CN104911237A - Method for synthesizing salidroside by attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers - Google Patents
Method for synthesizing salidroside by attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers Download PDFInfo
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Abstract
The invention provides a method for synthesizing salidroside by attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers. The method for synthesizing salidroside by the attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers is characterized by comprising the steps of extracting black brin seed beta-glucosaccharase enzyme liquid, preparing the attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers by an absorption-crosslinking method, and catalyzing and synthesizing the salidroside. According to the method, discarded black brin seeds are used as extraction raw materials of beta-glucosaccharase, the yield is large, the price is low, and acquiring is easy. The attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers replace free enzyme, the enzyme activity recovery rate is high, the environmental tolerance is also obviously improved, and products can be separated easily. The method generally has high enzyme activity recovery rate, good stability and reusability, and also has the advantages of high concentration of salidroside in the products. Convenience is provided for scale production.
Description
Technical field
The invention belongs to immobilized enzyme production field, relate to the method catalyzing and synthesizing rhodioside, be specifically related to a kind of method of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer synthesis rhodioside.
Background technology
Root of Kirilow Rhodiola is generally grown on Eurasian two areas, plateau, state, is Crassulaceae perennial herb or semishrub plant, has the good reputation of " plateau ginseng ".The whole world has Rhodida plant 96 kinds, main distribution and East Asia, the Central Asia, siberian and north America region.China has 73 kinds, is mainly distributed in southwest, northwest, North, Northeast China area.The principle active component of Root of Kirilow Rhodiola is rhodioside, and chemical name para hydroxybenzene ethyl-β-D-glucoside is the main active ingredient in the Root of Kirilow Rhodiola of current most study, is the primary evaluation index evaluating Root of Kirilow Rhodiola quality.Rhodioside has significant biologic activity, its effect is better than the Chinese medicine such as ginseng and Radix Et Caulis Acanthopanacis Senticosi commonly used, cross in removing body polyradical, anti-hypoxia, anti-ageing, resist myocardial ischemia, improve immunity of organisms etc. in there is unusual effect, therefore can play the dual function of radioprotective and antifatigue simultaneously.
The source of rhodioside mainly contains three kinds of approach at present, and first is plant origin; Second is chemosynthesis; 3rd is enzymatic clarification.2004, Tong etc. utilized Apple Seeds powder β-glucuroide to synthesize first for substrate with p-hydroxyphenylethanol and glucose, had reacted rear rhodioside concentration and can reach 11.8g/L, higher than chemical synthesis and plant origin method.Therefore, enzyme process becomes a kind of effective ways of synthesis rhodioside.Beta-glucosidase is present in occurring in nature widely, all finds that there is the existence of this enzyme in microorganism and vegeto-animal body.At present, beta-glucosidase can be applicable to improve drink drinks local flavor, produce oligomeric dragon gallbladder sugar, hydrolyzed soy bean isoflavone, synthesis of glycoside compounds and degraded cellulose.But at present the glucuroide of plant origin mainly comes from Semen Armeniacae Amarum, the higher and limits throughput of its price, is unfavorable for large-scale production and application.
In actual applications, the anti-ability of bothering of free enzyme factor is to external world more weak and be difficult to recycle, and fixation techniques for enzyme is extensively studied as the major project means improving enzyme stability and reusing and uses.Have a lot for immobilized solid support material, comprise natural macromolecular material, synthesized polymer material, inorganic materials and matrix material etc., its concave soil (also referred to as attapulgite) is a kind of inorganic materials, and its main component is a kind of multi-hole type chain stratiform zeopan class clay mineral.This inorganic mineral of recessed soil, not only cheap and easy to get, and possess the advantages such as physical strength is high, Heat stability is good, anti-organic solvent, antimicrobial corrosion.The preparation of crosslinking enzyme polymkeric substance does not need to carry out the loaded down with trivial details and crystallisation process of complexity to enzyme, and its preparation process is simple, cheaper starting materials.Fully utilize stability and physical strength that recessed soil absorption and the method prepared of crosslinking enzyme polymkeric substance can improve immobilized enzyme effectively, for it provides more wide application space.
In the technology of immobilized enzyme, recessed soil is widely used as the carrier of enzyme.Application number is the patent " a kind of detection method of the intracellular hydrogen peroxide based on horseradish peroxidase-recessed native nano composite material " of CN201110064051, publication number is the patent " the immobilized lipase-catalyzed dose of method preparing biofuel of organically-modified attapulgite " of CN102719498A, application number is that recessed soil is used for different enzyme immobilizatio by the patents such as the patent " a kind of preparation method of the attapulgite immobilized enzyme for transesterification reaction " of CN201110350307, all obtains good effect.Recessed soil is various enzyme immobilizatio, provides a kind of excellent carrier.Actually rare for the synthesis of the β-glucuroide of rhodioside at present, and the concentration of product salidroside is still lower, the enzyme rate of recovery alive is lower, cost is high, suitability for industrialized production cannot be met to the maximized demand of economic interests, therefore, exploitation has more high efficiency β-glucuroide for the synthesis of rhodioside and utilizes this enzyme to grope optimum reaction condition and have greatly market using value.
Summary of the invention
The object of the invention is the above-mentioned shortcoming for existing in prior art, a kind of method of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer synthesis rhodioside is provided, first in black cloth woods seed, beta-glucosidase enzyme liquid is extracted, then absorption-crosslinking is utilized to prepare recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of preparation is finally utilized to catalyze and synthesize rhodioside, there is the high enzyme rate of recovery alive, satisfactory stability and reusability, there is the advantage that the concentration of product salidroside is higher simultaneously.
For achieving the above object, the present invention takes following technical scheme:
The method of a kind of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer synthesis rhodioside of the present invention, comprise extract black cloth woods seed beta-glucosidase enzyme liquid, absorption-crosslinking prepares recessed native load black cloth woods seed beta-glucosidase cross-linked polymer and catalyzes and synthesizes rhodioside three part.
The method of above-mentioned recessed native load black cloth woods seed beta-glucosidase cross-linked polymer synthesis rhodioside, step comprises:
(1) black cloth woods seed beta-glucosidase enzyme liquid is extracted:
1) black cloth woods seed is shelled, broken wall pulverize, obtain particle diameter 100 object black cloth woods seed powder, then the NaAc_HAc buffer solution of pH5.4 is added according to 50g/L, add the polygalacturonase accounting for black cloth woods seed powder massfraction 1 ~ 2% again, mix, cultivate 1 ~ 2h in 40 DEG C of isothermal vibrations, gained solution centrifugal is separated, get supernatant liquor vacuum lyophilization, abrasive dust crosses 60 mesh sieves, obtains dry powder;
2) extract of 25 ~ 50 times of dry powder volumes is added in step 1) dry powder, at 4 DEG C, stir 1 h, centrifugation, collect extracting solution, add the ammonium sulfate powder of extracting solution quality 40%, stir 30 min, recentrifuge is separated, get supernatant liquor, add the ammonium sulfate powder of supernatant liquor quality 65%, stir 30 min, third time centrifugation, the precipitation of third time centrifugation is dissolved in damping fluid, and concentration of ordinary dissolution is 2.65 ~ 4.2g/L, leaves standstill 10 ~ 12h, 3kDa ultrafiltration membrance filter, obtains black cloth woods seed beta-glucosidase enzyme liquid;
(2) absorption-crosslinking prepares recessed native load black cloth woods seed beta-glucosidase cross-linked polymer:
The recessed soil of sour modification is added in step (1) black cloth woods seed beta-glucosidase enzyme liquid, the recessed native mass ratio of black cloth woods seed beta-glucosidase and sour modification is 1:20 ~ 30, add the Microcrystalline Cellulose of black cloth woods seed beta-glucosidase quality 0.2% again, stir 0.5 ~ 1h; Then add precipitation agent, the volume ratio of precipitation agent and black cloth woods seed beta-glucosidase enzyme liquid is 2 ~ 4:1, stirs 10 ~ 15 min; Then add glutaraldehyde, it is 20 ~ 40 mmol/L that glutaraldehyde adds final concentration; 8 ~ 10 DEG C are stirred crosslinking curing 1 ~ 2h; In reaction system, add distilled water termination reaction, centrifugal 10 ~ 15 min of 8000 ~ 10000 rpm, be precipitated as recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, 4 DEG C of preservations, for subsequent use;
(3) rhodioside is catalyzed and synthesized:
Phosphate buffer soln and the trimethyl carbinol prepare mixing solutions according to volume ratio 1:4, then add glucose and p-hydroxyphenylethanol, add concentration and be respectively 0.25mol/L and 2.5mol/L, mix, 50 DEG C of preheating 30 min; Add the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of the quality such as glucose, mixing, airtight, react in constant temperature oscillator, rotating speed 200 rpm, 45 ~ 55 DEG C of reaction 70 ~ 75 h, collect reaction solution, centrifugation, clear liquid is product salidroside.
In step (1), described extract is phosphate buffer soln, and concentration is 50 mM, pH is 7.4;
In step (1), the damping fluid of dissolution precipitation is Tris-HCl damping fluid, and concentration is 20 mM, pH is 7.4;
In step (1), the concentration of obtained black cloth woods seed beta-glucosidase enzyme liquid is 2.5 ~ 4.0g/L;
In step (2), the recessed soil of described sour modification, method of modifying is: recessed soil mixes with the HCl solution of 2 mol/L, and melting concn 0.5g/ml stirs 12 h, filters, and deionized water wash is extremely without Cl
﹣ion, room temperature in vacuo is dry;
In step (2), precipitation agent is 95% ethanol through ﹣ 20 DEG C of precoolings;
In step (2), the volume of distilled water is 5 ~ 10 times of whole reaction system volume;
In step (1) and (2), described mixing speed is 30r/min;
In step (3), described phosphate buffer soln, PH is 6.0;
In step (3), described reaction, preferably 50 DEG C of reaction 72 h.
In the inventive method, reaction recurve soil load black cloth woods seed beta-glucosidase cross-linked polymer is recyclable, recycling mode is: collect the precipitation that reaction solution centrifugation goes out, first with concentration to be 20 mM, pH be 7.4 Tris-HCl buffer solution 3 times, then add the phosphate buffer soln that 1.0 mLPH are 6.5, vibration makes it to become suspension liquid, leave standstill 10h, centrifugation, is precipitated as the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of recovery.
The method of the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer synthesis rhodioside of the present invention, its beneficial effect is:
1) present invention employs the extraction raw material of black cloth woods seed discarded in food processing process as beta-glucosidase, output is large, price is low, easily obtain, to these fruit stone recyclings, can environmental pollution be reduced, realize low-carbon (LC), green circulation economy;
2) recessed native load black cloth woods seed beta-glucosidase cross-linked polymer replaces resolvase, and the enzyme rate of recovery alive improves, and is also significantly improved to the tolerance level of environment.During preparation, recessed soil is through peracid modification, glutaraldehyde effect, obtains the immobilized enzyme that zymoprotein unit concentration is higher, simultaneously by add Microcrystalline Cellulose make it have good stability, physical strength high, recycle the high feature of number of times.
3) the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of the present invention concentration that catalyzes and synthesizes product salidroside is higher, recessed native load black cloth woods seed beta-glucosidase cross-linked polymer is recyclable, significantly improve the utilization ratio of enzyme, reduce production cost.In addition, reaction recurve soil load black cloth woods seed beta-glucosidase cross-linked polymer, through centrifugal namely separable, reduces the separating difficulty of product, for large-scale production is provided convenience condition.
Embodiment
Further illustrate the inventive method below in conjunction with specific embodiment, but embodiment does not limit in any form to the present invention.
Embodiment 1: extract black cloth woods seed beta-glucosidase enzyme liquid 1
1) black cloth woods seed is shelled, broken wall pulverize, obtain particle diameter 100 object black cloth woods seed powder, then the NaAc_HAc buffer solution of pH5.4 is added according to 50g/L, add the polygalacturonase accounting for black cloth woods seed powder massfraction 1% again, mix, cultivate 1h in 40 DEG C of isothermal vibrations, gained solution centrifugal is separated, get supernatant liquor vacuum lyophilization, abrasive dust crosses 60 mesh sieves, obtains dry powder;
2) (concentration is 50 mM to add the phosphate buffer soln of 25 times of dry powder volumes in step 1) dry powder, pH is 7.4) as extract, at 4 DEG C, 30r/min stirs 1 h, centrifugation, collect extracting solution, add the ammonium sulfate powder of extracting solution quality 40%, stir 30 min, recentrifuge is separated, get supernatant liquor, add the ammonium sulfate powder of supernatant liquor quality 65%, 30r/min stirs 30 min, three centrifugations, (concentration is 20 mM the precipitation of third time centrifugation to be dissolved in Tris-HCl damping fluid, pH is 7.4), concentration of ordinary dissolution is 2.65g/L, leave standstill 10h, 3kDa ultrafiltration membrance filter, obtain black cloth woods seed beta-glucosidase enzyme liquid,
The concentration of the black cloth woods seed beta-glucosidase enzyme liquid that embodiment 1 is obtained is 3.82g/L.
Embodiment 2: extract black cloth woods seed beta-glucosidase enzyme liquid 2
1) black cloth woods seed is shelled, broken wall pulverize, obtain particle diameter 100 object black cloth woods seed powder, then the NaAc_HAc buffer solution of pH5.4 is added according to 50g/L, add the polygalacturonase accounting for black cloth woods seed powder massfraction 2% again, mix, cultivate 2h in 40 DEG C of isothermal vibrations, gained solution centrifugal is separated, get supernatant liquor vacuum lyophilization, abrasive dust crosses 60 mesh sieves, obtains dry powder;
2) (concentration is 50 mM to add the phosphate buffer soln of 50 times of dry powder volumes in step 1) dry powder, pH is 7.4) as extract, at 4 DEG C, 30r/min stirs 1 h, centrifugation, collect extracting solution, add the ammonium sulfate powder of extracting solution quality 40%, 30r/min stirs 30 min, recentrifuge is separated, get supernatant liquor, add the ammonium sulfate powder of supernatant liquor quality 65%, stir 30 min, three centrifugations, (concentration is 20 mM the precipitation of third time centrifugation to be dissolved in Tris-HCl damping fluid, pH is 7.4), concentration of ordinary dissolution is 4.2g/L, leave standstill 12h, 3kDa ultrafiltration membrance filter, obtain black cloth woods seed beta-glucosidase enzyme liquid,
The concentration of the black cloth woods seed beta-glucosidase enzyme liquid that embodiment 2 is obtained is 4.01g/L.
Embodiment 3: extract black cloth woods seed beta-glucosidase enzyme liquid 3
1) black cloth woods seed is shelled, broken wall pulverize, obtain particle diameter 100 object black cloth woods seed powder, then the NaAc_HAc buffer solution of pH5.4 is added according to 50g/L, add the polygalacturonase accounting for black cloth woods seed powder massfraction 1.5% again, mix, cultivate 1.5h in 40 DEG C of isothermal vibrations, gained solution centrifugal is separated, get supernatant liquor vacuum lyophilization, abrasive dust crosses 60 mesh sieves, obtains dry powder;
2) (concentration is 50 mM to add the phosphate buffer soln of 35 times of dry powder volumes in step 1) dry powder, pH is 7.4) as extract, at 4 DEG C, 30r/min stirs 1 h, centrifugation, collect extracting solution, add the ammonium sulfate powder of extracting solution quality 40%, 30r/min stirs 30 min, recentrifuge is separated, get supernatant liquor, add the ammonium sulfate powder of supernatant liquor quality 65%, stir 30 min, three centrifugations, (concentration is 20 mM the precipitation of third time centrifugation to be dissolved in Tris-HCl damping fluid, pH is 7.4), concentration of ordinary dissolution is 3.0g/L, leave standstill 10 ~ 12h, 3kDa ultrafiltration membrance filter, obtain black cloth woods seed beta-glucosidase enzyme liquid,
The concentration of the black cloth woods seed beta-glucosidase enzyme liquid that embodiment 3 is obtained is 3.67g/L.
Embodiment 4: absorption-crosslinking prepares recessed native load black cloth woods seed beta-glucosidase cross-linked polymer 1
The recessed soil of sour modification is added in the black cloth woods seed beta-glucosidase enzyme liquid that Example 1 is obtained, the recessed native mass ratio of black cloth woods seed beta-glucosidase and sour modification is 1:20, add the Microcrystalline Cellulose of black cloth woods seed beta-glucosidase quality 0.2% again, 30r/min stirs 0.5h; Then add 95% ethanol through ﹣ 20 DEG C of precoolings as precipitation agent, the volume ratio of precipitation agent and black cloth woods seed beta-glucosidase enzyme liquid is that 2:1,30r/min stir 10 min; Then add glutaraldehyde, it is 20 mmol/L that glutaraldehyde adds final concentration; 8 DEG C are stirred crosslinking curing 1h; In reaction system, add 5 times of distilled water termination reactions of whole reaction system volume, the centrifugal 10min of 8000 rpm, be precipitated as recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, under being stored in 4 DEG C of environment; Recessed mud acid method of modifying be: recessed soil mixes with the HCl solution of 2 mol/L, and melting concn 0.5g/ml stirs 12 h, filters, and deionized water wash is to without Cl
﹣ion, room temperature in vacuo is dry.
Embodiment 5: absorption-crosslinking prepares recessed native load black cloth woods seed beta-glucosidase cross-linked polymer 2
The recessed soil of sour modification is added in the black cloth woods seed beta-glucosidase enzyme liquid that Example 2 is obtained, the recessed native mass ratio of black cloth woods seed beta-glucosidase and sour modification is 1:30, add the Microcrystalline Cellulose of black cloth woods seed beta-glucosidase quality 0.2% again, 30r/min stirs 1h; Then add 95% ethanol through ﹣ 20 DEG C of precoolings as precipitation agent, the volume ratio of precipitation agent and black cloth woods seed beta-glucosidase enzyme liquid is that 4:1,30r/min stir 15 min; Then add glutaraldehyde, it is 40 mmol/L that glutaraldehyde adds final concentration; 10 DEG C are stirred crosslinking curing 2h; In reaction system, add 10 times of distilled water termination reactions of whole reaction system volume, centrifugal 15 min of 10000 rpm, be precipitated as recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, under being stored in 4 DEG C of environment; Recessed mud acid method of modifying is: recessed soil mixes with the HCl solution of 2 mol/L, and melting concn 0.5g/ml stirs 12 h, filters, and deionized water wash is extremely without Cl
﹣ion, room temperature in vacuo is dry.
Embodiment 6: catalyze and synthesize rhodioside 1
Phosphate buffer soln and the trimethyl carbinol prepare mixing solutions according to volume ratio 1:4, and then add glucose and p-hydroxyphenylethanol, concentration is respectively 0.25mol/L and 2.5mol/L, mix, 50 DEG C of preheating 30 min, recessed native load black cloth woods seed beta-glucosidase cross-linked polymer prepared by the embodiment 4 adding the quality such as glucose, mixing, airtight, react in constant temperature oscillator, rotating speed 200 rpm, 50 DEG C of reaction 72h, collect reaction solution, centrifugation, clear liquid is product salidroside.
In embodiment 6 product, the concentration of rhodioside is 35.24 g/L.
Embodiment 7: catalyze and synthesize rhodioside 2
Phosphate buffer soln and the trimethyl carbinol prepare mixing solutions according to volume ratio 1:4, and then add glucose and p-hydroxyphenylethanol, concentration is respectively 0.25mol/L and 2.5mol/L, mix, 50 DEG C of preheating 30 min, recessed native load black cloth woods seed beta-glucosidase cross-linked polymer prepared by the embodiment 5 adding the quality such as glucose, mixing, airtight, react in constant temperature oscillator, rotating speed 200 rpm, 45 DEG C of reaction 70 h, collect reaction solution, centrifugation, clear liquid is product salidroside.
In embodiment 7 product, the concentration of rhodioside is 32.56 g/L.
Embodiment 8: catalyze and synthesize rhodioside 3
Phosphate buffer soln and the trimethyl carbinol prepare mixing solutions according to volume ratio 1:4, and then add glucose and p-hydroxyphenylethanol, concentration is respectively 0.25mol/L and 2.5mol/L, mix, 50 DEG C of preheating 30 min, recessed native load black cloth woods seed beta-glucosidase cross-linked polymer prepared by the embodiment 4 adding the quality such as glucose, mixing, airtight, react in constant temperature oscillator, rotating speed 200 rpm, 55 DEG C of reaction 75 h, collect reaction solution, centrifugation, clear liquid is product salidroside.
In embodiment 8 product, the concentration of rhodioside is 35.71 g/L.
Embodiment 9: use the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of recovery to catalyze and synthesize rhodioside
The recycling mode of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer is: be first 20 mM by concentration, pH is the Tris-HCl buffer solution 3 times of 7.4, add the phosphate buffer soln that 1.0 mLPH are 6.5 again, vibration makes it to become suspension liquid, leave standstill 10h, centrifugation, is precipitated as the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of recovery.
Catalyze and synthesize rhodioside method with embodiment 6, reclaim use 8 batches continuously, in product, the concentration of rhodioside is the stable yield of 31.67 ~ 35.68 g/L, rhodioside.
Comparative example 1: extract black cloth woods seed beta-glucosidase enzyme liquid, step is with reference to embodiment 1, and do not add polygalacturonase during extraction, other are with embodiment 1
The concentration of the black cloth woods seed beta-glucosidase enzyme liquid that comparative example 1 is obtained is 3.05g/L.
Comparative example 2: prepare recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, step is with reference to embodiment 4, do not add Microcrystalline Cellulose: and use obtained recessed native load black cloth woods seed beta-glucosidase cross-linked polymer to catalyze and synthesize rhodioside, step is with embodiment 6, and in obtained product, the concentration of rhodioside is 32.85 g/L.
Reclaim and use the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer in comparative example 2 to catalyze and synthesize rhodioside, recovery method is with embodiment 9, catalyze and synthesize rhodioside method with embodiment 6, continuous recovery use 3 batches, in product, the concentration of rhodioside is respectively 31.74g/L, 28.17g/L and 24.39g/L, and the concentration of rhodioside reduces and the recyclable number of times of polymkeric substance obviously reduces.
Recessed native load black cloth woods seed beta-glucosidase cross-linked polymer performance measurement prepared by the present invention
Enzyme determination of recovery rates method alive: the hydrolysis vigor of the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of sampling and measuring, with the hydrolysis vigor of resolvase liquid for 100%, calculate the relative hydrolysis vigor of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, i.e. the enzyme of the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer rate of recovery alive.
The measuring method of the hydrolysis vigor of resolvase liquid: get 0.1 mL resolvase liquid, be placed on 50 mM of 2.9 mL, the citric acid-Na of pH 7.0
2hPO
4in damping fluid, 45 DEG C hatch 3 min after, p-nitrophenyl-β-D-Glucose glycosides (pNPG) the 15.0 μ L adding 200 mM in system starts reaction, after 5 min, adds the Na of 1.0 M
2cO
3solution 3.0 mL termination reaction, and read the light absorption value of reaction solution at 405 nm immediately, the typical curve of the light absorption value contrast p-NP of gained, calculates the hydrolysis vigor of resolvase liquid.
The measuring method of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer hydrolysis vigor: get 0.1 mL recessed native load black cloth woods seed beta-glucosidase cross-linked polymer solution to be measured, be placed on 50 mM of 2.9 mL, the citric acid-Na of pH 7.0
2hPO
4in damping fluid, 45 DEG C hatch 3 min after, p-nitrophenyl-β-D-Glucose glycosides (pNPG) the 15.0 μ L adding 200 mM in system starts reaction, after 5 min, adds the Na of 1.0 M
2cO
3solution 3.0 mL termination reaction, and read the light absorption value of reaction solution at 405 nm immediately.The typical curve of the light absorption value contrast p-NP of gained, calculates recessed native load black cloth woods seed beta-glucosidase cross-linked polymer hydrolysis vigor.1 enzyme unit definition alive is: under the test conditions above, in 1 min, beta-glucoside enzymic hydrolysis discharges the enzyme amount required for 1 μm of ol p-NP.
The enzyme rate of recovery alive of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer prepared by the embodiment of the present invention is respectively: embodiment 4 is 75.7%, embodiment 5 is 76.5%, enzyme after reclaiming for 8 times in embodiment 9 rate of recovery alive is followed successively by 75.2%, 75.0%, 74.7%, 74.1%, 73.9%, 73.5%, 73.1% and 72.7%, the comparative example 2 enzyme rate of recovery alive is that the enzyme rate of recovery alive after reclaiming for 74.4%, 3 times is followed successively by 69.2%, 62.5% and 55.7%.
Resolvase and recessed native load black cloth woods seed beta-glucosidase cross-linked polymer performance comparison:
(1) optimum temperuture: the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer getting the preparation of appropriate resolvase liquid, comparative example 2 and embodiment 1 respectively, under being placed in the condition of 30 ~ 70 DEG C respectively, measure hydrolysis vigor, determine according to result, the optimum temperuture of resolvase is 55 DEG C, the optimum temperuture of recessed native load black cloth woods seed beta-glucosidase cross-linked polymer: comparative example 2 is 58 DEG C, and embodiment 1 is 60 DEG C.
(2) transformation period that enzyme is alive relatively: the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer getting the preparation of appropriate resolvase liquid, comparative example 2 and embodiment 1 respectively, 8 h are hatched at being placed in 60 DEG C respectively, one group of sample is taken out every 1 h, be placed in the refrigerator of 4 DEG C, after hatching end, measure remaining hydrolysis vigor, the hydrolysis vigor without the enzyme liquid of hatching process is defined as 100%.The transformation period that the relative enzyme of resolvase is lived is 60 min, and the transformation period that its relative enzyme of the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of embodiment 1 is lived is increased to 120 min, comparative example 2 be then 80min.
(3) pH tolerant: the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer getting the preparation of appropriate resolvase liquid, comparative example 2 and embodiment 1 respectively, is placed in 50 mM citric acid-Na of different pH value 3.0 ~ 8.0 respectively
2hPO
4in damping fluid, under the condition of 25 DEG C, hatch 24 h, measure remaining hydrolysis vigor, the hydrolysis vigor without the enzyme liquid of hatching process is defined as 100%.Resolvase pH tolerant is 5.0 ~ 7.5, recessed native load black cloth woods seed beta-glucosidase cross-linked polymer pH tolerant: comparative example 2 is 4.1 ~ 7.6, and embodiment 1 is 3.6 ~ 8.0.
Claims (10)
1. a method for recessed native load black cloth woods seed beta-glucosidase cross-linked polymer synthesis rhodioside, is characterized in that: comprise extract black cloth woods seed beta-glucosidase enzyme liquid, absorption-crosslinking prepares recessed native load black cloth woods seed beta-glucosidase cross-linked polymer and catalyzes and synthesizes rhodioside three part.
2. method according to claim 1, is characterized in that, step comprises:
(1) black cloth woods seed beta-glucosidase enzyme liquid is extracted:
1) black cloth woods seed is shelled, broken wall pulverize, obtain particle diameter 100 object black cloth woods seed powder, then the NaAc_HAc buffer solution of pH5.4 is added according to 50g/L, add the polygalacturonase accounting for black cloth woods seed powder massfraction 1 ~ 2% again, mix, cultivate 1 ~ 2h in 40 DEG C of isothermal vibrations, gained solution centrifugal is separated, get supernatant liquor vacuum lyophilization, abrasive dust crosses 60 mesh sieves, obtains dry powder;
2) extract of 25 ~ 50 times of dry powder volumes is added in step 1) dry powder, at 4 DEG C, stir 1 h, centrifugation, collect extracting solution, add the ammonium sulfate powder of extracting solution quality 40%, stir 30 min, recentrifuge is separated, get supernatant liquor, add the ammonium sulfate powder of supernatant liquor quality 65%, stir 30 min, third time centrifugation, the precipitation of third time centrifugation is dissolved in damping fluid, and concentration of ordinary dissolution is 2.65 ~ 4.2g/L, leaves standstill 10 ~ 12h, 3kDa ultrafiltration membrance filter, obtains black cloth woods seed beta-glucosidase enzyme liquid;
(2) absorption-crosslinking prepares recessed native load black cloth woods seed beta-glucosidase cross-linked polymer:
The recessed soil of sour modification is added in step (1) black cloth woods seed beta-glucosidase enzyme liquid, the recessed native mass ratio of black cloth woods seed beta-glucosidase and sour modification is 1:20 ~ 30, add the Microcrystalline Cellulose of black cloth woods seed beta-glucosidase quality 0.2% again, stir 0.5 ~ 1h; Then add precipitation agent, the volume ratio of precipitation agent and black cloth woods seed beta-glucosidase enzyme liquid is 2 ~ 4:1, stirs 10 ~ 15 min; Then add glutaraldehyde, it is 20 ~ 40 mmol/L that glutaraldehyde adds final concentration; 8 ~ 10 DEG C are stirred crosslinking curing 1 ~ 2h; In reaction system, add distilled water termination reaction, centrifugal 10 ~ 15 min of 8000 ~ 10000 rpm, be precipitated as recessed native load black cloth woods seed beta-glucosidase cross-linked polymer, 4 DEG C of preservations, for subsequent use;
(3) rhodioside is catalyzed and synthesized:
Phosphate buffer soln and the trimethyl carbinol prepare mixing solutions according to volume ratio 1:4, then add glucose and p-hydroxyphenylethanol, add concentration and be respectively 0.25mol/L and 2.5mol/L, mix, 50 DEG C of preheating 30 min; Add the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of the quality such as glucose, mixing, airtight, react in constant temperature oscillator, rotating speed 200 rpm, 45 ~ 55 DEG C of reaction 70 ~ 75 h, collect reaction solution, centrifugation, clear liquid is product salidroside.
3. method according to claim 2, is characterized in that: in step (1), and described extract is phosphate buffer soln, and concentration is 50 mM, pH is 7.4; The damping fluid of dissolution precipitation is Tris-HCl damping fluid, and concentration is 20 mM, pH is 7.4.
4. method according to claim 2, is characterized in that: in step (1), and the concentration of obtained black cloth woods seed beta-glucosidase enzyme liquid is 2.5 ~ 4.0g/L.
5. method according to claim 2, is characterized in that: in step (2), the recessed soil of described sour modification, and method of modifying is: recessed soil mixes with the HCl solution of 2 mol/L, and melting concn 0.5g/ml stirs 12 h, filters, and deionized water wash is extremely without Cl
﹣ion, room temperature in vacuo is dry.
6. method according to claim 2, is characterized in that: in step (2), and precipitation agent is 95% ethanol through ﹣ 20 DEG C of precoolings; The volume of distilled water is 5 ~ 10 times of whole reaction system volume.
7. method according to claim 2, is characterized in that: in step (1) and (2), described mixing speed is 30r/min.
8. method according to claim 2, is characterized in that: in step (3), described phosphate buffer soln, and PH is 6.0.
9. method according to claim 2, is characterized in that: in step (3), and described reaction is 50 DEG C of reaction 72 h.
10. the method according to the arbitrary claim of 1-9, it is characterized in that: reaction recurve soil load black cloth woods seed beta-glucosidase cross-linked polymer is recyclable, recycling mode is: collect the precipitation that reaction solution centrifugation goes out, first be 20 mM by concentration, pH is the Tris-HCl buffer solution 3 times of 7.4, add the phosphate buffer soln that 1.0 mLPH are 6.5 again, vibration makes it to become suspension liquid, leave standstill 10h, centrifugation, is precipitated as the recessed native load black cloth woods seed beta-glucosidase cross-linked polymer of recovery.
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Application publication date: 20150916 Assignee: Zibo Hefeng Seed Technology Co.,Ltd. Assignor: HUAIYIN INSTITUTE OF TECHNOLOGY Contract record no.: X2021990000798 Denomination of invention: A concave soil loaded black brin seed b- Synthesis of salidroside from glucosidase crosslinked polymer Granted publication date: 20170929 License type: Common License Record date: 20211220 |
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