CN106222159A - A kind of defying age co-immobilization enzyme and preparation method thereof - Google Patents
A kind of defying age co-immobilization enzyme and preparation method thereof Download PDFInfo
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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Abstract
The invention discloses a kind of defying age co-immobilization enzyme, including catalase and glucoseoxidase, the vigor of described catalase and glucoseoxidase ratio is for 8:1～15:1.The preparation method of defying age co-immobilization enzyme, including following preparation process: catalase and glucoseoxidase are made catalase and glucoseoxidase mixed enzyme solution by vigor than in the phosphate buffer being dissolved in, in mixed enzyme solution, add chitosan microball make mixed liquor；Mixed liquor stirring and adsorbing；Adding mass concentration percent is that glutaraldehyde solution carries out cross-linking reaction；Place balance to room temperature；Finally, add β D glucose, stand after electromagnetic agitation, standby.The present invention can remove, by catalase and glucoseoxidase synergism, the hydrogen peroxide that body surface is produced through bivalence electron reduction under the external condition effects such as ultraviolet by oxygen, reaches the purpose of slow down aging.
Description
Technical field
The present invention relates to a kind of co-curing enzyme and preparation method thereof, be specifically related to a kind of defying age co-immobilization enzyme and system thereof
Preparation Method.
Background technology
People's interior free yl is broadly divided into endogenous and exogenous free radical.Endogenous free radical mostlys come from body certainly
The metabolic activity of body, wherein based on aerobic metabolism.When the generation of interior free yl and remove unbalance after, internal too much from
A series of radical reaction can be caused by base, thus cause the reactions such as lipid peroxidation.People's aerobic vital movement creates
First free radical in human body: ultra-oxygen anion free radical (O2-·).Superoxide dismutase can remove superoxide anion freely
Base (O2-) generation hydrogen peroxide, and peroxidating Hydrogen Energy permeates cell membranes, this is ultra-oxygen anion free radical (O2-Less than)
's.Can generate, by Fenton's reaction, the hydroxyl radical free radical (OH) that oxidisability is higher after hydrogen peroxide enters cell.Hydroxyl
Free radical (OH) is the free radical that activity in vivo is the strongest, can pass through H-Abstraction reaction, molecular rearrangement, double bond conjugation and oxygen knot
Closing, H-Abstraction reaction forms lipid peroxide and lipid free radical (L), and is formed the chain of lipid peroxidation by this circulation
Reaction of propagation, causes constantly consuming and a large amount of generations of lipid peroxide of lipid molecular.Lipid peroxidation is in cell
Film, nucleic acid, protein and enzyme, cause irreversible damage, and these materials become oxide or peroxide and loses
Intrinsic effect, the continuous accumulation of these effects, promote body aging.Therefore, ultra-oxygen anion free radical (O2-) to cell
Damage be that the hydrogen peroxide formed by dismutation reaction is mediated.Meanwhile, with advancing age, a large amount of hydrogen peroxide are at head
Accumulation in Faing can hinder melanic formation in hair, causes hair the most gradually to bleach.
Exogenous free radical is mainly the oil smoke from cooking, radiation, smoking, excessive drinking, sunlight exposure etc., and the skin of people is every
It is in air, pollutes, radiates, in the environment such as ultraviolet, can form substantial amounts of free radical in atmosphere, under normal circumstances these
Free radical will soon disappear after producing, but when after the aggregates in itself and air, can form stable free radical, to human body
Skin even body causes erosion.Show as skin dark yellow, matt, lax nonelastic, even there is pigmentation, speckle occurs
Point.
Summary of the invention
The technical problem to be solved is often irradiated by computer radiate or ultraviolet and indulges in excessive drinking or stay up late
Crowd, it may appear that pachylosis, the phenomenon of pigmentation, it is therefore intended that a kind of defying age immobilized enzyme and preparation side thereof altogether are provided
Method, can remove body surface under the external condition effects such as ultraviolet by oxygen by catalase and glucoseoxidase synergism
Through the hydrogen peroxide that bivalence electron reduction produces, reach the purpose of slow down aging.
The present invention is achieved through the following technical solutions:
A kind of defying age co-immobilization enzyme, including catalase and glucoseoxidase, described catalase and Portugal
The vigor of glucoseoxidase is than for 8:1～15:1.
Preferably, the vigor proportioning of described catalase and glucoseoxidase is 9:1～12:1.
A kind of preparation method of defying age co-immobilization enzyme, including following preparation process:
A, catalase and glucoseoxidase are made peroxidating by vigor than in the phosphate buffer being dissolved in
Hydrogen enzyme and glucoseoxidase mixed enzyme solution；
B, the mixed enzyme solution prepared in step a add chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are stirred absorption；
D, after completing step c add glutaraldehyde solution carry out cross-linking reaction；
E, standing, balance is to room temperature；
F, after completing step e add β-D-Glucose, after electromagnetic agitation, stand, standby；
Wherein, in a step, the vigor of described hydrogen oxide enzyme and glucoseoxidase is than for 8:1～15:1 or 9:1～12:
1。
Preferably, in described a step, the pH of phosphate buffer is 3～8, and concentration is 0.1～0.5mol/L；Described c walks
In Zhou, mixed liquor is at 20～50 DEG C, under conditions of pH is 5～7, and stirring and adsorbing 40～55min；In described Step d, add penta 2
The mass concentration percent of aldehyde solution is 1.5～3.5%.
Preferably, described catalatic Rate activity be 50000U/mL, pH be 6.0；The ratio of described glucoseoxidase
Vigor is 10000U/g.
Preferably, described a step preparation catalase and the volume of glucoseoxidase mixed enzyme solution: in b step
The quality of chitosan microball is 5 (mL): 2 (g)～5 (mL): 4 (g).
The defying age co-curing enzyme of the present invention is acted on body surface, the oxidation resistant protective layer of densification can be formed, be conducive to clear
The hydrogen peroxide produced through bivalence electron reduction by oxygen under the external condition effects such as ultraviolet except body surface, it is to avoid free radical produces
Give birth to and remove unbalance, causing free chain reaction to accelerate the aging of human body skin；Meanwhile, can be decomposed by glucoseoxidase
β-D-Glucose in co-curing enzyme removes oxygen and the excess of oxygen of catalase breaks hydrogen peroxide generation of body surface excess,
And make body surface oxygen content reach dynamic equilibrium, keep elasticity of skin and pale；Glucoseoxidase produces when decomposing β-D-Glucose
Raw gluconic acid, gluconic acid has chelating copper ion characteristic, can help to inhibit the activity of Tyrosinase, and pre-anti-melanin is raw
Become, the appearance of prevention senile plaque.
The present invention compared with prior art, has such advantages as and beneficial effect:
The present invention is a kind of defying age co-immobilization enzyme and preparation method thereof, by catalase and glucoseoxidase
Synergism can remove the peroxidating that body surface is produced through valency bivalence electron reduction under the external condition effects such as ultraviolet by oxygen
Hydrogen, glucoseoxidase decomposes the β-D-Glucose in co-curing enzyme and removes oxygen and the catalase breaks mistake of body surface excess
The excess of oxygen that hydrogen oxide produces, and make body surface oxygen content reach dynamic equilibrium, keeps elasticity of skin and pale, reaches defying age
Purpose.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is made
Further describing in detail, the exemplary embodiment of the present invention and explanation thereof are only used for explaining the present invention, are not intended as this
The restriction of invention.
Embodiment 1
The preparation method of a kind of defying age co-immobilization enzyme, defying age co-immobilization enzyme, including catalase and Fructus Vitis viniferae
The vigor of carbohydrate oxidase, catalase and glucoseoxidase is than for 8:1.
Including following preparation process:
A, catalase and glucoseoxidase were made than in the phosphate buffer being dissolved in for 8:1 by vigor
Hydrogen oxide enzyme and glucoseoxidase mixed enzyme solution, wherein, the pH of phosphate buffer is 3, and concentration is 0.1mol/L；
B, the mixed enzyme solution prepared in step a add 3g chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are at 20 DEG C, under conditions of pH is 5, and stirring and adsorbing 40min；
D, after completing step c add mass concentration percent be the glutaraldehyde solution of 1.5%, the temperature conditions of 5 DEG C
Lower cross-linking reaction 2.5h；
E, placement balance are to room temperature；
F, after completing step e add 0.8g β-D-Glucose, after electromagnetic agitation 30min stand, standby.
Wherein, catalatic Rate activity be 50000U/mL, pH be 6.0；Described solid glucose is oxidasic than living
Power is 10000U/g；The catalase of a step preparation and the volume of glucoseoxidase mixed enzyme solution: chitosan in b step
The quality of microsphere is 5 (mL): 2 (g).
Embodiment 2
The preparation method of a kind of defying age co-immobilization enzyme, defying age co-immobilization enzyme, including catalase and Fructus Vitis viniferae
The vigor of carbohydrate oxidase, catalase and glucoseoxidase is than for 9:1.
Including following preparation process:
A, catalase and glucoseoxidase were made than in the phosphate buffer being dissolved in for 9:1 by vigor
Hydrogen oxide enzyme and glucoseoxidase mixed enzyme solution, wherein, the pH of phosphate buffer is 5, and concentration is 0.3mol/L；
B, the mixed enzyme solution prepared in step a add 3g chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are at 30 DEG C, under conditions of pH is 6.5, and stirring and adsorbing 45min；
D, after completing step c add mass concentration percent be the glutaraldehyde solution of 2.5%, the temperature conditions of 5 DEG C
Lower cross-linking reaction 2.5h；
E, placement balance are to room temperature；
F, after completing step e add 0.8g β-D-Glucose, after electromagnetic agitation 30min stand, standby.
Wherein, catalatic Rate activity be 50000U/mL, pH be 6.0；Described solid glucose is oxidasic than living
Power is 10000U/g；The catalase of a step preparation and the volume of glucoseoxidase mixed enzyme solution: chitosan in b step
The quality of microsphere is 5 (mL): 3 (g).
Embodiment 3
The preparation method of a kind of defying age co-immobilization enzyme, defying age co-immobilization enzyme, including catalase and Fructus Vitis viniferae
The vigor of carbohydrate oxidase, catalase and glucoseoxidase is than for 10:1.
Including following preparation process:
A, catalase and glucoseoxidase are made than in the phosphate buffer being dissolved in for 10:1 by vigor
Catalase and glucoseoxidase mixed enzyme solution, wherein, the pH of phosphate buffer is 5, and concentration is 0.3mol/L；
B, the mixed enzyme solution prepared in step a add 3g chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are at 30 DEG C, under conditions of pH is 6.5, and stirring and adsorbing 45min；
D, after completing step c add mass concentration percent be the glutaraldehyde solution of 2.5%, the temperature conditions of 5 DEG C
Lower cross-linking reaction 2.5h；
E, placement balance are to room temperature；
F, after completing step e add 0.8g β-D-Glucose, after electromagnetic agitation 30min stand, standby.
Wherein, catalatic Rate activity be 50000U/mL, pH be 6.0；Described solid glucose is oxidasic than living
Power is 10000U/g；The catalase of a step preparation and the volume of glucoseoxidase mixed enzyme solution: chitosan in b step
The quality of microsphere is 5 (mL): 3 (g).
Embodiment 4
The preparation method of a kind of defying age co-immobilization enzyme, defying age co-immobilization enzyme, including catalase and Fructus Vitis viniferae
The vigor of carbohydrate oxidase, catalase and glucoseoxidase is than for 12:1.
Including following preparation process:
A, catalase and glucoseoxidase are made than in the phosphate buffer being dissolved in for 12:1 by vigor
Catalase and glucoseoxidase mixed enzyme solution, wherein, the pH of phosphate buffer is 5, and concentration is 0.5mol/L；
B, the mixed enzyme solution prepared in step a add 3g chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are at 30 DEG C, under conditions of pH is 6.5, and stirring and adsorbing 45min；
D, after completing step c add mass concentration percent be the glutaraldehyde solution of 2.5%, the temperature conditions of 5 DEG C
Lower cross-linking reaction 2.5h；
E, placement balance are to room temperature；
F, after completing step e add 0.8g β-D-Glucose, after electromagnetic agitation 30min stand, standby.
Wherein, catalatic Rate activity be 50000U/mL, pH be 6.0；Described solid glucose is oxidasic than living
Power is 10000U/g；The catalase of a step preparation and the volume of glucoseoxidase mixed enzyme solution: chitosan in b step
The quality of microsphere is 5 (mL): 3 (g).
Embodiment 5
The preparation method of a kind of defying age co-immobilization enzyme, defying age co-immobilization enzyme, including catalase and Fructus Vitis viniferae
The vigor of carbohydrate oxidase, catalase and glucoseoxidase is than for 14:1.
Including following preparation process:
A, catalase and glucoseoxidase are made than in the phosphate buffer being dissolved in for 14:1 by vigor
Catalase and glucoseoxidase mixed enzyme solution, wherein, the pH of phosphate buffer is 5, and concentration is 0.5mol/L；
B, the mixed enzyme solution prepared in step a add 3g chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are at 30 DEG C, under conditions of pH is 6.5, and stirring and adsorbing 45min；
D, after completing step c add mass concentration percent be the glutaraldehyde solution of 2.5%, the temperature conditions of 5 DEG C
Lower cross-linking reaction 2.5h；
E, placement balance are to room temperature；
F, after completing step e add 0.8g β-D-Glucose, after electromagnetic agitation 30min stand, standby.
Wherein, catalatic Rate activity be 50000U/mL, pH be 6.0；Described solid glucose is oxidasic than living
Power is 10000U/g；The catalase of a step preparation and the volume of glucoseoxidase mixed enzyme solution: chitosan in b step
The quality of microsphere is 5 (mL): 3 (g).
Embodiment 6
The preparation method of a kind of defying age co-immobilization enzyme, defying age co-immobilization enzyme, including catalase and Fructus Vitis viniferae
The vigor of carbohydrate oxidase, catalase and glucoseoxidase is than for 15:1.
Including following preparation process:
A, catalase and glucoseoxidase are made than in the phosphate buffer being dissolved in for 15:1 by vigor
Catalase and glucoseoxidase mixed enzyme solution, wherein, the pH of phosphate buffer is 8, and concentration is 0.5mol/L；
B, the mixed enzyme solution prepared in step a add 3g chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are at 50 DEG C, under conditions of pH is 7, and stirring and adsorbing 55min；
D, after completing step c add mass concentration percent be the glutaraldehyde solution of 3.5%, the temperature conditions of 5 DEG C
Lower cross-linking reaction 2.5h；
E, placement balance are to room temperature；
F, after completing step e add 0.8g β-D-Glucose, after electromagnetic agitation 30min stand, standby.
Wherein, catalatic Rate activity be 50000U/mL, pH be 6.0；Described solid glucose is oxidasic than living
Power is 10000U/g；The catalase of a step preparation and the volume of glucoseoxidase mixed enzyme solution: chitosan in b step
The quality of microsphere is 5 (mL): 4 (g).
One, performance test:
24 people are carried out dermal application mode observation by sample, and (the maleest 16 people, female 8 people, and age are 28～30
Year), wherein programmer 6 people, builder 6 people, civilian post class 6 people, sells class 6 people, is divided into tri-groups of A, B, C to carry out contrast real
Test, often organize programmer, builder, civilian post class, sell each two people of class.Its occupation mode be get up every morning wash one's face after and evening
On sleep before smear.Builder is in the majority with physical work owing to being exposed to the sun for a long time, and pachylosis, the colour of skin is dark yellow；Programmer and civilian post
Class is the most long-time facing to computer radiate；Sell class predominantly drink dinner party more.Wherein smoking number reaches 12 people.Use real
The common immobilized enzyme executing example proportioning and the acquisition of preparation scheme is respectively used to three groups of personnel, and the time on probation is two weeks.By Fes Kai Er
Test, wherein before three groups of personnel are tried out by skin aggregative index measuring instrument and after being suitable for:
Moisture measurement scope: 0～99.9%；Ideal range: 40～60%；
Oil point measurement scope: 5～50%, its numerical value is included in moisture；Ideal range: 15～23%；
Coefficient of elasticity: 0～1；Ideal range: 0.45～0.55.
Test result data is the meansigma methods of this group membership.
Two, test result:
Table 1 A group skin quality test result
Table 2 B group skin quality test result
| Embodiment | Moisture (%) | Oil point (%) | Elastic |
| Before Shi Yong | 31 | 12 | 0.61 |
| 1 | 37 | 14 | 0.59 |
| 2 | 42 | 17 | 0.54 |
| 3 | 56 | 21 | 0.49 |
| 4 | 53 | 19 | 0.50 |
| 5 | 50 | 16 | 0.53 |
| 6 | 47 | 17 | 0.57 |
Table 3 C group skin quality test result
Being shown by the performance test results of table 1～3, it is dark yellow that tri-groups of personnel of A, B and C all show as skin before being suitable for,
Tarnish, is dried coarse nonelastic, has a small amount of pigmentation, speckle occur.Aoxidize by adjusting catalase and glucose
The proportioning of enzyme, when the vigor of catalase and glucoseoxidase is 10:1 than optimum proportioning, makes catalase and Fructus Vitis viniferae
Carbohydrate oxidase reach the suitableeest catalytic condition and catalytic efficiency, played anti-aging effects good.Below or above this proportioning, all
Can not produce a desired effect.Chitosan microball is remarkably improved associating enzyme effect by changing to enzymatic pathway, controls release, increases
Targeting, enhanced stability, minimizing stimulation etc..Defying age co-curing enzyme acts on body surface, can form the anti-oxidation protection of densification
Layer: catalase is conducive to removing what body surface was produced through bivalence electron reduction by oxygen under the external condition effects such as ultraviolet
Hydrogen peroxide, it is to avoid free radical produces and removes unbalance, causes free chain reaction to accelerate the aging of human body skin；Meanwhile,
Decompose the β-D-Glucose in co-curing enzyme by glucoseoxidase and remove oxygen and the catalase breaks of body surface excess
The excess of oxygen that hydrogen peroxide produces, and make body surface oxygen content reach dynamic equilibrium.Aoxidized by regulation hydrogen oxide enzyme and glucose
The vigor proportioning of enzyme, gives full play to the synergism of the two, makes speed and the Fructus Vitis viniferae of catalase catalytic decomposition hydrogen peroxide
The speed of carbohydrate oxidase catalysis oxidation of beta-D-Glucose reaches the dynamic equilibrium of recurrent state, reaches the purpose of defying age.Meanwhile,
Glucoseoxidase produces gluconic acid when decomposing β-D-Glucose, and gluconic acid has chelating copper ion characteristic, can help to press down
Having made the activity of Tyrosinase, pre-anti-melanin generates, the appearance of prevention senile plaque.Hence in so that skin quality test result table
Being now: cutaneous roughness is improved that skin has more gloss and elasticity, performance moist, pigmentation substantially subtracts
Gently.
One defying age co-curing enzyme of the present invention is primarily adapted for use in and is often irradiated by computer radiate and ultraviolet, indulge in excessive drinking or endure
The crowd at night, also has good regulation to make xerosis cutis, the crowd that coarse, fibrous tissue does not has toughness and pigmentation
With, it is coupled to cosmetics and uses, it will have more preferable effect.
Above-described detailed description of the invention, has been carried out the purpose of the present invention, technical scheme and beneficial effect further
Describe in detail, be it should be understood that the detailed description of the invention that the foregoing is only the present invention, be not intended to limit the present invention
Protection domain, all within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. done, all should comprise
Within protection scope of the present invention.
Claims (6)
1. a defying age co-immobilization enzyme, it is characterised in that include catalase and glucoseoxidase, described peroxidating
The vigor of hydrogen enzyme and glucoseoxidase is than for 8:1～15:1.
A kind of defying age co-immobilization enzyme the most according to claim 1, it is characterised in that described catalase and Fructus Vitis viniferae
The vigor proportioning of carbohydrate oxidase is 9:1～12:1.
3. the preparation method of a defying age co-immobilization enzyme, it is characterised in that include following preparation process:
A, catalase and glucoseoxidase are made catalase by vigor than in the phosphate buffer being dissolved in
With glucoseoxidase mixed enzyme solution；
B, the mixed enzyme solution prepared in step a add chitosan microball and makes mixed liquor；
C, mixed liquor b step prepared are stirred absorption；
D, after completing step c add glutaraldehyde solution carry out cross-linking reaction；
E, standing, balance is to room temperature；
F, after completing step e add β-D-Glucose, after electromagnetic agitation, stand, standby；
Wherein, in a step, the vigor of described hydrogen oxide enzyme and glucoseoxidase is than for 8:1～15:1 or 9:1～12:1.
The preparation method of a kind of defying age co-immobilization enzyme the most according to claim 3, it is characterised in that described a step
In, the pH of phosphate buffer is 3～8, and concentration is 0.1～0.5mol/L；In described step c, mixed liquor at 20～50 DEG C, pH
Under conditions of being 5～7, stirring and adsorbing 40～55min；In described Step d, the mass concentration percent adding glutaraldehyde solution is
1.5～3.5%.
The preparation method of a kind of defying age co-immobilization enzyme the most according to claim 3, it is characterised in that described peroxidating
The Rate activity of hydrogen enzyme be 50000U/mL, pH be 6.0；The Rate activity of described glucoseoxidase is 10000U/g.
The preparation method of a kind of defying age co-immobilization enzyme the most according to claim 3, it is characterised in that described a step
The catalase of preparation and the volume of glucoseoxidase mixed enzyme solution: in b step, the quality of chitosan microball is 5 (mL): 2
(g)～5 (mL): 4 (g).
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