CN109337892B - Method for immobilizing taro polyphenol oxidase by using fibroin - Google Patents
Method for immobilizing taro polyphenol oxidase by using fibroin Download PDFInfo
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
Abstract
The invention discloses a method for fixing taro polyphenol oxidase by fibroin, which comprises the following steps: the method comprises the steps of immobilizing the taro polyphenol oxidase by taking fibroin as a carrier, investigating immobilization temperature, immobilization time, pH value and solid-liquid ratio of PPO crude enzyme liquid to immobilized enzyme as adsorption conditions, detecting by an ultraviolet-visible spectrophotometry according to a Coomassie brilliant blue method, and determining the loading capacity, immobilized enzyme activity, activity recovery rate and enzyme adsorption content of the polyphenol oxidase in the immobilization process by using a standard curve and a calculation formula. The method has the advantages of low cost, simple operation and wide and easily-obtained raw materials, and the prepared immobilized enzyme has the stability of enzyme, keeps the high efficiency and specificity of the taro polyphenol oxidase, and has the characteristics of convenient separation and recovery, repeated use, good continuous operation controllability and the like.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of materials, and particularly relates to a method for immobilizing taro polyphenol oxidase by using fibroin, which is low in cost, simple to operate and capable of enabling the prepared immobilized enzyme to have enzyme stability, high efficiency and specificity.
[ background of the invention ]
The underground tuber of the perennial herb taro of the family arisaematis is a main food for people in tropical or subtropical regions, and has extremely high edible value and health-care function. However, polyphenol oxidase in taro tissues can easily cause enzymatic browning during processing, and seriously affect the appearance quality and commodity value of products, so the problem of browning in taro processing needs to be solved urgently.
In order to solve the problems, people use an immobilized enzyme technology to perform immobilized treatment on polyphenol oxidase, and the immobilized polyphenol oxidase can maintain the characteristics of high efficiency, specificity and mild enzyme catalytic reaction of the enzyme, has high operation and storage stability, improves reusability and the like, greatly reduces the use cost of the enzyme, and improves the production efficiency. The immobilization method of enzymes can be mainly classified into two categories, chemical and physical, according to their properties and uses, and can be carried out by entrapment, cross-linking, adsorption, covalent bonding, and the like. In the immobilized enzyme technology, the selection of the carrier material is an important factor for determining whether the enzyme can be immobilized successfully and the activity of the immobilized enzyme is high or low, and the property and the application range of the immobilized enzyme are directly influenced.
Fibroin is a novel carrier, and is an immobilized enzyme carrier with the advantages of mild reaction conditions, strong reactivity, good thermal safety and the like due to the special amino acid composition and crystal structure, so that the fibroin becomes a research hotspot. However, the study on the fibroin immobilized polyphenol oxidase is not much at present, and if the fibroin can be applied to the field of immobilized enzyme, the method has wide space and good prospect.
[ summary of the invention ]
The invention aims to solve the problems and provides the method for immobilizing the taro polyphenol oxidase by the fibroin, which has the advantages of low cost, simple operation and enzyme stability, high efficiency and specificity of the prepared immobilized enzyme.
The invention provides a method for fixing taro polyphenol oxidase by fibroin, which comprises the following steps:
a. preparing crude enzyme liquid of taro polyphenol oxidase: cutting taros into pieces, adding phosphate buffer solution, homogenizing according to the solid-to-liquid ratio of the taros to the phosphate buffer solution being 1:2, centrifuging the homogenized mixed solution, and standing to obtain supernatant which is taro polyphenol oxidase crude enzyme solution;
b. preparing silk fibroin: shearing silkworm cocoons into 1-2 cm2Placing the small silkworm cocoon pieces in 0.5% sodium carbonate (w/v is 1:50) solution, heating and degumming twice in boiling water, each time for 0.5h, and drying to obtain fibroin;
c. silk fibroin immobilized taro polyphenol oxidase: adding fibroin into crude enzyme liquid of the taro polyphenol oxidase to perform taro polyphenol oxidase immobilization, detecting by an ultraviolet-visible spectrophotometry according to a Coomassie brilliant blue method, and determining the loading capacity, immobilized enzyme activity, activity recovery rate and enzyme adsorption content of the polyphenol oxidase in the immobilization process by using a standard curve and a calculation formula.
In step c, the immobilization time is 20 min.
In the step c, the solid-to-liquid ratio of the silk fibroin to the crude enzyme solution of the taro polyphenol oxidase is 1: 25.
In the step c, the pH value of the reaction system solution is 7.
In step c, the immobilization temperature is 40 ℃.
In the step c, the absorption wavelength of the ultraviolet-visible spectrophotometry is 595 nm.
In the step c, the loading amount of the polyphenol oxidase in the immobilization process is equal to the protein concentration of the raw enzyme solution, and the immobilized enzyme is filtered to obtain the protein concentration.
In the step c, the activity of the immobilized enzyme is calculated according to the formula:
in the formula: delta A-change in absorbance over a period of time
N-ratio of total enzyme solution volume to extraction volume involved in the reaction
T-reaction time (min)
W-sample weight (g)
The calculation formula of the activity recovery rate is as follows:
in the formula: r-Activity of immobilized enzyme (U/g)
R-Activity of free enzyme under the same conditions (U/g)
Calculating formula of enzyme adsorption content:
in the formula: m-enzyme adsorption content mg/g
C1-unadsorbed concentration of supernatant (mg/mL)
C2-concentration of supernatant after adsorption (mg/mL)
m-mass of added material (g).
Preferably, the method for immobilizing the taro polyphenol oxidase by the silk fibroin comprises the following steps:
a. preparing crude enzyme liquid of taro polyphenol oxidase: cutting taros into blocks, adding phosphate buffer solution, and mixing the taros and the phosphate buffer solution according to the solid-to-liquid ratio of 1:2, homogenizing, centrifuging the homogenized mixed solution, and standing to obtain a supernatant, namely a crude taro polyphenol oxidase enzyme solution;
b. preparing silk fibroin: shearing silkworm cocoons into 1-2 cm2Placing the small silkworm cocoon pieces in 0.5% sodium carbonate (w/v is 1:50) solution, heating and degumming twice in boiling water, each time for 0.5h, and drying to obtain fibroin;
c. silk fibroin immobilized taro polyphenol oxidase: adding fibroin into crude enzyme liquid of the taro polyphenol oxidase at the reaction temperature of 40 ℃ and the pH value of the solution of 7 to perform taro polyphenol oxidase immobilization for 20min, wherein the solid-to-liquid ratio of the fibroin to the crude enzyme liquid of the taro polyphenol oxidase is 1:25, detecting by an ultraviolet-visible spectrophotometry according to a Coomassie brilliant blue method, and determining the loading capacity, the immobilized enzyme activity, the activity recovery rate and the enzyme adsorption content of the polyphenol oxidase in the immobilization process by using a standard curve and a calculation formula.
The invention has the beneficial effects that: the silk fibroin-immobilized taro polyphenol oxidase method of the invention adopts silk fibroin as a carrier, utilizes a microporous material with more carboxylic acid groups self-provided by silk, and adopts a physical method to adsorb crude enzyme liquid of the polyphenol oxidase to immobilize taro polyphenol oxidase. The method has the advantages of low cost, simple operation, wide and easily available raw materials, enzyme stability of the prepared immobilized enzyme, high efficiency and specificity of the taro polyphenol oxidase, convenient separation and recovery, repeated use, good continuous operation controllability and the like.
[ description of the drawings ]
FIG. 1 is a bovine serum albumin standard curve.
FIG. 2 is a graph showing the effect of immobilization time on the effect of immobilizing polyphenol oxidase.
FIG. 3 is a graph showing the effect of solid-liquid ratio on the immobilization effect of polyphenol oxidase.
FIG. 4 is a graph showing the effect of pH on the immobilization of polyphenol oxidase.
FIG. 5 is a graph showing the effect of the immobilization temperature on the immobilization effect of polyphenol oxidase.
[ detailed description ] embodiments
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited thereto.
1. Instrument and reagent
(1) The instrument comprises the following steps: 1524R centrifuge (Zhuhai Black horse medical instruments, Inc.), Evolution 300 UV-visible spectrophotometer (U.S. thermoelectric Co., Ltd.), PHS-3C acidimeter (Shanghai apparatus, electronic scientific instruments, Inc.).
(2) Reagent: coomassie brilliant blue reagent (shinyleaf fine chemical research institute of tianjin), disodium hydrogen phosphate dodecahydrate (analytically pure) (west longa science corporation), sodium dihydrogen phosphate (analytically pure) (meduo chemical reagent plant), sodium chloride (analytically pure) (meduo chemical reagent plant), and ethanol (95%) (meduo chemical reagent plant).
2. Test methods and results
The test method comprises the following steps:
(1) preparing crude enzyme liquid of taro polyphenol oxidase: cutting taro into pieces, adding phosphate buffer solution, homogenizing according to the solid-to-liquid ratio of taro to phosphate buffer solution of 1:2, centrifuging the mixed solution with a centrifuge, standing for 20min, and taking the supernatant to obtain crude enzyme solution of taro polyphenol oxidase.
(2) Preparing silk fibroin: shearing silkworm cocoons into 1-2 cm2Placing the small pieces of the silkworm cocoons into a 0.5% sodium carbonate (w/v is 1:50) solution, heating and degumming twice in boiling water for 0.5h each time, and drying to obtain the fibroin.
(3) Preparation of 0.01% Coomassie brilliant blue G250 solution: 0.1G of Coomassie brilliant blue G250 solid is accurately weighed, 50mL of 95% ethanol and 100mL of 85% concentrated phosphoric acid are added, and the volume is adjusted to 1L by using ultrapure water.
(4) Preparation of 0.1g/L bovine serum albumin solution: 0.1g of Bovine Serum Albumin (BSA) solid is accurately weighed, dissolved in ultrapure water and fixed to 1L to serve as standard protein liquid.
(5) And (3) reacting the fibroin with crude enzyme solution of the taro polyphenol oxidase to obtain the fibroin-immobilized taro polyphenol oxidase.
(6) Sample fluid protein concentration determination
Collecting filtrate 1mL when preparing taro polyphenol oxidase solution and fibroin immobilized polyphenol oxidase, respectively adding 4mL Coomassie brilliant blue, shaking, standing for 5min, and measuring absorbance at 595nm with ultraviolet-visible spectrophotometer (A)595) The concentration of polyphenol oxidase in the filtrate was calculated from the standard curve. Then: the loading amount of polyphenol oxidase in the immobilization process is equal to the protein concentration of the raw enzyme solution-the protein concentration of the immobilized enzyme filtrate.
(7) Enzyme Activity assay
When the adsorption amount of the fibroin immobilized taro polyphenol oxidase is measured, 5mL of G250 solution and 0.02mL of 0.9% sodium chloride solution are added into 0.08mL of sample supernatant after the adsorption time is finished, the mixture reacts for 3min, and then the light absorption value is measured at 595 nm. And then converting into the content of the adsorbed enzyme of the material according to a polyphenol oxidase standard curve.
Wherein, the calculation formula of the activity of the immobilized enzyme is as follows:
in the formula: delta A-change in absorbance over a period of time
N-ratio of total enzyme solution volume to extraction volume involved in the reaction
T-reaction time (min)
W-sample weight (g)
The calculation formula of the activity recovery rate is as follows:
in the formula: r-Activity of immobilized enzyme (U/g)
R-Activity of free enzyme under the same conditions (U/g)
Calculating formula of enzyme adsorption content:
in the formula: m-enzyme adsorption content mg/g
C1-unadsorbed concentration of supernatant (mg/mL)
C2-concentration of supernatant after adsorption (mg/mL)
m-mass of added material (g)
(8) Drawing a standard curve
7 clean colorimetric tubes are taken, numbered in sequence according to the sequence of the table 1, and reagents are added. Mixing, standing for 5min, measuring absorbance in ultraviolet-visible spectrophotometer with test tube No. 1 as blank control, and calculating absorbance (A) according to data in Table 1595) Plotting the ordinate and each standard concentration as the abscissa, and drawing a standard curve by a regression analysis method, wherein the standard curve of the bovine serum albumin at room temperature is obtained as shown in figure 1: a ═ 0.0091C +0.0131, R2＝0.9991。
TABLE 1 bovine serum albumin standard curve
Based on the method, the influence of the immobilization reaction time, the solid-liquid ratio, the pH value and the immobilization temperature on the immobilization effect of the polyphenol oxidase is researched, and the optimal condition of the immobilization effect of the fibroin immobilized taro polyphenol oxidase is obtained.
Results and discussion:
(1) effect of immobilization reaction time on the immobilization Effect of Polyphenol oxidase
The change curve of the influence on the enzyme adsorption content and the enzyme activity recovery rate (with the highest activity recovery rate of 100% in the same group and the ratio of the other activity recovery rates to the ratio) under the conditions that the reaction temperature is 25 ℃, the ratio of the crude enzyme solution of the fibroin and the taro polyphenol oxidase is 1:100, and the pH value of the solution is controlled to be 6.5 and under different immobilization reaction times (10, 20, 30, 40, 50, 60, 90 and 120min) is shown in figure 2.
As can be seen from FIG. 2, the enzyme adsorption content and the relative activity recovery rate of the fibroin-immobilized polyphenol oxidase did not vary greatly within 10min and 20min, and both reached the maximum values at the reaction time of 20 min. With the increase of time, the enzyme adsorption content and the relative activity recovery rate are not increased any more and are gradually reduced. Moreover, after a reaction time of 40min, the relative activity recovery decreases with time in a range of not very large, remaining between 60% and 67%.
The reaction time of 20min is selected as the optimal immobilization time by combining the enzyme adsorption content effect and the relative activity recovery rate of the silk immobilized polyphenol oxidase.
(2) Influence of solid-liquid ratio on immobilization effect of polyphenol oxidase
Under the conditions that the immobilization temperature is 25 ℃, the immobilization time is 20min and the pH value of the solution is 6.5, the change curves of the influence of different ratios (1:25, 1:50, 1:100, 1:150, 1:200 and 1: 300) of the fibroin and the crude enzyme solution of the PPO on the enzyme adsorption content and the enzyme activity recovery rate (the highest activity recovery rate of the same group is 100 percent, and the other activity recovery rates and the ratio thereof) are shown in FIG. 3.
As can be seen from FIG. 3, in terms of the enzyme adsorption content, the enzyme adsorption content gradually increased in the course of the reaction solid-to-liquid ratio changed from 1:25 to 1:100 and reached a maximum of 771.86mg/g at 1:100, and then the enzyme adsorption content gradually decreased without increasing with the increase in the solid-to-liquid ratio. However, from the viewpoint of the relative activity recovery rate, the relative activity recovery rate gradually decreases as the solid-liquid ratio increases. The adsorption load capacity of the fibroin carrier structure on the immobilized enzyme is related, and when the solid-liquid ratio is larger and larger, the adsorption solid-liquid ratio of the fibroin and the crude PPO (Poly-p-phenylene oxide) of the taro is saturated, so that the adsorption effect is influenced.
The immobilization effect and the relative activity recovery rate are integrated, and the optimal immobilization effect is selected when the solid-liquid ratio is 1: 25. However, in view of better analysis and integration of the above experiments, the following factors will be studied using a solid to liquid ratio of (1: 100).
(3) Effect of pH on the immobilization Effect of Polyphenol oxidase
Under the conditions that the reaction temperature is 25 ℃, the ratio of the fibroin to the crude enzyme solution of the taro is 1:100, and the immobilization time is 20min, the change curves of the influence of different solution values of pH (3.6, 4.5, 6.0, 6.5, 7.0, 7.5, 8.0) (original solution) on the enzyme adsorption content and the enzyme activity recovery rate (the highest activity recovery rate of the same group is 100%, and the other activity recovery rates and the ratio thereof) are shown in figure 4.
As can be seen from FIG. 4, the pH of the solution also has an effect on the serine-immobilized taro polyphenol oxidase. As can be seen from the graphs of the enzyme adsorption content and the relative activity recovery rate, the enzyme adsorption content and the relative activity recovery rate gradually increase with the increase of the pH value of the solution, and reach maximum values at a pH value of 7.5 and a pH value of 7 respectively. Then the pH value continues to increase, and the enzyme adsorption content and the relative activity recovery rate are not increased any more but reduced.
This is probably because in the case of the enzyme under peracid or alkalescence conditions, part of the molecular structure of the enzyme may be destroyed, the activity is reduced, the polyphenol oxidase is fixed in the silk fibroin, and some groups in the silk fibroin molecules may have structural changes. And (3) considering the adsorption content effect and the relative activity recovery rate of the immobilized enzyme comprehensively, and selecting the condition of pH value of 7 as the optimal immobilization effect condition.
(4) Effect of immobilization temperature on the immobilization Effect of Polyphenol oxidase
Taking the crude enzyme liquid of silk fibroin and the PPO of the taro in a ratio of 1:100, the immobilization time is 20min, the pH value of the solution is controlled to be 7, and the influence change curves of different immobilization temperatures (20, 25, 30, 35, 40, 45, 50, 55 and 60 ℃) on the enzyme adsorption content and the enzyme activity recovery rate (the highest activity recovery rate of the same group is 100%, and the other activity recovery rates and the ratio thereof) are shown in FIG. 5.
It can be seen from fig. 5 that the immobilization temperature is one of the main factors affecting the fibroin-immobilized polyphenol oxidase. Under different immobilization temperature environments, the enzyme adsorption content of the immobilized enzyme does not change greatly when the reaction temperature rises from 20 to 25 ℃, and reaches a maximum value (831.16mg/g) at 20 ℃, and the enzyme adsorption content does not increase any more but gradually decreases as the immobilization temperature is gradually increased along with the experiment. From the relative activity recovery, it can be seen that the activity recovery gradually increased and reached a maximum of 40 ℃ as the temperature was gradually increased from 20 ℃ to 40 ℃ and then gradually decreased as the temperature was increased. This is probably because, as the temperature rises, a part of the structure of the enzyme itself is destroyed and the enzyme activity gradually decreases, resulting in a decrease in adsorption capacity. And (3) considering the comprehensive relative activity recovery rate and adsorption content effect, and selecting 40 ℃ as the optimal immobilization temperature of the fibroin immobilized polyphenol oxidase.
(5) Orthogonal experiment for optimizing immobilization conditions of fibroin immobilized polyphenol oxidase
On the basis of a single-factor test, four main factors of immobilization temperature, immobilization time, solid-liquid ratio and pH value are selected as L9(34) The results of the orthogonality test and the analysis of the orthogonality results are shown in tables 2, 3 and 4.
TABLE 2 Silk immobilization polyphenol oxidase orthogonal factors Table
TABLE 3 Silk fibroin immobilized taro polyphenol oxidase L9 (3)4) Results of orthogonal experiments
Table 4 verification test of the study of fibroin-immobilized taro polyphenol oxidase
R according to Table 31And R2The value is that from the aspects of enzyme activity and enzyme adsorption content, various factors have more effect on the silk fibroin immobilized taroThe main and secondary influences of the effect of the phenol oxidase are respectively as follows: c>B>D>A、C>D>B>A; from the K values, different optimum combinations are obtained from the aspects of enzyme activity and enzyme adsorption content, respectively A2B3C2D3、A1B3C2D1. Neither of the two optimal protocols obtained in the orthogonal experiment were found in the 9 protocols of the orthogonal experiment and the two protocols had differences between the reaction temperature and the solid-to-liquid ratio, so that additional verification tests were required for the two optimal combinations, and the verification results are shown in fig. 4.
In order to verify the correctness of the optimal combination of the orthogonal test, the enzyme activity and the enzyme adsorption content of the residual liquid after the fibroin immobilized taro polyphenol oxidase are measured through the verification test. As can be seen from the data in Table 4, A1B3C2D1The combined enzyme activity and enzyme adsorption content effect are better than those of A2B3C2D3Is better. The comprehensive test data can select the reaction temperature of 35 ℃, the reaction time of 25min, the pH value of 7, the solid-liquid ratio of 1:15, the preparation method is the optimal combination for preparing the fibroin immobilized taro polyphenol oxidase.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (4)
1. A method for immobilizing taro polyphenol oxidase by silk fibroin is characterized by comprising the following steps:
a. preparing crude enzyme liquid of taro polyphenol oxidase: cutting taros into pieces, adding phosphate buffer solution, homogenizing according to the solid-to-liquid ratio of the taros to the phosphate buffer solution being 1:2, centrifuging the homogenized mixed solution, and standing to obtain supernatant which is taro polyphenol oxidase crude enzyme solution;
b. preparing silk fibroin: shearing silkworm cocoons into 1-2 cm2Placing the small pieces of silkworm cocoon in 0.5% carbonic acidHeating and degumming twice in boiling water in a sodium (w/v = 1:50) solution for 0.5h each time, and drying to obtain fibroin;
c. silk fibroin immobilized taro polyphenol oxidase: adding fibroin into crude enzyme liquid of the taro polyphenol oxidase at the reaction temperature of 40 ℃ and the pH value of the solution of 7 to perform taro polyphenol oxidase immobilization for 15min, wherein the solid-to-liquid ratio of the fibroin to the crude enzyme liquid of the taro polyphenol oxidase is 1:35, detecting by an ultraviolet-visible spectrophotometry according to a Coomassie brilliant blue method, and determining the loading capacity, immobilized enzyme activity, activity recovery rate and enzyme adsorption content of the polyphenol oxidase in the immobilization process by using a standard curve and a calculation formula.
2. The method for immobilizing conus polyphenol oxidase through silk fibroin according to claim 1, wherein in step c, the absorption wavelength of the ultraviolet-visible spectrophotometry is 595 nm.
3. The method for immobilizing taro polyphenol oxidase through silk fibroin according to claim 1, wherein in step c, the loading amount of polyphenol oxidase during the immobilization process = raw enzyme solution protein concentration-immobilized enzyme filtrate protein concentration.
4. The method for immobilizing taro polyphenol oxidase through silk fibroin according to claim 1, wherein in step c, the immobilized enzyme activity is calculated according to the formula:
enzyme activity (U/g) =
In the formula:
delta A-change in absorbance over a period of time
N-ratio of total enzyme solution volume to extraction volume involved in the reaction
T-reaction time (min)
W-sample weight (g)
The calculation formula of the activity recovery rate is as follows:
recovery (%) =
In the formula:
r-Activity of immobilized enzyme (U/g)
R-Activity of free enzyme under the same conditions (U/g)
Calculating formula of enzyme adsorption content:
enzyme adsorption content (mg/g) M =
In the formula:
m-enzyme adsorption content mg/g
C1-unadsorbed concentration of supernatant (mg/mL)
C2-concentration of supernatant after adsorption (mg/mL)
m-mass of added material (g).
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