阅读说明：本技术 一种连续流固定化甲酰甘氨酸生成酶反应器及其使用方法与应用 (Continuous flow immobilized formylglycine generating enzyme reactor and use method and application thereof ) 是由 贾凌云 彭强 任军 臧柏林 于 2019-09-05 设计创作，主要内容包括：本发明公开了一种连续流固定化甲酰甘氨酸生成酶反应器及其使用方法与应用,属于固定化酶技术领域。用于蛋白的定点醛基化修饰。本发明通过将甲酰甘氨酸生成酶固载到琼脂糖凝胶上,显著提高了其稳定性,简化了产物分离。通过发明一种固定化酶的原位重构方法,使得固定化甲酰甘氨酸生成酶能够连续使用,且反应体系中无需再加入铜离子,从而简化了反应体系,降低了应用成本。将固定化甲酰甘氨酸生成酶应用于连续流反应器,提高了固定化酶的催化效率,实现了自动化的连续催化,具有过程可控、产物均一等优点,具有较大的工业放大潜力。(The invention discloses a reactor for generating enzyme by continuous flow immobilized formylglycine and a using method and application thereof, belonging to the technical field of immobilized enzyme. The method is used for site-directed aldehyde modification of protein. According to the invention, the formylglycine generating enzyme is immobilized on the agarose gel, so that the stability of the formylglycine generating enzyme is obviously improved, and the product separation is simplified. By the in-situ reconstruction method of the immobilized enzyme, the immobilized formylglycine generating enzyme can be continuously used, and copper ions do not need to be added into a reaction system, so that the reaction system is simplified, and the application cost is reduced. The immobilized formylglycine generating enzyme is applied to the continuous flow reactor, so that the catalytic efficiency of the immobilized enzyme is improved, the automatic continuous catalysis is realized, and the method has the advantages of controllable process, uniform product and the like and has great industrial amplification potential.)

1. A continuous flow immobilized formylglycine generating enzyme reactor, comprising: comprises a pump and a reaction cavity, wherein the reaction cavity is filled with immobilized formylglycine generating enzyme, the immobilized formylglycine generating enzyme is used for immobilizing the formylglycine generating enzyme on a carrier matrix, and a reducing agent is also added into a reaction solution during continuous flow reaction.

2. The continuous-flow immobilized formylglycine generating enzyme reactor of claim 1, wherein: the pump comprises a micro peristaltic pump, a high-efficiency liquid phase pump and an injection pump.

3. The continuous-flow immobilized formylglycine generating enzyme reactor of claim 1, wherein: the formylglycine generating enzyme comprises a formylglycine generating enzyme derived from mycobacterium tuberculosis, a formylglycine generating enzyme derived from streptomyces coelicolor and a formylglycine generating enzyme derived from high-temperature bent monad.

4. The continuous-flow immobilized formylglycine generating enzyme reactor of claim 1, wherein: the carrier matrix comprises epoxy activated agarose microspheres, ferroferric oxide magnetic microspheres and silicon dioxide microspheres.

5. The continuous-flow immobilized formylglycine generating enzyme reactor of claim 1, wherein: the reducing agent added into the reaction solution comprises dithiothreitol, mercaptoethanol, tri (2-carboxyethyl) phosphine and mercaptoethylamine.

6. The method of using a continuous flow immobilized formylglycine generating enzyme reactor of claim 1, wherein: the method comprises the following steps:

(1) starting a pump, and washing the reaction cavity by using a reaction buffer solution;

(2) dissolving a sample with an aldehyde label in a reaction buffer solution, adding a reducing agent, pumping continuous flow immobilized formylglycine into a reaction cavity of an enzyme reactor, allowing the sample to flow through the reactor, and collecting liquid flowing out of the reactor to obtain the sample modified by aldehyde.

7. Use according to claim 6, characterized in that: the reaction buffer in the step (1) is 50mM triethanolamine, pH 9.0 and 50mM sodium chloride.

8. Use according to claim 6, characterized in that: the retention time of the sample flowing through the reactor in the step (2) is more than 4 minutes.

9. Use of the continuous flow immobilized formylglycine generating enzyme reactor according to any of the claims 1 to 5 for the site-directed aldehyde modification of nanobodies.

Technical Field

The invention relates to a reactor for generating enzyme by continuous flow immobilized formylglycine and a using method and application thereof, belonging to the technical field of immobilized enzyme.

Background

Protein site-directed modification is a core technology in protein engineering, and has wide application in the fields of bioengineering and biomedicine. Because aldehyde groups have excellent bioorthogonal reaction characteristics, site-directed aldehyde modification of proteins is an important component in the field of protein modification. Formylglycine Generating Enzyme (FGE) is a high-efficiency fixed-point aldehyde modification tool for proteins. As shown in fig. 1, it generates an aldehyde group by recognizing an aldehyde tag "CXPXR" fused to a specific site of a protein and catalyzing the conversion of cysteine (Cys) therein to formylglycine (fvy). FGE has been developed as a protein site-directed modification tool at present, for example, US utility model US20080187956a1 discloses for the first time the technique of FGE for site-directed aldehyde modification of protein, US invention patent US20160230205a1 discloses the technique of FGE for performing high-efficiency aldehyde conversion in vivo and in vitro. However, the current in vitro application of FGE is mainly limited by its low stability, high preparation and use costs, and complex reaction system, which make FGE difficult to be applied to large-scale industrial production.

An immobilized enzyme reactor is a technology for immobilizing enzyme on a carrier matrix and realizing product conversion by utilizing solid phase catalysis. Compared with free enzyme, the immobilized enzyme has the advantages of high stability, easy product separation and the like. The immobilized enzyme reactor is divided into a batch reactor and a continuous flow reactor. Continuous flow reactor refers to a technique in which a substrate is passed through an immobilized enzyme reactor to obtain a product under flowing conditions. The continuous flow reactor has the advantages of controllable process automation, on-line separation, good mass transfer effect and the like, and is widely applied to industrial production at present.

Disclosure of Invention

The invention aims to solve the technical problem of applying a continuous flow immobilized enzyme reactor to fixed point aldehyde modification of protein, thereby providing an immobilized enzyme reactor which is stable, efficient, lower in cost and easier to be used for industrial production amplification.

In order to solve the technical problem, the invention provides a continuous flow immobilized formylglycine generating enzyme reactor, which comprises a pump and a reaction cavity, wherein the reaction cavity is filled with immobilized formylglycine generating enzyme, the immobilized formylglycine generating enzyme is prepared by immobilizing formylglycine generating enzyme on a carrier matrix, and a reducing agent is also added into a reaction solution during continuous flow reaction.

Further, in the above technical scheme, the pump includes a micro peristaltic pump, a high performance liquid pump, and an injection pump.

Further, in the above technical solution, the formylglycine generating enzyme includes a formylglycine generating enzyme derived from mycobacterium tuberculosis, a formylglycine generating enzyme derived from streptomyces coelicolor, and a formylglycine generating enzyme derived from thermomonas flexus.

Further, in the above technical scheme, the carrier matrix includes epoxy-activated agarose microspheres, ferroferric oxide magnetic microspheres, and silica microspheres.

Further, in the above technical scheme, the reducing agent added to the reaction solution includes dithiothreitol, mercaptoethanol, tris (2-carboxyethyl) phosphine, and mercaptoethylamine.

The invention also provides a using method of the reactor for generating enzyme by continuous flow immobilized formylglycine, which comprises the following steps:

(1) starting a pump to enable the reaction buffer solution to wash the reaction cavity;

(2) dissolving a sample with an aldehyde label in a reaction buffer solution, adding a reducing agent, pumping continuous flow immobilized formylglycine into a reaction cavity of an enzyme reactor, allowing the sample to flow through the reactor, and collecting liquid flowing out of the reactor to obtain the sample modified by aldehyde.

Further, in the above technical solution, the reaction buffer in step (1) is 50mM triethanolamine, pH 9.0, 50mM sodium chloride.

Further, in the above technical solution, the retention time of the sample flowing through the reactor in the step (2) is greater than 4 minutes.

The invention also provides application of the continuous flow immobilized formylglycine generating enzyme reactor in site-specific hydroformylation modification of the nano antibody.

Advantageous effects of the invention

The invention provides an immobilized FGE, which is characterized in that common FGE with different sources is immobilized, the stability of in vitro catalysis is improved, and the product separation after catalytic reaction is facilitated.

The invention provides a continuous flow immobilization FGE reactor, which improves the catalytic efficiency of immobilized enzyme, realizes automatic and continuous protein modification, and has the advantages of controllable process, uniform product, easy amplification and the like.

Drawings

FIG. 1 is a schematic diagram of the catalytic principle of FGE.

FIG. 2 is an electrophoresis diagram showing the purification of each FGE; (FIG. a) is FGE (MtFGE) from Mycobacterium tuberculosis (Mycobacterium tuberculosis), (FIG. b) is FGE (TcFGE) from Thermomonospora curvata, and (FIG. c) is FGE (ScFGE) from Streptomyces coelicolor; in the figure, lane M is Marker protein, and lanes 1, 2, 3 and 4 are the conditions of whole bacteria expression, soluble expression, inclusion body expression and final purity, respectively.

FIG. 3a is a liquid phase separation of a substrate and a product polypeptide; FIG. 3b is a graph of mass spectrometric identification of the product; FIG. 3c is a mass spectrometric identification of a substrate.

FIG. 4 shows the retention of the activity of the immobilized enzyme relative to the free enzyme.

FIG. 5a is a fixed-point aldehyde modified electrophoresis diagram of a nanobody, wherein lanes 1, 2 and 3 are unmodified, modified and labeled with a fluorescent molecule Lucifer Yellow CH (LYC) with a hydrazide group, respectively; FIGS. 5b and 5c are the modified and labeled protein profiles, respectively.

FIG. 6 is a catalytic schematic of a continuous flow immobilized formylglycine generating enzyme reactor.

FIG. 7a is a graph of the effect of flow rate (retention time) in a continuous flow immobilized enzyme reactor on conversion; FIG. 7b shows the continuous use of a continuous flow immobilized enzyme reactor.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical companies.

Materials used in the following examples:

fge from Mycobacterium tuberculosis (Mycobacterium tuberculosis) (mtfge): the protein sequence is shown as SEQ ID NO. 1;

fge (scfge) from Streptomyces coelicolor: the protein sequence is shown as SEQ ID NO. 2;

fge (tcfge) from thermomomonas flexuosa (Thermomonospora curvata): the protein sequence is shown as SEQ ID NO. 3.