阅读说明：本技术 一种固定化酶制备γ-氨基丁酸的工艺 (Process for preparing gamma-aminobutyric acid by immobilized enzyme ) 是由 林金新 黄平 郭小雷 于 2020-05-13 设计创作，主要内容包括：本发明涉及氨基丁酸的制备技术领域,尤其涉及一种固定化酶制备γ-氨基丁酸的工艺。其包括以下步骤：制备固定化谷氨酸脱羧酶：制备离子液体再生纤维素载体；将海藻糖、普鲁兰多糖和氧化石墨烯混合后高速匀浆得到热稳定剂,并加入到谷氨酸脱羧酶液中得到热稳定处理后的酶液；向热稳定处理后的酶液中加入还原型谷胱甘肽,得到活性处理后的酶液；将离子液体再生纤维素载体加入到活性处理后的酶液中,密封、并置于25~30℃水浴震荡12~16h,然后取出载体、并用去离子冲洗,冷冻冻干得到固定化谷氨酸脱羧酶；以浓度为1~2%的谷氨酸溶液为底物,固定化谷氨酸脱羧酶为酶源,将谷氨酸转化为γ-氨基丁酸。通过本申请的工艺制备γ-氨基丁酸具有较高的收率。(The invention relates to the technical field of preparation of aminobutyric acid, and particularly relates to a process for preparing gamma-aminobutyric acid by using immobilized enzyme. Which comprises the following steps: preparation of immobilized glutamate decarboxylase: preparing an ionic liquid regenerated cellulose carrier; mixing trehalose, pullulan and graphene oxide, homogenizing at a high speed to obtain a heat stabilizer, and adding the heat stabilizer into a glutamic acid decarboxylase solution to obtain an enzyme solution subjected to heat stabilization treatment; adding reduced glutathione into the enzyme solution after the thermal stabilization treatment to obtain enzyme solution after the activity treatment; adding an ionic liquid regenerated cellulose carrier into the enzyme solution after the activity treatment, sealing, placing in a water bath at 25-30 ℃, oscillating for 12-16 h, taking out the carrier, washing with deionized water, freezing and freeze-drying to obtain immobilized glutamic acid decarboxylase; glutamic acid solution with the concentration of 1-2% is used as a substrate, immobilized glutamate decarboxylase is used as an enzyme source, and glutamic acid is converted into gamma-aminobutyric acid. The process for preparing the gamma-aminobutyric acid has higher yield.)

1. A process for preparing gamma-aminobutyric acid by immobilized enzyme is characterized by comprising the following steps: the method comprises the following steps:

preparation of immobilized glutamate decarboxylase: a. preparing an ionic liquid regenerated cellulose carrier; b. mixing trehalose, pullulan and graphene oxide, homogenizing at a high speed to obtain a heat stabilizer, and adding the heat stabilizer into a glutamic acid decarboxylase solution to obtain an enzyme solution subjected to heat stabilization treatment; c. adding reduced glutathione into the enzyme solution subjected to the thermal stabilization treatment, and enabling the mass concentration of the reduced glutathione to be 0.3-0.8 mg/mL to obtain an enzyme solution subjected to activity treatment; d. adding an ionic liquid regenerated cellulose carrier into the enzyme solution after the activity treatment, sealing, placing in a water bath at 25-30 ℃, oscillating for 12-16 h, taking out the carrier, washing with deionized water, freezing and freeze-drying to obtain immobilized glutamic acid decarboxylase;

and (2) converting the glutamic acid into gamma-aminobutyric acid by taking a glutamic acid solution with the concentration of 1-2% as a substrate and taking the immobilized glutamic acid decarboxylase as an enzyme source.

2. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 1, wherein the immobilized enzyme comprises the following steps: in the step (2), the immobilized glutamate decarboxylase is filled in a reaction column, and the glutamic acid solution flows through the reaction column at the flow rate of 1.1-1.3V column volume/h at the temperature of 35-40 ℃.

3. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 1, wherein the immobilized enzyme comprises the following steps: in the step (1), the method further comprises the following steps: adding the ionic liquid regenerated cellulose carrier into a sodium periodate solution, taking out after modification, directly freezing and freeze-drying to obtain a sodium periodate modified carrier, and adding the modified carrier into an enzyme solution after active treatment.

4. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 3, wherein the immobilized enzyme comprises the following steps: adopting a sodium periodate solution with the concentration of 0.5-1 mol/L, wherein the adding proportion of the ionic liquid regenerated cellulose carrier to the sodium periodate is 1 g: (0.05-0.1) mol.

5. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 3, wherein the immobilized enzyme comprises the following steps: the freeze-drying conditions are as follows: and (3) carrying out freeze drying for 18-24 h at-85 to-75 ℃ by using liquid nitrogen.

6. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 1, wherein the immobilized enzyme comprises the following steps: in the step b, the concentration of the glutamic acid decarboxylase liquid is 4-7 mg/mL.

7. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 1, wherein the immobilized enzyme comprises the following steps: the mixing mass ratio of the glutamic acid decarboxylase to the ionic liquid regenerated cellulose carrier is (0.25-0.3): 1.

8. the process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 1, wherein the immobilized enzyme comprises the following steps: the processing step of the step a comprises the following steps: dissolving cellulose in ionic liquid, placing the ionic liquid at 100-120 ℃, magnetically stirring and dissolving the ionic liquid for 5-7 hours, and standing the solution for 12-16 hours to obtain an ionic liquid/cellulose mixed solution; then dripping the ionic liquid/cellulose mixed solution into deionized water by using an injector to ensure that the ionic liquid/cellulose mixed solution is regenerated into small balls; and washing the pellets by using deionized water, and naturally airing to obtain the ionic liquid regenerated cellulose carrier.

9. The process for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 8, wherein the immobilized enzyme comprises the following steps: the adding mass ratio of the cellulose to the ionic liquid is (10-15): 100.

10. the method for preparing gamma-aminobutyric acid by using immobilized enzyme according to claim 8The process is characterized in that: the ionic liquid is BMIM]Cl、[C₄MIM]One of Cl.

Technical Field

The invention relates to the technical field of preparation of aminobutyric acid, and particularly relates to a process for preparing gamma-aminobutyric acid by using immobilized enzyme.

Background

Gamma-aminobutyric acid (GABA) is a naturally occurring functional amino acid, has the functions of treating hypertension, epilepsy and Parkinson's disease, preventing arteriosclerosis, regulating arrhythmia, easing pain, resisting skin aging and the like, and has very important values in medical treatment and health care. GABA in organisms is mostly produced by the decarboxylation of glutamate catalyzed by glutamate decarboxylase (GAD), the coenzyme of which is pyridoxal 5-phosphate. The GABA preparation method as a principle of medicines or health products mainly comprises 3 methods of chemical synthesis, separation and extraction and biological synthesis, and the biological synthesis method becomes a mainstream technology. The biosynthesis method is to convert glutamic acid into GABA by using immobilized glutamate decarboxylase (IGAD).

The productivity of the biosynthesis method is determined by the activity of the immobilized enzyme to a great extent, the immobilization of the enzyme is to bind the enzyme on a carrier material to separate the enzyme from the whole body, but the enzyme can still react with a substrate, and the immobilized enzyme not only has the characteristics of specificity, high efficiency and the like of the enzyme, but also has the advantages of being recyclable, reusable and the like, and has longer service life and storage life than free enzyme. The carrier material is an important part of the immobilized enzyme, the structure and performance of the carrier material have great influence on various performances of the immobilized enzyme, and most of the commonly used immobilized carriers at present are hydrogel, carbon fiber, chitosan and the like.

For example, patent No. CN103045666A discloses a method for preparing gamma-aminobutyric acid by using magnetic microsphere immobilized rice bran glutamate decarboxylase. Extracting glutamic acid decarboxylase from a rice bran enzyme extracting solution, and carrying out particle reaction on the glutamic acid decarboxylase and prepared magnetic chitosan microspheres to obtain magnetic immobilized glutamic acid decarboxylase; mixing the immobilized enzyme with the reaction liquid, adding 0.01-0.05mol/L sodium glutamate as a substrate, and reacting in a shaking table to obtain the gamma-aminobutyric acid. The magnetic chitosan is used as a carrier, the carrier is convenient to separate and recycle from a reaction system only through magnetism, the effect of improving enzyme activity is not achieved, and the yield of the gamma-aminobutyric acid is low.

Disclosure of Invention

The invention aims to solve the problems and provides a process for preparing gamma-aminobutyric acid by using immobilized enzyme.

The technical scheme for solving the problems is to provide a process for preparing gamma-aminobutyric acid by immobilized enzyme, which comprises the following steps:

(1) preparation of immobilized glutamate decarboxylase: a. preparing an ionic liquid regenerated cellulose carrier; b. mixing trehalose, pullulan and graphene oxide, homogenizing at a high speed to obtain a heat stabilizer, and adding the heat stabilizer into a glutamic acid decarboxylase solution to obtain an enzyme solution subjected to heat stabilization treatment; c. adding reduced glutathione into the enzyme solution subjected to the thermal stabilization treatment, and enabling the mass concentration of the reduced glutathione to be 0.3-0.8 mg/mL to obtain an enzyme solution subjected to activity treatment; d. adding an ionic liquid regenerated cellulose carrier into the enzyme solution after the activity treatment, sealing, placing in a water bath at 25-30 ℃, oscillating for 12-16 h, taking out the carrier, washing with deionized water, freezing and freeze-drying to obtain immobilized glutamic acid decarboxylase;

(2) and (2) converting the glutamic acid into gamma-aminobutyric acid by taking a glutamic acid solution with the concentration of 1-2% as a substrate and taking the immobilized glutamic acid decarboxylase as an enzyme source.

Wherein, the heat stabilizer that pullulan, trehalose and oxidation graphite alkene formed can provide the layer of protecting water at the enzyme periphery for the enzyme is difficult to be because of the dehydration deformation, does benefit to the stability of maintaining enzyme molecular space structure.

Substances that enhance the activity of enzymes and accelerate the enzymatic reaction are called activators. The activating agent has a plurality of types, including (i) inorganic cations such as sodium ions, potassium ions, copper ions, calcium ions and the like; ② inorganic anions such as chloride ion, bromide ion, iodide ion, sulfate ion phosphate ion and the like; ③ organic compounds, such as vitamin C, cysteine, reductive glutathione, etc. The application adopts reduced glutathione as an enzyme stimulant, can stimulate the activity of enzyme, improve the enzyme activity to improve the yield of gamma-aminobutyric acid, and improve the use effect of the produced gamma-aminobutyric acid.

Meanwhile, the graphene oxide can adsorb and fix glutathione molecules, glutathione is oxidized in situ on the graphene oxide and self-assembled to form colloidal particles, and the colloidal particles can be used as carriers of enzymes to further improve the enzyme activity.

Preferably, in the step (2), the immobilized glutamate decarboxylase is filled in the reaction column, and the glutamic acid solution flows through the reaction column at a flow rate of 1.1-1.3V column volume/h at 35-40 ℃.

Preferably, the step (1) further comprises the steps of: adding the ionic liquid regenerated cellulose carrier into a sodium periodate solution, taking out the carrier after modification, directly freezing and freeze-drying to obtain a sodium periodate modified carrier, and adding the modified carrier into an enzyme solution after active treatment.

The carrier is modified by sodium periodate, the oxidation of the sodium periodate can break the continuous diol group of cellulose to generate aldehyde group (-CHO), and the aldehyde group can be reacted with amino group (-NH) in enzyme molecule₂) And the covalent bond is formed, so that the enzymatic activity of the immobilized enzyme can be improved. Meanwhile, the modified carrier is not washed by deionized water and is directly freeze-dried, so that part of sodium periodate is attached to the carrier, when the carrier containing the sodium periodate is added into enzyme liquid containing colloidal particles formed by graphene oxide and glutathione, the structure of the reducing glutathione in the colloidal particles contains an active sulfhydryl-SH and is easy to oxidize and dehydrogenate; sodium periodate selectively oxidizes sulfides to sulfoxides, which can be oxidized to sulfones at elevated temperatures or with the addition of excess sodium periodate. Therefore, under the selective oxidation action of sodium periodate, sulfydryl is oxidized into sulfoxide group or even sulfone group, the sulfoxide group and the sulfone group have strong electron withdrawing property, cellulose has electron donating group-OH, enzyme has electron donating group-NH₂. Therefore, the colloidal particles can also become an intermediate connection structure except taking the physical adsorption of the gel as the carrier of the enzyme, and are respectively combined with the cellulose electron-donating groups and the enzyme electron-donating groups through the electron-withdrawing groups, so that the enzyme is firmly loaded on the carrier, and the immobilization stability and the enzyme activity are effectively improved.

Preferably, the method adopts a sodium periodate solution with the concentration of 0.5-1 mol/L, and the adding proportion of the ionic liquid regenerated cellulose carrier to the sodium periodate is 1 g: (0.05-0.1) mol.

As a preferred aspect of the present invention, the freeze-drying conditions are: and (3) carrying out freeze drying for 18-24 h at-85 to-75 ℃ by using liquid nitrogen.

Preferably, in step b, the concentration of the glutamate decarboxylase solution is 4-7 mg/mL.

Preferably, the glutamic acid decarboxylase and the ionic liquid regenerated cellulose carrier are mixed according to the mass ratio of (0.25-0.3): 1.

preferably, the processing step of step a comprises: dissolving cellulose in ionic liquid, placing the ionic liquid at 100-120 ℃, magnetically stirring and dissolving the ionic liquid for 5-7 hours, and standing the solution for 12-16 hours to obtain an ionic liquid/cellulose mixed solution; then dripping the ionic liquid/cellulose mixed solution into deionized water by using an injector to ensure that the ionic liquid/cellulose mixed solution is regenerated into small balls; and washing the pellets by using deionized water, and naturally airing to obtain the ionic liquid regenerated cellulose carrier.

Preferably, the adding mass ratio of the cellulose to the ionic liquid is (10-15): 100.

preferably, the ionic liquid is selected from [ BMIM ] Cl or [ C4MIM ] Cl.

The invention has the beneficial effects that:

1. according to the application, the reductive glutathione stimulates the enzyme activity, so that the enzyme activity can be improved, the yield of the gamma-aminobutyric acid can be improved, and the using effect of the produced gamma-aminobutyric acid can be improved.

2. The application takes the regenerated cellulose pretreated by the ionic liquid as an immobilized enzyme carrier, and has the advantages of high immobilization rate, easy recovery, easy biodegradation and environmental protection.

3. The gamma-aminobutyric acid is continuously prepared by filling the reaction column, and the preparation speed is high.

Detailed Description

The following are specific embodiments of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.