CN100999726A - Preparation process of ficin - Google Patents
Preparation process of ficin Download PDFInfo
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- CN100999726A CN100999726A CN 200610163292 CN200610163292A CN100999726A CN 100999726 A CN100999726 A CN 100999726A CN 200610163292 CN200610163292 CN 200610163292 CN 200610163292 A CN200610163292 A CN 200610163292A CN 100999726 A CN100999726 A CN 100999726A
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Abstract
The present invention relates to ficin preparing process and belongs to the field of biotechnology. The ficin preparing process includes the following steps: washing fresh fig, adding 0.9 % concentration NaCl solution of 200-500 ml, homogenating, soaking, filtering to eliminate residue, centrifuging for 10 min, adding tan liquor slowly, setting at 4 deg.c for 1-2 hr, centrifuging for 10 min, collecting the precipitate, dissolving in buffering liquid, centrifuging, collecting the precipitate, twice repeating, dissolving in phosphate buffer, and freeze drying. The present invention has the advantages of simple technological process, high yield, no pollution, and may be used in mass production of ficin.
Description
Technical field
The invention belongs to biological technical field, the ficin that particularly utilizes the biotechnology preparation to have the high protein enzymic activity.
Background technology
Ficin (Ficin:EC 3.4.22.3) belongs to proteolytic ferment, mainly is present in Ficus carica L. natural gum, Fructus Fici Chinese olive (Brunswike, Ficus Carica) and the leaf.Ficin is the prozyme that contains various ingredients.Ficin and papoid, bromeline are similar, belong to thiol proteinase, and essential sulfydryl is contained at its position, active centre, can be that multiple reductive agent such as halfcystine activates.About ficin (prozyme) molecular weight 25000-26000; Iso-electric point 9.0.It can be with peptide bond, fat key and the amido linkage of various natural proteins of specificity hydrolysis widely and synthetic substrate.Millet cake improvement, the meat that can be used for foodstuffs industry softens, milk-product solidify; The modifying agent of livestock industry nutrient fodder; Aspect medicine industry, as antiphlogistic, digestive enhancers, wormer etc.; It simultaneously also is the common agents in the life science.Therefore, ficin has huge application potential.
Walti at first obtained the ficin crystallization from Fructus Fici natural gum in 1938.Wong in 1964 and Liener have determined near the aminoacid sequence the enzyme active center sulfydryl, find that its and papoid (Light) and bromeline (Murachi) have homology highly.Nineteen sixty-eight Englund gets F.Glabrata natural gum, separates obtaining the component of multiple tool protease activity through the CM-cellulose displacement chromatography, and has measured the amino acid composition of ficin component III; N-terminal amino acid is Leu, and molecular weight is 25500 ± 750.
Williams separated gummy liquid in 1969, after ammonium sulfate, sodium-chlor are saltoutd, obtained the ficin of various ingredients through the CM-cellulose ion exchange chromatography.Kortt discovered in 1974, contained the ficin that has 6 kinds of components at least in the F.glabrata natural gum, separated obtaining 3 kinds of ficins (Ficin) by ammonium sulfate precipitation, CM-cellulose ion exchange chromatography, Sephadex G-75.The same year, Anderson adopted the affinity chromatography method, separated obtaining two kinds of ficins.
The nineties, the domestic prozyme that has the investigator to obtain ficin with ethanol sedimentation, ultrafiltration.Also have the investigator to obtain the prozyme of ficin through the acetone dehydration with ethanol segmentation extracting, post precipitation.
Related data shows, relevant ficin separation and Extraction, and external many uses are saltoutd, the method for column chromatography obtains ficin, because its complicated process of preparation, the cycle is long, and the loss alive of total enzyme is bigger, therefore these methods only limit to prepared in laboratory, are not suitable for scale operation; The preparation of domestic literature report ficin adopts organic solvent segmentation extracting, precipitation and ultrafiltration membrance filter to obtain target product more.These method productive rates are low, the cost height.If the scale operation ficin needs to reclaim ethanol, acetone and other organic solvent, the wasting manpower and material resources, and organic solvent is inflammable and explosive, and being used for industrial production will have great potential safety hazard.
Summary of the invention
The invention provides a kind of preparation method of ficin, low to solve the productive rate that exists in the existing production method, the cost height, need to reclaim ethanol, acetone and other organic solvent, the wasting manpower and material resources, and organic solvent is inflammable and explosive, is used for the problem that industrial production will have great potential safety hazard.The technical scheme that the present invention takes is:
Select fresh Fructus Fici Chinese olive 100g to clean, adding 200～500mL 0.9% contains the NaCl solution of 1～5mMEDTA and 1～5mMCys, homogenate, soaked 1～3 hour, slagging-off is filtered, centrifugal 10 minutes of 4000～6000rpm, slowly adding tan-liquor to final concentration in supernatant liquor is 0.2～0.5%, placed 1～2 hour for 4 ℃, 4000～6000rpm abandons supernatant liquor after centrifugal 10 minutes, collecting precipitation pH7.0,0.1M contain the phosphoric acid buffer dissolving of 1～5mM EDTA and 1～5mM Cys, centrifugal 10 minutes of 4000～6000rpm, abandoning supernatant, repeat twice, with 30～50mL pH7.0,0.1M contains the phosphoric acid buffer dissolving of 1～5mM EDTA and 1～5mMCys, lyophilize.
The invention has the advantages that, provide a kind of technology simple, the productive rate height, safety non-pollution, mass producible prepare the method for ficin.The present invention is a raw material with fresh Fructus Fici Chinese olive (Brunswike, Ficus Carica), adopts biotechnology, adds an amount of protective material, with 0.9% sodium-chlor extracting, with tannic acid precipitation target product, through lyophilize, obtains ficin.This technology cost is low, the productive rate height, and the cycle is short, good reproducibility, environmentally safe is fit to mass production, has huge application potential.
Description of drawings
Fig. 1. the SDS----PAGE electrophoretogram of ficin.
12% separation gel, 5% concentrates glue, Coomassie brilliant blue dyeing.Sample is from left to right:
1. ficin; 2. ficin; 3. ficin (U.S. Sigma);
4. ficin; 5. standard molecular weight Marker (Pharmacia).
Embodiment
Embodiment 1:
Select fresh Fructus Fici Chinese olive 100g to clean, adding 200mL 0.9% contains the NaCl solution of 5mM EDTA and 5mMCys, homogenate was soaked 3 hours, and slagging-off is filtered, centrifugal 10 minutes of 6000rpm, slowly adding tan-liquor to final concentration in supernatant liquor is 0.2%, 4 ℃ of placement 2 hours, and 6000rpm is after centrifugal 10 minutes, abandon supernatant liquor, collecting precipitation pH7.0,0.1M contain the phosphoric acid buffer dissolving of 5mM EDTA and 5mMCys, centrifugal 10 minutes of 6000rpm, abandoning supernatant, repeat twice, use 30mL pH7.0,0.1M contains the phosphoric acid buffer dissolving of 5mM EDTA and 5mMCys, lyophilize obtains the ficin powder.
Embodiment 2
Select fresh Fructus Fici Chinese olive 100g to clean, adding 350mL 0.9% contains the NaCl solution of 3mM EDTA and 3mMCys, homogenate, soaked 2 hours, slagging-off is filtered, centrifugal 10 minutes of 5000rpm, slowly adding tan-liquor to final concentration in supernatant liquor is 0.3%, placed 1.5 hours for 4 ℃, 5000rpm abandons supernatant liquor after centrifugal 10 minutes, collecting precipitation pH7.0,0.1M contain the phosphoric acid buffer dissolving of 3mM EDTA and 3mMCys, centrifugal 10 minutes of 5000rpm, abandoning supernatant, repeat twice, with 40mL pH7.0,0.1M contains the phosphoric acid buffer dissolving of 3mMEDTA and 3mMCys, lyophilize.
Embodiment 3
Select fresh Fructus Fici Chinese olive 100g to clean, adding 500mL 0.9% contains the NaCl solution of 1mM EDTA and 1mMCys, homogenate, soaked 1 hour, slagging-off is filtered, centrifugal 10 minutes of 4000rpm, slowly adding tan-liquor to final concentration in supernatant liquor is 0.5%, placed 1 hour for 4 ℃, 4000rpm abandons supernatant liquor after centrifugal 10 minutes, precipitation pH7.0,0.1M contain the phosphoric acid buffer dissolving of 1mM EDTA and 1mMCys, centrifugal 10 minutes of 4000rpm, abandoning supernatant, repeat twice, with 50mL pH7.0,0.1M contains the phosphoric acid buffer dissolving of 1mM EDTA and 1mMCys, lyophilize.
Experimental example: the detection of ficin
(1) determination of activity of ficin
With reference to American Pharmacopeia Kunitz method ficin being carried out enzymic activity detects.Unit of enzyme activity is defined as: under prescribed condition, per minute makes 280nm place absorption value rising 0.001 be defined as activity unit, i.e. a 1U.After measured:
The work of sample enzyme is 95000～120000U/g solid.
(2) molecular weight and purity detecting
With reference to the 3rd one of 2005 editions Chinese Pharmacopoeia, adopt 5% concentrated glue, 12% separation gel, the pH discontinuous system is carried out the SDS-polyacrylamide gel electrophoresis, ficin with Pharmacia lower molecular weight standard protein and U.S. Sigma company is a standard control, the molecular weight and the purity of examination product.
Referring to Fig. 1, from the SDS----PAGE electrophoretogram as seen, the ficin that this technology is extracted, 1,2,4 swimming lanes among the figure, consistent with the ficin enzyme molecular weight of U.S. Sigma company.
Claims (1)
1, a kind of preparation method of ficin, it is characterized in that may further comprise the steps: select fresh Fructus Fici Chinese olive 100g to clean, adding 200～500mL 0.9% contains the NaCl solution of 1～5mM EDTA and 1～5mMCys, homogenate, soaked 1～3 hour, slagging-off is filtered, centrifugal 10 minutes of 4000～6000rpm, slowly adding tan-liquor to final concentration in supernatant liquor is 0.2～0.5%, 4 ℃ of placements 1～2 hour, behind centrifugal 10 minutes of 4000～6000rpm, abandon supernatant liquor, collecting precipitation pH7.0,0.1M contain the phosphoric acid buffer dissolving of 1～5mM EDTA and 1～5mM Cys, centrifugal 10 minutes of 4000～6000rpm, abandoning supernatant repeats twice, with 30～50mL pH7.0,0.1M contain the phosphoric acid buffer dissolving of 1～5mM EDTA and 1～5mM Cys, lyophilize.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101632926A CN100439495C (en) | 2006-12-22 | 2006-12-22 | Preparation process of ficin |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101632926A CN100439495C (en) | 2006-12-22 | 2006-12-22 | Preparation process of ficin |
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| CN100999726A true CN100999726A (en) | 2007-07-18 |
| CN100439495C CN100439495C (en) | 2008-12-03 |
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| CNB2006101632926A Expired - Fee Related CN100439495C (en) | 2006-12-22 | 2006-12-22 | Preparation process of ficin |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104431287A (en) * | 2014-12-31 | 2015-03-25 | 临沂格瑞食品有限公司 | Ficus whey poly-peptides and preparation method thereof |
| CN107619793A (en) * | 2017-11-08 | 2018-01-23 | 青岛农业大学 | A kind of aspergillus screened from fig |
| CN108991113A (en) * | 2018-06-22 | 2018-12-14 | 金少举 | A kind of preparation method of soybean peptide Sodium guanylate beverage |
| CN115637211A (en) * | 2022-09-05 | 2023-01-24 | 祝雪花 | A kind of ficin extracting equipment and its application method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104988129A (en) * | 2015-08-07 | 2015-10-21 | 齐鲁工业大学 | Extraction method of efficient active enzymes of fig latex |
-
2006
- 2006-12-22 CN CNB2006101632926A patent/CN100439495C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104431287A (en) * | 2014-12-31 | 2015-03-25 | 临沂格瑞食品有限公司 | Ficus whey poly-peptides and preparation method thereof |
| CN107619793A (en) * | 2017-11-08 | 2018-01-23 | 青岛农业大学 | A kind of aspergillus screened from fig |
| CN108991113A (en) * | 2018-06-22 | 2018-12-14 | 金少举 | A kind of preparation method of soybean peptide Sodium guanylate beverage |
| CN115637211A (en) * | 2022-09-05 | 2023-01-24 | 祝雪花 | A kind of ficin extracting equipment and its application method |
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| CN100439495C (en) | 2008-12-03 |
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