WO2019091454A1

WO2019091454A1 - Enzyme complex and self-assembly catalytic nanowires

Info

Publication number: WO2019091454A1
Application number: PCT/CN2018/114833
Authority: WO
Inventors: 门冬; 张先恩; 魏翠华
Original assignee: 中国科学院武汉病毒研究所
Priority date: 2017-11-09
Filing date: 2018-11-09
Publication date: 2019-05-16
Also published as: CN108004226A

Abstract

Provided is the use of a fibroblast protein in enhancing enzyme catalytic activity and/or enzyme stability. The present invention displays enzyme molecules with high density and arrayed on the surface of the nanostructure of the fibroblast protein by the self-assembly of the structure of a fibrillar protein to form a linear nanostructure having catalytic capacity. The nanostructure simulates the natural regionalization status of intracellular enzyme molecules, thereby improves the enzyme catalytic activity and stability without modifying the enzyme molecules themselves.

Description

Enzyme complex and self-assembled catalytic nanowire

Technical field

The invention relates to an enzyme catalyst, in particular to an enzyme complex and a self-assembled catalytic nanowire.

Background technique

As a biocatalyst, enzymes have been widely used in various fields of production and life. In recent decades, with the continuous technological breakthrough of enzyme engineering, it has become more and more widely used in industry, agriculture, medical and health, energy development and environmental engineering. In the research and application of the enzyme catalytic process, it is always expected that the higher the activity and stability of the enzyme, the better the catalytic performance. How to improve the catalytic ability of enzyme protein is one of the research hotspots in academia, and it is also an urgent problem to be solved in industrial production practice. Traditional methods for enhancing the catalytic ability of enzyme proteins mainly include chemical modification and molecular enzyme engineering.

Enzymatic chemical modification is a method of applying chemical methods mainly including 1) a site-directed mutagenesis method for introducing a specific molecule at a specific site of an enzyme protein; 2) using a bifunctional group reagent such as glutaraldehyde, PEG, etc. Crosslinking technique for covalent cross-linking between different peptide chains in the molecule or in the molecule; 3) Small molecule compound modification by chemical modification of the active site of the enzyme or the side chain group other than the active site by a small molecule compound Various chemical modifications are applied to the enzyme molecule, and the enzyme protein is molecularly modified by cleavage, splicing and chemical modification of the side chain group to change its physical and chemical properties and biological activity. The chemical modification of the enzyme is mainly through the introduction or removal of the chemical group, and most of the modification occurs at the active site or the essential group of the enzyme, so that not only the covalent structure of the enzyme protein is changed, but also the activity of the enzyme protein is deepened with the degree of modification. The gradual weakening, that is, the enzymatic chemical modification can not effectively improve the catalytic efficiency of the enzyme itself, but will reduce the enzyme activity.

Molecular enzyme engineering is the use of genetic engineering and protein engineering methods and techniques to study the relationship between the cloning and expression of enzyme genes, the structure and function of enzyme proteins, and based on this, the enzyme molecules are redesigned and oriented. Molecular enzyme engineering needs to start from one or more existing parent enzymes, through gene mutation and recombination, to construct a library of artificial mutant enzymes, and finally obtain evolutionary enzymes with certain characteristics through certain screening or selection methods. The molecular enzyme engineering method not only depends on the perfection of the structural biological data of the parent enzyme molecule, but also requires time-consuming and laborious construction of the artificial mutant enzyme library, and the later screening process is more complicated and cumbersome.

Comprehensive enzyme chemical modification and molecular enzyme engineering techniques, both work focused on processing a single enzyme molecule, without considering the overall distribution of enzyme molecules in living cells. More and more studies have shown that enzyme molecules are not uniformly dispersed in bacteria as in solution, but clusters exist. This cluster of enzyme molecules in the form of supramolecular aggregates has higher catalytic activity. However, the enzyme molecules expressed by gene cloning often appear in a monodisperse form, which may be one of the reasons why the prepared enzyme molecule has a lower activity than the natural state enzyme molecule.

Summary of the invention

The invention provides a fibrin protein for overcoming the shortcomings of the enzymes and stability of the enzymes existing in the prior art, the preparation is complicated, the cost is high, and the reagents, materials or methods suitable for the enzyme are required for specific enzymes. Use in enhancing enzyme catalytic activity and/or enzyme stability. The fibrillar protein of the present invention is amyloidogenesis fibrils (amyloid fibrin), referred to as fibrin.

In order to achieve the object of the present invention, the present invention adopts the following technical solutions:

One aspect of the invention provides the use of fibrin for enhancing enzyme catalytic activity and/or enzyme stability.

Illustratively, the fibrin forms an enzyme complex with the enzyme, and the enzyme is directly or indirectly attached to the fibrin.

Illustratively, the enzyme is linked to the fibrillar by a linker peptide and/or a linker protein.

In a specific embodiment of the invention, the enzyme is linked to the fibrillar by a linker peptide.

In a specific embodiment of the invention, the fibrillar forms a fibrous nanostructure.

In a specific embodiment of the invention, the fibrin and the enzyme complex formed by the enzyme self-assemble to form a fibrous nanostructure.

In a specific embodiment of the invention, the fibrous nanostructures are formed by fibroin self-assembly.

Illustratively, the fibrillar can be any protein capable of forming a fibrous form and capable of being linked to an enzyme.

Illustratively, the fibrin is yeast prion protein or amyloid. For example, it may be yeast prion protein Sup35, amyloid Ure2, silk fibroin or the like.

Illustratively, the fibrin is yeast prion Sup35, preferably, the fibrin is a self-assembling domain formed by amino acids 1-61 of the yeast prion Sup35.

In a specific embodiment of the invention, the enzyme is selected from the group consisting of a protease, a nuclease or an artificial enzyme.

In a specific embodiment of the invention, the enzyme is selected from the group consisting of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a synthetase.

Illustratively, oxidoreductases such as glucose oxidase, catalase, hydrogenase, oxygenase, and the like.

Illustratively, transferases such as transmethylase, transaminase, hexokinase, phosphorylase, and the like. Specifically, such as sitagliptin transaminase.

Illustratively, hydrolases such as esterases, glycosylases, peptidases, nucleases and the like. Specifically, such as methyl parathion hydrolase.

Illustratively, lyases such as aldolase, hydratase, deaminase, carbonic anhydrase, pyruvate decarboxylase, alkaline phosphatase, and the like.

Illustratively, isomerases such as racemates, epimerases, mutases, and the like.

Illustratively, a synthetase such as an aminoacyl tRNA synthetase, a glutamine synthetase, a trehalose synthase, or the like.

In a specific embodiment of the invention, the enzyme comprises an enzyme that uses carbohydrates, proteins, fats, nucleic acids, and alkaloids as substrates.

Illustratively, carbohydrate-based enzymes such as amylase, glucose isomerase, lactase, chymosin, cellulase, glycanase, lactase, etc.; protein-based enzymes such as bromelain , pepsin, trypsin, etc.; enzymes such as lipase, esterase, etc.; and enzymes such as alkaline phosphatase with nucleic acid and alkaloid as substrates.

Another aspect of the present invention provides a method for preparing the above enzyme complex, which comprises merging fibrin with an enzyme molecule by molecular cloning or fusing a fibrin, a linker peptide (or street protein), an enzyme molecule to form a fusion protein gene, and The fusion protein gene is expressed.

In a specific embodiment of the present invention, the preparation method of the above enzyme complex comprises the following steps:

The fibrin, the linker peptide and the enzyme molecule are fused to form a fusion protein gene by molecular cloning, and the fusion protein gene is expressed and purified.

In a specific embodiment of the invention, the fibrin is a yeast prion self-assembling domain Sup35, and the enzyme is methyl parathion hydrolase MPH (or sitagliptin transaminase ATA-117). Specific preparation methods of the enzyme complex include:

The yeast prion self-assembling domain Sup35 and the gene mph of methyl parathion hydrolase MPH (or the gene ata-117 of sitagliptin transaminase ATA-117) are fused by a ligation peptide (for example, GGGGS) by molecular cloning. Fusion protein gene Sup35-mph (or Sup35-ata-117);

The fusion protein gene Sup35-mph (or Sup35-ata-117) was cloned into an expression vector, and the expression vector was transferred into an expression host, and the fusion protein Sup35-MPH (or Sup35-ATA-117) was induced and purified.

In a specific embodiment, the expression and purification of the fusion protein gene can be carried out by a method conventionally used in the art for purification of protein expression, for example, the fusion protein gene is cloned into an expression vector, and the expression vector is transferred into an expression host for cultivation, and activation to logarithm After the growth period, the induced albumin is added, and after being crushed and purified, the fusion protein is obtained. In the present invention, the type and class of the expression vector and the expression host are not limited, and a vector and a host conventionally used for genetic modification in the art may be used. Specifically, the expression vector may be pET-28, pET-32, pET-15 or pET-11, etc.; the expression host may be selected from Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium, Saccharomyces cerevisiae, Pichia or Mammalian cells.

In a specific embodiment, a linker protein can be substituted for the linker peptide, which can also reduce the effect of steric hindrance that the fusion protein may have.

A further aspect of the present invention provides a self-assembled catalytic nanowire comprising the enzyme complex of the present invention or the enzyme complex prepared by the above preparation method, wherein more than 50% of the fibrin protein in the enzyme complex is directly Or indirectly linked to an enzyme.

Illustratively, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of fibrin is directly or indirectly linked to an enzyme, The specific ratio can be determined based on actual production needs and costs.

Illustratively, fibrillar is linked to the enzyme by a linker peptide and/or a linker protein.

Illustratively, the linker peptide is GGGGS.

In a specific embodiment of the invention, fibroin self-assembles to form fibrous nanostructures.

In still another aspect, the present invention provides a method for preparing a self-assembled catalytic nanowire, comprising the following steps:

Mixing a part of the above enzyme complex or a mixture of the partial enzyme complex and the fibrin protein without enzyme complexing as a monomer to form a nanowire, and breaking the nanowire into a nanowire fragment by ultrasonication to prepare a nanowire seed ;

The prepared nanowire seed is mixed with another partial enzyme complex or a mixture of the partial enzyme complex and the fibrin protein without enzyme complexation, and seed-induced self-assembly is performed to form a self-assembled catalytic nanowire.

In a specific embodiment of the invention, the enzyme complex is Sup35-MPH (or Sup35-ATA-117), and the preparation of the self-assembled catalytic nanowire comprises the following steps:

A portion of Sup35-MPH (or Sup35-ATA-117) was incubated at 4 °C for one week to form nanowires, and the nanowires were broken into nanowire fragments by ultrasound to prepare Sup35-MPH seeds (or Sup35-ATA- 117 seeds);

The prepared Sup35-MPH seed (or Sup35-ATA-117 seed) is mixed with another part of Sup35-MPH (or Sup35-ATA-117) in a certain molar ratio, and mixed and incubated at 4 ° C for seed-induced self-assembly. The catalytic nanowires are assembled, and 100% of the Sup35 in the nanowire is directly or indirectly connected to MPH (or ATA-117).

A portion of Sup35-MPH (or Sup35-ATA-117) was incubated at 4 °C for one week to form nanowires, and the nanowires were broken into nanowire fragments by ultrasound to prepare Sup35-MPH seeds (or Sup35-ATA- 117 seeds).

Another portion of Sup35-MPH (or Sup35-ATA-117) is mixed with Sup35 of uncomplexed Sup35-MPH (or Sup35-ATA-117) in a molar ratio to form a mixed monomer.

The prepared Sup35-MPH seed (or Sup35-ATA-117 seed) was mixed with the above mixed monomer in a certain molar ratio, and mixed and incubated at 4 ° C to perform seed-induced self-assembly to form a self-assembled catalytic nanowire. The ratio of Sup35 in the prepared nanowires or the ratio of MPH (or ATA-117) to the linker can be controlled by adjusting the ratio of Sup35-MPH (or Sup35-ATA-117) to Sup35 in the mixed monomer.

In another specific embodiment of the present invention, the enzyme complex is Sup35-MPH (or Sup35-ATA-117), and the preparation of the self-assembled catalytic nanowire comprises the following steps:

A mixture of a part of Sup35-MPH (or Sup35-ATA-117) and Sup35 which is not compounded with Sup35-MPH (or Sup35-ATA-117) in a certain molar ratio is used as a monomer and is incubated at 4 ° C for one week to form a nanometer. Sup35-MPH seeds (or Sup35-ATA-117 seeds) were prepared by disrupting the nanowires into nanowire fragments by ultrasound.

Put another part of Sup35-MPH (or Sup35-ATA-117) with Sup35 that is not complexed with Sup35-MPH (or Sup35-ATA-117) (there are no separate Sup35 protein molecules present, only Sup35-MPH and Sup35-ATA are present -117 protein molecules or nanowires formed by molecules) are mixed in a certain molar ratio to form a mixed monomer.

The prepared Sup35-MPH seed (or Sup35-ATA-117 seed) was mixed with the above mixed monomer in a certain molar ratio, and mixed and incubated at 4 ° C to perform seed-induced self-assembly to form a self-assembled catalytic nanowire. The Sup35 can be controlled by adjusting the ratio of Sup35-MPH (or Sup35-ATA-117) to Sup35 in the seed and/or the ratio of Sup35-MPH (or Sup35-ATA-117) to Sup35 in the mixed monomer. The ratio of MPH (or ATA-117) is linked directly or via a linker peptide.

The invention adopts the seed-induced self-assembly method, and the seed can rapidly induce the fusion protein with the self-assembled domain to be assembled at the end thereof, and can realize the nanometer by controlling the assembly ratio of the seed and the monomer (or mixed monomer) fusion protein. Line length control.

In still another aspect, the present invention provides the use of the above enzyme complex or self-assembled catalytic nanowire as a catalyst.

In still another aspect, the invention provides the use of fibrin in the preparation of self-assembled catalytic nanowires for enhancing enzyme catalysis and/or enzyme stability.

Still another aspect of the invention provides the use of fibrous nanostructures to enhance enzyme catalytic activity and/or stability.

In still another aspect, the present invention provides a method of increasing enzyme activity and/or stability, comprising:

(1) directly or indirectly linking the enzyme to fibrin to obtain an enzyme complex;

(2) The enzyme complex obtained in (1) is self-assembled into a nanowire, thereby allowing the enzyme to be displayed on a self-assembled nanowire.

Illustratively, the present invention has at least one of the following advantages:

The present invention connects the self-assembly domain of fibroblast, such as yeast prion Sup35, to the gene of the enzyme by gene manipulation, and binds the enzyme molecule to the C-terminus of the yeast prion Sup35 through gene expression, and utilizes the yeast 朊 protein Sup35 self-assembling domain. The polymerization forms a nanostructure, and a large number of enzyme molecules are displayed in a high density and array on the surface of the nanostructure to form a linear nanostructure having catalytic ability. The nanostructure mimics the natural localization state of the enzyme molecule, thereby increasing the catalytic activity and/or stability of the enzyme without altering the enzyme molecule itself. For example, in the embodiment of the present invention, compared with the freely dispersed free MPH in the solution, the MPH combined with the fibrin-forming protein has a Michaelis constant K _m less than 1/5 of the free enzyme, and the catalytic constant K _{cat is} increased by 1 The maximum rate V _{max is} increased by 26.5 times and the specific activity is increased by 4.8 times, which significantly improves the affinity and catalytic ability of the methyl parathion hydrolase MPH with the substrate; for the sitagliptin transaminase ATA-117, While increasing the conversion rate of sitagliptin transaminase ATA-117 to the substrate, the substrate conversion time is shortened, and the substrate conversion rate can be increased by about 30% in the same reaction time to achieve the highest substrate conversion. The time-dependent self-assembled catalytic nanowire Sup35-ATA-117 is more than four times shorter than the free enzyme ATA-117.

DRAWINGS

Figure 1. Schematic diagram of the fusion protein Sup35-MPH (Sup35-ATA-117) expression and nanowire enzyme protein self-assembly structure in the examples of the present invention.

Figure 2. SDS-MPH enzyme complex and Sup35-ATA-117 enzyme complex SDS-PAGE gel electrophoresis detection in the examples of the present invention.

3A is an electron micrograph of a Sup35-MPH nanowire formed by self-assembly in an embodiment of the present invention.

3B is an electron micrograph of a Sup35-ATA-117 nanowire formed by self-assembly in an embodiment of the present invention.

Figure 4A is a graph showing changes in product OD ₄₀₅ values over time during the Sup35-MPH reaction of the free enzyme MPH and nanowire states in the examples of the present invention.

Fig. 4B is a graph showing the results of kinetic comparison of the enzyme Mp and Sup35-MPH of the free enzyme MPH and the nanowire state in the examples of the present invention.

Fig. 5 is a graph showing the results of comparison of the catalytic ability of the free enzyme ATA-117 and the enzyme protein Sup35-ATA-117 in the nanowire state in the examples of the present invention.

Detailed ways

Description: In the specific embodiments and examples of the present invention, the abbreviations "MPH", "ATA-117", "Sup35-MPH", and "Sup35-ATA-117" are case-insensitive and can represent fusion genes, fusion proteins or self-assembly. Nanowires, the specific meanings are understood in terms of context.

"Sup35-MPH" can directly link the yeast prion protein Sup35 to MPH, or it can represent the yeast prion protein Sup35 and MPH by ligation peptide or street protein. The specific meaning is understood according to the context.

"Sup35-ATA-117" can directly link the yeast prion protein Sup35 to ATA-117, or it can represent the yeast prion protein Sup35 and ATA-117 through a linker peptide or a linker protein, and the specific meaning is understood according to the context.

The linker peptide in a specific embodiment of the present invention can be replaced with a adaptor protein as needed. For example, a leucine zipper or the like.

The enzyme complex, the self-assembled catalytic nanowire of the present invention and a preparation method thereof are further illustrated by taking Sup35-MPH/Sup35-ATA-117 as an example in the following with reference to specific examples.

Example 1 Preparation of Sup35-MPH Enzyme Complex

1. Sup35-MPH enzyme complex cloning

The yeast prion self-assembling domain (Sup35-1-61 amino acid, Sup35 for specific sequence, see SEQ ID NO.1) and methyl parathion hydrolase (MPH 36-331) by molecular cloning The amino acid, the specific sequence is shown in SEQ ID NO. 2, and the fusion protein Sup35-MPH is formed by fusion ligation of a flexible linker peptide (see SEQ ID NO. 3 for specific sequence), and then the His tag is fused at its N-terminus. A single methyl parathion hydrolase (MPH) was used as a control. Specific steps are as follows:

1) Using plasmid pET28a as a template, PCR amplification using primer MPH-Primer-1 to obtain plasmid pET28a containing primer MPH-Primer-1 sequence;

2) Using the MPH gene fragment as a template and PCR amplification using the primer MPH-Primer-3, the N-terminal His-tag MPH gene fragment was obtained;

3) The plasmid pET28a-Sup35 ^1-61 –Linker–MPH-His _tag with C-terminal His _tag in the laboratory (see the literature: Mendong, multi-functional nanowire based on 朊 protein self-assembly and ultra-sensitive Biosensing, PhD thesis) as a template, PCR amplification using MPH-Primer-2, N-terminal Sup35 ^1-61- MPH fragment with His-tag;

4) homologous recombination of the gene fragments obtained in steps 1) and 2) to obtain the expression vector pET28a-His-MPH;

5) The homologous recombination reaction with the gene fragments obtained in steps 1) and 3) provides the expression vector pET28a-His _tag -Sup35 ^1-61 -Linker-MPH.

2. Expression and purification of Sup35-MPH enzyme complex

The expression vectors pET28a-His-MPH and pET28a-His _tag -Sup35 ^1-61 -Linker-MPH were transformed into E. coli expression strain BL21, respectively, and positive clones were picked up by kanamycin resistance plates. The picked positive clone E. coli BL21 was secondarily activated into kanamycin-resistant LB medium, and cultured at 37 ° C, shaking at 200 rpm to logarithmic growth phase (OD value of about 0.5). To the culture, IPTG at a final concentration of 1 mM and kanamycin at a working concentration of 50 μM (Kan's final concentration of 50 μg/ml) were added, and the protein expression was induced by shaking culture at 120 °C for 8 hours at 25 °C. The cells were collected by centrifugation at 8000 rpm for 5 minutes, and the cells were sonicated, centrifuged at 10,000 × g for 30 minutes to remove cell debris, and the supernatant protein was purified by affinity chromatography to obtain purified MPH and Sup35-MPH (as shown in Fig. 1). ). MPH is a free methyl parathion hydrolase protein as a control.

It is known to those skilled in the art that the specific parameters (e.g., concentration, time, temperature, etc.) in the expression and purification of the protein in the above embodiments of the present invention are not intended to limit the present invention, and those skilled in the art can adjust according to actual needs.

3. Self-assembled catalytic nanowire Sup35-MPH controllable preparation

(1) Preparation of Sup35-MPH seed: After incubating a part of the enzyme complex Sup35-MPH as a monomeric protein at 4 ° C for one week, the long nanowire was broken into nanowire fragments by the shear force generated by ultrasound. Seeds are prepared which can rapidly induce the fusion of a fusion protein with a self-assembling domain at its end.

(2) Preparation of Sup35-MPH nanowire: The prepared Sup35-MPH seed was mixed with another Sup35-MPH enzyme complex at a certain molar ratio, and incubated at 4 ° C for 8 hours to induce seed induction. Quick assembly (as shown in Figure 1). The molar ratio of the Sup35-MPH seed to the Sup35-MPH enzyme complex may be any ratio, and the ratio of 1:4 is selected in the present embodiment. The free methyl parathion hydrolase protein (MPH) was used as a control.

Example 2 Detection of Sup35-MPH Enzyme Complex and Nanowires

The MPH and Sup35-MPH prepared in Example 1 were subjected to SDS-PAGE electrophoresis, and the obtained gel was subjected to Coomassie blue staining, and the results are shown in Fig. 2. As can be seen from Fig. 2, between 30 KDa and 40 KDa, there are clear and distinct bands near 40 KDa, which correspond to the free methyl parathion hydrolase protein (MPH) and Sup35-MPH enzyme complex, respectively. Electrophoresis showed no obvious heteroprotein bands, indicating that the purity and expression levels of the methyl parathion hydrolase protein (MPH) and Sup35-MPH enzyme complex prepared in Example 1 were high.

The self-assembled catalytic nanowire Sup35-MPH prepared in Example 1 was subjected to electron microscopic examination, and the results are shown in Fig. 3A. As can be seen from FIG. 3A, the concentration and length distribution of the nanowires formed by self-assembly are uniform.

Example 3 Preparation of Sup35-ATA-117 Enzyme Complex

1. Sup35-ATA-117 enzyme complex clone

The yeast prion self-assembling domain (S1-6-amino acid of Sup35, Sup35 for short, specific sequence is shown in SEQ ID NO.1) and sitagliptin transaminase (ATA-117) by molecular cloning, see SEQ ID for specific sequence Shown by NO.4) The fusion protein Sup35-ATA-117 was formed by fusion ligation of a flexible linker peptide (see the specific sequence shown in SEQ ID NO. 3), and then the His tag was fused at its N-terminus. A separate sitagliptin transaminase (ATA-117) was used as a control. Specific steps are as follows:

1) Using plasmid pET28a as a template, PCR amplification using primer ATA-Primer-1 to obtain pET28a vector containing primer ATA-Primer-1 sequence;

2) Using the plasmid PUC57-ATA-117 as a template, PCR amplification was performed using the primer ATA-Primer-2 to obtain a His-tag His _tag -ATA-117 gene fragment with N-terminal;

3) Using plasmid pET28a-His _tag -Sup35 ^1-61 -Linker-MPH as a template, PCR amplification with primer ATA-Primer-3 was performed to obtain His-tag His _tag- Sup35 ^1-61- Linker gene with N-terminal Fragment

4) Using the primer ATA-Primer-4 as a template and using PUC57-ATA-117 as a template, the Linker-ATA-117 gene fragment was obtained;

5) using the His _tag- Sup35 ^1-61- Linker fragment obtained in the step 3) and the Linker-ATA-117 fragment obtained in the step 4) as a template, and PCR amplification using the primer ATA-Primer-5, and amplifying the Sup35 ^{1- 61-} Linker-ATA-117 gene fragment;

6) homologous recombination of the gene fragments obtained in steps 1) and 2) to obtain the expression vector pET28a-His _tag -ATA-117;

7) The gene fragment obtained in steps 1) and 5) is homologously recombined to obtain an expression vector pET28a-His _tag -Sup35 ^1-61 -ATA-117.

2. Expression and purification of Sup35-ATA-117 enzyme complex

The expression vectors pET28a-His _tag -ATA-117, pET28a-His _tag -Sup35 ^1-61 -ATA-117 were transformed into E. coli expression strain BL21, respectively, and positive clones were picked up by kanamycin resistance plates. The picked positive clone E. coli BL21 was secondarily activated into kanamycin-resistant LB medium, and cultured at 37 ° C, shaking at 200 rpm to logarithmic growth phase (OD value of about 0.5). IPTG at a final concentration of 1 mM and kanamycin at a working concentration of 50 μM were added to the culture, and protein expression was induced by shaking culture at 25 ° C, 120 rpm for 8 hours. The cells were collected by centrifugation at 8000 rpm for 5 minutes, and the cells were sonicated, centrifuged at 10,000 × g for 30 minutes to remove cell debris, and the supernatant protein was purified by affinity chromatography to obtain purified ATA-117 and Sup35-ATA-117 (eg, Figure 1). ATA-117 is a free sitagliptin transaminase protein as a control.

3. Self-assembled catalytic nanowire Sup35-ATA-117 controllable preparation

(1) Preparation of Sup35-ATA-117 seed: A part of the enzyme complex Sup35-ATA-117 was incubated as a monomeric protein for one week at 4 ° C, and the long nanowires were interrupted by the shear force generated by ultrasound. Seeds are prepared as nanowire fragments, which can rapidly induce fusion of fusion proteins with self-assembled domains at their ends.

(2) Preparation of Sup35-ATA-117 nanowire: The prepared Sup35-ATA-117 seed was mixed with another part of Sup35-ATA-117 enzyme complex at a certain molar ratio, and incubated at 4 ° C for 8 hours. It causes seed-induced rapid assembly (as shown in Figure 1). The molar ratio of the Sup35-ATA-117 seed to the Sup35-ATA-117 enzyme complex may be any ratio, and the ratio of 1:6 is selected in the present embodiment. The free sitagliptin transaminase protein (ATA-117) was used as a control.

Example 4 Detection of Sup35-ATA-117 Enzyme Complex and Nanowires

The ATA-117 and Sup35-ATA-117 prepared in Example 3 were subjected to SDS-PAGE electrophoresis, and the obtained gel was subjected to Coomassie blue staining, and the results are shown in Fig. 2. As can be seen from Fig. 2, there is a clear and distinct band between 50KDa and 60KDa near 40KDa, which corresponds to the free sitagliptin transaminase protein (ATA-117) and Sup35-ATA-117 enzyme complex, respectively. Electrophoresis showed no obvious heteroprotein bands, indicating that the purity and expression levels of the sitagliptin transaminase protein (ATA-117) and Sup35-ATA-117 complex prepared in Example 3 were high.

The self-assembled catalytic nanowire Sup35-ATA-117 prepared in Example 3 was subjected to electron microscopic examination, and the results are shown in Fig. 3B. As can be seen from FIG. 3B, the distribution of the concentration and length of the nanowires formed by self-assembly is uniform.

The enzyme kinetics and enzyme stability analysis of the self-assembled catalytic nanowires Sup35-MPH and Sup35-ATA-117 prepared in Example 1 and Example 3, respectively, were carried out.

Example 5 Study on Kinetics and Stability of Self-Assembled Catalytic Nanowire Sup35-MPH

The self-assembled catalytic nanowire Sup35-MPH (referred to as SMPH) prepared in Example 1 can catalyze the reaction of a substrate such as methyl parathion, the product is yellow, and has light absorption at a wavelength of 405 nm, and is detected by a microplate reader. The production of the product and the change of the amount of the product were analyzed to analyze the enzymatic kinetics and stability of the self-assembled catalytic nanowire Sup35-MPH. The experimental results are shown in Fig. 4A and Fig. 4B. The free MPH was used as a control (referred to as MPH).

As can be seen from Fig. 4A, the reaction rate of the free enzyme MPH is much lower than that of the nanowire state enzyme Sup35 ^1-61 -MPH, SMPH, and the SMPH reaches the equilibrium point of the reaction in a short time.

The upper left graph of Fig. 4B is a graph showing the results of the Michaelis constant test. From the upper left graph of Fig. 4B, it can be seen that the Michaelis constant K _{m of} the nanowire state enzyme SMPH is 5.4 times lower than that of the free enzyme MPH, that is, the nanowire state enzyme SMPH is easier to The substrate binds and its affinity with the substrate is significantly enhanced.

The upper right panel of Fig. 4B is a graph showing the results of the catalytic constant test. It can be seen from the upper right panel of Fig. 4B that the catalytic constant K _{cat of the} nanowire state enzyme SMPH is doubled compared with the free enzyme MPH, that is, the substrate concentration is saturated, the nanowire The state enzyme SMPH catalyzes the same reaction at a much higher rate than the free enzyme MPH.

The lower left panel of Fig. 4B is a graph of the maximum catalytic rate test results. As can be seen from the lower left panel of Fig. 4B, under the same conditions, the maximum rate _{Vmax of the} nanowire state enzyme SMPH is 26.5 times higher than that of the free enzyme MPH.

The lower right panel of Fig. 4B is a graph showing the results of enzyme activity test. As can be seen from the lower right panel of Fig. 4B, the specific activity of the nanowire state enzyme SMPH is 4.8 times higher than that of the free enzyme MPH, that is, per milligram of nanowire. The number of enzyme activities contained in the state enzyme SMPH protein is much higher than the number of enzyme activity units per mg of free enzyme MPH.

In summary, the self-assembled catalytic nanowire Sup35-MPH has significantly improved catalytic properties and stability compared to free MPH.

Example 6 Study on Kinetics and Stability of Self-Assembled Catalytic Nanowire Sup35-MPH

The self-assembled catalytic nanowire Sup35-ATA-117 prepared in Example 3 can catalyze the conversion of sitagliptin intermediate 4 (the sitagliptin precursor ketone) to sitagliptin. The production of sitagliptin and the change of product amount were detected by HPLC to analyze the catalytic ability of self-assembled catalytic nanowire Sup35-ATA-117. The experimental results are shown in Fig. 5. The free ATA-117 enzyme was used as a control.

It can be seen from Fig. 5 that the substrate conversion rate of the self-assembled catalytic nanowire Sup35-ATA-117 is significantly higher than that of the free enzyme ATA-117. The substrate conversion rate can be increased by up to 30% in the same reaction time, and the self-assembly catalytic nanowire Sup35-ATA-117 which is the highest substrate conversion rate is more than four times shorter than the free enzyme ATA-117.

In summary, fibroin (such as Sup35) can improve the catalysis and stability of the enzyme molecule. Fibroblasts assemble into protein nanostructures using their own properties, and display the enzyme molecules on the surface of protein nanostructures in a high density and array to form catalytic nanostructures. The nanostructure mimics the natural localization state of the intracellular enzyme molecule, thereby improving the catalytic activity and stability of the enzyme without changing the enzyme molecule itself.

The method or application provided by the present invention can be used as directed evolution of enzyme molecules, referred to as enzyme directed evolution. In the process of natural evolution, enzyme molecules ensure the ability of the enzyme to adapt to any changes in the environment, but natural evolution has neither a specific direction nor a specific target. It spontaneously occurs during the reproduction and survival of the entire organism. The natural evolution of enzymes is not manifested in the continuous improvement of the activity and stability of an enzyme molecule, but in the adaptability of the organism as a whole, and the enhancement of its regulatory ability. Therefore, it is usually only required for the enzyme to perform specific biological functions in the organism. Specificity.

Directed evolution technology enables the long evolutionary process in nature to be simulated in the laboratory, enabling humans to modify enzyme molecules according to their own wishes and needs, and even to design new enzyme molecules (new proteins) that did not exist in nature. ). Compared with natural evolution, the directed evolution of enzyme molecules is entirely under human control, allowing the enzyme molecules to evolve toward the specific targets that people expect.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., which are within the spirit and principles of the present invention, should be included in the scope of the present invention. within.

Claims

The use of fibrin in enhancing enzyme catalytic activity and/or enzyme stability.
The use according to claim 1, wherein the fibrin forms an enzyme complex with the enzyme, and the enzyme is directly or indirectly linked to the fibrin, preferably, the enzyme is linked to a peptide and/or on the street. The protein is linked to the fibrillar.
The use according to claim 1 or 2, wherein the fibrin, or an enzyme complex formed by the fibrin and the enzyme, self-assembles to form a fibrous nanostructure, the fibrin protein being yeast prion protein or Amyloid, preferably, the fibrin is yeast prion Sup35; and optionally, the enzyme is selected from the group consisting of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a synthetase.
The use according to Claim 3, wherein the enzyme is selected from the group consisting of glucose oxidase, amylase, pepsin, alkaline phosphatase, cellulase, glycanase, lactase, lipase, esterase, methyl sulfonate Phosphorus hydrolase or sitagliptin transaminase, preferably, the enzyme is methyl parathion hydrolase or sitagliptin transaminase.
In an application according to any one of claims 2 to 4, the method of preparing the enzyme complex comprises or consists of the following steps:

By molecular cloning, the fibrin is fused with the enzyme molecule or the fibrin, the linker (or street protein), and the enzyme molecule are fused to form a fusion protein gene, and the fusion protein gene is expressed.
A self-assembling catalytic nanowire comprising the enzyme complex of any one of claims 2 to 4 or the enzyme complex prepared according to claim 5, wherein more than 50% of the fibrin of the enzyme complex is directly or The enzyme is indirectly attached, preferably 60% or 70% or 80% or 90% or more of fibrin is directly or indirectly linked to the enzyme.
The self-assembled catalytic nanowire of claim 6 wherein the fibrillar self-assembles to form a fibrous nanostructure.
The method for preparing a self-assembled catalytic nanowire according to claim 6 or 7, comprising the steps of:

Mixing a part of the enzyme complex or a mixture of the partial enzyme complex and the fibrin protein without the enzyme complex as a monomer, forming a nanowire, and breaking the nanowire into a nanowire fragment by ultrasonication to prepare a nanowire seed;

The prepared nanowire seed is mixed with another partial enzyme complex or a mixture of the partial enzyme complex and the fibrin protein without the enzyme complex, and seed-induced self-assembly is formed to form a self-assembled catalytic nanowire.

The enzyme complex described herein is the enzyme complex of any one of claims 2 to 4 or the enzyme complex of claim 5.
An enzyme complex according to any one of claims 2 to 4 or an enzyme complex prepared by the preparation method according to claim 5 or the self-assembled catalytic nanowire according to claim 6 or 7 or as Use of the self-assembled catalytic nanowire prepared by the preparation method of claim 8 as a catalyst.
Use of fibrin in the preparation of self-assembled catalytic nanowires for enhancing enzyme catalysis and/or enzyme stability, preferably, said self-assembled catalytic nanowires as claimed in claim 6 or 7 or as claimed 8 self-assembled catalytic nanowires prepared.
The use of fibrous nanostructures to enhance enzyme catalytic activity and/or enzyme stability.
A method of increasing enzyme activity and/or stability, comprising:

(1) directly or indirectly linking the enzyme to fibrin to obtain an enzyme complex;

(2) The enzyme complex obtained in (1) is self-assembled into a nanowire, thereby allowing the enzyme to be displayed on a self-assembled nanowire.