阅读说明：本技术 一种固定化海藻糖合酶催化麦芽糖产海藻糖的方法 (Method for producing trehalose by catalyzing maltose through immobilized trehalose synthase ) 是由 于令君 郝云飞 李莉 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种固定化海藻糖合酶催化麦芽糖产海藻糖的方法,本发明将海藻糖合酶通过双重吸附-交联法固定于介孔分子筛SBA-15上,用于催化麦芽糖产海藻糖,固定化方法简单、时间短、无污染、条件温和,固定化后的海藻糖合酶pH稳定性和热稳定性明显改善,且具有良好的酶学特性和可重复操作性,有利于酶的使用和储存,同时海藻糖合酶固定化时使用粗酶液,无需纯化,使用10批次后,仍能保持初始酶活的70％以上,节省用酶成本,具有十分可观的工业化应用前景。(The invention discloses a method for producing trehalose by catalyzing maltose through immobilized trehalose synthase, which is characterized in that the trehalose synthase is immobilized on mesoporous molecular sieve SBA-15 by a double adsorption-crosslinking method and is used for catalyzing the maltose to produce trehalose, the immobilization method is simple, the time is short, no pollution is caused, the condition is mild, the pH stability and the thermal stability of the immobilized trehalose synthase are obviously improved, the trehalose synthase has good enzymology characteristic and repeatable operability, the use and the storage of enzyme are facilitated, meanwhile, crude enzyme liquid is used during the immobilization of the trehalose synthase, the purification is not required, more than 70 percent of the initial enzyme activity can be still kept after 10 batches of the trehalose synthase are used, the enzyme cost is saved, and the trehalose synthase has very great industrial application prospect.)

1. A method for producing trehalose from maltose by using immobilized trehalose synthase as a catalyst, which is characterized by comprising the following steps:

(1) preparation of free enzyme solution

Inoculating an enzyme-producing strain into 30mL of TB culture medium, shaking the flask at 37 ℃ and 200r/min until the OD value is 0.6, adding an inducer IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 1mmol/L, fermenting for 20h, centrifuging the obtained fermentation liquor, collecting thalli, washing the thalli by using a phosphate buffer solution, then carrying out resuspension, carrying out ultrasonic crushing and centrifugation, and taking the supernatant to obtain a trehalose synthase enzyme solution;

(2) adsorption

Suspending 0.1g of mesoporous molecular sieve carrier in trehalose synthase enzyme solution prepared by 5ml of phosphate buffer solution, and oscillating for a certain time at 4 ℃;

(3) cross-linking

Adding 1.0-2.0% glacial acetic acid solution of chitosan with mass fraction of 1.0-2.0% into the immobilized enzyme system obtained in the step (2), adding glutaraldehyde aqueous solution with mass fraction of 0.5-2.0% of the same volume, shaking for a certain time, taking out, centrifuging for 5-10min to remove attached free enzyme, transferring supernatant, washing and precipitating with phosphate buffer solution, centrifuging again, repeatedly washing with the phosphate buffer solution to obtain immobilized trehalose synthase, drying at low temperature, and storing at 4 ℃;

(4) immobilized enzyme reaction

And (3) mixing 1g of the immobilized trehalose synthase obtained in the step (3) with 9ml of 0.03mol/L phosphate buffer, adding 20ml of 10% maltose solution, placing in a water bath at 25 ℃ for reaction, sampling at regular intervals within 5-48 h, boiling in boiling water for 10min to terminate the reaction, and measuring the content of trehalose by using an HPLC method.

2. The method for producing trehalose from maltose by using the immobilized trehalose synthase as the catalyst according to claim 1, wherein the enzyme-producing strain in step (1) is selected from streptomyces roseus or arthrobacter, the strain is fermented at 25 ℃ and 200r/min, and the strain is collected by centrifuging the fermentation broth at 800r/min for 5 min.

3. The method of claim 1, wherein the mesoporous molecular sieve in step (2) is SBA-15, and has a pore size of 6-11 nm.

4. The method for producing trehalose from maltose by using the immobilized trehalose synthase as claimed in claim 1, wherein the step (2) is performed by shaking with a cyclotron shaker or a constant temperature shaker.

5. The method for producing trehalose from maltose by using immobilized trehalose synthase according to claim 1, wherein the pH of the phosphate buffer in step (3) is 7.2.

6. The method for producing trehalose from maltose by using immobilized trehalose synthase as claimed in claim 1, wherein step (3) is performed by HPLC using NH₂Column (5 μm × 250mm × 4.6mm), mobile phase acetonitrile: 75 parts of water: 25, the flow rate is 1.0ml/min, the sample injection amount is 5 mu l, the column temperature is room temperature, and the carrier after reaction can be recycled after being washed by buffer solution.

7. The method for producing trehalose by catalyzing maltose by using the immobilized trehalose synthase as claimed in claim 1, wherein the optimal immobilization conditions of the trehalose synthase are as follows: pH6.0, enzyme feed amount 62.5mg/g, immobilization 12 h.

8. The method of claim 1, wherein the immobilized trehalose synthase obtained by using the optimal immobilization conditions can maintain more than 70% of the initial enzyme activity after 10 batches of immobilized trehalose synthase is used.

Technical Field

The invention belongs to the technical field of enzyme immobilization, and particularly relates to a method for producing trehalose by catalyzing maltose through immobilizing trehalose synthase by taking mesoporous molecular sieve SBA-15 as a carrier.

Background

Trehalose is a disaccharide formed by linking two glucopyranose monomers through alpha, alpha-1, -1 glycosidic bonds, and is a stress metabolite produced by organisms under environmental stress conditions. It has protective effect on biological membrane, protein and nucleic acid under adverse circumstances. The trehalose synthase can generate trehalose in one step by taking cheap maltose as a substrate, has short reaction flow, is easy to adjust, does not need to coexist with phosphate, and has low cost when acting on the substrate maltose. Trehalose synthase in a free state has poor stability, is easy to inactivate and cannot be recycled, and is not easy to be separated from a product after reaction, but the defects can be effectively overcome by combining an immobilization technology.

The mesoporous molecular sieve is a novel material with a pore size between that of micropores and macropores and with a huge surface area and a three-dimensional pore structure. It has excellent properties not possessed by other porous materials: the highly ordered pore structure has single pore size distribution, wide pore size range, various mesoporous shapes, adjustable pore wall composition and properties, and optimized synthesis conditions, and can obtain high heat stability and hydrothermal stability. The chitosan is also a porous net structure, so that SBA-15 adsorbed with protein can be combined with the chitosan again, and the immobilized particles are adsorbed in the net structure of the chitosan again, thereby achieving double adsorption of the immobilized enzyme. However, enzyme molecules are easy to fall off only by the immobilization of enzyme protein through adsorption, so that a bifunctional reagent can be added for further crosslinking, and the chitosan molecules contain free amino groups and are easy to indirectly and covalently bond with the enzyme through a chemical crosslinking agent.

The trehalose synthase is fixed on the mesoporous molecular sieve SBA-15 by a double adsorption-crosslinking method for catalyzing maltose to produce trehalose, the immobilization method is simple, short in time, free of pollution, mild in condition, highly ordered in pore channels of the SBA-15 molecular sieve, uniform in surface property and controllable, and capable of enhancing the stability and activity of the enzyme, so that the enzyme immobilization process is predictable, the pH stability and the thermal stability of the immobilized trehalose synthase are obviously improved, the immobilized trehalose synthase has good enzymological characteristics and repeatability, the use and the storage of the enzyme are facilitated, and meanwhile, a crude enzyme solution is used during the immobilization of the trehalose synthase, the purification is not needed, and the trehalose synthase has a very considerable industrial application prospect.

Disclosure of Invention

The invention aims to provide a method for producing trehalose from maltose by using immobilized trehalose synthase as a catalyst. In order to achieve the purpose, the invention adopts the following technical scheme:

(1) preparation of free enzyme solution

Inoculating an enzyme-producing strain into 30mL of TB culture medium, shaking the flask at 37 ℃ and 200r/min until the OD value is 0.6, adding an inducer IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 1mmol/L, fermenting for 20h, centrifuging the obtained fermentation liquor, collecting thalli, washing the thalli by using a phosphate buffer solution, then carrying out resuspension, carrying out ultrasonic crushing and centrifugation, and taking the supernatant to obtain a trehalose synthase enzyme solution;

(2) adsorption

0.1g of mesoporous molecular sieve carrier is suspended in trehalose synthase enzyme solution prepared by 5ml of phosphate buffer solution and is shaken for a certain time at 4 ℃.

(3) Cross-linking

Adding 1.0-2.0% glacial acetic acid solution of chitosan with mass fraction of 1.0-2.0% into the immobilized enzyme system obtained in the step (2), adding glutaraldehyde aqueous solution with mass fraction of 0.5-2.0% of the same volume, shaking for a certain time, taking out, centrifuging for 5-10min to remove attached free enzyme, transferring supernatant, washing and precipitating with phosphate buffer solution, centrifuging again, repeatedly washing with the phosphate buffer solution to obtain immobilized trehalose synthase, drying at low temperature, and storing at 4 ℃.

(4) Immobilized enzyme reaction

And (3) mixing 1g of the immobilized trehalose synthase obtained in the step (3) with 9ml of 0.03mol/L phosphate buffer, adding 20ml of 10% maltose solution, placing in a water bath at 25 ℃ for reaction, sampling at regular intervals within 5-48 h, boiling in boiling water for 10min to terminate the reaction, and measuring the content of trehalose by using an HPLC method.

Further preferably, the enzyme-producing strain in step (1) is selected from Streptomyces roseus or Arthrobacter, and is fermented at 25 ℃ and 200r/min, and the fermentation liquid is centrifuged at 800r/min for 5min to collect the thallus.

More preferably, the mesoporous molecular sieve in the step (2) is SBA-15, and the pore size of the mesoporous molecular sieve is 6-11 nm.

Further preferably, the step (2) is performed by oscillating using a cyclotron oscillator or a constant temperature oscillator.

Further preferably, the pH of the phosphate buffer in the step (3) is 7.2.

Further preferably, the high performance liquid chromatograph in the step (3) is selected from NH₂Column (5 μm × 250mm × 4.6mm), mobile phase acetonitrile: 75 parts of water: 25, the flow rate is 1.0ml/min, the sample injection amount is 5 mu l, the column temperature is room temperature, and the carrier after reaction can be recycled after being washed by buffer solution.

Further preferably, the optimal immobilization conditions for trehalose synthase are: pH6.0, enzyme feed amount 62.5mg/g, immobilization 12 h.

Further preferably, the immobilized trehalose synthase obtained by the optimal immobilization conditions can be maintained at 70% or more of the initial enzyme activity after 10 batches of the immobilized trehalose synthase are used.

The method for immobilizing the trehalose synthase has the following beneficial effects:

(1) the crude enzyme liquid prepared by fermentation can be directly used during the immobilization of trehalose synthase, and purification is not needed, so that the cost is saved, the immobilization efficiency is improved, and the production flow is simplified.

(2) The free enzyme is fixed on the mesoporous molecular sieve SBA-15, and the stability and the activity of the enzyme can be enhanced due to the highly ordered pore channels, the uniform surface property and the controllability of the SBA-15 molecular sieve, so that the enzyme immobilization process can be predicted.

(3) And combining the SBA-15 adsorbed with the protein with the chitosan to ensure that the immobilized particles are adsorbed in the net structure of the chitosan again to achieve double adsorption of the immobilized enzyme, and adding a bifunctional group reagent for further crosslinking.

The trehalose synthase is fixed on the mesoporous molecular sieve SBA-15 by a double adsorption-crosslinking method for catalyzing maltose to produce trehalose, the immobilization method is simple, short in time, free of pollution and mild in condition, the pH stability and the thermal stability of the immobilized trehalose synthase are obviously improved, the trehalose synthase has good enzymological characteristics and repeatable operability, the trehalose synthase is favorable for use and storage of enzyme, meanwhile, a crude enzyme solution is used during immobilization of the trehalose synthase, purification is not needed, more than 70% of the initial enzyme activity can be still maintained after 10 batches of trehalose synthase are used, the enzyme cost is saved, and the trehalose synthase has a very considerable industrial application prospect.

Drawings

FIG. 1 shows the effect of immobilization time on enzyme activity of immobilized enzyme

FIG. 2 is a graph showing the effect of pH on the immobilization effect

FIG. 3 is a graph showing the effect of enzyme supply on enzyme activity of immobilized enzyme

FIG. 4 shows the optimum reaction pH of immobilized enzyme and free enzyme

FIG. 5 shows the pH stability of immobilized and free enzymes

FIG. 6 shows the optimum reaction temperature for immobilized enzyme and free enzyme

FIG. 7 is a comparison of the thermostability of immobilized and free enzymes

FIG. 8 shows the operational stability of immobilized enzyme

Detailed Description

The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples, and the present invention can be implemented or applied by various embodiments, and all other examples obtained by a person of ordinary skill in the art without any inventive work are within the scope of the present invention.

(1) Preparation of free enzyme solution

Inoculating an enzyme-producing strain into 30mL of TB culture medium, shaking the flask at 37 ℃ and 200r/min until the OD value is 0.6, adding an inducer IPTG (isopropyl-beta-thiogalactoside) until the final concentration is 1mmol/L, fermenting for 20h, centrifuging the obtained fermentation liquor, collecting thalli, washing the thalli by using a phosphate buffer solution, then carrying out resuspension, carrying out ultrasonic crushing and centrifugation, and taking the supernatant to obtain a trehalose synthase enzyme solution;

(2) adsorption

0.1g of mesoporous molecular sieve carrier is suspended in trehalose synthase enzyme solution prepared by 5ml of phosphate buffer solution and is shaken for a certain time at 4 ℃.

(3) Cross-linking

Adding 1.0-2.0% glacial acetic acid solution of chitosan with mass fraction of 1.0-2.0% into the immobilized enzyme system obtained in the step (2), adding glutaraldehyde aqueous solution with mass fraction of 0.5-2.0% of the same volume, shaking for a certain time, taking out, centrifuging for 5-10min to remove attached free enzyme, transferring supernatant, washing and precipitating with phosphate buffer solution, centrifuging again, repeatedly washing with the phosphate buffer solution to obtain immobilized trehalose synthase, drying at low temperature, and storing at 4 ℃.

(4) Immobilized enzyme reaction

And (3) mixing 1g of the immobilized trehalose synthase obtained in the step (3) with 9ml of 0.03mol/L phosphate buffer, adding 20ml of 10% maltose solution, placing in a water bath at 25 ℃ for reaction, sampling at regular intervals within 5-48 h, boiling in boiling water for 10min to terminate the reaction, and measuring the content of trehalose by using an HPLC method.

Definition of enzyme activity unit: the amount of enzyme required to produce 1. mu.g trehalose per hour in the reaction system was 1 enzyme activity unit.

Example 1

Optimization of immobilization conditions

Sequentially inspecting the enzyme activities of the immobilized enzymes under the conditions of different immobilization time, different pH PBS buffer solutions and different enzyme supply amounts, and selecting the optimal immobilization pH, adsorption time and enzyme supply amount according to the influence of the enzyme activities on the immobilization effect.

(1) Influence of immobilization time on enzyme activity of immobilized enzyme

Relative enzyme activities of the immobilized trehalose synthase were measured at immobilization times of 2, 4, 8, 12, 16, and 20h, respectively, and the effect of the immobilization time on the activity of the immobilized enzyme was examined, and the results are shown in FIG. 1.

As shown in figure 1, as the immobilization time of trehalose synthase on an SBA-15 carrier is increased, the relative enzyme activity basically shows a linear rising trend, when the immobilization time reaches 12h, the relative enzyme activity reaches the maximum, the immobilization time is continuously prolonged, the relative enzyme activity does not rise any more, and the adsorption quantity reaches saturation, and the activity of the enzyme is lost along with the time prolongation, so that the relative enzyme activity slightly decreases, and the optimal immobilization time is selected to be 12 h.

(2) Influence of pH on enzymatic Activity of immobilized enzymes

Immobilized trehalose synthases were prepared in PBS buffer solutions of different pH (pH 2, 3, 4, 5, 6, 7, respectively) and the effect of pH on the enzymatic activity of the immobilized enzymes was examined, and the results are shown in FIG. 2.

As shown in figure 2, when the pH is between 2 and 6, the relative enzyme activity is higher, and when the pH exceeds 6, the relative enzyme activity is sharply reduced. The mediator SBA-15 synthesized using a nonionic surfactant had a pH that did not significantly affect its immobilization, and the immobilized enzyme obtained at pH6 was most active for immobilization of trehalose synthase on SBA-15, and therefore the optimum pH was selected to be 6.0.

(3) Influence of enzyme supply on enzyme activity of immobilized enzyme

Pure trehalose synthase solution is diluted by PBS buffer solution with pH of 6.0 by different times, and then fixed on SBA-15 for 12h respectively, the immobilized enzyme activity under different enzyme supply is measured, the enzyme supply is the mass ratio of pure enzyme to carrier, the influence of the enzyme supply on the immobilized enzyme activity is inspected, and the result is shown in figure 3.

As shown in fig. 3, when the enzyme supply amount is 62.5mg/g, the relative enzyme activity of the immobilized enzyme is the largest, and when the enzyme supply amount is too large, the activity of the immobilized enzyme is reduced, because too many enzyme molecules are loaded in the pore channels of the molecular sieve, thereby limiting the diffusion and transmission of substances in the pore channels, and therefore, the optimal enzyme supply amount is selected to be 62.5 mg/g.

The optimal immobilization conditions are obtained as follows; the enzyme was immobilized at pH6.0 for 12 hours under the condition of an enzyme feed amount of 62.5 mg/g.

Example 2

Physicochemical Properties of immobilized enzymes

Preparing immobilized enzyme under the optimal immobilization condition, comparing with free enzyme with the same coupling protein content, and investigating the physicochemical property of the free enzyme.

(1) The optimum reaction pH value and pH value stability of the immobilized enzyme.

As shown in FIG. 4, the optimum pH of the free enzyme was 7.0, and the optimum pH of the immobilized trehalose synthase was 7.5, which was shifted to the right side compared with the free enzyme, and it was found that the optimum pH stability range of the immobilized enzyme was broader than that of the free enzyme.

As shown in figure 5, the pH stability range of the free enzyme is narrow, the pH value is too high or too low between 6.6 and 7.4, and the influence on the activity of the free enzyme is great, but after the immobilized enzyme is placed in a buffer solution with the pH value of 2 to 7 for 1 hour, the activity of the residual enzyme is measured to reach 45 to 90 percent of the highest activity, which is still higher, which indicates that the placement of the immobilized trehalose synthase is not obviously influenced by the pH value, and the pH value range is wider, thus indicating that the immobilized trehalose synthase has better pH value stability.

(3) The optimum reaction temperature and thermal stability of the immobilized enzyme.

As can be seen from FIGS. 6 and 7, the optimum temperature for the free enzyme is 25 to 55 ℃ and the optimum reaction temperature is 45 ℃. The enzyme can be stably stored at 25-45 ℃, the enzyme activity loss is more when the temperature is higher than 45 ℃, and the enzyme activity is almost completely lost when the temperature is higher than 55 ℃; the optimum temperature of the immobilized enzyme is 35 ℃, when the optimum temperature is higher than 35 ℃, the activity loss of the immobilized enzyme is small, the activity loss can reach more than 60 percent of the highest enzyme activity, the denaturation of the enzyme is usually the result of the change of a tertiary structure, certain unstable groups oxidize other physical changes of the activated protein at high temperature, and under certain conditions, the thermal stability of the enzyme can be increased by the immobilization effect through increasing the rigidity of molecules.

(4) Operational stability of immobilized enzymes

As shown in figure 8, the enzyme activity of the immobilized enzyme is gradually reduced along with the increase of the use times at the optimum reaction temperature of 35 ℃, the enzyme activity loss is relatively large after the immobilized enzyme is used for 4 times, the residual enzyme activity is about 75 percent of the original residual enzyme activity, the residual enzyme activity difference is not large after the immobilized enzyme is used for 10 times, and more than 70 percent of the original enzyme activity is still kept, so the immobilized trehalose synthase has good operation stability and can be repeatedly used.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention should not be limited thereby, and modifications and equivalent variations made by those skilled in the art can be made without departing from the scope of the present invention.