阅读说明：本技术 一种固定化青霉素g酰化酶及其制备方法 (Immobilized penicillin G acylase and preparation method thereof ) 是由 陈振斌 柳春丽 周永山 张定军 李慧 吴翠玲 于 2020-05-26 设计创作，主要内容包括：本发明一种固定化青霉素G酰化酶及其制备方法,属于固定化酶技术领域。所述固定化青霉素G酰化酶为具有纳米囊性结构的Fe<Sub>3</Sub>O<Sub>4</Sub>@PTA-PGA/PDA,是由Fe<Sub>3</Sub>O<Sub>4</Sub> NPs固定化PGA和包覆在所述Fe<Sub>3</Sub>O<Sub>4</Sub> NPs固定化PGA表面的PDA涂层制成,所述Fe<Sub>3</Sub>O<Sub>4</Sub> NPs的表面负载有PTA。制备方法为：用TA对Fe<Sub>3</Sub>O<Sub>4</Sub> NPs进行表面修饰,制备得Fe<Sub>3</Sub>O<Sub>4</Sub>@PTA NPs；将所述Fe<Sub>3</Sub>O<Sub>4</Sub>@PTA NPs作为载体对PGA进行固定化,制备得Fe<Sub>3</Sub>O<Sub>4</Sub>@PTA-PGA NPs；用DA对所述Fe<Sub>3</Sub>O<Sub>4</Sub>@PTA-PGA NPs进行表面包覆,即得。本发明的固定化青霉素G酰化酶具有良好的pH稳定性和温度耐受性,且操作稳定性和重复利用率较好,具有很高的工业应用价值。(The invention relates to an immobilized penicillin G acylase and a preparation method thereof, belonging to the technical field of immobilized enzymes. The immobilized penicillin G acylase is Fe with a nano-cystic structure 3 O 4 @ PTA-PGA/PDA, is made of Fe 3 O 4 NPs immobilized PGA and coated on the Fe 3 O 4 PDA coating of NPs immobilized PGA surface, Fe 3 O 4 The surface of the NPs is loaded with PTA. The preparation method comprises the following steps: using TA for Fe 3 O 4 NPs are subjected to surface modification to prepare Fe 3 O 4 @ PTA NPs; subjecting said Fe to 3 O 4 The @ PTA NPs are used as carriers to immobilize PGA to prepare Fe 3 O 4 @ PTA-PGA NPs; the DA is used for the Fe 3 O 4 And coating the surface of the @ PTA-PGA NPs to obtain the product. The immobilized penicillin G acylase has good pH stability and temperature tolerance, and has operation stability and repeatabilityThe utilization rate is good, and the industrial application value is high.)

1. An immobilized penicillin G acylase having a nanocapsular structure comprising:

Fe₃O₄NPs immobilized PGA, said Fe₃O₄The surface of the NPs is loaded with PTA; and

coated with the Fe₃O₄NPs immobilized PDA coating of PGA surface.

2. A method for preparing immobilized penicillin G acylase, which is characterized by comprising the following steps:

using TA for Fe₃O₄NPs are subjected to surface modification to prepare Fe₃O₄@PTA NPs；

Subjecting said Fe to₃O₄The @ PTA NPs are used as carriers to immobilize PGA to prepare Fe₃O₄@PTA-PGA NPs；

Using DA for said Fe₃O₄Coating the surface of the @ PTA-PGA NPs to prepare Fe₃O₄@ PTA-PGA/PDA, namely the immobilized penicillin G acylase of the invention.

3. The method according to claim 2, wherein Fe is produced₃O₄@ PTA NPs, including:

mixing Fe₃O₄NPs are dispersed in distilled water and N is introduced into a constant temperature water bath at 40 DEG C₂30min；

To Fe₃O₄Adding aqueous TA solution to aqueous dispersions of NPs and adding N₂Reacting for 2 hours under protection;

separating the product after the reaction is finished, washing the separated product with distilled water until the absorbance of a washing liquid for washing the product at 275nm is less than 0.005, and determining the absorbance by adopting an ultraviolet spectrophotometry to obtain Fe₃O₄@PTA NPs。

4. The method of manufacturing according to claim 3, further comprising:

at a temperature of 40 ℃ for the prepared Fe₃O₄@ PTA NPs were vacuum dried to constant weight.

5. The method according to claim 2, wherein Fe is produced₃O₄@ PTA-PGA/PDA, comprising:

mixing Fe₃O₄@ PTA-PGA NPs dispersed in PBS buffer;

to Fe₃O₄Adding DA into the @ PTA-PGANPs/PBS solution, and carrying out polymerization reaction for 4-9h in a constant-temperature water bath at 25 ℃ under stirring;

after the reaction is finished, separating the product, washing the separated product by using PBS buffer solution until the absorbance of the eluent for washing the product at 507nm is less than 0.005, and measuring the absorbance by adopting an ultraviolet spectrophotometry to obtain Fe₃O₄@PTA-PGA/PDA。

6. The method of manufacturing according to claim 5, further comprising:

at a temperature of 40 ℃ for the prepared Fe₃O₄@ PTA-PGA/PDA was vacuum dried.

7. The method according to claim 5, wherein the concentration of the PBS buffer is 40mmol/mL, and the pH is 8.0.

8. The process according to claim 5, wherein the polymerization is carried out for a period of 4 hours.

9. Immobilized penicillin G acylase according to claim 1, characterized in that Fe₃O₄The average particle size of the NPs was 9.13 nm.

Technical Field

The invention belongs to the technical field of immobilized enzyme preparation, and particularly relates to immobilized penicillin G acylase and a preparation method thereof, in particular to Fe with a nanocapsule structure₃O₄@ PTA-PGA/PDA and preparation method thereof.

Background

Penicillin G acylase, abbreviated as PGA, is mainly used for producing β -lactam antibiotics. The free penicillin G acylase has many defects in industrial catalytic application, such as easy loss of catalytic activity, difficult recovery of free enzyme, difficult separation and purification of products and difficult realization of continuous operation in the production process. The immobilized penicillin G acylase overcomes the defects of the free enzyme, not only can maintain the catalytic activity of the free enzyme, but also can improve the operation stability, the production process is easy to realize continuous operation, is easy to separate from the product and can be repeatedly used, and the immobilized penicillin G acylase is widely researched at home and abroad and has made good progress. However, the preparation and/or selection of better performing supports to meet the requirements of industrial applications and to reduce mass transfer limitations remains a key to the further utilization of penicillin G acylases.

Researches show that the microenvironment of polyhydroxy is favorable for maintaining the active conformation of the PGA, and Tannic Acid (TA) is polyphenol containing galloyl and is used for constructing the microenvironment of polyhydroxy on the surface of a carrier, so that enzyme can be immobilized on the surface of the carrier through physical adsorption, and the enzyme activity of the immobilized PGA is improved. However, since the carrier is adsorbed to PGA by van der waals force and hydrogen bonding, the interaction is weak, and PGA is easily dropped during actual operation, which seriously affects recycling thereof. In addition, the enzyme activity of the immobilized PGA prepared by the conventional adsorption method is usually only 80% or less of the initial activity after repeated use for 6 times, for example, Xue and the like adopt Co to increase a weak acid binding site (Co/Si is 0.01) in an MCM-48 framework, and the enzyme activity of the immobilized penicillin G acylase can be maintained at about 80% after 6 times of use. Therefore, how to further improve the operation stability and the recycling rate of the immobilized penicillin G acylase prepared by the adsorption method is a difficult problem which needs to be solved urgently.

Disclosure of Invention

In order to solve the above problems, the present invention provides an immobilized penicillin G acylase and a method for preparing the same, which reduces the falling off of immobilized PGA and improves the operational stability and the recycling rate of immobilized PGA by means of surface modification of a carrier and coating a PDA coating on the surface of immobilized PGA.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides an immobilized penicillin G acylase, which is a nano cystic structure and comprises the following components:

Fe₃O₄NPs immobilized PGA, said Fe₃O₄The surface of the NPs is loaded with PTA; and

coated with the Fe₃O₄NPs immobilized PDA coating of PGA surface.

The invention also provides a preparation method of the immobilized penicillin G acylase, which comprises the following steps:

using TA for Fe₃O₄NPs are subjected to surface modification to prepare Fe₃O₄@PTA NPs；

Subjecting said Fe to₃O₄@ PTA NPs as a carrier, immobilizing PGA, and preparing immobilized PGA (Fe)₃O₄@PTA-PGA NPs)；

The DA is used for the Fe₃O₄Coating the surface of the @ PTA-PGA NPs to prepare Fe₃O₄@ PTA-PGA/PDA, which is the immobilized penicillin G acylase of the invention.

Preferably, Fe is prepared₃O₄@ PTA NPs, including:

mixing Fe₃O₄NPs are dispersed in distilled water and N is introduced into a constant temperature water bath at 40 DEG C₂30min；

To Fe₃O₄Adding aqueous TA solution to aqueous dispersions of NPs and adding N₂Reacting for 2 hours under protection;

separating the product, and washing the separated product with distilled water for several times until washingThe absorbance of the washing liquid of the product at 275nm is less than 0.005, and the absorbance is measured by adopting an ultraviolet spectrophotometry, namely Fe is obtained₃O₄@PTA NPs。

Preferably, Fe is prepared₃O₄@ PTA NPs, further including:

at a temperature of 40 ℃ for the prepared Fe₃O₄@ PTA NPs were vacuum dried to constant weight.

Preferably, Fe is prepared₃O₄@ PTA-PGA/PDA, comprising:

mixing Fe₃O₄@ PTA-PGA NPs dispersed in PBS buffer;

to Fe₃O₄Adding DA into the @ PTA-PGANPs/PBS solution, and carrying out polymerization reaction for 4-9h in a constant-temperature water bath at 25 ℃ under stirring;

separating the product, washing the separated product with PBS buffer solution for multiple times until the absorbance of the eluent for washing the product at 507nm is less than 0.005, and measuring the absorbance by adopting an ultraviolet spectrophotometry to obtain Fe₃O₄@PTA-PGA/PDA。

Preferably, Fe having a nanocapsular structure is prepared₃O₄@ PTA-PGA/PDA, also includes:

at a temperature of 40 ℃ for the prepared Fe₃O₄@ PTA-PGA/PDA was vacuum dried.

Preferably, the concentration of the PBS buffer is 40mmol/mL, and the pH is 8.0.

Preferably, the Fe is prepared₃O₄The polymerization time of @ PTA-PGA/PDA was 4 h.

Preferably, the Fe₃O₄The average particle size of the NPs was 9.13 nm.

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

the immobilized penicillin G acylase prepared by the invention has a nano-capsular structure, has good pH stability and temperature tolerance, has good operation stability and reutilization rate, and has high industrial application value. Experiments show that the enzyme activity retention rate of the immobilized penicillin G acylase after 90 days of storage is 87.1%, 94.1% of relative enzyme activity is still retained after 12 times of repeated use, and the recovery rate of the immobilized penicillin G acylase is 98.0%.

The preparation method is simple and feasible, mild in condition, high in biocompatibility and capable of realizing large-scale production.

Drawings

FIG. 1 shows Fe prepared in this example₃O₄The structure schematic diagram of @ PTA-PGA/PDA;

FIG. 2 shows the preparation of Fe according to the present invention₃O₄The flow chart of @ PTA-PGA/PDA;

FIG. 3 is Fe according to an embodiment of the present invention₃O₄NPs、Fe₃O₄@PTA NPs、Fe₃O₄@ PTA-PGA NPs and Fe₃O₄FTIR spectrum of @ PTA-PGA/PDA;

FIG. 4 shows Fe according to an embodiment of the present invention₃O₄NPs、Fe₃O₄@PTA NPs、Fe₃O₄@ PTA-PGA NPs and Fe₃O₄An XRD spectrogram of @ PTA-PGA/PDA;

FIG. 5 shows Fe according to an embodiment of the present invention₃O₄NPs、Fe₃O₄@PTA NPs、Fe₃O₄@ PTA-PGA NPs and Fe₃O₄A hysteresis loop of @ PTA-PGA/PDA;

FIG. 6 shows free PGA and Fe according to an embodiment of the present invention₃O₄@PTA-PGA NPs、Fe₃O₄EAR of @ PTA-PGA/PDA changes with the change of catalytic pH value;

FIG. 7 shows free PGA and Fe according to an embodiment of the present invention₃O₄@PTA-PGA NPs、Fe₃O₄The EAR of @ PTA-PGA/PDA changes with the catalytic temperature;

FIG. 8 shows free PGA and Fe according to the embodiment of the present invention under a temperature environment of 60 ℃₃O₄@PTA-PGA NPs、Fe₃O₄EAR profile with incubation time for @ PTA-PGA/PDA;

FIG. 9 shows the free PGA at pH 4 and the results of the examples of the present inventionFe₃O₄@PTA-PGA NPs、Fe₃O₄EAR profile with incubation time for @ PTA-PGA/PDA;

FIG. 10 free PGA at pH 10 and Fe as described in the examples of the present invention₃O₄@PTA-PGA NPs、Fe₃O₄EAR profile with incubation time for @ PTA-PGA/PDA;

FIG. 11 shows free PGA and Fe according to an embodiment of the present invention₃O₄@PTA-PGA NPs、Fe₃O₄R of @ PTA-PGA/PDA_aA profile over storage time;

FIG. 12 is Fe according to an embodiment of the present invention₃O₄@ PTA-PGANPs and Fe₃O₄R of @ PTA-PGA/PDA_aThe change condition along with the use times;

FIG. 13 is Fe according to an embodiment of the present invention₃O₄R of @ PTA-PGA/PDA_eThe change condition along with the use times;

FIG. 14 is Fe according to the example of the present invention₃O₄The change curve of the PDA coating amount of @ PTA-PGA/PDA along with the polymerization reaction time;

FIG. 15 shows free PGA, Fe according to an embodiment of the present invention₃O₄@ PTA-PGA NPs and Fe prepared at different polymerization times₃O₄The change curve of the absorbance of the @ PTA-PGA/PDA and 6-APA in the residual liquid along with the catalytic time;

FIG. 16 shows Fe according to an embodiment of the present invention₃O₄@ PTA-PGA NPs and Fe₃O₄EA and EAR of @ PTA-PGA/PDA are shown along with the change curve of polymerization reaction time;

FIG. 17 shows free PGA, Fe according to the present example₃O₄@ PTA-PGA NPs and Fe prepared at polymerization times of 4h and 15h, respectively₃O₄The morphological structure schematic diagram of @ PTA-PGA/PDA;

fig. 18 is Fe as described in the examples of the present invention in an environment with pH 4₃O₄EAR and R of @ PTA-PGA/PDA_aThe curve as a function of the polymerization time;

fig. 19 is Fe as described in the examples of the present invention in an environment of pH 10₃O₄EAR and R of @ PTA-PGA/PDA_aThe curve as a function of the polymerization time;

FIG. 20 shows Fe in the example of the present invention at a temperature of 60 ℃₃O₄EAR and R of @ PTA-PGA/PDA_aThe curve as a function of the polymerization time;

in the figure, EA-enzyme activity, EAR-enzyme activity recovery rate, R_aRelative enzyme activity, R_eRecovery of immobilized PGA.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

Referring to FIGS. 1 to 2, FIG. 1 provides a schematic structural diagram of an immobilized penicillin G acylase of the present invention, and FIG. 2 provides a flow chart of a preparation process of an immobilized penicillin G acylase of the present invention.

Referring to fig. 1, the present invention provides an immobilized penicillin G acylase, which is a nanocapsular structure comprising:

Fe₃O₄NPs immobilized PGA, said Fe₃O₄The surface of the NPs is loaded with PTA; and

coated with the Fe₃O₄NPs immobilized PDA coating of PGA surface.

The source of all raw materials in the invention is not particularly limited, and the raw materials are commercially available or prepared by a conventional method disclosed in the field.

In the examples of the present invention, TA (tannin) was obtained from Shanghai Michelin reagent, Inc. and had a purity of 98%.

In the examples of the present invention, Fe was used₃O₄The average particle diameter of the NPs is 9.13nm, and the NPs are prepared by adopting a reverse microemulsion method.

Tannic acid is TA, a galloyl-containing polyphenol, and belongs to catechol derivatives. The invention adopts TA to carry Fe₃O₄NPs are subjected to surface modification to construct Fe₃O₄The polyhydroxy microenvironment on the surface of the NPs improves the enzyme activity of the immobilized PGA.

Further, the present invention utilizes oxidation of TA from poly-p-Fe₃O₄Surface modification at Fe by NPs₃O₄Forming bionic adhesion layer on the surface of NPs, and immobilizing PGA by physical adsorption to obtain immobilized PGA (Fe)₃O₄@PTA-PGANPs)。

Dopamine, DA for short, is a catechol derivative. The present invention utilizes the adhesion of DA to the immobilized PGA (Fe)₃O₄@ PTA-PGA NPs) to form a protective coating layer, thereby obtaining immobilized penicillin G acylase (Fe) with nanocapsule structure₃O₄@ PTA-PGA/PDA), the shedding of the immobilized PGA is reduced, and the operation stability and the repeated utilization rate of the immobilized PGA are improved.

Referring to fig. 2, the present invention also provides a method for preparing immobilized penicillin G acylase, comprising the steps of:

using TA for Fe₃O₄NPs are subjected to surface modification to prepare Fe₃O₄@PTA NPs；

Subjecting said Fe to₃O₄@ PTA NPs as carrier, immobilizing PGA, and preparing immobilized PGA (Fe)₃O₄@PTA-PGA NPs)；

The DA is used for the Fe₃O₄Coating the surface of the @ PTA-PGA NPs to prepare Fe₃O₄@ PTA-PGA/PDA, which is the immobilized penicillin G acylase of the invention.

In an embodiment of the invention, the production of Fe₃O₄@ PTA NPs, including:

mixing Fe₃O₄NPs are dispersed in distilled water and N is introduced into a constant temperature water bath at 40 DEG C₂30min；

To Fe₃O₄Adding aqueous TA solution to aqueous dispersions of NPs and adding N₂Reacting for 2 hours under protection;

after the reaction is finished, the reaction is carried out onSeparating the product, washing the separated product with distilled water for multiple times until the absorbance of the eluent for washing the product at 275nm is less than 0.005, and measuring the absorbance by adopting an ultraviolet spectrophotometry to obtain Fe₃O₄@PTA NPs。

In an embodiment of the invention, Fe is prepared₃O₄@ PTA NPs, further including:

at a temperature of 40 ℃ for the prepared Fe₃O₄@ PTA NPs were vacuum dried to constant weight.

In an embodiment of the invention, Fe is prepared₃O₄@ PTA-PGA/PDA, comprising:

mixing Fe₃O₄Dispersing the @ PTA-PGANPs in PBS buffer to obtain Fe₃O₄@ PTA-PGANPs/PBS solution;

to the Fe₃O₄Adding DA into the @ PTA-PGANPs/PBS solution, and carrying out polymerization reaction for 4-9h in a constant-temperature water bath at 25 ℃ under stirring;

separating the product after the reaction is finished, washing the separated product for multiple times by using PBS buffer solution until the absorbance of the eluent at 507nm is less than 0.005 by adopting an ultraviolet spectrophotometry, and obtaining the Fe with the nanocapsular structure₃O₄@PTA-PGA/PDA。

In an embodiment of the invention, Fe is prepared₃O₄@ PTA-PGA/PDA, also includes:

at a temperature of 40 ℃ for the prepared Fe₃O₄@ PTA-PGA/PDA was vacuum dried.

In an embodiment of the present invention, the PBS buffer has a concentration of 40mmol/mL and a pH of 8.0.

In an embodiment of the invention, Fe is prepared₃O₄The polymerization time of @ PTA-PGA/PDA was 4 h.

In an embodiment of the invention, the Fe₃O₄The average particle size of the NPs was 9.13 nm.

The present invention will be further illustrated with reference to the following examples.