Liquid is concentrated into right amount, freeze-drying, obtains carrageenan oligosaccharide.

2. the method for claim 1, it is characterised in that the concentration of described homogenate is 3-5% (w/v).

3. the method for claim 1, it is characterised in that described step 2) according to the U/g's of mass ratio 1.0-3.5 ten thousand Ratio adds cellulase.

4. the method for claim 1, it is characterised in that described step 3) according to 0.5-11.0U/g centipede algaes Amount addition high temperature carrageenase.

5. the method for claim 1, it is characterised in that the amino acid sequence of described high temperature carrageenase is SEQ ID NO:1。

6. the method for claim 1, it is characterised in that described step 4) activated carbon in activated-charcoal column is first to use After watery hydrochloric acid immersion, then remove watery hydrochloric acid, with soaked in absolute ethyl alcohol, then pour out absolute ethyl alcohol and add distilled water immersion, finally will Activated carbon dry for standby.