CN107177585B

CN107177585B - A kind of enzyme immobilized carrier and preparation method thereof

Info

Publication number: CN107177585B
Application number: CN201710513613.9A
Authority: CN
Inventors: 陈振斌; 王旭东; 刘冬蕾; 李轲; 张霞云; 李慧
Original assignee: Lanzhou University of Technology
Current assignee: Lanzhou University of Technology
Priority date: 2017-06-29
Filing date: 2017-06-29
Publication date: 2021-04-20
Anticipated expiration: 2037-06-29
Also published as: CN107177585A

Abstract

The invention discloses an enzyme immobilization carrier and a preparation method thereof, belonging to the field of material science and engineering, comprising PDEA-b-HEMA-b-GMA, wherein GMA accounts for 12-92% of the mole percentage range of temperature sensitive monomers, HEMA accounts for 12-84% of the mole percentage range of temperature sensitive monomers, LCST is 33-39 ℃, the enzyme activity range of immobilized PGA is 660-1030U/g, the corresponding free enzyme amount range 903-1090U, and the approximate range of the enzyme activity recovery rate is 73-95%, and after being repeatedly used for 10 times, the enzyme activity retention rate is 79%.

Description

Enzyme immobilization carrier and preparation method thereof

Technical Field

The invention belongs to the field of material science and engineering, and relates to an enzyme immobilization carrier and a preparation method thereof.

Background

Immobilized enzyme (Immobilized enzyme) is a new technology developed in the 60 s of the 20 th century. The immobilized enzyme is an enzyme which functions as a catalyst in a certain spatial range and can be used repeatedly and continuously. The core problem of the process for producing 6-Aminopenicillanic acid (6-APA) by an enzyme method is the preparation technology of high-efficiency immobilized Penicillin acylase (PGA), and the design and preparation of a carrier material are the key points of the PGA immobilization technology.

Therefore, from the perspective of carrier microenvironment design, the present invention prepares an enzyme immobilized carrier with high enzyme activity recovery rate and suitable Lower critical sol/gel phase transition temperature (LCST).

Disclosure of Invention

The invention aims to synthesize a temperature-sensitive block copolymer carrier with a suitable low critical sol/gel phase transition temperature, and prepare the immobilized enzyme with high enzyme activity recovery rate by designing the microenvironment of the carrier.

The invention designs the main chain structure of a polymer carrier into a Thermo-sensitive polymer (TP) which can generate sol/gel phase transition and has proper Low Critical Solution Temperature (LCST), thereby realizing PGA immobilization and catalytic conversion in a sol state and recycling from a liquid environment in a gel state.

The carrier is a block polymer prepared by a Reversible Addition Fragmentation Transfer (RAFT) polymerization method. The carrier comprises three parts of a temperature-sensitive monomer N, N-diethylacrylamide, a microenvironment functional monomer beta-hydroxyethyl methacrylate and a target spot functional acrylic monomer glycidyl methacrylate.

The carrier microenvironment is provided by a hydroxyl functional group monomer which is beneficial to PGA to exert catalytic activity, an immobilized target is provided by an epoxy group which can be covalently bonded with amine groups on the PGA at room temperature through click reaction (the high-grade structure of the PGA is not damaged, and the catalytic activity of the PGA is not influenced), and the carrier is provided by temperature-sensitive polymer poly N, N-diethylacrylamide which can generate sol/gel phase transition and has proper LCST. The immobilized penicillin acylase carrier prepared based on the design can realize the immobilization and catalytic conversion of the enzyme in a sol state and the recovery and reuse in a gel state.

The temperature-sensitive polymer carrier synthesized by the invention has the advantages that the molar percentage ranges of monomers providing immobilized targets and monomers providing microenvironment for keeping PGA activity respectively account for 12-92% and 12-84% of the temperature-sensitive monomers, the approximate range of LCST of the temperature-sensitive carrier is 33-39 ℃, the approximate range of enzyme activity of immobilized PGA is 660-1030U/g (enzyme activity of unit mass immobilized on the carrier), the approximate range of free enzyme loading capacity is 903-1090U (free enzyme amount immobilized on the carrier), and the approximate range of enzyme activity recovery rate is 73-95%.

The preparation method of the enzyme immobilization carrier comprises the following steps:

step 1, preparation of temperature-sensitive triblock copolymer carrier

1) Synthesizing a temperature-sensitive polymer macromolecule RAFT reagent: taking Azodiisobutylcyanide (AIBN), trithiodocosanoic acid-2-cyanoisopropyl ester (CPDTC) as a RAFT reagent and a temperature-sensitive monomer N, N-Diethylacrylamide (DEA) in a molar ratio of 1:20:1000, and the mass ratio of the temperature-sensitive monomer to Acenaphthylene (ACE) is 1.2700:0.0035, weighing the temperature-sensitive monomer with a certain mass, taking ethyl acetate as a solvent to prepare a solution with a concentration of 50 percent (mass percentage), introducing nitrogen for 30min, stirring and reacting in a sunflower oil bath at 65 ℃ for a certain time, and then precipitating a crude product three times (the ethyl acetate is the solvent, and the normal-alkane is the precipitator) to obtain a macromolecular RAFT reagent with a certain molecular weight;

2) synthesis of diblock copolymer: keeping the molar ratio among AIBN, the temperature-sensitive polymer macromolecule RAFT reagent and functional monomer beta-Hydroxyethyl methacrylate (HEMA) providing microenvironment to be 1:20:1000, preparing a solution with the concentration of 50 percent (mass percentage concentration) by taking N, N-Dimethylformamide (DMF) as a solvent, introducing nitrogen for 30min, stirring and reacting in an oil bath at 65 ℃ for a given time, and carrying out precipitation for three times (DMF as the solvent and diethyl ether as a precipitator) to obtain the diblock copolymer with different monomer ratios.

3) Synthesis of triblock copolymer: the same as 2), copolymerizing the obtained diblock RAFT reagent with Glycidyl Methacrylate (GMA) to obtain triblock copolymers (DMF is solvent and diethyl ether is precipitant) with different monomer ratios.

Step 2, immobilization of PGA

Weighing a certain amount of the synthesized triblock copolymer carrier, adding the triblock copolymer carrier into a given reactor, adding free PGA enzyme solution which is 50 times of the mass of the block copolymer and 10 percent (the proenzyme solution is diluted to 10 percent according to the volume ratio), sealing, and vibrating in a dark place for 24 hours at the temperature of 25 ℃; separating free enzyme residual solution, washing the immobilized PGA with phosphate buffer solution until penicillin G is added into the washing solution, and developing with PDAB for a given time, and then measuring the absorbance of the solution to be less than 0.005; drying in a vacuum oven at 35 deg.c for 48 hr, and weighing to obtain immobilized PGA.

Compared with the prior art, the invention has the beneficial effects that:

the invention synthesizes a temperature-sensitive triblock copolymer carrier with suitable low critical sol/gel phase transition temperature (LCST), and when the temperature is lower than the LCST, the PGA plays a catalytic activity in a stretched state; above the LCST, the carrier shrinks to facilitate its recycling from the liquid environment. More importantly, the microenvironment of the carrier is constructed by functional group monomers (functional monomers containing hydroxyl) which are beneficial to the PGA to exert catalytic activity, and the immobilized target is designed to be target monomers containing epoxy groups which can realize covalent bonding with the PGA at room temperature through click reaction.

Drawings

FIG. 1 is a 6-APA standard curve;

FIG. 2 is a flow chart of immobilized PGA.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

1. Block copolymer¹H NMR detection

Preparing each block copolymer sample into a solution with the concentration of 10.00mg/mL, using deuterated Dimethyl sulfoxide (DMSO-d) as a solvent, and performing nuclear magnetic resonance hydrogen spectroscopy (¹H NMR).

Block copolymer temperature sensitivity detection

A block copolymer sample is prepared into an extremely dilute aqueous solution with the concentration of 0.01mg/mL, and the temperature sensitivity is represented by static fluorescence spectrum detection (LS-55 type fluorescence spectrum analyzer, Perkin-Elmer company, USA. the detection conditions are that the excitation wavelength is 290nm, the scanning speed is 240nm/min, the excitation and emission slits are both 10nm, the scanning emission wavelength range is 330nm-430nm, and the fluorescence intensity is measured at 380 nm).

2. Immobilization of penicillin acylase and determination of enzyme activity, enzyme loading capacity and enzyme activity recovery rate

Immobilization as shown in fig. 2, a certain amount of the synthesized triblock copolymer carrier is weighed and added into a given reactor, then free PGA enzyme solution which is 10% of the mass of 50 times of the block copolymer (the proenzyme solution is diluted to 10% by volume) is added, sealing is carried out, and the mixture is vibrated for 24 hours in a dark place at the temperature of 25 ℃; separating free enzyme residual solution, washing the immobilized PGA with phosphate buffer solution until penicillin G is added into the washing solution, and detecting the absorbance of the solution to be less than 0.005 after the solution is developed for a given time by p-dimethylaminobenzaldehyde (PDAB); drying in a vacuum oven at 35 deg.c for 48 hr, and weighing to obtain immobilized PGA.

Determination of enzyme activity, enzyme loading amount and enzyme activity recovery rate a certain amount of immobilized PGA reacted with 10% (wt%) of 50 times amount of penicillin G potassium for 5 minutes, and the reaction solution was diluted by a certain amount. Taking 0.5mL of supernatant, adding 3.5mL of PDAB solution for developing for 3 minutes, determining the content of 6-APA by a PDAB color developing method, and calculating the enzyme activity, the enzyme loading amount and the enzyme activity recovery rate according to the formulas (1), (2) and (3).

AC＝a_v0-a_v (2)

In the formula, a_gRepresenting the activity (U/g) of the immobilized PGA, C representing the concentration (mM) of 6-APA, V representing the volume (mL) of 6-APA, m representing the mass (g) of PGA, t representing the reaction time of the immobilized PGA with the substrate, AC representing the enzyme loading amount, a_v0Represents the enzyme activity (U/mL), a_vRepresenting the activity (U/mL) of the immobilized raffinate enzyme, and representing the recovery rate of the enzyme activity by AR; v₁Represents the volume of enzyme solution used for immobilizing PGA.

3. Examples of the embodiments

The immobilized PGA obtained by taking PDEA-b-HEMA-b-GMA as a carrier according to the ratio of each structural unit of 100:72:24(DHG7224) has the LCST of 39 ℃, the immobilized enzyme activity of 790U/g, the corresponding free enzyme load of 915U and the enzyme activity recovery rate of 86%.

The immobilized PGA obtained by using DGH2462 as a carrier has the LCST of 36 ℃, the activity of the immobilized enzyme is 765U/g, the corresponding free enzyme loading capacity is 900U, and the recovery rate of the enzyme activity is 85 percent.

The immobilized PGA obtained by using DHG4724 as a carrier has the LCST of 36 ℃, the activity of the immobilized enzyme of 820U/g, the corresponding free enzyme loading amount of 910U and the enzyme activity recovery rate of 90 percent.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims

1. an enzyme immobilization carrier is characterized in that, the carrier structure is the triblock copolymer of PDEA-b-HEMA-b-GMA, and wherein GMA accounts for the molar percentage scope of thermosensitive monomer to be 12-92%, The molar percentage of HEMA in thermosensitive monomer ranges from 12-84%, its LCST is 33-39°C, the immobilized PGA enzyme activity range is 660-1030U/g, the corresponding free enzyme loading range is 903-1090U, and the enzyme activity range is 903-1090U. The recoveries ranged from 73-95%, and after 10 reuses, the enzymatic activity retention was 79%.

2. The enzyme-immobilized carrier according to claim 1, wherein the ratio of each structural unit in PDEA-b-HEMA-b-GMA is 100:47:24, and its LCST is 36°C, and the immobilized penicillin acylates The corresponding free enzyme amount of the enzyme was 910U, the enzyme activity was 820U/g, and the recovery rate of the enzyme activity was 90%.

3. a preparation method of the described enzyme immobilization carrier of claim 1, is characterized in that, comprises the following steps:

Preparation of Thermosensitive Triblock Copolymer Carrier

1) Synthesis of macromolecular RAFT reagent PDEA: take the molar ratio of AIBN to CPDTC and DEA as 1:20:1000, and the mass ratio of DEA to ACE as 1.2700:0.0035, weigh a certain mass of DEA, the mass fraction of DEA is 50%, the acetic acid Ethyl ester is used as solvent, put into a test tube, ultrasonicated for 1-2 minutes, put in a magnet, sealed, vented with nitrogen, and reacted with magnetic stirring in a 65°C oil bath for a certain period of time, and then the crude product is dissolved three times, ethyl acetate is used as a solvent, n-decane is used as a precipitant to obtain PDEA, a macromolecular RAFT reagent with a certain molecular weight;

2) Synthesis of diblock copolymer PDEA-b-HEMA with different monomer ratios: with different molar ratios of AIBN and macromolecular RAFT reagent PDEA to monomer HEMA, weigh a certain mass of monomers and add different In the test tube, DMF is the solvent, the monomer and PDEA account for 5% of the total mass fraction of the solvent, ultrasonic for 1-2 minutes, put in a magnet, seal, pass nitrogen, and react with magnetic stirring in a 65°C oil bath for different times, and dissolve for three times. , DMF is the solvent, and ether is the precipitant, that is, the diblock copolymer PDEA-b-HEMA with different monomer ratios is obtained;

3) Synthesis of triblock copolymer PDEA-b-HEMA-b-GMA with different monomer ratios: with different molar ratios of AIBN and diblock copolymer PDEA-b-PHEMA and monomer GMA, weigh a certain amount of each. The mass of monomers were added to different test tubes, DMF was the solvent, and the total mass fraction of the monomer and the diblock copolymer was 5% of the solvent, ultrasonicated for 1-2 minutes, put into the magnet, sealed, and purged with nitrogen. 65 ℃ oil bath magnetic stirring reaction for different time, dissolved three times, DMF as solvent, diethyl ether as precipitant, namely triblock copolymer PDEA-b-HEMA-b-GMA with different monomer ratios.