阅读说明：本技术 一种纳米材料固定化脂肪酶的方法 (Method for immobilizing lipase by using nano material ) 是由 陈可泉 刘逸 李辉 郭兴 乔荣梅 陆秋豪 曾金磊 李春秋 欧阳平凯 于 2019-11-26 设计创作，主要内容包括：本发明公开了一种纳米材料固定化脂肪酶的方法。该方法先制备固定化酶,再将固定化酶重悬后重新加入CALB、Zn<Sup>2+</Sup>和二甲基咪唑溶液,离心后的纳米固定化脂肪酶,经过预冻后,继续冻干称重,即可计算出蛋白固载量。与现有技术相比,采用该方法固定化脂肪酶后,在固载率有提高的情况下仍然保持较高的酶活,方便重复利用,降低生产成本,提高经济效益。(The invention discloses a method for immobilizing lipase by using a nano material. The method comprises the steps of firstly preparing immobilized enzyme, then re-suspending the immobilized enzyme and then adding CALB and Zn again 2+ And (3) carrying out pre-freezing on the nano immobilized lipase after centrifugation and the dimethyl imidazole solution, and continuously carrying out freeze-drying and weighing to calculate the protein immobilization amount. Compared with the prior art, after the lipase is immobilized by adopting the method, the high enzyme activity is still kept under the condition of improving the immobilization rate, the reutilization is convenient, the production cost is reduced, and the economic benefit is improved.)

1. A method for immobilizing lipase by using nano materials is characterized by comprising the following steps:

step 1, determining the protein concentration of lipase CALB to be 4 g/L, and respectively preparing NaDC and CoCl with equal molar ratio₂Adding lipase CALB at room temperature, stirring at low speed, and sequentially adding NaDC and Co²⁺Stirring at low speed for 20-40 min, centrifuging at high speed for 2 times to obtain immobilized lipase, collecting supernatant, and measuringProtein content of the supernatant;

step 2, according to the following steps of 1: zn is arranged in a molar ratio of 4²⁺And a dimethyl imidazole solution, the immobilized lipase after centrifugal washing is re-suspended by pure water and then is placed on a magnetic stirrer again, the rotating speed is increased to 6000-8000 turns, CALB is added again after uniform stirring, and Zn is sequentially added²⁺Mixing with dimethyl imidazole solution, stirring for 30 min, centrifuging to obtain supernatant, washing with MiNi-Q for three times to obtain immobilized lipase of nanometer material, collecting supernatant, and measuring protein content in the supernatant;

and 3, pre-freezing the nano material immobilized lipase, putting the nano material immobilized lipase into a freeze dryer for freeze drying, weighing the nano material immobilized lipase after freeze drying, and calculating the protein immobilization amount.

2. The method of claim 1, wherein the low-speed stirring speed in step 1 is 200-300 rpm.

3. The method for immobilizing lipase by using nano-materials as claimed in claim 1, wherein the rotation speed of the high-speed centrifugation in step 1 is 2000-4000 rpm.

4. The method for immobilizing lipase by using nano material as claimed in claim 1, wherein Zn prepared in step 2²⁺Is anhydrous zinc acetate.

5. The method for immobilizing lipase by using nano materials as claimed in claim 1, wherein the rotation speed of centrifugal washing in the step 2 is 8000 rpm.

6. The method for immobilizing lipase by using nanomaterials as claimed in claim 1, wherein the lyophilized material in step 3 is in a powder form.

Technical Field

The invention belongs to the technical field of immobilized enzyme preparation, and particularly relates to a method for immobilizing lipase by using a nano material.

Background

Lipases are one of important industrial enzyme preparations, can catalyze reactions such as lipolysis, ester exchange and ester synthesis, and are widely applied to industries such as oil processing, food, medicine and daily chemical industry. Lipases from different sources have different catalytic characteristics and catalytic activities. Wherein, the mass production of the lipase with transesterification or esterification function for organic phase synthesis has important significance for synthesizing fine chemical chiral compounds through enzyme catalysis. At present, most of esterification reactions are high-temperature acid-base catalysis, and the problems of toxic by-products, harsh reaction conditions, high energy consumption, environmental pollution and the like exist. The enzymatic synthesis can overcome the defects, has mild reaction conditions, low energy consumption and strong catalytic reaction specificity, is not easy to generate byproducts, is environment-friendly and is a development direction of green chemical industry. Free enzymes have a general problem of being difficult to function in organic phase reactions, being extremely easy to inactivate, having poor reusability, and the like. The common solution is immobilization, which generally increases stability after enzyme immobilization, is easy to separate from a reaction system, is easy to control, can be repeatedly used, is convenient to transport and store, and is beneficial to automatic production.

The patent CN102839166A utilizes macroporous resin to immobilize lipase, because of high organic matter residue, difficult pretreatment and too large pores, the patent CN101205532A utilizes alginic acid-calcium carbonate hybrid gel to immobilize β -glucuronidase, and because of the characteristics of complex preparation process, enzyme activity loss and the like, the immobilization of the lipase is not facilitated.

The nano material has a large specific surface area, the nano material has great advantages as an immobilized material, the immobilized enzyme is easy to carry out, the immobilized amount of the enzyme is improved, the nano material can be quickly separated out and is convenient to recycle, and the immobilized material has good biocompatibility, but the MOFS (metal organic framework compound) uses the dimethyl imidazole as a reagent in the preparation process, the pH value of the aqueous solution of the dimethyl imidazole is about 11, the damage to the enzyme activity is large, and meanwhile, because the synthesis speed of the MOFS is high, part of the enzyme solution is dissociated outside and cannot be combined on the nano material, and finally, the immobilized rate is low.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for immobilizing lipase by using a nano material, which overcomes the problems that the existing free lipase is difficult to be immobilized by a single MOFS material and the enzyme activity is low. After the lipase is immobilized by the method, the high enzyme activity is still maintained under the condition of improving the immobilization rate, the reuse is convenient, the production cost is reduced, and the economic benefit is improved.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a method for immobilizing lipase by using nano materials comprises the following steps:

step 1, determining that the protein concentration of lipase CALB (Candida antarctica lipase B) is 4 g/L, and respectively preparing NaDC (sodium deoxycholate) and CoCl with equal molar ratio₂(cobalt chloride), firstly adding CALB (Candida antarctica lipase B) at room temperature, stirring at low speed, and then sequentially adding NaDC (sodium deoxycholate) and Co²⁺Stirring at low speed for 30 min, centrifuging at high speed, washing for 2 times to obtain immobilized lipase, collecting supernatant, and measuring protein content of the supernatant;

step 2, according to the following steps of 1: zn is arranged in a molar ratio of 4²⁺And a dimethyl imidazole solution, the immobilized lipase after centrifugal washing is re-suspended by pure water and then is placed on a magnetic stirrer again, the rotating speed is adjusted to 6000-²⁺Mixing with dimethyl imidazole solution, stirring for 30 min, centrifuging to obtain supernatant, washing with MiNi-Q (ultrapure water) for three times to obtain immobilized lipase, collecting supernatant, and measuring protein content in the supernatant;

and 3, pre-freezing the nano material immobilized lipase, putting the nano material immobilized lipase into a freeze dryer for freeze drying, weighing the nano material immobilized lipase after freeze drying, and calculating the protein immobilization amount.

The improvement is that the speed of the low-speed stirring in the step 1 is 200-300 turns.

As an improvement, the rotating speed of the high-speed centrifugation in the step 1 is 2000-4000 revolutions, and the rotating speed is too high to easily disintegrate the cholate core formed firstly.

The calculation method for calculating the protein immobilization amount in the step 3 comprises the following steps: the total mass of added enzyme was a, the mass of enzyme measured in the supernatant was B (mass of not immobilized enzyme either), C was the mass of immobilized enzyme, C = a-B, assuming the final mass of the material was D, the calculation method of the immobilization amount E was E = C/D, and the freeze-dried immobilized enzyme was stored in a refrigerator at 4 ℃ for future use.

As a refinement, Zn provided in step 2²⁺Is anhydrous zinc acetate.

As a modification, the rotation speed of the centrifugal washing in the step 2 is 8000 revolutions.

As a modification, the material after freeze-drying in step 3 is in a powder form.

Has the advantages that:

compared with the prior art, the method for immobilizing lipase by using nano materials has the advantages that:

(1) the preparation process of the carrier is simple, the cost is low,

(2) toxic and harmful byproducts are not generated in the preparation process, and the preparation process is environment-friendly;

(3) the specific surface area of the carrier is increased, which is beneficial to the fixation of the enzyme and the stability after the fixation;

(4) firstly, synthesizing a hydrophobic cholate core, which is beneficial to protecting lipase and preventing the lipase from losing enzyme activity in an alkaline environment;

(5) the material can be separated from the product after centrifugation, so that the product can be conveniently separated and detected, and the recycling of enzyme is facilitated;

(5) the thermal stability of the immobilized lipase is improved, the enzyme activity of the free enzyme is only preserved by 45 percent at the constant temperature of 80 ℃ for 2 hours, and the enzyme activity of the immobilized lipase can be preserved by more than 80 percent.

Drawings

FIG. 1 is a comparison of activity of immobilized lipase of nanomaterials at different temperatures;

FIG. 2 is a comparison of activity of immobilized lipase of nanomaterials at different pH;

FIG. 3 is a schematic structural diagram of the nanomaterial-immobilized lipase of the present invention, wherein the 1-nanomaterial-immobilized lipase, 2-cholate core, and 3-ZIF-8 framework.

Detailed Description

The invention is further described with reference to specific examples.

A method for immobilizing lipase by using nano materials comprises the following steps:

step 1, determining that the protein concentration of lipase CALB (Candida antarctica lipase B) is 4 g/L, and respectively preparing NaDC (sodium deoxycholate) and CoCl with equal molar ratio₂(cobalt chloride), firstly adding CALB (Candida antarctica lipase B) at room temperature, stirring at low speed, and then sequentially adding NaDC (sodium deoxycholate) and Co²⁺Stirring at 200-300 rpm for 30 min, centrifuging at high speed and washing for 2 times to obtain immobilized enzyme, collecting supernatant, and measuring protein content of the supernatant;

step 2, the molar ratio of the raw materials is 1:4 configuration of Zn²⁺And a dimethyl imidazole solution, the immobilized enzyme after centrifugal washing is re-suspended by pure water and then is placed on a magnetic stirrer again, the rotating speed is adjusted to 6000-8000 turns, 2-3ml of CALB (candida antarctica lipase B) is added again after even stirring, and then Zn is sequentially added²⁺Mixing with dimethyl imidazole solution, stirring for 30 min, centrifuging to obtain supernatant, washing with MiNi-Q (ultrapure water) for three times to obtain immobilized lipase, collecting supernatant, and measuring protein content in the supernatant;

step 3, pre-freezing the washed material, putting the material into a freeze dryer for freeze drying, weighing the material after freeze drying, and calculating the protein immobilization amount, wherein the calculation method comprises the following steps: the total mass of added enzyme was a, the mass of enzyme measured in the supernatant was B (mass of not immobilized enzyme either), C was the mass of immobilized enzyme, C = a-B, assuming the final mass of the material was D, the calculation method of the immobilization amount E was E = C/D, and the freeze-dried immobilized enzyme was stored in a refrigerator at 4 ℃ for future use.

The rotating speed of the high-speed centrifugation in the step 1 is 2000-4000 revolutions, and the formed cholate core is easy to disintegrate if the rotating speed is too high.