阅读说明：本技术 一种糖用固定化酶的活力恢复的方法 (Method for recovering activity of immobilized enzyme for sugar ) 是由 米运宏 韦武林 蓝伯广 陈贞 于 2021-10-08 设计创作，主要内容包括：一种糖用固定化酶活力恢复方法,通过纯净水清洗活力下降50%-60%的糖用固定化酶,真空度负0.04-负0.06Mpa下搅拌处理20-30min,再壳聚糖吸附3-5小时后用浓度为0.2-0.5mol/L的乙酸调节使壳聚糖溶解、过滤分离、再次用纯净水清洗2-3次,沥干水后,用铝复合膜包装封口,置于钴60辐照,剂量为2-5KGy,然后置于温度2-5℃放置24小时,即检测得到糖用固定化酶活力恢复到初始活力的77-83%。(A method for restoring activity of immobilized enzyme for sugar comprises the steps of cleaning immobilized enzyme for sugar with activity reduced by 50% -60% by purified water, stirring for 20-30min under the condition that the vacuum degree is minus 0.04-minus 0.06Mpa, adsorbing chitosan for 3-5 h, adjusting by using acetic acid with the concentration of 0.2-0.5mol/L to dissolve chitosan, filtering and separating, cleaning by purified water for 2-3 times, draining water, packaging and sealing by using an aluminum composite membrane, irradiating by using cobalt 60 with the dose of 2-5KGy, and then placing at the temperature of 2-5 ℃ for 24 h to obtain the immobilized enzyme for sugar with activity restored to 77-83% of the initial activity by detection.)

1. A method for recovering the activity of immobilized enzyme for sugar is characterized by comprising the following steps:

(1) washing immobilized sugar with activity reduced by 50-60% with 2-3 times of purified water at 37-42 deg.C and conductivity no greater than 12us/cm for 2-3 times;

(2) adding 1.2-1.5 times of purified water, stirring at vacuum degree of minus 0.04-minus 0.06Mpa, temperature of 37-42 deg.C and rotation speed of =35-40r/min for 20-30min, filtering to obtain filtrate, and washing the filtrate with purified water for 2-3 times;

(3) adding the filtrate into 2 times volume of purified water, adjusting the temperature to 26-28 ℃, adding 1.3-1.7% of chitosan (W/W), stirring at the rotation speed of =35-45r/min for 3-5 hours, adjusting the chitosan to be dissolved by using acetic acid with the concentration of 0.2-0.5mol/L, filtering and separating, adding 1.6-1.8 times of purified water by weight into the filtrate, adjusting the pH value to be 7.2-7.3 by using 0.1-0.3mol/L of sodium hydroxide solution, standing for 45-50min, filtering, cleaning the filtrate for 2-3 times by using purified water with the water temperature of 30-33 ℃ and the conductivity of less than or equal to 12us/cm, draining, packaging and sealing by using an aluminum composite membrane;

(5) placing the mixture in cobalt 60 for irradiation, wherein the irradiation dose is 2-5 KGy;

(6) standing at 2-5 deg.C for 24 hr to obtain immobilized enzyme with sugar activity restored to 77-83% of original activity.

2. The immobilized enzyme for sugars of claim 1 is an immobilized sucrose invertase, an immobilized fructosyltransferase, an immobilized glucose oxidase, or an immobilized psicose C3 isomerase.

Technical Field

The invention relates to a method for recovering activity of immobilized enzyme for sugar, in particular to a method for recovering activity by using the steps of washing with purified water, negative pressure treatment, chitosan adsorption and cobalt 60 irradiation.

Background

The enzyme is an active protein, a high-efficiency biocatalyst and has high catalytic efficiency. The enzyme catalysis reaction has the advantages of substrate specificity, high catalysis efficiency, mild reaction conditions and the like, and is widely applied to biological products, food industry, environmental protection, biomass energy, medicines and the like. Because the liquid free enzyme has poor stability in the using process, the application temperature range is narrow and the liquid free enzyme is easy to inactivate; the enzyme reaction and the product are mixed together, are difficult to separate, are not beneficial to recycling, and have high production cost. Thus, immobilized enzymes (immobilized enzymes) are enzymes that are bound by a carrier in a certain space range to perform catalytic action and can be used repeatedly and continuously. The immobilized enzyme can be repeatedly used for many times, the product is easy to separate, the stability is obviously improved, and the immobilized enzyme can be repeatedly used for a long time. The carrier of the immobilized enzyme is porous and has adsorption performance such as active carbon, kaolin, alumina, silica gel, microporous glass, porous ceramic, cellulose, collodion, CMC, DEAE-cellulose, DEAE-sephadex, chitosan, ion exchange resin and the like. After the enzyme is immobilized, the carriers with the adsorption function adsorb other impurities in a reaction system in the process, so that the immobilized enzyme is reduced from contacting with a substrate to reduce the activity of the immobilized enzyme. In particular, the immobilized enzyme for sugar has various proteins, colloids and pigments in impurities in sugar liquor, and the activity of the immobilized enzyme for sugar is easy to lose due to the fact that the growth of microorganisms is easily caused by adding sugar. Besides that the immobilized weak enzyme is easy to fall off from the carrier and the activity of the immobilized enzyme is lost due to the influence of ions in the reaction, the immobilized enzyme is reduced due to the adsorption of impurities by the carrier and the contamination of the immobilized enzyme by bacteria. The invention relates to a vitality recovery method for immobilized enzyme for sugar, which aims at reducing vitality of the immobilized enzyme for sugar caused by impurity influence, bacterial contamination and ion influence in the process of immobilizing the enzyme for sugar.

Disclosure of Invention

The method solves the problem that the activity of the immobilized enzyme for sugar is restored after the activity is reduced in the production process, improves the production efficiency and reduces the production cost. The invention aims to provide a method for recovering the activity of immobilized enzyme for sugar, which has the advantages of reasonable process, energy conservation, environmental protection, good recovery effect and prolonged service life of the immobilized enzyme.

The invention realizes the aim through the following technical scheme: a method for recovering the activity of immobilized enzyme for sugar comprises the following steps:

(1) taking immobilized enzyme for sugar with activity reduced by 50-60%, and cleaning with purified water 2-3 times of 2-3 times at 37-42 deg.C and conductivity no more than 12 us/cm;

(2) adding 1.2-1.5 times of purified water with conductivity of 12us/cm or less, stirring at vacuum degree of minus 0.04-minus 0.06Mpa, temperature of 37-42 deg.C and rotation speed of 35-40r/min for 20-30min, filtering to obtain filtrate, and washing the filtrate with purified water for 2-3 times;

(3) adding the filtrate into 2 times volume of purified water, adjusting the temperature to 26-28 ℃, adding 1.3-1.7% of chitosan (W/W), stirring at the rotation speed of =35-45r/min for 3-5 hours, adjusting the chitosan to be dissolved by using acetic acid with the concentration of 0.2-0.5mol/L, filtering and separating, adding 1.6-1.8 times of purified water by weight into the filtrate, adjusting the pH value to be 7.2-7.3 by using 0.1-0.3mol/L of sodium hydroxide solution, standing for 45-50min, filtering, cleaning the filtrate for 2-3 times by using purified water with the water temperature of 30-33 ℃ and the conductivity of less than or equal to 12us/cm, draining, packaging and sealing by using an aluminum composite membrane;

(5) irradiating with cobalt 60 at an irradiation dose of 2-5 KGy;

(6) standing at 2-5 deg.C for 24 hr to obtain immobilized enzyme with sugar activity restored to 77-83% of original activity.

The invention has the outstanding advantages that:

1. desorbing the impurities adsorbed by the immobilized carrier again by using negative pressure, releasing the impurities, recovering the performance of the carrier again, increasing the contact area of the substrate and the enzyme, and improving the activity of the immobilized enzyme for sugar;

2. the chitosan is used for adsorbing ions, metals and the like adsorbed by the immobilized enzyme for the sugar, and the chitosan solution has a sterilization function, so that the influence of the ions, the metals, the bacteria and the like on the immobilized enzyme for the sugar is reduced, and the activity of the immobilized enzyme for the sugar is improved again;

3. the irradiation of cobalt 60 has certain bactericidal effect on the immobilized enzyme for sugar due to the radiation effect. Meanwhile, the enzyme originally affected by ions is displaced and distorted, and the enzyme is changed back to the original shape after low-level radiation of cobalt 60, so that the activity is restored again.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples.

The activity detection of the immobilized enzyme for the sugar is carried out according to the enterprise standard of the company.

Example 1

The conductivity of the pure water is 8us/cm, and the immobilized enzyme for sugar is immobilized glucose oxidase.

(1) Taking immobilized enzyme for sugar with activity reduced by 60%, washing with purified water at 37 deg.C for 2 times, and adding purified water 2 times the weight of the immobilized enzyme;

(2) adding 1.2 times of purified water, stirring at vacuum degree of minus 0.04Mpa at 37 deg.C and rotation speed =35r/min for 25min, filtering to obtain filtrate, and washing the filtrate with purified water for 2-3 times;

(3) adding the filtrate into 2 times volume of purified water, adjusting the temperature to 28 ℃, adding 1.6% chitosan (W/W), stirring for 2 hours at the rotation speed =40r/min, adjusting the chitosan to be dissolved by using acetic acid with the concentration of 0.3mol/L, filtering and separating, adding 1.6 times weight of purified water into the filtrate, adjusting the pH value to 7.2 by using 0.12mol/L sodium hydroxide solution, standing for 50 minutes, filtering, cleaning the filtrate for 2 times by using the purified water at the water temperature of 30 ℃, draining water, packaging and sealing by using an aluminum composite membrane;

(5) irradiating with cobalt 60 at an irradiation dose of 2 KGy;

(6) after 24 hours of standing at a temperature of 2 ℃, the activity of the immobilized glucose oxidase was recovered to 77% of the initial activity.

Example 2

Another embodiment of the method for recovering the activity of the immobilized enzyme for sugar comprises the following steps:

the conductivity of the pure water is 5us/cm, and the immobilized enzyme for sugar is immobilized sucrose invertase.

(1) Taking immobilized enzyme for sugar with activity reduced by 60%, washing with purified water at 40 deg.C for 3 times;

(2) adding 1.5 times of purified water, stirring at vacuum degree of minus 0.06Mpa, temperature of 42 deg.C and rotation speed =40r/min for 30min, filtering to obtain filtrate, and washing the filtrate with purified water for 3 times;

(3) adding the filtrate into 2 times volume of purified water, adjusting the temperature to 26 ℃, adding 1.3% chitosan (W/W), stirring for 3 hours at the rotation speed of =35r/min, adjusting the chitosan to be dissolved by using acetic acid with the concentration of 0.25mol/L, filtering and separating, adding 1.8 times weight of purified water into the filtrate, adjusting the pH value to 7.3 by using 0.15mol/L sodium hydroxide solution, standing for 45 minutes, filtering, cleaning the filtrate for 2 times by using the purified water at the water temperature of 32 ℃, draining, packaging and sealing by using an aluminum composite membrane;

(5) irradiating with cobalt 60 at a dose of 4 KGy;

(6) after 24 hours at a temperature of 5 ℃, the activity of the immobilized sucrose invertase was recovered to 78% of the original activity.

Example 3

In yet another embodiment of the method for recovering the activity of the immobilized enzyme for sugar, the method comprises the following steps:

the conductivity of the pure water is 10us/cm, and the immobilized enzyme for sugar is immobilized fructosyltransferase.

(1) Taking immobilized enzyme for sugar with activity reduced by 53%, washing with purified water at 38 deg.C for 2 times, and adding 3 times of purified water;

(2) adding 1.4 times of purified water, stirring at 42 deg.C and rotation speed =35r/min under vacuum degree of minus 0.05Mpa for 25min, filtering to obtain filtrate, and washing the filtrate with purified water for 2 times;

(3) adding the filtrate into 2 times volume of purified water, adjusting the temperature to 27 ℃, adding 1.5% chitosan (W/W), stirring for 4 hours at the rotating speed =44r/min, adjusting the chitosan to be dissolved by using acetic acid with the concentration of 0.3mol/L, filtering and separating, adding 1.7 times weight of purified water into the filtrate, adjusting the pH value to 7.3 by using 0.3mol/L sodium hydroxide solution, standing for 50 minutes, filtering, cleaning the filtrate for 3 times by using the purified water with the water temperature of 33 ℃, draining the water, packaging and sealing by using an aluminum composite membrane;

(5) irradiating with cobalt 60 at a dose of 3 KGy;

(6) after 24 hours at 4 ℃ the activity of the immobilized fructosyltransferase was found to return to 83% of the initial activity.

Example 4

In yet another embodiment of the method for recovering the activity of the immobilized enzyme for sugar, the method comprises the following steps:

the conductivity of the pure water is 3us/cm, and the immobilized enzyme for sugar is immobilized psicose C3 isomerase.

(1) Taking immobilized enzyme for sugar with activity reduced by 55%, washing with purified water at 40 deg.C for 3 times, and adding 2 times of purified water;

(2) adding 1.3 times of purified water, stirring at vacuum degree of minus 0.06Mpa, temperature of 40 deg.C and rotation speed =40r/min for 25min, filtering to obtain filtrate, and washing the filtrate with purified water for 3 times;

(3) adding the filtrate into 2 times volume of purified water, adjusting the temperature to 28 ℃, adding 1.7% chitosan (W/W), stirring for 3 hours at the speed of =40r/min, adjusting the chitosan to be dissolved by using acetic acid with the concentration of 0.2mol/L, filtering and separating, adding 1.5 times weight of purified water into the filtrate, adjusting the pH value to 7.2 by using 0.2mol/L sodium hydroxide solution, standing for 50 minutes, filtering, cleaning the filtrate for 3 times by using the purified water with the water temperature of 32 ℃, draining water, packaging and sealing by using an aluminum composite membrane;

(5) irradiating with cobalt 60 at a dose of 5 KGy;

(6) after 24 hours of standing at 5 ℃, the activity of the immobilized psicose C3 isomerase was recovered to 79% of the original activity.