CN108715842B - Preparation method of high-activity beta-glucosidase - Google Patents
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Abstract
The invention discloses a preparation method of high-activity beta-glucosidase, which adopts a pulse ultrasonic probe to carry out ultrasonic treatment on a beta-D-glucosidase enzyme liquid; the temperature of the ultrasonic treatment is 30-42 ℃, and the sound intensity is 12.1-242.04W/cm2. The method adopts a pulse ultrasonic probe to carry out ultrasonic treatment on the beta-D-glucosidase enzyme liquid, and can obviously improve the activity of the beta-glucosidase under specific conditions.
Description
Technical Field
The invention relates to the technical field of fruit and vegetable processing, in particular to a preparation method of high-activity beta-glucosidase.
Background
Beta-glucosidase is a flavor enhancing enzyme, also called beta-D-glucoside hydrolase (beta-GC for short); the enzyme does not need any coenzyme and is a real hydrolase; can act on most natural glucoside, and can release bonded aroma substances by hydrolysis; it is used as a flavouring or flavouring enzyme in many food products.
Especially for fruit and vegetable products, the beta-glucosidase has wide application prospect, and a plurality of fruits such as grapes, strawberries, oranges, pineapples, apricots, peaches, plums and the like have bonded aroma substances. The release of glycosidically bonded aroma precursor materials is believed to be an effective method for enhancing and maintaining good aroma qualities of fruits and their processed products. Endogenous beta-glucosidase naturally exists in many fruits, and for example, the peel, pulp and juice of citrus are found to contain beta-glucosidase, and the enzyme content and activity are different due to different parts. However, the content and the activity of endogenous enzymes in fruits and vegetables are often lower, flavor precursor substances cannot be sufficiently hydrolyzed, and the flavor of fruit and vegetable products can be greatly improved if proper treatment is carried out by adding exogenous beta-glucosidase.
Although the aroma substances released by the enzymatic hydrolysis approach the aroma of fruits, the enzymatic hydrolysis has the disadvantage of requiring a long time (at least 48 h); therefore, how to increase the activity of the odorant enzyme to shorten the hydrolysis time becomes a new research focus.
At present, methods for improving enzyme activity mainly comprise genetic engineering, mutagenesis screening of enzyme-producing strains, addition of metal ions, high-temperature treatment, application of immobilized enzymes and the like. However, the safety of gene food is yet to be further investigated, and the development of gene engineering is still incomplete. The cost for screening high-yield enzyme strains through mutagenesis is high, the production process is complicated and far from meeting the requirement of industrial preparation, and the application of adding metal ions, high-temperature treatment and immobilized enzyme in the actual production of beverages still has many limitations. Therefore, the development of a safe, efficient, low-cost, pollution-free method for improving the enzyme activity is an urgent solution.
The ultrasonic technology is a novel non-thermal food processing technology, and has the advantages of improving the food quality, improving the food safety, modifying the food and the like. In recent years, many researchers have applied this to the improvement of enzyme activity in the food industry: haile Ma et al (Ma H, Huang L, Jia J, et al. Effect of energy-pumped ultrasound on Alcalase [ J ]. Ultrasonics Sonochhemistry, 2011,18(1):419-24.) study showed that ultrasound had an effect on alkaline protease activity. The highest Alcalase activity was achieved when the samples were sonicated for 4 minutes with an energy polymerization of 80W, wherein the enzyme activity increased 5.8% over the control. The results of Wenjuan Qu et al (Qu W, Ma H, Liu B, et al. enzymolysis reaction kinetics and thermal simulations of degraded wheat germ protein with ultrasonic pretreatment [ J ]. Ultrasonics biochemistry, 2013,20(6):1408-13.) show that temperature and ultrasound have a positive effect on the enzymatic hydrolysis of wheat germ protein (DWGP). Compared with the traditional enzymolysis, the probe and bath type ultrasonic pretreatment enzymolysis obviously improves the reaction rate constant and the enzymolysis efficiency, and improves the polypeptide yield. Stephen Barton et al (Barton S, Bullock C, Weir D. the effects of ultrasound on the activities of sodium glycerosidase enzymes of industrial activities [ J ]. Enzyme & microbiological Technology,1996,18(3): 190-. The results showed that the reaction rate was increased and the activity of sucrose hydrolase converting enzyme was significantly increased (37% at 0.9 μm) under the ultrasonic conditions.
However, no report on the effect of sonication on β -glucosidase activity has been reported.
Disclosure of Invention
The invention provides a preparation method of high-activity beta-glucosidase, which remarkably improves the activity of the beta-glucosidase and the decomposition capacity of the beta-glucosidase on aroma substances in fruit and vegetable juice by an ultrasonic treatment means.
The specific technical scheme is as follows:
a preparation method of high-activity beta-glucosidase adopts a pulse ultrasonic probe to carry out ultrasonic treatment on a beta-D-glucosidase enzyme solution; the temperature of the ultrasonic treatment is 30-42 ℃, and the sound intensity is 12.1-242.04W/cm2。
Experiments show that the optimum temperature of the beta-D-glucosidase is reduced from 50 ℃ to 40 ℃ under the action of the ultrasonic wave, and the main reason of analysis can be as follows: the cavitation of ultrasound changes the conformation of beta-D-glucosidase, which may in turn change the biological activity of the enzyme molecules, and the thermal effect of the ultrasound may also cause the selection of the enzyme molecules for the ambient temperature. In the range of lower action temperature, the activity of the beta-D-glucosidase is not obviously changed along with the change of the temperature, but the enzyme activity is slightly improved under the action of the ultrasonic wave; when the temperature exceeds the optimum temperature, the activity of the beta-D-glucosidase shows a trend of decreasing, and the inhibition effect of ultrasonic treatment on the enzyme activity is stronger than that of a control group, which shows that the activity of the beta-D-glucosidase is easily inhibited at higher temperature.
In addition, the experiment also shows that when the sound intensity is less than 12.1, the enzyme activity enhancing effect is weak, and when the sound intensity is more than 242.04, the enzyme can be inhibited along with the increase of the sound intensity. The reason for the analysis may be: the cavitation of the ultrasonic wave can moderately change the conformation of the beta-D-glucosidase, and the moderate change can be favorable for improving the enzyme activity of the beta-D-glucosidase; when the power is at a higher level, the strong cavitation can generate high temperature and high pressure to cause great change and even damage to the space structure of the beta-D-glucosidase, the mechanical action of ultrasonic waves destroys polypeptide chains in an enzyme molecular structure along with the continuous enhancement of the power, a large amount of free radicals can be generated to attack enzyme molecules to generate chemical change, and the two can cause the enzyme activity to be inhibited and even inactivated.
Preferably, the pH of the beta-D-glucosidase enzyme solution is 4 to 5 in the ultrasonic treatment. The pH value of the enzyme solution of the beta-D-glucosidase is 7, and the pH value is adjusted by 0.1mol/L citric acid-trisodium citrate buffer solution, and the pH value can influence the enzyme activity.
Preferably, the duty ratio of the ultrasonic treatment is 30-50%. Tests show that the duty ratio is too low (less than 30%) to reduce the working efficiency, and too high (more than 50%) to release energy generated by cavitation effect and damage the instrument.
Preferably, the sound intensity of the ultrasonic treatment is 12.1-242.. 04.W/cm2。
Preferably, the pulse time of the ultrasonic treatment is 2 to 5 seconds. Tests show that the cavitation effect is reduced when the pulse time is too short (less than 2S), and the action effect of the ultrasonic waves is reduced when the energy generated by the cavitation effect is not released due to too long pulse time (more than 5S).
Preferably, the frequency of the ultrasonic treatment is 16 to 100 kHz. The ultrasonic wave is a mechanical wave with a frequency exceeding the upper limit of human hearing, and can be divided into three frequency ranges of 16-100 kHz (low-frequency high-energy ultrasonic), 100 kHz-1 MHz (high-frequency ultrasonic) and 1-10 MHz (diagnostic ultrasonic). The ultrasonic frequency is inversely proportional to the size of the cavitation bubbles. Therefore, the low-frequency high-energy ultrasound (16 to 100kHz) generates large cavity intensity, so that a high-temperature and high-pressure cavity region is formed. Cavitation does not occur in the megahertz range as the cavitation intensity gradually diminishes with increasing frequency.
Preferably, the total duration of the ultrasonic treatment is 1-13 minutes.
Preferably, the probe extends into a position 1cm below the liquid level of the reaction liquid, and the liquid level is kept at 2-6 cm. It was found experimentally that different liquid level heights lead to different sound field distributions. The sound field of the ultrasonic wave is characterized by a standing wave when the liquid level is 2cm to 6cm in height, and the ratio of the reflected wave gradually decreases as the height increases, and reaches 0 when the liquid level reaches 6cm in height, and thereafter, from 6cm to 12cm, the sound field of the ultrasonic wave is characterized by a traveling field, and the ultrasonic wave is attenuated to zero just when the ultrasonic wave reaches 6cm in height, so that the total average sound intensity decreases as the height increases.
More preferably, the temperature of the ultrasonic treatment is 40 ℃, and the sound intensity is 60.51W/cm2The duty cycle was 50%, the pulse time was 2s, the pH was 5, and the total duration was 3 minutes.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method adopts a pulse ultrasonic probe to carry out ultrasonic treatment on the beta-D-glucosidase enzyme liquid, and can obviously improve the activity of the beta-glucosidase and the capability of the beta-glucosidase for decomposing aroma substances in the fruit and vegetable juice under specific conditions.
(2) In the prior art, enzyme treatment of beta-D-glucosidase needs 48 hours, so that the use of the beta-D-glucosidase is limited; the invention firstly applies the ultrasonic technology to the improvement of the enzyme activity of the beta-D-glucosidase, and the enzyme after ultrasonic treatment only needs 8 hours, so that the aroma enhancement treatment of the beta-D-glucosidase on fruit and vegetable juice products becomes possible.
(3) In the prior art, the setting of ultrasonic condition parameters is too extensive, the enzyme activity cannot be accurately controlled, the process cannot be repeated, and the method cannot be used for industrial amplification production; the method accurately controls the ultrasonic conditions; for example: expression of acoustic energy: the sound energy is generally expressed by rated power in the field, but the parameter is extremely inaccurate, because the rated power is only partially converted into sound energy, the conversion rates of different instruments are different, and the volume of a processed sample can influence the sound energy, the sound energy is accurately expressed by the sound intensity obtained by a thermodynamic measurement method, and the sound energy is not influenced by the instrument and the volume of the sample; pulse mode: the influence of the pulse mode on the enzyme activity is considered, and the pulse mode can also influence the acoustic energy, the cavitation effect and the instrument; level height: the invention considers the influence of the liquid level height on the enzyme activity, the liquid level height can influence the distribution of the sound field, and the influence on the enzyme activity is great. Ultrasound methods used in the prior art
Drawings
FIG. 1 is a graph showing the effect of the time of sonication on the activity of beta-GC in example 2.
FIG. 2 is a graph showing the effect of the temperature of sonication on the activity of β -GC enzyme in example 2.
FIG. 3 is a graph showing the effect of the sound intensity of ultrasonic treatment on the activity of beta-GC enzyme in example 2.
FIG. 4 is a graph showing the effect of pH on beta-GC enzyme activity of the sonicated media of example 2.
FIG. 5 is a graph showing the effect of duty cycle ratio of ultrasonic treatment on the activity of beta-GC enzyme in example 2.
FIG. 6 is a graph showing the effect of the liquid level depth of the ultrasonic treatment on the activity of beta-GC enzyme in example 2.
Detailed Description
The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.
Method for measuring enzyme activity:
enzyme activity is defined as: the enzyme amount of 1 mu mol p-nitrophenol released per minute is one beta-D-glucosidase activity unit (U) under certain analysis conditions by taking nitrophenyl-beta-D galactopyranoside (pNPG) as a substrate.
Adding 0.1mL diluted enzyme solution into 10mL test tube with stopper, adding 0.3mL pH5.0 citric acid-trisodium citrate buffer solution, adding 0.2mL pNPG, keeping temperature at 50 deg.C for 10min, adding 2mL Na2CO3The reaction was terminated with the solution, and distilled water was added to 5 mL.
Blank controls were: after the addition of the enzyme, 2ml of Na was added2CO3The enzyme is inactivated and buffer and substrate are added. And (3) measuring a light absorption value at 400nm, determining the production amount of the p-nitrophenol, and calculating the enzyme activity.
Calculating the formula: x ═ cV/tV'
In the formula: x: beta-D-glucosidase activity, U/mL; c: concentration of p-nitrophenol, mmol/L; v: the final volume of the reaction solution is mL; v': the amount of enzyme solution, mL; t: action time, min.
Example 1
A preparation method of high-activity beta-glucosidase comprises the following specific steps:
(1) preparation of beta-D-glucoside hydrolase solution: weighing a proper amount of beta-D-glucosidase from almond (6U/mg) Sigma, dissolving in 0.1mol/L citric acid-trisodium citrate buffer solution with pH of 5 to prepare an enzyme solution with the concentration of 0.04mmol, and storing in a refrigerator at 4 ℃ for later use.
(2) The beta-D-glucoside hydrolase solution is treated by probe type pulse ultrasonic wave of 20kHz, and the ultrasonic treatment process is as follows: the probe is extended to 1cm below the liquid level, so that the height of the ultrasonic probe extending into the liquid level is consistent during each treatment, and the control action conditions are strictly consistent. The liquid level is kept at 5cm, the pulse time is 2s, the duty ratio is 30 percent, the temperature is 35 ℃, and the sound intensity is 100.23W/cm2Treatment time 5 minutes.
The enzyme activity of the obtained beta-glucosidase is 0.25 mu/ml through enzyme activity determination.
Comparative example 1
A preparation method of high-activity beta-glucosidase comprises the following specific steps:
(1) preparation of beta-D-glucoside hydrolase solution: an appropriate amount of beta-D-glucosidase was weighed and dissolved in 0.1mol/L citric acid-trisodium citrate buffer solution with pH5 from the company of Sigma, almond (6U/mg) to prepare an enzyme solution with a concentration of 0.04 mmol.
The enzyme activity of the obtained beta-glucosidase is 0.16 mu/ml through enzyme activity determination.
Example 2
(1) Preparation of beta-D-glucoside hydrolase solution: weighing a proper amount of beta-D-glucosidase from almond (6U/mg) Sigma, dissolving in 0.1mol/L citric acid-trisodium citrate buffer solution with pH of 5 to prepare an enzyme solution with the concentration of 0.04mmol, and storing in a refrigerator at 4 ℃ for later use.
(2) A single-factor experimental design method is adopted, the influence of different ultrasonic powers, ultrasonic time, ultrasonic temperatures, ultrasonic medium pH values, ultrasonic duty ratios and liquid level heights on the enzyme activity of the beta-D-glucosidase is mainly researched, and the change rule of the enzyme activity of the beta-D-glucosidase under the action of different ultrasonic conditions is explored (the other steps and parameters are the same as those in example 1).
(a) Influence of ultrasonic intensity on activity of beta-D-glucosidase
Fixing ultrasonic wave for 10min, duty ratio 33.33%, pH5 of ultrasonic wave medium, temperature 40 deg.C of ultrasonic wave medium, setting ultrasonic wave power at 1%, 3%, 5%, 10%, 20%, 30% and 40% of total power (950W), respectively, and measuring sound intensity according to thermodynamics method to obtain corresponding ultrasonic sound intensity of 12.10,36.31,60.51,121.02,242.04,363.06and 484.08W/cm 2.
(b) Effect of ultrasound time on beta-D-glucosidase Activity
The fixed ultrasonic sound intensity is 60.51W/cm2The duty ratio was 33.33%, the ultrasonic medium pH was 5, the ultrasonic medium temperature was 40 ℃, and the ultrasonic time was set to 1, 5, 10, 15, 20, and 25min, respectively.
As shown in fig. 1, the activity of β -D-glucosidase shows a tendency of slightly increasing and then slowly decreasing with the increase of the ultrasound time, and it can be seen that the ultrasound treatment in a shorter time is beneficial to the development of β -D-glucosidase molecules to a more optimal structure, and if the ultrasound treatment time is too long, the structure of the enzyme molecules may be damaged by the long-time action of the ultrasonic shear and the shock wave, and the enzyme molecules may be broken into fragments and tend to an unfavorable conformation.
(c) Effect of ultrasound temperature on beta-D-glucosidase Activity
The fixed ultrasonic sound intensity is 60.51W/cm2Ultrasonic time is 10min, duty ratio is 33.33%, and ultrasonic medium temperature is set to be 20, 30, 40, 50 and 60 ℃.
(d) Influence of pH value of ultrasonic medium on activity of beta-D-glucosidase
The fixed ultrasonic sound intensity is 60.51W/cm2Ultrasonic time 10min, duty ratio 33.33%, ultrasonic medium temperature 40 ℃, ultrasonic medium buffer solution pH 3, 4, 5, 6and 7 respectively (figure 4).
(e) Effect of ultrasonic duty cycle on beta-D-glucosidase Activity
The fixed ultrasonic sound intensity is 60.51W/cm2pH5 of ultrasonic medium, ultrasonic mediumThe mass temperature was 40 ℃ and the ultrasound duty cycles were set at 33.33, 40, 50, 66.67 and 100%, respectively (actual ultrasound time was 3min) (fig. 5).
(f) Effect of liquid level on beta-D-glucosidase Activity
The fixed ultrasonic sound intensity is 60.51W/cm2The ultrasonic medium pH5 and the ultrasonic medium temperature were 40 ℃ and the ultrasonic liquid level heights were set to 2.00, 4.00, 6.00, 8.00, 10.00 and 12.00, respectively (FIG. 6).
As shown in FIG. 3, the activity of beta-D-glucosidase is significantly improved by the action of ultrasound with low intensity, and the activity of beta-D-glucosidase is reduced with the continuous increase of ultrasound intensity, when the ultrasound intensity exceeds 242.04W/cm2After that, the enzyme activity is inhibited.
Example 3
The treatment of the beta-glucosidase was performed under the optimal conditions for each factor obtained in example 2, as follows:
(1) preparation of beta-D-glucoside hydrolase solution: weighing a proper amount of beta-D-glucosidase from almond (6U/mg) Sigma, dissolving in 0.1mol/L citric acid-trisodium citrate buffer solution with pH of 5 to prepare an enzyme solution with the concentration of 0.04mmol, and storing in a refrigerator at 4 ℃ for later use.
(2) The beta-D-glucoside hydrolase solution is treated by probe type pulse ultrasonic wave of 20kHz, and the ultrasonic treatment process is as follows: the probe is extended to 1cm below the liquid level, so that the height of the ultrasonic probe extending into the liquid level is consistent during each treatment, and the control action conditions are strictly consistent. The liquid level is kept at 4cm, the pulse time is 2s, the duty ratio is 50 percent, the temperature is 40 ℃, and the sound intensity is 60.51W/cm2pH5, total treatment time 3 minutes.
Under the condition of optimal factors (i.e. pulse time of 2s, duty ratio of 50%, temperature of 40 deg.C, sound intensity of 60.51W/cm)2pH5, total processing time of 3 minutes, liquid level height of 4cm), the enzyme activity of the obtained beta-glucosidase is 0.40 mu/ml through enzyme activity determination.
The β -glucosidase prepared in this example and the β -glucosidase prepared in comparative example 1 were placed in fresh orange juice for enzymatic hydrolysis, and after the treatment, the content of aroma substances in the fresh orange juice was analyzed by GC-MS method, and the results are shown in table 1.
TABLE 1 comparison of aroma content of freshly squeezed orange juice under different conditions
Claims (1)
1. A preparation method of high-activity beta-glucosidase is characterized in that a pulse ultrasonic probe is adopted to carry out ultrasonic treatment on beta-D-glucosidase enzyme liquid with pH of 5 and concentration of 0.04 mmol; the ultrasonic treatment temperature is 40 ℃, and the sound intensity is 60.51W/cm2The duty ratio is 50%, the pulse time is 2s, the frequency is 20kHz, and the total duration is 3 minutes; the ultrasonic probe extends into a position 1cm below the liquid level of the reaction liquid, and the liquid level is kept at 4 cm.
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| CN101942406A (en) * | 2010-08-12 | 2011-01-12 | 淮海工学院 | Marine nocardiopsissp.HY-G and beta-glucosidase produced by same |
| CN103710333A (en) * | 2013-12-21 | 2014-04-09 | 华中科技大学 | Carrier for immobilization as well as preparation method thereof and immobilized beta-glucosaccharase |
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| KR20100109262A (en) * | 2009-03-31 | 2010-10-08 | 국민대학교산학협력단 | A method for hydrolizing cellulose by the digestive enzymes of reticulitermessperatus |
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| CN101942406A (en) * | 2010-08-12 | 2011-01-12 | 淮海工学院 | Marine nocardiopsissp.HY-G and beta-glucosidase produced by same |
| CN103710333A (en) * | 2013-12-21 | 2014-04-09 | 华中科技大学 | Carrier for immobilization as well as preparation method thereof and immobilized beta-glucosaccharase |
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| Title |
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| "定点突变技术提高β-葡萄糖苷酶活性";李沛华等;《基因组学与应用生物学》;20160613;第35卷(第8期);摘要 * |
| "超声波对多酚氧化酶酶活力的影响及其机理";王文宗等;《食品科学》;20100901;第31卷(第17期);摘要，第331页左栏第1段，第332页第1.3.2小节 * |
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