CN100390278C - Fructus momordicae protease and extraction method thereof - Google Patents
Fructus momordicae protease and extraction method thereof Download PDFInfo
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- CN100390278C CN100390278C CNB200410040965XA CN200410040965A CN100390278C CN 100390278 C CN100390278 C CN 100390278C CN B200410040965X A CNB200410040965X A CN B200410040965XA CN 200410040965 A CN200410040965 A CN 200410040965A CN 100390278 C CN100390278 C CN 100390278C
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- Prior art keywords
- clear liquid
- momordica grosvenori
- fructus momordicae
- drying
- enzyme powder
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- 108091005804 Peptidases Proteins 0.000 title abstract description 12
- 238000000605 extraction Methods 0.000 title abstract description 8
- 239000004365 Protease Substances 0.000 title abstract description 7
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 28
- 239000000843 powder Substances 0.000 claims abstract description 19
- 235000011171 Thladiantha grosvenorii Nutrition 0.000 claims abstract description 18
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 241001409321 Siraitia grosvenorii Species 0.000 claims abstract description 8
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- 238000004108 freeze drying Methods 0.000 claims abstract description 6
- 238000001556 precipitation Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 16
- 230000002797 proteolythic effect Effects 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 3
- 239000004744 fabric Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001641 gel filtration chromatography Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 2
- 239000002893 slag Substances 0.000 claims description 2
- 235000019750 Crude protein Nutrition 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 8
- 102000035195 Peptidases Human genes 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 239000005018 casein Substances 0.000 abstract description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 abstract description 3
- 235000021240 caseins Nutrition 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 238000007710 freezing Methods 0.000 abstract description 3
- 230000008014 freezing Effects 0.000 abstract description 3
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 2
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 abstract 5
- 235000019833 protease Nutrition 0.000 abstract 4
- 108090000526 Papain Proteins 0.000 abstract 1
- 238000004440 column chromatography Methods 0.000 abstract 1
- 239000010985 leather Substances 0.000 abstract 1
- 235000019834 papain Nutrition 0.000 abstract 1
- 229940055729 papain Drugs 0.000 abstract 1
- 235000019419 proteases Nutrition 0.000 abstract 1
- 238000005185 salting out Methods 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 abstract 1
- 238000001694 spray drying Methods 0.000 abstract 1
- 244000185386 Thladiantha grosvenorii Species 0.000 description 11
- 235000009815 Momordica Nutrition 0.000 description 9
- 241000218984 Momordica Species 0.000 description 9
- 244000189799 Asimina triloba Species 0.000 description 3
- 235000006264 Asimina triloba Nutrition 0.000 description 3
- 235000009467 Carica papaya Nutrition 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 244000099147 Ananas comosus Species 0.000 description 2
- 235000007119 Ananas comosus Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 241000370738 Chlorion Species 0.000 description 1
- 240000006053 Garcinia mangostana Species 0.000 description 1
- 235000017048 Garcinia mangostana Nutrition 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a fructus momordicae proteinase and an extraction method thereof, wherein the fructus momordicae proteinase is prepared by using fresh fructus momordicae through the steps of juicing, filtering, ultrafiltration or salting out, organic solvent precipitation, vacuum freezing or spray drying, preparing crude enzyme powder, dissolving the crude enzyme powder with distilled water, carrying out column chromatography, dialyzing to remove salt, and freeze drying. The enzyme has strong hydrolytic activity to casein, the activity of crude enzyme can reach 150 ten thousand units/g, which is about twice of the activity of crude enzyme of papain, and the momordica grosvenori proteinase and the extraction thereof increase one more plant proteinase resource in China. Meanwhile, the invention has great significance for the deep processing of the momordica grosvenori, the improvement of the comprehensive utilization level of the momordica grosvenori and the increase of the economic benefit of the momordica grosvenori on the development of the enzymology research and the bioscience and the development of the industries of food, medical treatment, leather making, chemical industry and the like which use protease.
Description
(1) technical field:
The invention belongs to plant protease, particularly momordica grosvenori proteolytic and extracting method thereof.
(2) background technology:
Grosvenor Momordica is the distinctive cucurbitaceous plant of China, it is reported that Grosvenor Momordica contains 6～38% carbohydrate and 1～4% sweeteners higher 300 times than sucrose sweetness that easily utilize for absorption of human body, also contain compositions such as abundant vitamins C, protein, have very high economic worth and edibleness.The development and use of Grosvenor Momordica have caused domestic and international scientific worker's extensive attention.
At present, Grosvenor Momordica is mainly sold with the oven dry fruit.Profound development and use mainly are the extractions of mangosteen sweetener and are protective foodss such as the Grosvenor Momordica electuary made of base-material, Grosvenor Momordica fruit treasure, beverage made of Momordica grosvenorii Swingle with the Fructus Monordicae extract.China's plant protease resource mainly depends on minority tropical plants such as pawpaw, pineapple, Fructus Fici for a long time, does not see that as yet report is arranged and contain the very strong proteolytic enzyme of vigor in the Grosvenor Momordica.
(3) summary of the invention:
The objective of the invention is to disclose a kind of momordica grosvenori proteolytic that from fresh Fructus Momordicae, extracts and extracting method thereof.
The extracting method step of momordica grosvenori proteolytic is:
(1) picks out the fresh Fructus Momordicae that does not go mouldy, clean up and drain;
(2) squeezing, filter cloth filters, and centrifugal removing slag gets clear liquid;
(3) clear liquid obtains thick enzyme powder, method one by in following three kinds of methods any one: clear liquid micro-pore-film filtration, filtrate must be held back ultrafiltrated through the ultra-filtration membrane ultrafiltration, vacuum lyophilization or spraying drying, just thick enzyme powder; Method two: clear liquid adds organic solvent, staticly settle filtration after, gained precipitation drying, just thick enzyme powder; Method three: clear liquid adds ammonium sulfate, and after staticly settling, the gained precipitation desalts through dialysis, in the dialysis tubing solution after vacuum lyophilization or spraying drying, just thick enzyme powder;
(4) get thick enzyme powder with suitable dissolved in distilled water, centrifugal remove insolubles after, carry out gel filtration chromatography, collect and have the part that enzyme is lived, lyophilize, just momordica grosvenori proteolytic.
With the momordica grosvenori proteolytic that the aforesaid method extraction separation goes out, the experiment proved that casein is had the intensive hydrolytic activity.Employing is that the ultraviolet spectrophotometry of reaction substrate is measured the vigor of enzyme with the casein, and thick enzyme activity can reach 1,500,000 units/gram, is about the twice of the thick enzyme activity of pawpaw albumen on the market, and is higher through the momordica grosvenori proteolytic vigor of separation and purification.The discovery of momordica grosvenori proteolytic and extraction make China many again a plant protease resource, of the present invention applying will change the situation that China's plant protease resource long-term mainly depends on minority plant resourceses such as pawpaw, pineapple.The present invention simultaneously is to the development of enzymology and bio-science, to industrial expansions such as the food of using proteolytic enzyme, medical treatment, process hides, chemical industry, to the deep processing of Grosvenor Momordica, improve Grosvenor Momordica level of comprehensive utilization, increase its economic benefit and all be of great importance.
(4) specific embodiment:
Utilize ripe fresh arhat fruit fruit extraction separation momordica grosvenori proteolytic, concrete processing step is:
(1) thick enzyme extraction: choose new fresh Fructus Momordicae 1000g, clean and drain, press extracting juice, filter cloth filters, centrifugal, and (4000r/min 15min), collects supernatant liquor, and clear liquid obtains thick enzyme powder by in following three kinds of methods any one.Method one: with the 0.45um micro-pore-film filtration of supernatant liquor with Φ 150mm, filtrate is through the ultrafiltration of hollow tube ultra-fine filter, the ultrafiltrated that must dam, this ultrafiltrated carries out drying freezing freezing below-40 ℃ with vacuum freeze drier, white powder be thick enzyme powder 15.8g; Method two: clear liquid adds precooled ethanol to 70% (v/v), leave standstill for some time after, remove by filter solution, gained precipitate through vacuum lyophilization, must buff powder be thick enzyme powder 20.5g; Method three: it is 0.7 that clear liquid adds ammonium sulfate to saturation ratio, staticly settle filtration after, must precipitate and desalt, and dialysis tubing solution is carried out lyophilize get white thick enzyme powder 12.3g through dialysis.
(2) being further purified of enzyme: earlier with gel chromatography column (Φ 25 * 700mm) distilled water balance, get above-mentioned thick enzyme powder 1g again and be dissolved in 10ml distilled water, centrifugal (4000r/min, 10min) get supernatant liquor 5ml upper prop, successively with each wash-out 40min of NaCl solution (flow velocity is 1mL/min) of distilled water and 0.1,0.2,0.3,0.4,0.5mol, elutriant detects (detecting) by UV-detector under the 250nm wavelength, collect by the peak, get the common 30ml of tool enzymic activity, dialyse with dialysis tubing, to using AgNO
3Detection with solution for vacuum lyophilize in the dialysis tubing, gets 0.034g white momordica grosvenori proteolytic less than chlorion.
Claims (2)
1. momordica grosvenori proteolytic, it is characterized in that: through squeezing, filter and remove residue is after the clear liquid via hole diameter is the micro-pore-film filtration of 0.45 μ m by fresh Fructus Momordicae for it, again through ultrafiltration, drying, or clear liquid adding ethanol staticly settle the filtration after drying, or clear liquid adding ammonium sulfate to saturation ratio is 0.7, the crude protein enzyme that precipitate, dialyse, desalt, drying obtains, again through water dissolution, centrifugal, gel filtration chromatography, lyophilize are made.
2. the extracting method of a momordica grosvenori proteolytic is characterized in that the processing step that extracts is:
(1) picks out the fresh Fructus Momordicae that does not go mouldy, clean up and drain;
(2) squeezing, filter cloth filters, and centrifugal removing slag gets clear liquid;
(3) clear liquid obtains thick enzyme powder, method one by in following three kinds of methods any one: clear liquid is with the micro-pore-film filtration of aperture 0.45 μ m, and filtrate must be held back ultrafiltrated through the ultra-filtration membrane ultrafiltration, vacuum lyophilization or spraying drying, just thick enzyme powder; Method two: clear liquid adds ethanol to 70% (v/v), staticly settle filtration after, gained precipitation drying, just thick enzyme powder; Method three: clear liquid adds ammonium sulfate to saturation ratio and reaches 0.7, and after staticly settling, the gained precipitation desalts through dialysis, in the dialysis tubing solution after vacuum lyophilization or spraying drying, just thick enzyme powder;
(4) get thick enzyme powder with suitable dissolved in distilled water, centrifugal remove insolubles after, carry out gel filtration chromatography, collect and have the part that enzyme is lived, lyophilize, momordica grosvenori proteolytic.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410040965XA CN100390278C (en) | 2004-10-31 | 2004-10-31 | Fructus momordicae protease and extraction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410040965XA CN100390278C (en) | 2004-10-31 | 2004-10-31 | Fructus momordicae protease and extraction method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1766099A CN1766099A (en) | 2006-05-03 |
| CN100390278C true CN100390278C (en) | 2008-05-28 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB200410040965XA Expired - Fee Related CN100390278C (en) | 2004-10-31 | 2004-10-31 | Fructus momordicae protease and extraction method thereof |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088006A (en) * | 2013-01-09 | 2013-05-08 | 广西南剑生物科技有限公司 | Method for extracting protease from squeezed juice of sisal |
| CN113215137A (en) * | 2021-06-11 | 2021-08-06 | 江苏格局生物医药科技有限公司 | Preparation method of enzyme for pharmaceutical industry |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1415749A (en) * | 2002-10-16 | 2003-05-07 | 叶高瑞 | Method for preparing albumen and chymogen of carica papaya |
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- 2004-10-31 CN CNB200410040965XA patent/CN100390278C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1415749A (en) * | 2002-10-16 | 2003-05-07 | 叶高瑞 | Method for preparing albumen and chymogen of carica papaya |
Non-Patent Citations (2)
| Title |
|---|
| 罗汉果成份及其开发利用. 曹东宁.河南科学，第17卷第suppl期. 1999 |
| 罗汉果成份及其开发利用. 曹东宁.河南科学，第17卷第suppl期. 1999 * |
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| Publication number | Publication date |
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| CN1766099A (en) | 2006-05-03 |
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