CN103333869A - Method for extracting and preparing peroxidase from stem and leaves of sweet potato - Google Patents
Method for extracting and preparing peroxidase from stem and leaves of sweet potato Download PDFInfo
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- CN103333869A CN103333869A CN2013102688030A CN201310268803A CN103333869A CN 103333869 A CN103333869 A CN 103333869A CN 2013102688030 A CN2013102688030 A CN 2013102688030A CN 201310268803 A CN201310268803 A CN 201310268803A CN 103333869 A CN103333869 A CN 103333869A
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Abstract
The invention discloses a method for extracting and preparing peroxidase from the stems and leaves of sweet potato. The method comprises the following steps of: performing homogenate extraction on the stems and/or leaves of sweet potato by use of water or a phosphate buffer solution; centrifuging or filtering to obtain protein crude enzyme extraction liquid; precipitating the extraction liquid by use of ammonium sulfate or an organic solvent to obtain separated and purified peroxidase; and drying to obtain high-activity peroxidase powder. The method disclosed by the invention further develops the sweet potato and turns waste into wealth, is energy-saving and environment-friendly and is simple to operate; and the production cost of the product is low, and large-scale production is facilitated.
Description
Technical field
The present invention relates to a kind of preparation and extractive technique of protein, relate in particular to the method for extracting and prepare peroxidase a kind of stem from sweet potato and the leaf.
Background technology
According to Food and Argriculture OrganizationFAO's statistics, the cultivated area of China sweet potato reaches 3,680,000 hm
2, accounting for about 60% of world's sweet potato total area, ultimate production is 0.81 hundred million ton, accounts for about 80% of Gross World Product.Because the establishing in large scale of sweet potato has produced a large amount of byproducts---stem and the leaf of sweet potato.China produces 70000000000 kilograms on sweet potato stem, 72,000,000,000 kilograms of Rhizoma Dioscoreae esculentae leaf per year.
The stem of sweet potato and leaf contain a large amount of nutritive substances, crude protein content is higher than the content of rice, barley up to 16.5-17.1%, is close with the content of corn, also contain abundant vitamin A, B, C and mineral substance iron, calcium etc., be described as " vegetables queen " by Vegetable Research center, Asia.China some areas are edible as vegetables with the young stem and leaf of sweet potato.Peasant household is used as feed with sweet potato leaves and stems and sweet potato root tuber.But these consumptions are all fewer, and the nutritive ingredient utilization ratio is low.At present, most sweet potato leaves and stems is used as refuse and abandons, and has caused a large amount of wastings of resources, has also caused environmental pollution simultaneously.
Summary of the invention
The objective of the invention is, in order to overcome the deficiencies in the prior art, it is the method for feedstock production high reactivity peroxidase that a kind of stem with sweet potato and leaf are provided, and stem and the leaf of sweet potato are made recycling, turns waste into wealth, and improves the economic worth of sweet potato industry.
For reaching above technical purpose, the technical solution used in the present invention is as follows:
A kind of from sweet potato stem and leaf extract and prepare the method for peroxidase, it may further comprise the steps:
(1) prepares stem and/or the blade-section of fresh sweet potato;
(2) stem of described sweet potato and/or the extracting solution of blade-section and its 0.5～2.5 times of volume are carried out homogenate, lixiviate, cleer and peaceful precipitation in the separation, its supernatant is the superoxide enzyme extract;
(3) adding solid ammonium sulfate to saturation concentration in described superoxide enzyme extract is 40%～90%, or to add organic solvent to its final concentration be 50%～80%, the precipitation zymoprotein, and cleer and peaceful precipitation in the separation, it is precipitated as peroxidase;
(4) the peroxidase kept dry that described step (3) is obtained.
Particularly, the extracting solution in the described step (2) is any one of tap water, distilled water, deionized water or phosphate buffered saline buffer; Extraction time is 1 hour.
Further, the organic solvent in the described step (3) is ethanol or acetone; Sedimentation time is 1 hour.
Further, the drying means of described step (4) be lyophilize, vacuum-drying or spray-dired any one.
Preferably, carry out under 4 ℃ or room temperature described step (2)～(4).
More preferably, also comprise step (5), the organic solvent in the described step (3) is reclaimed.Particularly, it adopts underpressure distillation to reclaim described organic solvent.
Compared with prior art, the present invention has following advantage:
A) the present device less investment is simple to operate, and the products production cost is low, is convenient to scale operation;
B) production process is pollution-free, less energy-consumption, zero release.Organic solvent can recycle and reuse fully with methods such as underpressure distillation, and the dry-matter after the distillation (being mainly soluble saccharide) can be made animal or microbial nutrition as byproduct, and the sweet potato leaves and stems slag can be used for feed or organic fertilizer production;
C) the present invention has efficiently utilized sweet potato stem and leaf class waste, turns waste into wealth, and has improved value of exploiting and utilizing and the Industrial economic benefit of sweet potato;
D) through the superoxide enzyme assay, the peroxidase in sweet potato stem and the leaf has high reactivity, can be used as the raw material of preparation high reactivity peroxidase.
Description of drawings
Fig. 1 extracts and prepares the schema of the method for peroxidase from the stem of sweet potato and leaf for the present invention.
Fig. 2 be with the present invention from the stem of sweet potato and leaf, extract and the method that prepare peroxidase from the enzymic activity comparison diagram of the peroxidase of the different organ extraction of sweet potato.
Fig. 3 extracts from the stem of sweet potato and leaf for the present invention and prepares in the method for peroxidase the precipitation situation comparison diagram of peroxidase under the ammonium sulfate of different concns.
Fig. 4 extracts from the stem of sweet potato and leaf for the present invention and prepares in the method for peroxidase the precipitation situation comparison diagram of peroxidase under Different concentrations of alcohol.
Fig. 5 extracts from the stem of sweet potato and leaf for the present invention and prepares in the method for peroxidase the precipitation situation comparison diagram of peroxidase under the acetone of different concns.
Fig. 6 extracts and prepares the purity testing situation of the peroxidase powder that the method for peroxidase obtains from the stem of sweet potato and leaf for the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail.
Embodiment 1
Shown in the schema of Fig. 1, the present invention extracts from the stem of sweet potato and leaf and to prepare the concrete steps of method of peroxidase as follows:
(1) stem and/or the blade-section of the sweet potato that preparation 100g is fresh;
(2) be the phosphate buffered saline buffer homogenate of 1:1 with the stem of described sweet potato and/or blade-section and its solid-to-liquid ratio, lixiviate is after 1 hour, through centrifugal or filter back acquisition superoxide enzyme extract;
(3) adding solid ammonium sulfate to saturation concentration in described superoxide enzyme extract is 40%～90%, or adds ethanol or acetone to its final concentration is 50%～80%, precipitate after 1 hour, and cleer and peaceful precipitation in the separation, it is precipitated as peroxidase;
(4) peroxidase that described step (3) is obtained obtains high reactivity peroxidase enzyme powder after lyophilize;
(5) adopt underpressure distillation to reclaim ethanol or acetone in the supernatant of described step (3), at last small organic molecule.
Above step is all carried out under 4 ℃ or room temperature.
Embodiment 2
Superoxide enzyme extract, the peroxidase throw out of step (3) acquisition and the peroxidase enzyme powder that step (4) obtains that step (2) among the described embodiment 1 is obtained detect peroxidase enzymic activity and separation and purification effect by spectrophotometry and non-denaturing polyacrylamide running gel autography method respectively, and its result is as follows:
Please refer to Fig. 2, from the different organ of sweet potato, extract peroxidase with method of the present invention, comprise the piece root of sweet potato, new stem and old stem and young leaves and Lao Ye, the result shows, from the old stem of sweet potato and blade (no matter new with old), the content of the activated peroxidase that particularly extracts in the old stem and prepare is the highest, thereby has determined the position of drawing materials of the most suitable extraction peroxidase in the sweet potato.
Please refer to Fig. 3～5, " stoste " among the figure refers to the superoxide enzyme extract that step among the embodiment 1 (2) obtains, the peroxidase of all the other swimming lanes is the ammonium sulfate through different concns, peroxidase throw out behind ethanol or the acetone precipitation, the result shows, peroxidase with ammonium sulfate during as precipitation agent the optimal concentration of ammonium sulfate be 60%～90%, the optimal concentration of ethanol is 70%～80% during as precipitation agent with ethanol, the optimal concentration of acetone is 60%～80% during as precipitation agent with acetone, peroxidase protein band in the precipitation agent precipitate and separate of above-mentioned optimal concentration is clear, complete, and the active coloring color is darker, shows that the peroxidase through precipitate and separate still keeps high catalytic activity.
Please refer to Fig. 6, behind the further concentrate drying of the peroxidase of precipitate and separate, its enzymic activity still keeps.
In addition, with the peroxidase that spectrophotometry obtains behind 70% ethanol sedimentation, the protein content of this peroxidase enzyme powder is 23.22mg/g enzyme powder, and the peroxidase enzymic activity is 128.69U/mg albumen.
In sum, the present invention extracts from the stem of sweet potato and leaf and the method for preparing peroxidase is further developed sweet potato, turn waste into wealth, and energy-conserving and environment-protective, simple to operate, the products production cost is low, is convenient to scale operation.
Above-described embodiment is preferred implementation of the present invention; but not merely be restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, all be included within protection scope of the present invention.
Claims (9)
1. method of extracting and prepare peroxidase from the stem of sweet potato and leaf is characterized in that it may further comprise the steps:
(1) prepares stem and/or the blade-section of fresh sweet potato;
(2) stem of described sweet potato and/or the extracting solution of blade-section and its 0.5～2.5 times of volume are carried out homogenate, lixiviate, cleer and peaceful precipitation in the separation, its supernatant is the superoxide enzyme extract;
(3) adding solid ammonium sulfate to saturation concentration in described superoxide enzyme extract is 40%～90%, or to add organic solvent to its final concentration be 50%～80%, the precipitation zymoprotein, and cleer and peaceful precipitation in the separation, it is precipitated as peroxidase;
(4) the peroxidase kept dry that described step (3) is obtained.
As claimed in claim 1 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: the extracting solution in the described step (2) is any one of tap water, distilled water, deionized water or phosphate buffered saline buffer.
As claimed in claim 2 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: the extraction time in the described step (2) is 1 hour.
As claimed in claim 1 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: the organic solvent in the described step (3) is ethanol or acetone.
As claimed in claim 4 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: the sedimentation time in the described step (3) is 1 hour.
As claimed in claim 1 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: the drying means of described step (4) be lyophilize, vacuum-drying or spray-dired any one.
As claim 1-6 any one described from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: carry out under 4 ℃ or room temperature described step (2)～(4).
As claimed in claim 7 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: also comprise step (5), the organic solvent in the described step (3) is reclaimed.
As claimed in claim 8 from sweet potato stem and leaf extract and prepare the method for peroxidase, it is characterized in that: described organic solvent is reclaimed in the employing underpressure distillation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134590A (en) * | 2018-08-27 | 2019-01-04 | 辽宁大学 | A kind of extracting method and its application of Sweet Potato albumen |
| CN111139227A (en) * | 2019-12-31 | 2020-05-12 | 毛秋霞 | Method for extracting and purifying peroxidase |
Citations (3)
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| CN1369557A (en) * | 2002-03-18 | 2002-09-18 | 华南农业大学 | Process for extracting peroxidase from tender leaves of sugar cane |
| CN1384879A (en) * | 1999-10-29 | 2002-12-11 | 韩国生命工学研究院 | peroxidase genomic gene derived from ipomoea batatas and promoter thereof |
| CN1928078A (en) * | 2006-10-10 | 2007-03-14 | 山西职工医学院 | Extraction method of peroxidase |
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- 2013-06-28 CN CN2013102688030A patent/CN103333869A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384879A (en) * | 1999-10-29 | 2002-12-11 | 韩国生命工学研究院 | peroxidase genomic gene derived from ipomoea batatas and promoter thereof |
| CN1369557A (en) * | 2002-03-18 | 2002-09-18 | 华南农业大学 | Process for extracting peroxidase from tender leaves of sugar cane |
| CN1928078A (en) * | 2006-10-10 | 2007-03-14 | 山西职工医学院 | Extraction method of peroxidase |
Non-Patent Citations (5)
| Title |
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| 付伟丽等: "甘薯叶过氧化物酶的分离纯化及其部分性质研究", 《食品科学》 * |
| 吴娟等: "红薯过氧化物酶的纯化与性质测定", 《湖南农业科学》 * |
| 杨贤松: "紫色甘薯不同组织器官的同工酶研究", 《资源开发与市场》 * |
| 王志兵等: "豆壳过氧化物酶几种提取方法的比较研究", 《食品工业科技》 * |
| 陈燕珍等: "木薯叶过氧化物酶的分离纯化及其同工酶的研究", 《安徽农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109134590A (en) * | 2018-08-27 | 2019-01-04 | 辽宁大学 | A kind of extracting method and its application of Sweet Potato albumen |
| CN111139227A (en) * | 2019-12-31 | 2020-05-12 | 毛秋霞 | Method for extracting and purifying peroxidase |
| CN111139227B (en) * | 2019-12-31 | 2021-07-30 | 毛秋霞 | Method for extracting and purifying peroxidase |
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