CN102408481A - Preparation method for pea aspartic acid protease inhibitor - Google Patents
Preparation method for pea aspartic acid protease inhibitor Download PDFInfo
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- CN102408481A CN102408481A CN2011103735133A CN201110373513A CN102408481A CN 102408481 A CN102408481 A CN 102408481A CN 2011103735133 A CN2011103735133 A CN 2011103735133A CN 201110373513 A CN201110373513 A CN 201110373513A CN 102408481 A CN102408481 A CN 102408481A
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Abstract
The invention discloses a preparation method for a pea aspartic acid protease inhibitor, and belongs to the technical field of a plant extraction biological activity product. The method comprises the following steps of: directly grinding pea into powder; leaching with water; performing high-speed centrifugation; thermally treating supernatant; performing high-speed centrifugation again; separating the purified aspartic acid protease inhibitor from the supernatant by a DEAE (Diethylaminoethyl)-52 ion exchange column chromatography; and then freeze-drying under vacuum to obtain the aspartic acid protease inhibitor dry powder. The aspartic acid protease inhibitor prepared by the invention has higher inhibition activity on pepsin and rennin in aspartic acid protease family, is directly extracted from pea, is low in cost and safe in application, and has broad application prospect in the fields of food additives, medicines, agriculture and the like.
Description
Technical field
A kind of preparation method of pea asparaginic acid protease inhibitors; Be to adopt pea to pulverize specifically, use flooding, high speed centrifugation; Thermal treatment; Supernatant obtains purer asparaginic acid protease inhibitors goods through the DEAE-52 ion exchange chromatography, belongs to plant extract bio-active products technical field.
Background technology
Biologically active substance is meant that some food contains the compound of multiple biologically active, can with the body effect after cause various biological effects.They are of a great variety, and carbohydrate, lipid, protein and peptide class, sterols, vegeto-alkali, glucoside or the like are arranged.They mainly are present in the plant food, and what have is favourable to the people, and what have is harmful.In prevention and treatment disease, fields such as food, biologically active substance is still a problem that is worth research, how to develop the favourable one side of bio-active substance confrontation human body, and avoiding or eliminating its deleterious one side is worth us further to go to inquire into.From pea, isolated the various active material at present both at home and abroad.
Thereby proteinase inhibitor is one type to have combination and makes protein inactivation play inhibiting active substance to intravital some enzyme of biology.Natural protease inhibitor is a kind of small molecular protein or polypeptide that can the arrestin hydrolytic enzyme activities, and very extensively all there is existence in its source in a lot of vegeto-animal histoorgans and certain micro-organisms.Difference according to the active site of the target protein enzyme of its inhibition roughly can be divided into following several kinds: serpin (Serpin): like trypsin inhibitor (Trypsin inhibitor IT); Cystatin (Cys-C): like antipain; Asparaginic acid protease inhibitors: like the hiv protease suppressor factor, pepstatin, protease A suppressor factor and blood vessel tension peptide protoenzyme inhibitor; Inhibitors of metalloproteinase: like carboxyl peptide enzyme inhibitor, angiotensin-convertion enzyme inhibitor (ACE) etc.Proteinase inhibitor has very application prospects in fields such as food, medicine and agriculturals.For example proteinase inhibitor has protease activities in the insect gut of inhibition, can be used as biological pesticide or utilizes genetically engineered to improve the insect-resistance of plant.
Aspartate protease is one type of important protein lytic enzyme, and its active site is made up of two asparagicacid residues.Aspartate protease has the active function of aspects such as the maturation, adjust blood pressure of digest food albumen, degradation antigen, viral protein and enzyme, and these proteolytic enzyme are to cause one of the pathogenic factors of several kinds of refractory diseases in the world such as AIDS, malignant tumour, hypertension.Understand asparaginic acid protease inhibitors in depth, certain help will be arranged treating these stubborn diseases.
The asparaginic acid protease inhibitors great majority of report all are through the chemical method synthetic both at home and abroad at present; Often can not get guaranteeing to have limited its application because of its pharmacology toxicity and security; Asparaginic acid protease inhibitors extensively is present in vegeto-animal histoorgan and the certain micro-organisms, and therefore this type of proteinase inhibitor of exploitation receives people's attention day by day from natural product.
Pisum leguminous plants, formal name used at school are Pisum sativum L..China is maximum in the world pea plantation state, produces 5000000 tons of peas per year, accounts for 40.7% of Gross World Product.The contained vegetal pole horn of plenty of pea; It can not only provide most glucide (starch content is about 50%); And be to supply one of key protein resource that people are edible and animal is feeding, pea contains 20%～24% protein, and amino acid whose ratio is than balance.At present, most pea is used to make bean vermicelli on the market.Bean vermicelli mainly is the starch that utilizes in the pea, and the protein in the pea is just along with sewage has been discharged, and not only contaminate environment has also caused the waste of protein resource simultaneously.The Research Significance of the deep processing of therefore, carrying out the pea resource and comprehensive utilization aspect comprehensively is great.
Summary of the invention
The object of the invention provides a kind of preparation method of pea asparaginic acid protease inhibitors, adopts pea to pulverize, and uses flooding; High speed centrifugation; Thermal treatment, high speed centrifugation obtain thick asparaginic acid protease inhibitors, again through the DEAE-52 ion exchange chromatography; Obtain purer asparaginic acid protease inhibitors, to the pepsic optimal inhibition reaction times be 1 hour; Better heat stability, 100 ℃ of insulations suppress activity and almost have no loss after 1 hour; The pH stable range is 2.0～7.0.It is carried out the substrate specificity analysis; Know that this suppressor factor has higher inhibition active to stomach en-and the rennin that is all aspartic acid family proteolytic enzyme, several kinds of proteolytic enzyme (papoid, trypsinase, neutral protease etc.) that belong to other family are not almost suppressed active.
Technical scheme of the present invention: a kind of preparation method of pea asparaginic acid protease inhibitors; The employing pea is pulverized, and uses flooding, high speed centrifugation; Supernatant is heat-treated; High speed centrifugation again, supernatant gets the asparaginic acid protease inhibitors of purifying through the DEAE-52 ion exchange chromatography, gets asparaginic acid protease inhibitors dry powder through vacuum lyophilization again; Its technology is:
The preparation of A pea supernatant:
(1) pea: commercially available;
(2) flooding: after pea is pulverized, under 4 ℃, carry out lixiviate 2 h by g/mL 1:4 (w/v) with water, during every stir once at a distance from half a hour.With 8000 r/min, 4 ℃ of following centrifugal 30 min, get supernatant after the lixiviate;
(3) thermal treatment: supernatant is carried out 60 ℃ of following water-bath 20 min, and 8000 r/min, 4 ℃ of following centrifugal 30 min get supernatant; Protein is about 0.98 ~ 1.12 with the concentration ratio of sugar in the supernatant, stomach en-is suppressed unit be about 0.97 ~ 1.03 IU/mL, the recovery 86.5% ~ 89.4%;
B separates asparaginic acid protease inhibitors:
(4) ion exchange chromatography: DEAE-52 ion-exchange chromatography specification is 30 cm * 2.2 cm, and level pad is the sodium phosphate buffer (pH6.8) of 10 mmol/L.Elution mode: behind 2 times of column volumes of level pad wash-out, add successively with level pad again and contain different concns 0.2,0.4; 0.6; 0.8 the phosphoric acid buffer of mol/L NaCl carries out stepwise elution, flow velocity is 1.5 mL/min, sample size 20 mL; 280 nm wavelength ultraviolet detection; Result such as Fig. 1 show: the peak liquid of collecting under the phosphoric acid buffer gradient of 0.2 mol/L NaCl has stronger inhibition activity to aspartate protease, and the peak of collecting under other gradient then unrestraint is active, collects stomach en-is had the active peak 1 of higher inhibition;
Protein is about 2.16 ~ 2.45 with the concentration ratio of sugar in the peak 1 collection liquid, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 1.56 ~ 1.87 mg/mL, stomach en-is suppressed unit be about 8.93 ~ 9.42 IU/mL, the recovery 70.4 ~ 73.8 %.
(5) be dried to powder: the peak 1 that adopts vacuum freezing drying method that the DEAE-52 ion exchange chromatography is collected down with the phosphoric acid buffer gradient elution that contains 0.2 mol/L NaCl carries out lyophilize, is asparaginic acid protease inhibitors dry powder.
The suppressor factor substrate specificity is analyzed: be made into the solution of 5 mg/mL with the freezing powder of suppressor factor that obtains in above-mentioned (5), measure its inhibition activity to different proteolytic enzyme, result such as Fig. 2.
Know by Fig. 2; Stomach en-in the aspartate protease family that from pea, is produced and rennin all have stronger inhibition active; Suppress unit and be respectively 9.13 IU/mL and 8.57 IU/mL; To belonging to the papoid of halfcystine family proteolytic enzyme, the trypsinase of Serine family and the neutral protease etc. that belongs to metalloprotease family nearly all suppress active.
The enzyme activity determination method: stomach en-, trypsinase, papoid vitality test adopt conventional protease activity amylograph; The rennin vitality test adopts the Arima method.
Enzyme is lived and defined: in the time of 40 ℃, PM decomposition oxyphorase produces the required enzyme amount of 1 μ mol tyrosine and is enzyme unit alive, and unit representes with U.
Suppress unit definition: measure enzyme according to aforesaid method and live; Press inhibiting rate=(suppressing preferment work-inhibition back enzyme lives)/inhibition preferment alive * 100% and calculate inhibiting rate; Regulation reaches 10% inhibitor activity to the stomach en-inhibiting rate and is one and suppresses unit that unit representes with IU under these conditions.
IC
50Value defined: under optimum condition, a certain amount of proteolytic enzyme inhibiting rate reached 50% o'clock suppressor factor addition.
Useful effect of the present invention: raw material pea of the present invention can use the pea processing waste material to replace, and can eliminate the pollution of pea source mill to surrounding enviroment like this, prevents the wasting of resources, turns waste into wealth.The prepared suppressor factor specificity of the present invention suppresses the activity of aspartate protease, directly from pea, extracts to have and draws materials easily, and cost is low, and impurity is less relatively, is widely used, and does not influence numerous advantages such as security of food.Employing thermal treatment extraction asparaginic acid protease inhibitors is not only easy, easy to operate, cost is low, and is easy to suitability for industrialized production, in fields such as medicine, foodstuff additive and the prevention and control of plant diseases, pest control good application prospects is arranged.
Description of drawings
The DEAE-52 ion-exchange chromatography figure of Fig. 1 pea vat liquor.
Fig. 2 pea asparaginic acid protease inhibitors suppresses effect relatively to different proteolytic enzyme.
Preparation technology's schema of a kind of pea asparaginic acid protease inhibitors of Fig. 3.
The specific examples mode
After 50 g peas are cleaned baking immediately, pulverize, add ultrapure water 200 mL, put into refrigerator, under 4 ℃, carry out lixiviate 2 h by the ratio of 1:4 (w/v), during every at a distance from stirring half a hour once.After lixiviate is intact, vat liquor with 8000 r/min, 4 ℃ of following centrifugal 30 min, is got supernatant 175 mL; Supernatant is carried out water-bath 20 min under 60 ℃, be cooled to room temperature, 8000 r/min, 4 ℃ of following centrifugal 30 min; Get supernatant 160 mL; Record protein and be about 0.98, stomach en-is suppressed unit be about 0.97 IU/mL, the recovery 87.6% with sugared concentration ratio; Supernatant being carried out ion exchange chromatography, get the thick suppressor factor of 20 mL at every turn and carry out the DEAE-52 ion exchange chromatography, is 30 cm * 2.2 cm, and level pad is the sodium phosphate buffer (pH 6.8) of 10 mmol/L.Elution mode: behind 2 times of column volumes of level pad wash-out, the phosphoric acid buffer that adds the NaCl of 0.2,0.4,0.6,0.8 mol/L different concns successively with level pad again carries out stepwise elution, and flow velocity is 1.5 mL/min, 280 nm wavelength ultraviolet detection.Collect the peak 1 under the phosphoric acid buffer gradient of 0.2 mol/L NaCl, the concentration ratio that records protein and sugar is about 2.35, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 1.56 mg/mL, stomach en-is suppressed unit be about 9.42 IU/mL, the recovery 72.8 %.
After 50 g peas are cleaned baking immediately, pulverize, add ultrapure water 200 mL, put into refrigerator, under 4 ℃, carry out lixiviate 2 h by the ratio of 1:4 (w/v), during every at a distance from stirring half a hour once.After lixiviate is intact, vat liquor with 8000 r/min, 4 ℃ of following centrifugal 30 min, is got supernatant 175 mL; Supernatant is carried out water-bath 20 min under 60 ℃, be cooled to room temperature, 8000 r/min, 4 ℃ of following centrifugal 30 min; Get supernatant 160 mL; Record protein and be about 1.05, stomach en-is suppressed unit be about 0.99 IU/mL, the recovery 88.4% with sugared concentration ratio; Supernatant being carried out ion exchange chromatography, get the thick suppressor factor of 20 mL at every turn and carry out the DEAE-52 ion exchange chromatography, is 30 cm * 2.2 cm, and level pad is the sodium phosphate buffer (pH 6.8) of 10 mmol/L.Elution mode: behind 2 times of column volumes of level pad wash-out, the phosphoric acid buffer that adds the NaCl of 0.2,0.4,0.6,0.8 mol/L different concns successively with level pad again carries out stepwise elution, and flow velocity is 1.5 mL/min, 280 nm wavelength ultraviolet detection.Collect the peak 1 under the phosphoric acid buffer gradient of 0.2 mol/L NaCl, the concentration ratio that records protein and sugar is about 2.36, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 1.74 mg/mL, stomach en-is suppressed unit be about 9.16 IU/mL, the recovery 72.9 %.
Embodiment 3
After the 50g pea cleaned baking immediately, pulverize, add ultrapure water 200 mL, put into refrigerator, under 4 ℃, carry out lixiviate 2 h by the ratio of 1:4 (w/v), during every at a distance from stirring half a hour once.After lixiviate is intact, vat liquor with 8000 r/min, 4 ℃ of following centrifugal 30 min, is got supernatant 175 mL; Supernatant is carried out water-bath 20 min under 60 ℃, be cooled to room temperature, 8000 r/min, 4 ℃ of following centrifugal 30 min; Get supernatant 160 mL; Record protein and be about 1.06, stomach en-is suppressed unit be about 0.98IU/mL, the recovery 87.4% with sugared concentration ratio; Supernatant being carried out ion exchange chromatography, get the thick suppressor factor of 20 mL at every turn and carry out the DEAE-52 ion exchange chromatography, is 30 cm * 2.2 cm, and level pad is the sodium phosphate buffer (pH 6.8) of 10 mmol/L.Elution mode: behind 2 times of column volumes of level pad wash-out, the phosphoric acid buffer that adds the NaCl of 0.2,0.4,0.6,0.8 mol/L different concns successively with level pad again carries out stepwise elution, and flow velocity is 1.5 mL/min, 280 nm wavelength ultraviolet detection.Collect the peak 1 under the phosphoric acid buffer gradient of 0.2 mol/L NaCl, the concentration ratio that records protein and sugar is about 2.45, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 1.87 mg/mL, stomach en-is suppressed unit be about 8.93 IU/mL, the recovery 73.8 %.
Claims (1)
1. the preparation method of a pea asparaginic acid protease inhibitors; It is characterized in that: adopt pea to pulverize, use flooding, high speed centrifugation; Supernatant is heat-treated; High speed centrifugation again, supernatant gets the asparaginic acid protease inhibitors of purifying through the DEAE-52 ion exchange chromatography, gets asparaginic acid protease inhibitors dry powder through vacuum lyophilization again; Its technology is:
The preparation of A pea supernatant:
(1) pea: commercially available;
(2) flooding: after pea is pulverized, under 4 ℃, carry out lixiviate 2 h by g/mL 1:4, with 8000 r/min, 4 ℃ of following centrifugal 30 min, get supernatant after the lixiviate with water;
(3) thermal treatment: supernatant is carried out 60 ℃ of following water-bath 20 min, and 8000 r/min, 4 ℃ of following centrifugal 30 min get supernatant;
B separates asparaginic acid protease inhibitors:
(4) ion exchange chromatography: DEAE-52 ion-exchange chromatography specification is 30 cm * 2.2 cm, and level pad is the sodium phosphate buffer of pH 6.8,10 mmol/L; Elution mode: behind 2 times of column volumes of level pad wash-out, add successively with level pad again and contain different concns 0.2,0.4; 0.6; 0.8 the phosphoric acid buffer of mol/L NaCl carries out stepwise elution, flow velocity is 1.5 mL/min, and sample size is 20 mL; 280 nm wavelength ultraviolet detection are collected the peak liquid of wash-out under the phosphoric acid buffer gradient contain 0.2 mol/L NaCl;
(5) be dried to powder: the peak liquid of collecting with the phosphoric acid buffer gradient elution that contains 0.2 mol/L NaCl in the DEAE-52 ion exchange chromatography is carried out lyophilize, promptly get asparaginic acid protease inhibitors dry powder.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275149A (en) * | 2008-05-08 | 2008-10-01 | 江南大学 | Preparation for asparaginic acid protease inhibitors |
| CN101597332A (en) * | 2009-07-14 | 2009-12-09 | 江南大学 | A kind of preparation method of potato asparaginic acid protease inhibitors |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101275149A (en) * | 2008-05-08 | 2008-10-01 | 江南大学 | Preparation for asparaginic acid protease inhibitors |
| CN101597332A (en) * | 2009-07-14 | 2009-12-09 | 江南大学 | A kind of preparation method of potato asparaginic acid protease inhibitors |
Non-Patent Citations (4)
| Title |
|---|
| GHALEB M. ABUEREISH: "Pepsin inhibitor from roots of Anchusa Strigosa", 《PHYTOCHEMISTRY》 * |
| SIMON A. CATER ET AL.: "Aspartic proteinase inhibitors from tomato and potato are morepotent against yeast proteinase A than cathepsin D", 《BIOCHIMICA BIOPHYSICA ACTA》 * |
| 田亚平等: "大豆蛋白酶A抑制剂的提取和性质初探", 《食品与发酵工业》 * |
| 陈荣等: "天冬氨酸蛋白酶家族的分类、功能和演化", 《鲁东大学学报( 自然科学版)》 * |
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