CN1156490C

CN1156490C - High-purity biliprotein separating process

Info

Publication number: CN1156490C
Application number: CNB011273704A
Authority: CN
Inventors: 王广策; 孙海宝; 马圣媛; 曾呈奎
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2001-09-03
Filing date: 2001-09-03
Publication date: 2004-07-07
Anticipated expiration: 2021-09-03
Also published as: CN1344723A

Abstract

The present invention relates to a method for separating high purity phycobiliprotein, particularly to a method for separating and purifying high quality phycobiliprotein products. For simplifying technology, accelerating a speed and reducing costs, the method mainly comprises the following steps: the extract of algae is prepared by a hypotonic method; hydroxyapatite is mixed with an algae crude extract; other components of the algae crude extract in supernatant liquid are removed; the hydroxyapatite combined with the phycobiliprotein is further washed by using phosphate buffer solution with low concentration to remove other components; phosphate buffer solution with moderate concentration is used for removing phycocyanin or phycoerythrin from the hydroxyapatite by washing, and the phycocyanin or the phycoerythrin are collected; then, phosphate buffer solution with high concentration is used for eluting the hydroxyapatite to obtain a component of allophycocyanin. The present invention has the advantage of enhancement of the yield and the purity of the phycobiliprotein and is favorable for producing the phycobiliprotein with high purity in a large-scale industry mode and reducing costs for separation and purification.

Description

The separation method of phycobiliprotein

(1) technical field

The present invention relates to a kind of simpler, more economical and phycobiliprotein goods of high-quality, the high added value of high-efficiency method separation and purification more, belonging to from marine organisms separation and purification has the novel method of economic worth material, is exactly a kind of separation method of phycobiliprotein specifically.

(2) background technology

Phycobiliprotein can be widely used in medical diagnosis, clinical medicine etc.The phycobiliprotein kind is many, and fluorescence is strong, be easy to combinations such as same vitamin H, antibody, so phycobiliprotein can be used as fluorescent probe of new generation; Phycobiliprotein has some unique curative effects, for example as the photosensitizers of photodynamic tumor treatment, improves body's immunological function etc.; Replace synthetic food color, the additive as foods and cosmetics not only can avoid the artificial color side effect possible to human body, and phycobiliprotein itself just has extremely strong nourishing function.External many companies invest and develop phycobiliprotein in succession, and the product price is very considerable.For example, the high-purity biliprotein retail price that Sigma company produces is about about 120 dollars every milligram, and the fluorescent probe of phycobiliprotein mark is (a Sigma company calendar year 2001 products catalogue) about 150 dollars every milligram.

About the patent of the application facet of phycobiliprotein mainly contains United States Patent (USP) U.S.PatentNo.4859582 and U.S.Patent No.4542104, in order to of the application of protection phycobiliprotein as fluorescent probe; United States Patent (USP) U.S.Patent No.5163898 and Chinese patent CN1091976A protection Spirulina phycocyanin are as the application of oncotherapy photosensitizers.Chinese patent CN1106414A has introduced the method for separating Phycocyanins, C-from spirulina with CN1130028A, more than two used methods of patent all are the rough methods of Spirulina phycocyanin, though it is simple, but the Phycocyanins, C-purity that obtains is extremely low, only can be as foodstuff additive, at all can not be as biochemical reagents.The separation method of high-purity biliprotein commonly used substantially all is according to document Biochemistry at present, 13,2960 (1974) and Journal of Cell Biology, 93,981 (1982) methods that provide, promptly at first using the polymer-phycobilisome of the method separation and purification phycobiliprotein of sucrose density gradient centrifugation, is phycobiliprotein with the phycobilisome depolymerization, uses the method purifying phycobiliprotein of gel-filtration (Sephadex G-200) again.There is following shortcoming in these methods:

1, waste time and energy with sucrose density gradient ultracentrifugation, because the equipment price costliness of ultracentrifuge, a lot of units do not have the equipment of ultracentrifuge, are not suitable for extensive separation and purification phycobiliprotein with ultracentrifugal method simultaneously;

2, employed parting material Sephadex G-200 costs an arm and a leg with the method for gel-filtration the time;

3, oversize with the used time of the method for gel-filtration, only the balance chromatography column just needs the time in an about week;

4, the yield with above-mentioned method separation and purification phycobiliprotein is too low, and general yield is less than 1%.

Just because of these defectives of above-mentioned isolation technique, make that the phycobiliprotein price is too expensive on the world market.

(3) summary of the invention

The objective of the invention is to above-mentioned defective, a kind of separation method of high-purity biliprotein is provided, simplify separating technology at prior art, accelerate velocity of separation, improve yield, reduce cost, thereby help the highly purified phycobiliprotein of large-scale industrial production.

The present invention adopts hypotonic method to prepare the algae crude extract, uses the sorbent material of homemade hydroxyapatite as phycobiliprotein.Not that hydroxyapatite is packed in the chromatography column, separate phycobiliprotein again, but the crude extract of homemade hydroxyapatite and algae is mixed in proportion, phycobiliprotein in the crude extract fully is adsorbed on the hydroxyapatite, thereby avoided residue and viscous polysaccharide in the crude extract to stop up the difficult problem of chromatography column.Concrete steps are as follows:

One, the extract for preparing algae with hypotonic way;

Two, hydroxyapatite and algae crude extract are mixed, phycobiliprotein in the crude extract is adsorbed onto on the hydroxyapatite, adopt the method for natural sedimentation or low-speed centrifugal to make the precipitation of hydroxyapatite that is combined with phycobiliprotein, remove other components of algae crude extract in the supernatant liquor;

Three, further be combined with the hydroxyapatite 2 to 3 times of phycobiliprotein with 0.001M to 0.005M phosphate buffered saline buffer (pH6.8) washing, with remove not in conjunction with or in conjunction with unstable other components;

Four, the phosphate buffered saline buffer (pH6.8) with 0.010M to 0.050M elutes Phycocyanins, C-or phycoerythrin from hydroxyapatite, collects Phycocyanins, C-or phycoerythrin;

Five, again with 0.05M to 0.15M phosphate buffered saline buffer (pH6.8) wash-out hydroxyapatite, obtain the allophycocyanin component.The phycobiliprotein goods yield of gained is at least 20%, and its yield scope is 20%--60%.

If the phycobiliprotein product that will obtain from step 4, five, repeating step two, three, four, five again, the absorption and the elution process of repetition and hydroxyapatite, the phycobiliprotein purity that obtains will reach or reach 4--6, be higher than the high-purity biliprotein product that external major company sells.

Use above method can reach two purposes effectively: (1), phycobiliprotein is separated from the algae crude extract; (2), with different phycobiliprotein (as Phycocyanins, C-, phycoerythrin and allophycocyanin etc.) separately.

The present invention compared with prior art mainly has following advantage:

1, hypotonic method is adopted in the extracting of algae inclusion, does not need special equipment (as the ultrasonic disruption instrument etc.), and hydroxyapatite can ownly prepare simultaneously, thereby very economical;

2, do not need ultracentrifugation, dress chromatography column and on chromatography column complicated operations such as application of sample, do not need very time-consuming processes such as balance and elution chromatography post simultaneously yet, generally only need one day the time just can complete operation, so simple to operation;

3, cost is low, if do not comprise labor cost, preresearch estimates only is 1/20th of an external like product;

4, yield height can reach more than 20% at least, is 20 times of the present phycobiliprotein separation method yield of using always, is fit to large-scale industrial production.

(4) embodiment is described in further detail method of the present invention by three embodiment.

Embodiment 1

Get the spirulina plalensis dry powder of 20 grams available from Shandong Province's Dongying city Shengli Oil Field.Add distilled water 200ml, placed 24 hours under 4 ℃ of conditions in the refrigerator, low-speed centrifugal removes residue, and supernatant liquor is the spirulina crude extract, and cumulative volume is 151ml, OD ₆₂₀=46.660 (containing thick phycobiliprotein amount is 1003.26 milligrams); Take by weighing 1 gram hydroxyapatite, be placed in the centrifuge tube with cover, with 0.001M phosphate buffered saline buffer balance, add 4ml spirulina crude extract (contain the Phycocyanins, C-amount and be about 26.576 milligrams) then, centrifuge tube turned upside down 10 minutes makes it abundant absorption, and low-speed centrifugal removed supernatant liquor in 2 minutes; Be adsorbed with twice of the hydroxyapatite of phycobiliprotein with the washing of above-mentioned damping fluid; The phosphate buffered saline buffer wash-out Phycocyanins, C-that adds 0.030M again, to elutriant colourless till, merge elutriant, cumulative volume is 37ml, OD ₆₂₀=1.133, contain Phycocyanins, C-and be about 5.969 milligrams; The yield that obtains Phycocyanins, C-from the spirulina crude extract is about 22.460%.If carry out hydroxyapatite absorption and elution process again, the purity A of Phycocyanins, C- ₆₂₀/ A ₂₈₀Can reach 4.5, and the purity of the Phycocyanins, C-that Sigma company is sold only is 4.4 (Sigma company product catalogues in 2000), is higher than the purity of the Phycocyanins, C-that Sigma company sold.

Embodiment 2

The treatment step of front is identical with embodiment 1, without the phosphate buffered saline buffer of 0.003M, and uses phosphate buffered saline buffer wash-out with 0.05M instead, be eluted to hydroxyapatite colourless till; The elutriant cumulative volume is 52ml, OD ₆₂₀=0.909, contain Phycocyanins, C-and be about 6.732 milligrams; The yield that obtains Phycocyanins, C-from the spirulina crude extract is about 25.330%.If carry out hydroxyapatite absorption and elution process again, the purity A of Phycocyanins, C- ₆₂₀/ A ₂₈₀Also can reach more than 4.

Embodiment 3

Ceramium kondoi picks up from Hui Spring gulf, Qingdao, with behind seawater and the tap water washing frond 2 times, adds isopyknic tap water and soaks 24 hours respectively, filters the residue of removing frond, after measured the OD of crude extract filtrate ₅₆₀=3.808; Take by weighing 2 gram hydroxyapatites, be placed in the centrifuge tube with cover, with 0.001M phosphate buffered saline buffer (pH6.8) balance, add 16ml Ceramium kondoi crude extract (contain the phycoerythrin amount and be about 7.4mg) then, centrifuge tube turned upside down 10 minutes makes it abundant absorption, and low-speed centrifugal removed supernatant liquor in 2 minutes; Be adsorbed with twice of the hydroxyapatite of phycobiliprotein with the washing of above-mentioned damping fluid; The phosphate buffered saline buffer wash-out phycoerythrin that adds 0.030M again, to elutriant colourless till, merge elutriant, cumulative volume is 72ml, OD ₅₆₅=0.504, contain phycoerythrin and be about 4.4mg; The yield that obtains phycoerythrin from the Ceramium kondoi crude extract is about 59%.If carry out hydroxyapatite absorption and elution process again, the purity A of phycoerythrin ₅₆₅/ A ₂₈₀Can reach more than 4.It is generally acknowledged phycoerythrin A ₅₆₅/ A ₂₈₀More than 3.5, be exactly very pure, and the product of the phycoerythrin that external Sigma company is sold does not provide concrete purity.

Claims

1, a kind of separation method of phycobiliprotein is characterized in that comprising following each step:

One, the crude extract for preparing algae with hypotonic way;

Three, be the hydroxyapatite 2 to 3 times that the washing of 6.8 phosphate buffered saline buffer is combined with phycobiliprotein further with 0.001M to 0.005M, pH value, with remove not in conjunction with or in conjunction with unstable other components;

Four, be that 6.8 phosphate buffered saline buffer elutes Phycocyanins, C-or phycoerythrin from hydroxyapatite with 0.010M to 0.050M, pH value, collect Phycocyanins, C-or phycoerythrin;

Five, with 0.05M to 0.15M, pH value be 6.8 phosphate buffered saline buffer wash-out hydroxyapatite again, obtain the allophycocyanin component, the phycobiliprotein goods yield 20%--60% of gained.

2, according to the separation method of the described phycobiliprotein of claim 1, it is characterized in that: the phycobiliprotein product that will obtain from step 4, five is repeating step two, three, four, five again, the absorption and the elution process of repetition and hydroxyapatite, the phycobiliprotein purity that obtains can reach 4-4.5.