CN109337892A - The method of the fixed taro polyphenol oxidase of fibroin - Google Patents
The method of the fixed taro polyphenol oxidase of fibroin Download PDFInfo
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Abstract
The invention discloses a kind of methods of the fixed taro polyphenol oxidase of fibroin, this method comprises the following steps: immobilizing using fibroin as carrier to taro polyphenol oxidase, the solid-to-liquid ratio for investigating immobilization temperature, immobilization time, pH value and PPO crude enzyme liquid and immobilised enzymes is adsorption conditions, according to Coomassie Brilliant Blue, it is detected by UV-VIS spectrophotometry, using standard curve and calculation formula, the load capacity of polyphenol oxidase in immobilization process, immobilized enzyme, activity recovery and enzyme absorption content are determined.The features such as cost is relatively low by the present invention, and easy to operate, raw material is easy to get extensively, and immobilised enzymes obtained has the stability of enzyme, and keeps efficient, the specificity of taro polyphenol oxidase, and has and separate and recover convenient, reuse, and continuous operation controllability is good.
Description
[technical field]
The invention belongs to Material Fields, and in particular to and it is a kind of at low cost, easy to operate, and the enzyme obtained that solidifies is with enzyme
The method of the fixed taro polyphenol oxidase of fibroin of stability, high efficiency, specificity.
[background technique]
The underground stem tuber of taro category Southern Star section herbaceos perennial taro is some torrid zones or the master of subtropical zone people
Food is wanted, there is high edible value and health-care effect.But the polyphenol oxidase work in-process in taro tissue is very easy to
Cause enzymatic browning, seriously affect the exterior quality and commodity value of product, it would therefore be highly desirable to solve brown stain in the processing of taro class
The problem of.
To solve the above-mentioned problems, people utilize enzyme immobilization technology, immobilize processing to polyphenol oxidase, pass through
The polyphenol oxidase of immobilization, can not only keep the efficient single-minded and mild enzymic catalytic reaction characteristic of enzyme, and operation storage is steady
The advantages that qualitative height, reusability improves, greatly reduces its use cost and improves production efficiency.And according to the property of enzyme and
Purposes, enzyme immobilizatio method can be mainly divided into chemistry and two macrotaxonomy of physics, fixing means can by investment, be crosslinked
Method, absorption method and covalent coupling method etc. carry out.Wherein, in enzyme immobilization technology, the selection of carrier material is to determine enzyme energy
An important factor for no success immobilization and immobilized enzyme height, it directly affects the property of immobilised enzymes and using model
It encloses.
Fibroin is that one kind has because it is with special amino acid composition and crystalline texture as a kind of novel carrier
The fixed enzyme vector for the advantages that reaction condition is mild, reactivity is strong, thermal safety is good and become research hotspot.But at present for
The research of fibroin fixing polyphenol oxidase is few, as can by fibroin be applied to immobilised enzymes field will have wide space and
Fine prospect.
[summary of the invention]
Present invention seek to address that the above problem, and it is at low cost, easy to operate to provide one kind, and solidification enzyme obtained has enzyme
Stability, high efficiency, specificity the fixed taro polyphenol oxidase of fibroin method.
A kind of method of the fixed taro polyphenol oxidase of fibroin provided by the invention, this method comprises the following steps:
A, the preparation of taro polyphenol oxidase crude enzyme liquid: by taro stripping and slicing, phosphate buffer is added, by taro and phosphoric acid
Salt buffer solid-to-liquid ratio is that 1:2 carries out homogeneous, the mixed liquor of homogeneous is centrifuged, after standing, supernatant is that taro is more
Phenol oxidase crude enzyme liquid；
B, silk cocoon the preparation of fibroin: is cut into 1~2cm2Small pieces, silk cocoon small pieces are placed in 0.5% sodium carbonate (w/v=
1:50) in solution, degumming is heated twice in boiling water, each 0.5h, fibroin obtained by drying；
C, the fixed taro polyphenol oxidase of fibroin: fibroin is added into taro polyphenol oxidase crude enzyme liquid and carries out taro polyphenol
Enzyme immobilization is aoxidized, and according to Coomassie Brilliant Blue, is detected by UV-VIS spectrophotometry, utilizes standard curve and meter
Formula is calculated, determines that the load capacity of polyphenol oxidase in immobilization process, immobilized enzyme, activity recovery and enzyme absorption contain
Amount.
In step c, the immobilization time is 20min.
In step c, the solid-to-liquid ratio of the fibroin and taro polyphenol oxidase crude enzyme liquid is 1:25.
In step c, the pH value of the reaction system solution is 7.
In step c, the immobilization temperature is 40 DEG C.
In step c, the absorbing wavelength of the UV-VIS spectrophotometry is 595nm.
In step c, the load capacity of polyphenol oxidase=original enzyme liquid protein concentration-immobilised enzymes in the immobilization process
Filter out protein concentration.
In step c, the calculation formula of the immobilized enzyme:
In formula: △ A --- the change of certain a period of time internal absorbance
N --- the ratio between the extraction volume that total enzyme solution volume is reacted with participation
T --- the reaction time (min)
W --- example weight (g)
The calculation formula of activity recovery:
In formula: r --- the vigor (U/g) of immobilised enzymes
R --- the vigor (U/g) of resolvase under the same terms
The calculation formula of enzyme absorption content:
In formula: M --- enzyme adsorbs content mg/g
C1 --- the unadsorbed concentration of supernatant (mg/mL)
C2 --- the concentration (mg/mL) after supernatant absorption
The quality (g) of the material of m --- addition.
Preferably, a kind of method of the fixed taro polyphenol oxidase of fibroin includes the following steps:
A, the preparation of taro polyphenol oxidase crude enzyme liquid: by taro stripping and slicing, phosphate buffer is added, by taro and phosphoric acid
Salt buffer solid-to-liquid ratio is that 1:2 carries out homogeneous, the mixed liquor after homogeneous is centrifuged, after standing, supernatant is taro
Polyphenol oxidase crude enzyme liquid；
B, silk cocoon the preparation of fibroin: is cut into 1~2cm2Small pieces, silk cocoon small pieces are placed in 0.5% sodium carbonate (w/v=
1:50) in solution, degumming is heated twice in boiling water, each 0.5h, fibroin obtained by drying；
C, the fixed taro polyphenol oxidase of fibroin: being 40 DEG C in reaction temperature, under the pH value of solution is 7, to taro polyphenol
Fibroin is added in oxidizing ferment crude enzyme liquid and carries out taro polyphenol oxidase enzyme immobilization 20min, the fibroin and taro polyphenol oxidase
The solid-to-liquid ratio of crude enzyme liquid is 1:25, and according to Coomassie Brilliant Blue, is detected by UV-VIS spectrophotometry, utilize standard
Curve and calculation formula determine the load capacity of polyphenol oxidase, immobilized enzyme, activity recovery and enzyme in immobilization process
Adsorb content.
The invention has the benefit that the method for the fixed taro polyphenol oxidase of fibroin of the invention is by being to carry with fibroin
Body, using the poromerics for the relatively multi-carboxy acid group that silk is provided for oneself, with physical method come the side of adsorbing polyphenols oxidizing ferment crude enzyme liquid
Method carries out the fixed taroization polyphenol oxidase of fibroin.Cost is relatively low for this method, easy to operate, and raw material is easy to get extensively, and is made
Immobilised enzymes there is the stability of enzyme, and keep efficient, the specificity of taro polyphenol oxidase, and there is separation and recovery just
The features such as prompt, reuse, continuous operation controllability is good.
[Detailed description of the invention]
Fig. 1 is bovine serum albumin standard curve.
Fig. 2 is influence of the immobilization time to polyphenol oxidase immobilization effect.
Fig. 3 is influence of the solid-to-liquid ratio to polyphenol oxidase immobilization effect.
Fig. 4 is influence of the pH value to polyphenol oxidase immobilization effect.
Fig. 5 is influence of the immobilization temperature to polyphenol oxidase immobilization effect.
[specific embodiment]
Further detailed description is done to the present invention combined with specific embodiments below, the present invention is not limited in any way.
1, instrument, reagent
(1) instrument: 1524R centrifuge (Heima Medical Instrument Co., Ltd., Zhuhai City), 300 ultraviolet-visible of Evolution
Spectrophotometer (Thermal Power Co., Ltd of the U.S.), PHS-3C acidometer (Shanghai INESA Scientific Instrument Co., Ltd.).
(2) reagent: Coomassie brilliant blue reagent (fine chemistry industry research institute is recovered in Tianjin), disodium hydrogen phosphate dodecahydrate
(analysis is pure) (western Gansu Province science limited liability company), sodium dihydrogen phosphate (analysis of two moisture is pure) (Chengdu Ke Long chemical reagent factory),
Sodium chloride (analysis is pure) (Chengdu Ke Long chemical reagent factory), ethyl alcohol (95%) (Chengdu Ke Long chemical reagent factory).
2, test method and result
Test method:
(1) preparation of taro polyphenol oxidase crude enzyme liquid: by taro stripping and slicing, being added phosphate buffer solution, by taro and
Phosphate buffer solid-to-liquid ratio is that 1:2 carries out homogeneous, and mixed liquor is centrifuged with centrifuge, and after standing 20min, taking supernatant is taro
Head polyphenol oxidase crude enzyme liquid.
(2) silk cocoon the preparation of fibroin: is cut into 1~2cm2Small pieces, silk cocoon small pieces are placed in 0.5% sodium carbonate (w/v=
1:50) in solution, degumming is heated twice in boiling water, and each 0.5h obtains fibroin after drying.
The preparation of (3) 0.01% Coomassie brilliant blue G250 solution: accurately weighing Coomassie brilliant blue G250 solid 0.1g, is added
95% ethyl alcohol 50mL, 85% concentrated phosphoric acid 100mL, be settled to 1L with ultrapure water.
(4) preparation of 0.1g/L bovine serum albumen solution: accurately weighing bovine serum albumin (BSA) solid 0.1g, and use is ultrapure
Simultaneously constant volume is to 1L for water dissolution, as standard protein liquid.
(5) fibroin is reacted with taro polyphenol oxidase crude enzyme liquid and fixes taro polyphenol oxidase to get to the fibroin
Enzyme.
(6) sample liquid determination of protein concentration
Filter liquor respectively takes 1mL when prepared by taro polyphenol oxidase enzyme solutions and fibroin immobile polyphenol oxidase, respectively plus
Enter 4mL Coomassie brilliant blue, shake up, stand 5min, surveys absorbance (A at 595nm with ultraviolet-visible spectrophotometer595), pass through
Standard curve calculates polyphenol oxidase enzyme concentration in filtrate.Then: the load capacity of polyphenol oxidase=protoenzyme liquid eggs in immobilization process
White matter concentration-immobilised enzymes filters out protein concentration.
(7) enzyme assay
When measuring fibroin immobilization taro polyphenol oxidase adsorbance, the sample supernatant after adsorption time is taken
The G250 solution of addition 5mL measures extinction after reacting 3min with 0.02mL0.9% sodium chloride solution at 595nm in 0.08mL
Value.Material adsorptive enzyme content is converted into further according to polyphenol oxidase standard curve.
Wherein, the calculation formula of immobilized enzyme:
In formula: △ A --- the change of certain a period of time internal absorbance
N --- the ratio between the extraction volume that total enzyme solution volume is reacted with participation
T --- the reaction time (min)
W --- example weight (g)
The calculation formula of activity recovery:
In formula: r --- the vigor (U/g) of immobilised enzymes
R --- the vigor (U/g) of resolvase under the same terms
The calculation formula of enzyme absorption content:
In formula: M --- enzyme adsorbs content mg/g
C1 --- the unadsorbed concentration of supernatant (mg/mL)
C2 --- the concentration (mg/mL) after supernatant absorption
The quality (g) of the material of m --- addition
(8) standard curve is drawn
7 clean colorimetric cylinders are taken, by 1 sequence number consecutively of table and reagent is added.5min is stood after mixing, is tried with No. 1
Pipe is blank control, absorbance is surveyed in ultraviolet-visible spectrophotometer, according to the data of table 1, with absorbance (A595) it is vertical
Coordinate, each normal concentration are abscissa mapping, and regression analysis draws standard curve, and such as Fig. 1 obtains bovine serum albumin at room temperature
Standard curve are as follows: A=0.0091C+0.0131, R2=0.9991.
1 bovine serum albumin standard curve of table
Based in this approach, research immobilized reactant time, solid-to-liquid ratio, pH value and immobilization temperature are to polyphenol oxidase
Immobilization effect influence, obtain the optimal conditions of the immobilization effect of the fixed taro polyphenol oxidase of fibroin.
Results and discussion:
(1) influence of the immobilized reactant time to the immobilization effect of polyphenol oxidase
It is 25 DEG C in reaction temperature, takes fibroin and taro polyphenol oxidase crude enzyme liquid ratio is 1:100, control solution value pH
Under conditions of 6.5, content is adsorbed to enzyme under different immobilized reactant times (10,20,30,40,50,60,90,120min)
And the influence of activity recovery (with the same group of most highly active rate of recovery for 100%, the rest activity rate of recovery and its ratio) changes
Curve is as shown in Figure 2.
As can be seen from Figure 2, the enzyme absorption content and the relative activity rate of recovery of fibroin immobile polyphenol oxidase are in 10min
And interior amplitude of variation is not very big in 20min, and has all reached maximum value when reacting 20min.With the increase of time, enzyme
Absorption content and the relative activity rate of recovery not only no longer increase but also gradually reduce.And in reaction time 40min after, with
The amplitude of the increase relative activity rate of recovery decline of time is not very greatly, to be maintained between 60% -67%.
When rate selection reaction is examined in the enzyme absorption content effect of comprehensive silk immobile polyphenol oxidase and relative activity recycling
Between be 20min be the best immobilization time.
(2) influence of the solid-to-liquid ratio to the immobilization effect of polyphenol oxidase
Immobilization temperature be 25 DEG C, immobilization time 20min, solution value pH be 6.5 under conditions of, different fibroins with
The ratio (1:25,1:50,1:100,1:150,1:200,1:300) of taro PPO crude enzyme liquid adsorbs content to enzyme and enzymatic activity is returned
Influence change curve such as Fig. 3 institute of yield (with the same group of most highly active rate of recovery for 100%, the rest activity rate of recovery and its ratio)
Show.
As can be seen from Figure 3, from the point of view of adsorbing content with enzyme, enzyme is inhaled during reacting solid-to-liquid ratio and being changed by 1:25 to 1:100
Attached content is gradually increased and reaches maximum value 771.86mg/g in 1:100, then adsorbs content with the increase enzyme of solid-liquid ratio
Not only it is gradually reduced instead without increasing.But from the point of view of the another relative activity rate of recovery, with the increase relative activity of the ratio of solid-liquid
The rate of recovery gradually decreases.This may be related to the absorption load-bearing capacity of immobilised enzymes due to the structure of fibroin carrier itself, when solid
When liquor ratio is increasing, fibroin and taro PPO crude enzyme liquid absorption solid-to-liquid ratio reach saturation, influence whether adsorption effect.
Comprehensive immobilization effect and the relative activity rate of recovery, choosing when solid-to-liquid ratio is 1:25 is best fixed effect.But it examines
Consider more preferable analysis and combine experiment above, factor below will use solid-to-liquid ratio to carry out development test for (1:100).
(3) influence of the pH value to the immobilization effect of polyphenol oxidase
It is 25 DEG C in reaction temperature, fibroin and taro crude enzyme liquid ratio are 1:100, under conditions of immobilization time 20min,
Different solution value pH (3.6,4.5,6.0,6.5,7.0,7.5,8.0) (original solution) adsorbs content and activity recovery to enzyme
The influence change curve of (with the same group of most highly active rate of recovery for 100%, the rest activity rate of recovery and its ratio) is as shown in Figure 4.
Figure 4, it is seen that solution ph also has an impact to fibroin immobilization taro polyphenol oxidase.Contained by enzyme absorption
Amount and relative activity Recovery curve figure are it is found that adsorb content and the relative activity rate of recovery also therewith with the increase enzyme of solution ph
It is gradually increased, and reaches maximum value when pH value is 7.5 and pH value is 7 respectively.Subsequent pH value continues to increase, enzyme adsorb content and
The relative activity rate of recovery not only no longer increases to be reduced instead.
This may be since enzyme is in the condition of peracid or alkali excessively, and the moieties structure of enzyme may be destroyed, living
Property weaken, polyphenol oxidase is fixed on inside fibroin, and it is related to may have occurred variation to structure for certain groups in fibroin molecule.It is comprehensive
Immobilised enzymes adsorbs content effect and the relative activity rate of recovery considers, chooses the condition that pH value is 7, is optimal fixed effect item
Part.
(4) influence of the immobilization temperature to the immobilization effect of polyphenol oxidase
It taking fibroin and taro PPO crude enzyme liquid ratio is 1:100, the immobilization time is 20min, and control solution value pH value is 7,
Different immobilization temperature (20,25,30,35,40,45,50,55,60 DEG C) adsorb content and activity recovery (with same group to enzyme
The most highly active rate of recovery is 100%, the rest activity rate of recovery and its ratio) influence change curve it is as shown in Figure 5.
As can be seen from Figure 5 immobilization temperature is to influence one of the principal element of fibroin immobile polyphenol oxidase.?
Under different immobilization temperature environments, the enzyme absorption when reaction temperature rises to 25 DEG C from 20 of the adsorptive enzyme content of immobilised enzymes contains
Amount variation less, and reaches maximum value (831.16mg/g) at 20 DEG C, as the temperature that immobilizes of test is gradually increased, enzyme
Absorption content is not further added by, and is gradually reduced instead.It can be seen that temperature gradually rises to from 20 DEG C from the relative activity rate of recovery
At 40 DEG C, activity recovery is gradually increased and is reaching 40 DEG C of maximum values, hereafter gradually drops with the rising activity recovery of temperature
It is low.This is main reasons is that may be because of the rising with temperature, the part-structure of enzyme itself is destroyed, and enzymatic activity is gradually
It reduces, adsorption capacity is caused to weaken.The comprehensive relative activity rate of recovery and absorption content effect consider, choose 40 DEG C and are used as fibroin solid
Surely change the optimal immobilization temperature of polyphenol oxidase.
(5) rigid condition of optimization of orthogonal test fibroin immobile polyphenol oxidase
On the basis of single factor experiment, immobilization temperature, immobilization time, four solid-to-liquid ratio, pH value principal elements are chosen
It is L9(34) orthogonal test, orthogonal interpretation of result is as shown in table 2, table 3 and table 4.
2 silk immobile polyphenol oxidase quadrature factor table of table
3 fibroin immobilization taro polyphenol oxidase L9 (3 of table4) Orthogonal experiment results
The verification test of 4 fibroin immobilization taro polyphenol oxidase of table research
According to the R of table 31And R2Value, from the point of view of enzyme activity and enzyme absorption content, each factor is to fibroin immobilization taro polyphenol
Oxidizing ferment influential effect primary and secondary is respectively: C > B > D > A, C > D > B > A；From each K value it is found that in terms of by enzyme activity and enzyme absorption content
Different optimal combination is obtained, is A respectively2B3C2D3、A1B3C2D1.This two groups of preferred plans obtained in orthogonal test all do not have
In 9 schemes of orthogonal test and two schemes have differences between reaction temperature and solid-to-liquid ratio, it is therefore desirable in addition to this
Two optimal combination carry out verification test, and verification result is as shown in Figure 4.
In order to verify the correctness of orthogonal test optimal combination, by verification test, from the fixed taro polyphenol of measurement fibroin
It is considered in terms of enzyme activity and enzyme the absorption content of raffinate after oxidizing ferment.It can be seen that and know by 4 data of table, A1B3C2D1Combination
Enzyme activity and enzyme absorption content effect will compare A2B3C2D3Will get well.35 DEG C of reaction temperature may be selected in compbined test data, instead
25min between seasonable, pH value=7, solid-to-liquid ratio 1:15, for the optimal combination of fibroin immobilization taro polyphenol oxidase preparation.
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow person skilled in the art
Scholar cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all according to the present invention
Equivalent change or modification made by Spirit Essence, should be covered by the protection scope of the present invention.
Claims (9)
1. a kind of method of the fixed taro polyphenol oxidase of fibroin, which is characterized in that this method comprises the following steps:
A, the preparation of taro polyphenol oxidase crude enzyme liquid: by taro stripping and slicing, being added phosphate buffer, slow by taro and phosphate
Fliud flushing solid-to-liquid ratio is that 1:2 carries out homogeneous, the mixed liquor of homogeneous is centrifuged, after standing, supernatant is taro polyphenol oxygen
Change enzyme crude enzyme liquid；
B, silk cocoon the preparation of fibroin: is cut into 1~2cm2Small pieces, silk cocoon small pieces are placed in 0.5% sodium carbonate (w/v=1:50)
In solution, degumming is heated twice in boiling water, each 0.5h, fibroin obtained by drying；
C, the fixed taro polyphenol oxidase of fibroin: fibroin is added into taro polyphenol oxidase crude enzyme liquid and carries out taro polyphenol oxidase
Enzyme immobilization, and according to Coomassie Brilliant Blue, it is detected by UV-VIS spectrophotometry, using standard curve and is calculated public
Formula determines the load capacity of polyphenol oxidase in immobilization process, immobilized enzyme, activity recovery and enzyme absorption content.
2. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that described solid in step c
Surely changing the time is 20min.
3. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that in step c, the silk
The solid-to-liquid ratio of element and taro polyphenol oxidase crude enzyme liquid is 1:25.
4. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that described anti-in step c
The pH value for answering system solution is 7.
5. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that described solid in step c
Surely changing temperature is 40 DEG C.
6. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that in step c, the purple
Outside-visible spectrophotometry absorbing wavelength is 595nm.
7. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that described solid in step c
The load capacity of polyphenol oxidase=original enzyme liquid protein concentration-immobilised enzymes filters out protein concentration during fixedization.
8. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that described solid in step c
Surely change the calculation formula of enzyme activity:
In formula: △ A --- the change of certain a period of time internal absorbance
N --- the ratio between the extraction volume that total enzyme solution volume is reacted with participation
T --- the reaction time (min)
W --- example weight (g)
The calculation formula of activity recovery:
In formula: r --- the vigor (U/g) of immobilised enzymes
R --- the vigor (U/g) of resolvase under the same terms
The calculation formula of enzyme absorption content:
In formula: M --- enzyme adsorbs content mg/g
C1 --- the unadsorbed concentration of supernatant (mg/mL)
C2 --- the concentration (mg/mL) after supernatant absorption
The quality (g) of the material of m --- addition.
9. the method for the fixed taro polyphenol oxidase of fibroin as described in claim 1, which is characterized in that this method includes as follows
Step:
A, the preparation of taro polyphenol oxidase crude enzyme liquid: by taro stripping and slicing, being added phosphate buffer, slow by taro and phosphate
Fliud flushing solid-to-liquid ratio is that 1:2 carries out homogeneous, the mixed liquor after homogeneous is centrifuged, after standing, supernatant is taro polyphenol
Oxidizing ferment crude enzyme liquid；
B, silk cocoon the preparation of fibroin: is cut into 1~2cm2Small pieces, silk cocoon small pieces are placed in 0.5% sodium carbonate (w/v=1:50)
In solution, degumming is heated twice in boiling water, each 0.5h, fibroin obtained by drying；
C, the fixed taro polyphenol oxidase of fibroin: being 40 DEG C in reaction temperature, under the pH value of solution is 7, to taro polyphenol oxidase
Fibroin is added in enzyme crude enzyme liquid and carries out taro polyphenol oxidase enzyme immobilization 20min, the fibroin and the thick enzyme of taro polyphenol oxidase
The solid-to-liquid ratio of liquid is 1:25, and according to Coomassie Brilliant Blue, is detected by UV-VIS spectrophotometry, utilize standard curve
And calculation formula, determine the load capacity of polyphenol oxidase in immobilization process, immobilized enzyme, activity recovery and enzyme absorption
Content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811427743.1A CN109337892B (en) | 2018-11-27 | 2018-11-27 | Method for immobilizing taro polyphenol oxidase by using fibroin |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811427743.1A CN109337892B (en) | 2018-11-27 | 2018-11-27 | Method for immobilizing taro polyphenol oxidase by using fibroin |
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| CN109337892B CN109337892B (en) | 2021-11-19 |
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