CN102367467A - Preparation method for aspartic protease inhibitor in enzymatic hydrolysis products of soybean protein isolate - Google Patents
Preparation method for aspartic protease inhibitor in enzymatic hydrolysis products of soybean protein isolate Download PDFInfo
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- CN102367467A CN102367467A CN2011103755828A CN201110375582A CN102367467A CN 102367467 A CN102367467 A CN 102367467A CN 2011103755828 A CN2011103755828 A CN 2011103755828A CN 201110375582 A CN201110375582 A CN 201110375582A CN 102367467 A CN102367467 A CN 102367467A
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Abstract
The invention provides a preparation method for an aspartic protease inhibitor in enzymatic hydrolysis products of soybean protein isolate, which belongs to the field of separation and purification of biological products. The invention relates to enzymatic hydrolysis of soybean protein isolate with papain for obtainment of a crude product of the aspartic protease inhibitor. The aspartic protease inhibitor is prepared from the crude product by carrying out heat treatment and high speed centrifugation on an enzymatic hydrolysate and carrying out DEAE-52 ion exchange chromatography and separation on an obtained supernatant, and dry powder of the aspartic protease inhibitor is obtained by carrying out vacuum freeze drying on the obtained aspartic protease inhibitor. The aspartic protease inhibitor prepared in the invention has high inhibitory activity on pepsin and chymosin in the family of aspartic protease; the aspartic protease inhibitor extracted from the natural processing material soybean protein isolate which has undergone enzymatic hydrolysis has good specificity, good stability, safety in application and an application prospect in a wide variety of fields, e.g., medicines and health care, food additives, control of insect diseases, etc.
Description
Technical field
The preparation method of the asparaginic acid protease inhibitors in a kind of soybean protein isolate enzymolysis product; Be to adopt papoid enzymatic hydrolysis of soybean protein isolates under given conditions specifically; Enzymolysis solution is through bakingout process, high speed centrifugation, DEAE-52 ion exchange chromatography; Obtain the asparaginic acid protease inhibitors of purifying, belong to biological products separating and purifying technology field.
Background technology
Every field such as zymin has been widely used in food, brewages, pharmacy, organic acid, Dian Fentang, fodder industry, weaving, leather, washing composition and healthcare products; Application Areas more and more widely; Food and people's life is closely related; Therefore enzyme is worth more and more importantly in Application in Food Industry, is curing, is obtaining very big embodiment in milk preparation, meat processing, novel nourishing food and the proteinic hydrolysis at present.
Enzymolysis protein matter is produced polypeptide has become current popular research, can obtain the active polypeptide of difference in functionality through different enzyme butt formulas, especially develops some oligopeptides materials, becomes a frontier in the protein research.
Aspartate protease belongs to a kind of proteolyze enzyme family, is distributed widely in nature, and existence is all arranged in virus, fungi, plant and the animal.This family mainly comprises stomach en-, cathepsin D, protease A, Angiotensinogenase and virus of AIDS proteolytic enzyme etc., and they all contain the avtive spot of the asparagicacid residue of two high conservatives.Some aspartate protease has been proved participates in some pathologic process, and for example, HIV-1 proteolytic enzyme plays an important role in the reproduction process of virus of AIDS, and the transfer of tumour cell is the different proteolytic enzyme of demand also.In addition, they participate in many physiological processs, as: food digestion; The lysosome degraded; Signal transduction pathways etc. also can be reconciled the initial invasion step of infectious biological, because proteolytic enzyme catalysis peptide bond hydrolysis is irreversible basically; Therefore, play an important role in the proteinase inhibitor accurate activity of supervisory network on guaranteeing the room and time of proteolytic enzyme widely.
The proteinase inhibitor general reference has inhibiting one type of material to proteolytic enzyme, can participate in proteic enzymolysis activity in the control volume, and is significant to keeping normal vital movement.Have and discover that some asparaginic acid protease inhibitors are useful to HIV and hypertensive clinical treatment, in cancer, obesity, cardiovascular disorder, inflammation also has the potential using value in nerve degenerative diseases and various transmissible disease and the parasitosis.Mostly the asparaginic acid protease inhibitors of report is through the chemical method synthetic at present, and security can not get guaranteeing, therefore studies the suppressor factor that from natural biological, extracts and will have bigger actual application value.
The utilization of China's animal/vegetable protein has had very big development, has obtained using widely like Sunlover 10, casein, whey-protein etc.Soybean as one of three big crops; Annual Sunlover 10 quantity of producing is very big, and protein contnt utilizes the proteolyze after biological enzyme technology will be purified more than 90% in the soybean protein isolate; The polypeptide of preparation biologically active is the another kind of approach that improves the Sunlover 10 added value.
Summary of the invention
The object of the invention provides under the soybean protein isolate specified conditions a kind of preparation method of asparaginic acid protease inhibitors in the enzymolysis product; Adopt papoid enzymatic hydrolysis of soybean protein isolates under given conditions; Enzymolysis solution is heat-treated, and high speed centrifugation obtains thick asparaginic acid protease inhibitors, again through the DEAE-52 ion exchange chromatography; Obtain purer asparaginic acid protease inhibitors, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 56.5 ~ 58.6 μ g/mL, the recovery 75% ~ 79%, to the pepsic optimal inhibition reaction times be 20 min; In 40 ℃～90 ℃ scopes, the pepstatin good thermal stability; The pH stable range is 2～11.It is carried out the substrate specificity analysis; Know that this suppressor factor all has higher inhibition active to stomach en-and the rennin that is all aspartic acid family proteolytic enzyme, and several kinds of proteolytic enzyme (neutral protease of the trypsinase of the papoid of halfcystine family, Serine family, metalloprotease family etc.) that belong to other family are not nearly all suppressed active.
Technical scheme of the present invention: the preparation method of the asparaginic acid protease inhibitors in a kind of soybean protein isolate enzymolysis product; Adopt papoid enzymatic hydrolysis of soybean protein isolates under given conditions; Enzymolysis solution is heat-treated; High speed centrifugation, 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:
A screening enzyme is optimized enzymatic hydrolysis condition:
(1) soybean protein isolate: available from AY Mantianxue Food Manufacturing Co., Ltd.;
(2) enzymolysis: soybean protein isolate solution 100 mL of configuration quality concentration 5%; Live papoid, flavor protease, trypsinase identical time of enzymolysis under optimum temperuture, pH condition separately of unit (the proteolytic enzyme amount that every gram soybean protein isolate adds is 8000U) with same enzyme respectively; The result is as shown in Figure 1, three kinds of different protease hydrolyzed soybean protein isolate 200 min.Peak a, b, the pairing inhibition activity of c are respectively: 3.6,1.5,0.9 IU/mL.Papain enzymolysis Sunlover 10 60 min, a place has maximum inhibition active to stomach en-at the peak.So choosing the product of papain enzymolysis soybean protein isolate 60 min is research object;
(3) papain enzymolysis soybean protein isolate: confirm that papoid is the righttest proteolytic enzyme of enzymatic hydrolysis of soybean protein isolates, enzymatic hydrolysis condition is: the papoid amount that every gram soybean protein isolate adds is 8000U, and pH 6.5.55 ℃ of following water enzyme digestion 60 min;
The preparation of supernatant behind the B soybean protein isolate enzymolysis:
(4) high speed centrifugation: enzymolysis solution with 8000 r/min, 4 ℃ of following centrifugal 30 min, is got supernatant;
(5) thermal treatment: supernatant is carried out 70 ℃ of following water-bath 20 min, and centrifugal 30 min of 8000 r/min get supernatant;
The separation preparation of C asparaginic acid protease inhibitors:
(6) ion exchange chromatography: DEAE-52 ion-exchange chromatography specification 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, add respectively 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. 2 show: the stomach en-that the peak of collecting under the phosphoric acid buffer gradient of 0.2 mol/L NaCl 1,2 pairs at peak belong to aspartic acid family proteolytic enzyme has stronger inhibition activity, and the peak of collecting under other gradient then unrestraint is active;
(7) be dried to powder: adopt vacuum freezing drying method that the peak liquid of collecting down 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, be asparaginic acid protease inhibitors dry powder.
The suppressor factor substrate specificity is analyzed: the solution that is made into 1 mg/mL with the freezing powder of suppressor factor that obtains in the above-mentioned steps (7); It is active to measure its inhibition to different proteolytic enzyme; The suppressor factor that from enzymatic hydrolysis of soybean protein isolates liquid, is produced all has stronger inhibition active to stomach en-in two kinds of aspartate protease families and rennin; Suppress unit and be respectively 8.3 IU/mL and 9.2 IU/mL, papoid, trypsinase, neutral protease etc. are not almost suppressed active.
Suppressor factor thermal stability analysis: the solution that is made into 1 mg/mL with the freezing powder of suppressor factor that obtains in the above-mentioned steps (7); At different temperature condition held 30 min postcooling to room temperature; It is active to pepsic inhibition to measure it; The result is presented in 40 ℃～90 ℃ scopes, this suppressor factor good thermal stability.Higher temperature stability all has big benefit to extraction, processing and preservation.
Confirming of optimal inhibition reaction times: when suppressor factor and stomach en-reaction times were lower than 20 min, inhibiting rate increased with the increase in reaction times, and inhibiting rate reached maximum when the reaction times was 20 min, and the time is longer to be influenced very little to inhibiting rate.The double influence of synthesis measuring time and inhibiting rate, selecting 20 min is suppressor factor and pepsic the best use of time.
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.
Description of drawings
The different proteolytic enzyme of Fig. 1 to the enzymolysis product of soybean protein isolate to pepsic inhibition specific activity.
The enzymolysis product DEAE-52 ion-exchange chromatography figure of Fig. 2 soybean protein isolate.
The preparation flow of the asparaginic acid protease inhibitors in a kind of soybean protein isolate enzymolysis product of Fig. 3.
The specific examples mode
Embodiment 1
Soybean protein isolate solution 300 mL of configuration 5%, the solution of 1. getting 100 mL adds the papoid of 40000 U enzyme amounts, and pH transfers to 6.5, and water-bath 200 min under 55 ℃ of conditions are every at a distance from 20 min, survey it to pepsic inhibition activity; 2. the solution of getting 100 mL adds the flavor protease of 40000 U enzyme amounts, and pH transfers to 3.0, and water-bath 200 min under 37 ℃ of conditions are every at a distance from 20 min, surveys it to pepsic inhibition activity; 3. the solution of getting 100 mL adds the trypsinase of 40000 U enzyme amounts, and pH transfers to 7.5, and water-bath 200 min under 37 ℃ of conditions are every at a distance from 20 min, surveys it to pepsic inhibition activity.The result shows papain enzymolysis soybean protein isolate 60 min, has maximum inhibition active to stomach en-, and suppressing activity is 3.6 IU/mL; Flavor protease enzymatic hydrolysis of soybean protein isolates 60 min have maximum inhibition active to stomach en-, and suppressing activity is 1.5 IU/mL; Trypsin digestion soybean protein isolate 120min has maximum inhibition active to stomach en-, and suppressing activity is 0.9 IU/mL, is research object so choose the product of papain enzymolysis soybean protein isolate 60 min.
Dispose 5% soybean protein isolate solution 100 mL, add the papoid of 40000 U enzyme amounts, transfer pH 6.5; Water-bath 60 min under 55 ℃ of conditions with 8000 r/min, 4 ℃ of following centrifugal 30 min, get supernatant 98 mL; Supernatant is carried out water-bath 20 min under 70 ℃, be cooled to room temperature, 8000 r/min, 4 ℃ of following centrifugal 30 min; Get supernatant 92 mL, pepsic inhibition activity is about 3.3 IU/mL, supernatant is carried out ion exchange chromatography; Getting 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.Collection contains the peak 1 under the phosphoric acid buffer gradient of 0.2 mol/L NaCl, and peak 2 is totally 35 mL, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 56.5 μ g/mL, and pepsic inhibition activity is about 8.5 IU/mL, and the recovery is about 77 %.
Embodiment 3
Dispose 5% soybean protein isolate solution 150 mL, add the papoid of 60000 U enzyme amounts, transfer pH 6.5; Water-bath 60 min under 55 ℃ of conditions with 8000 r/min, 4 ℃ of following centrifugal 30 min, get supernatant 147 mL; Supernatant is carried out water-bath 20 min under 70 ℃, be cooled to room temperature, 8000 r/min, 4 ℃ of following centrifugal 30 min; Get supernatant 138 mL, pepsic inhibition activity is about 3.4 IU/mL, supernatant is carried out ion exchange chromatography; Getting 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, peak 2 is totally 35 mL, to the 503nhibiting concentration IC of stomach en-(2800U)
50Value is about 57.6 μ g/mL, and pepsic inhibition activity is about 8.6 IU/mL, and the recovery is about 76 %.
Claims (1)
1. the preparation method of the asparaginic acid protease inhibitors in the soybean protein isolate enzymolysis product; It is characterized by: adopt the papain enzymolysis soybean protein isolate; Enzymolysis solution is heat-treated; High speed centrifugation, 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:
A screening enzyme is optimized enzymatic hydrolysis condition:
(1) soybean protein isolate: commercially available;
(2) enzymolysis: soybean protein isolate solution 100 mL of configuration quality concentration 5%; Live papoid, flavor protease, trypsinase identical time of enzymolysis under optimum temperuture, pH condition separately of unit with same enzyme respectively; The research enzymolysis product is active to pepsic inhibition, confirms to select for use papoid;
(3) papain enzymolysis soybean protein isolate: confirm that papoid is the righttest proteolytic enzyme of enzymatic hydrolysis of soybean protein isolates; Enzymatic hydrolysis condition is: the papoid amount that every gram soybean protein isolate adds is 8000U; 6.5,55 ℃ of following water enzyme digestion 60 min of pH;
The preparation of supernatant behind the B soybean protein isolate enzymolysis:
(4) high speed centrifugation: enzymolysis solution with 8000 r/min, 4 ℃ of following centrifugal 30 min, is got supernatant;
(5) thermal treatment: supernatant is carried out 70 ℃ of following water-bath 20 min, and centrifugal 30 min of 8000 r/min get supernatant;
The separation preparation of C asparaginic acid protease inhibitors:
(6) 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 the phosphoric acid buffer that contains different concns 0.2,0.4,0.6,0.8 mol/L NaCl successively respectively with level pad again and carry out stepwise elution; Flow velocity is 1.5 mL/min; Sample size is 20 mL, 280 nm wavelength ultraviolet detection;
(7) be dried to powder: adopt vacuum freezing drying method that the peak liquid of collecting down 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, be asparaginic acid protease inhibitors dry powder.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115777941A (en) * | 2022-12-17 | 2023-03-14 | 寿光市沃野化工有限责任公司 | Method for preparing compound amino acid by soybean enzymolysis |
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| 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 |
| CN102115774A (en) * | 2010-11-30 | 2011-07-06 | 华东理工大学 | Method for preparing plant polypeptide by enzyme process |
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| 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 |
| CN102115774A (en) * | 2010-11-30 | 2011-07-06 | 华东理工大学 | Method for preparing plant polypeptide by enzyme process |
Non-Patent Citations (1)
| Title |
|---|
| TAKAHASHI S. ET AL.: "Isolation of human renin inhibitor from soybean Soyasaponin I is the novel human renin inhibitor in soybean", 《BIOSCI. BIOTECHNOL. BIOCHEM》, vol. 72, no. 12, 7 December 2008 (2008-12-07), pages 3232 - 3236 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115777941A (en) * | 2022-12-17 | 2023-03-14 | 寿光市沃野化工有限责任公司 | Method for preparing compound amino acid by soybean enzymolysis |
| CN115777941B (en) * | 2022-12-17 | 2023-08-29 | 寿光市沃野化工有限责任公司 | Method for preparing compound amino acid by soybean enzymolysis |
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