CN108004226A - Multienzyme complex and self assembly catalytic nanometer line - Google Patents
Multienzyme complex and self assembly catalytic nanometer line Download PDFInfo
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- CN108004226A CN108004226A CN201711099649.3A CN201711099649A CN108004226A CN 108004226 A CN108004226 A CN 108004226A CN 201711099649 A CN201711099649 A CN 201711099649A CN 108004226 A CN108004226 A CN 108004226A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
Abstract
The present invention provides application of the fibroblast cells in enhancing enzymatic activity and/or enzyme stability.The present invention by enzyme molecule high density, is illustrated in the surface of fibroblast cells nanostructured to array, forms the wire nanostructured with catalytic capability by the self assembly of fiber-like protein structure.The nanostructured simulates the natural areas state of desmoenzyme molecule, so that in the case where not being changed in itself to enzyme molecule, improves the catalytic activity and stability of enzyme.For example, the Michaelis constant K of the Sup35 MPH of nano wire statemThe 1/5 of the free MPH of deficiency, catalytic constant Kcat1 times is improved, maximum rate Vmax26.5 times are improved, Rate activity improves 4.8 times；Compared to resolvase ATA 117, within the same reaction time, the substrate conversion efficiency of the Sup35 ATA 117 of nano wire state can improve 30% or so, and the time for reaching maximum conversion shortens more than 4 times.
Description
Technical field
The present invention relates to enzyme catalyst, more particularly to a kind of multienzyme complex and self assembly catalytic nanometer line.
Background technology
Enzyme is widely used to the every field for producing, living as a kind of biocatalyst.In recent decades, with
Enzyme engineering constantly technical breakthrough, gets in the application of industry, agricultural, medical and health, energy development and environmental project etc.
Come more extensive.In the research and application process to enzymatic process, people always it is expected that the vigor of enzyme and stability get over Gao Yue
It is good, and possess good catalytic performance.How to lift the catalytic capability of zymoprotein is one of research hotspot of academia, while
The problem of being in the urgent need to address in industrial practice.The method of traditional various lifting zymoprotein catalytic capabilities mainly includes
Chemical modification, molecular enzyme engineering etc..
Chemical Modification of Enzyme is that applied chemistry method mainly includes 1) being repaiied in zymoprotein special site introducing specific molecular
The directed mutagenesis method of decorations；2) using bifunctional group reagent such as glutaraldehyde, PEG by between zymoprotein molecule, between subunit
Or intramolecular difference peptide chain part, carry out the crosslinking technological of covalent cross-linking；3) using micromolecular compound to zymophore or
The methods of micromolecular compound modification that side-chain radical outside active site is chemically modified, implements a variety ofization to enzyme molecule
Modification is learned, molecular modification is carried out to zymoprotein by the chemical modification of the cutting of main chain, montage and side-chain radical, to change its reason
Change property and bioactivity.Chemical Modification of Enzyme is mainly the introducing or removing by chemical group, and modification mostly occurs in enzyme
On active site or essential group, so zymoprotein covalent structure is changed, and cause zymoprotein activity with
The intensification of degree of modification and gradually weaken, i.e. Chemical Modification of Enzyme can not effectively improve the catalytic efficiency of enzyme itself, on the contrary can
Reduce enzyme activity.
Molecular enzyme engineering is the methods and techniques using genetic engineering and protein engineering, studies clone and the table of enzyme gene
Reach, the Structure and Function of zymoprotein, and based on it enzyme molecule is redesigned and oriented machining.Molecule enzyme work
Journey needs, from one or more existing parent enzymes, by the mutation and restructuring of gene, to build an artificial mutation
Enzyme storehouse, the evolution enzyme with some characteristics is finally obtained by certain screening or system of selection.Molecular enzyme engineering method is not only
Dependent on parent's enzyme molecule structure biology data it is perfect, it is also necessary to it is time-consuming and laborious to build artificial mutation enzyme storehouse, later stage
Screening process be even more complicated cumbersome.
From the point of view of comprehensive Chemical Modification of Enzyme and molecular enzyme engineering technology, the work of the two, which all concentrates on, processes single enzyme molecule,
Overall distribution situation of the enzyme molecule in living cells is not considered.More and more researchs show enzyme molecule in bacterial body
It is not dispersed as in solution, but existing for cluster, it is this to occur enzyme point in the form of supramolecular aggregation
Submanifold has the catalytic activity of higher.But often gone out by the enzyme molecule expressed by gene cloning out in the form of monodispersed
Existing, this is probably prepared enzyme molecule one of active the reason for reducing compared with native state enzyme molecule.
The content of the invention
The present invention be overcome the catalysis characteristics of enzyme existing in the prior art and stability improve it is limited, prepare it is complicated, into
This height and for specific enzyme need find be suitable for the reagent, material or method of the enzyme the shortcomings of, there is provided fibroblast cells
Application in enhancing enzymatic activity and/or enzyme stability.Fibroblast cells of the present invention are amyloidogenesis
Fibrils (amyloid fibroblast cells), abbreviation fibroblast cells.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme that：
One aspect of the present invention provides application of the fibroblast cells in enhancing enzymatic activity and/or enzyme stability.
Exemplarily, the fibroblast cells form multienzyme complex with the enzyme, and the enzyme is directly or indirectly connected to institute
State fibroblast cells.
Exemplarily, the enzyme is connected to the fibroblast cells by connecting peptide and/or adaptor protein.
In the embodiment of the invention, the enzyme is connected to the fibroblast cells by connecting peptide.
In the embodiment of the invention, the fibroblast cells form fiber-like nanostructures.
In the embodiment of the invention, the fibroblast cells and the multienzyme complex self assembly shape of enzyme formation
Into fiber-like nanostructures.
In the embodiment of the invention, the fiber-like nanostructures are self-assembly of by fibroblast cells.
Exemplarily, the fibroblast cells can form threadiness, and the albumen that can be connected with enzyme to be any.
Exemplarily, the fibroblast cells are Yeast prion protein or amyloid.It may be, for example, Yeast prion protein
Sup35, amyloid Ure2, fibroin albumen etc..
Exemplarily, the fibroblast cells are Yeast prion protein Sup35, it is preferable that fibroblast cells are yeast protein egg
The self-assembled structures domain that the 1-61 amino acids of white Sup35 are formed.
In the embodiment of the invention, the enzyme is selected from protease, nuclease or artificial enzyme.
In the embodiment of the invention, the enzyme is selected from oxidoreducing enzyme, transferase, hydrolase, lyase, different
Structure enzyme or synzyme.
Exemplarily, oxidoreducing enzyme such as glucose oxidase, catalase, hydrogenase, oxygenase etc..
Exemplarily, transferase such as methylferase, transaminase, hexokinase, phosphorylase etc..Specifically, such as Xi Talie
Spit of fland transaminase.
Exemplarily, hydrolase such as esterase, glycosyl enzyme, peptase, nuclease etc..Specifically, such as Methyl Parathion Hydrolase.
Exemplarily, lyase such as aldolase, hydrase, deaminase, carbonic anhydrase, pyruvate decarboxylase, alkaline phosphatase
Enzyme etc..
Exemplarily, isomerase such as racemase, epimerase, mutase etc..
Exemplarily, synzyme such as aminoacyl tRNA synthetase, glutamine synthelase, trehalose synthetase etc..
In the embodiment of the invention, the enzyme is included with carbohydrate, protein, fat, nucleic acid, biology
Alkali is the enzyme of substrate.
Exemplarily, the enzyme such as amylase using carbohydrate as substrate, glucose isomerase, lactase, renin are fine
The plain enzyme of dimension, dextranase, lactase etc.；Enzyme such as bromelain, pepsin, trypsase etc. using protein as substrate；With
Fat is enzyme such as lipase, esterase etc. of substrate；Using nucleic acid, alkaloid as enzyme such as alkaline phosphatase of substrate etc..
Another aspect of the present invention provides the preparation method of above-mentioned multienzyme complex, it includes passing through molecular cloning, will be into fiber
Albumen merges with enzyme molecule or merges and to form fusion protein base fibroblast cells, connection peptide (or street corner albumen), enzyme molecule
Cause, and the antigen-4 fusion protein gene is expressed.
In the embodiment of the invention, the preparation method of above-mentioned multienzyme complex comprises the steps of：
By molecular cloning, fibroblast cells, connection peptide, enzyme molecule are merged and to form antigen-4 fusion protein gene, and this is melted
Hop protein gene is expressed, purified.
In the embodiment of the invention, the fibroblast cells are Yeast prion protein self-assembled structures domain Sup35,
The enzyme is Methyl Parathion Hydrolase MPH (or Xi Talieting transaminase ATA-117).The specific preparation method bag of multienzyme complex
Include：
By molecular cloning by the gene of Yeast prion protein self-assembled structures domain Sup35 and Methyl Parathion Hydrolase MPH
Mph (or gene ata-117 of Xi Talieting transaminases ATA-117) by connect peptide (such as：GGGGS) fusion connection is formed and melted
Hop protein gene Sup35-mph (or Sup35-ata-117)；
Antigen-4 fusion protein gene Sup35-mph (or Sup35-ata-117) is cloned into expression vector, expression vector is turned
Enter in expressive host and cultivate, induced expression fusion protein S up35-MPH (or Sup35-ATA-117) is simultaneously purified.
In a specific embodiment, expression, the purifying of antigen-4 fusion protein gene can use this area conventionally used for protein expression
The method of purifying, such as antigen-4 fusion protein gene is cloned into expression vector, expression vector is transferred in expressive host and is cultivated, is activated
Induction vindication albumen is added after to exponential phase, through crushing, obtaining fusion protein after purification.Wherein, the present invention carries expression
Body, the species of expressive host and classification are not construed as limiting, and carrier and host of this area conventionally used for genetic modification can be selected.Specifically
, expression vector can be pET-28, pET-32, pET-15 or pET-11 etc.；Expressive host may be selected from Escherichia coli, withered grass gemma
Bacillus, bacillus megaterium, corynebacteria, saccharomyces cerevisiae, Pichia pastoris or mammalian cell.
In a specific embodiment, adaptor protein can replace connection peptide, and it is that may be present can equally to reduce fusion protein
The influence of steric hindrance.
Another aspect of the invention provides a kind of self assembly catalytic nanometer line, it includes the multienzyme complex or above-mentioned in the present invention
Multienzyme complex made from preparation method, wherein, more than 50% fibroblast cells direct or indirect connection in the multienzyme complex
There is enzyme.
Exemplarily, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or
100% fibroblast cells direct or indirect connection has enzyme, and specific ratio can be determined according to needs of production and cost.
Exemplarily, fibroblast cells are connected to enzyme by connecting peptide and/or adaptor protein.
Exemplarily, the connection peptide is GGGGS.
In the embodiment of the invention, fibroblast cells are self-assembly of fiber nanostructured.
Further aspect of the present invention provides the preparation method of self assembly catalytic nanometer line, includes the following steps：
Mutually mixed by above-mentioned a part of multienzyme complex or by the part multienzyme complex fibroblast cells compound with not having enzyme
Close the mixture formed to be incubated as monomer, form nano wire, nano wire is broken for by nano wire fragment by ultrasound, preparation is received
Rice noodles seed；
By the nano wire seed prepared with another part multienzyme complex or the part multienzyme complex with there is no enzyme compound
Fibroblast cells mix to be formed mixture mixing be incubated, carry out Seed inducement be self-assembly of self assembly catalytic nanometer
Line.
In the specific embodiment of the present invention, the multienzyme complex is Sup35-MPH (or Sup35-ATA-
117), the preparation of self assembly catalytic nanometer line includes the following steps：
A part of Sup35-MPH (or Sup35-ATA-117) is placed in 4 DEG C as monomer to be incubated after a week, forms nanometer
Line, is broken for nano wire fragment by nano wire by ultrasound, prepares Sup35-MPH seeds (or Sup35-ATA-117 seeds)；
By the Sup35-MPH seeds prepared (or Sup35-ATA-117 seeds) and another part Sup35-MPH (or
Sup35-ATA-117) mix according to certain mol proportion example, be incubated in 4 DEG C of mixing, carry out Seed inducement and be self-assembly of self assembly
Catalytic nanometer line, 100% Sup35 direct or indirect connections have MPH (or ATA-117) in the nano wire.
In the specific embodiment of the present invention, the multienzyme complex is Sup35-MPH (or Sup35-ATA-
117), the preparation of self assembly catalytic nanometer line includes the following steps：
A part of Sup35-MPH (or Sup35-ATA-117) is placed in 4 DEG C as monomer to be incubated after a week, forms nanometer
Line, is broken for nano wire fragment by nano wire by ultrasound, prepares Sup35-MPH seeds (or Sup35-ATA-117 seeds).
By another part Sup35-MPH (or Sup35-ATA-117) and non-composite S up35-MPH (or Sup35-ATA-
117) Sup35 is mixed to form mix monomer according to certain mol proportion example.
By the Sup35-MPH seeds prepared (or Sup35-ATA-117 seeds) and above-mentioned mix monomer according to necessarily rubbing
You mix ratio, are incubated in 4 DEG C of mixing, carry out Seed inducement and are self-assembly of self assembly catalytic nanometer line.Can be by adjusting mixed
It is direct to close Sup35 in the nano wire prepared by the ratio control of Sup35-MPH (or Sup35-ATA-117) and Sup35 in monomer
Or the ratio of MPH (or ATA-117) is connected by connecting peptide.
In another specific embodiment of the present invention, the multienzyme complex is Sup35-MPH (or Sup35-ATA-
117), the preparation of self assembly catalytic nanometer line includes the following steps：
By a part of Sup35-MPH (or Sup35-ATA-117) and non-composite S up35-MPH (or Sup35-ATA-117)
Sup35 according to the mixture that certain mol proportion example is mixed to form as monomer be placed in 4 DEG C be incubated after a week, formed nano wire,
Nano wire is broken for by nano wire fragment by ultrasound, prepares Sup35-MPH seeds (or Sup35-ATA-117 seeds).
By another part Sup35-MPH (or Sup35-ATA-117) and non-composite S up35-MPH (or Sup35-ATA-
117) Sup35 (here there is no any individually Sup35 protein moleculars, only exists Sup35-MPH and Sup35-ATA-117
The nano wire that protein molecular or molecule are formed) according to certain mol proportion example it is mixed to form mix monomer.
By the Sup35-MPH seeds prepared (or Sup35-ATA-117 seeds) and above-mentioned mix monomer according to necessarily rubbing
You mix ratio, are incubated in 4 DEG C of mixing, carry out Seed inducement and are self-assembly of self assembly catalytic nanometer line.Can be by adjusting kind
Sup35-MPH in the ratio of Sup35-MPH (or Sup35-ATA-117) and Sup35 and/or mix monomer in son (or Sup35-
ATA-117) Sup35 connects MPH (or ATA- directly or by peptide be connected in the nano wire prepared with the control of the ratio of Sup35
117) ratio.
The present invention uses Seed inducement self-assembling method, and seed, which can quickly induce fusion, melting for self-assembled structures domain
Hop protein is assembled in its end, by controlling the assembling ratio of seed and monomer (or mix monomer) fusion protein, it can be achieved that to receiving
The control of rice noodles length.
Further aspect of the present invention provides the application of above-mentioned multienzyme complex or self assembly catalytic nanometer line as catalyst.
Further aspect of the present invention provide fibroblast cells prepare be used to strengthening enzyme catalysis and/enzyme stability from group
Fill the application in catalytic nanometer line.
Further aspect of the present invention provides fiber-like nanostructures answering in strengthening enzymatic activity and/or there be not stability
With.
Further aspect of the present invention provides a kind of method for increasing enzymatic activity and/or stability, it includes：
(1) enzyme is directly or indirectly connected to fibroblast cells and obtains multienzyme complex；
(2) multienzyme complex obtained in (1) is self-assembled into nano wire, so that the enzyme is illustrated in self-assembled nanometer
On line.
One of exemplary, the present invention at least has the advantage that：
The self assembly of fibroblast cells such as Yeast prion protein Sup35 is determined domain and enzyme by the present invention by genetically manipulated
Gene be connected, enzyme molecule is incorporated in the C-terminal of Yeast prion protein Sup35 by gene expression, utilizes Yeast prion protein
The polymerization in Sup35 self-assembled structures domain forms nanostructured, and by substantial amounts of enzyme molecule high density, be illustrated in nanometer to array
The surface of structure, forms the wire nanostructured with catalytic capability.The nanostructured simulates the natural areas of enzyme molecule
State, so that in the case where not being changed in itself to enzyme molecule, improves the catalytic activity and/or stability of enzyme.For example, this
It is normal with the MPH being combined into fibroblast cells, its Michaelis compared to the free MPH freely disperseed in solution in inventive embodiments
Number KmThe 1/5 of insufficient resolvase, catalytic constant Kcat1 times is improved, maximum rate Vmax26.5 times are improved, Rate activity improves
4.8 times, considerably improve the affinity and catalytic capability of Methyl Parathion Hydrolase MPH and substrate；For Xi Talieting
Transaminase ATA-117, while conversion ratios of the raising Xi Talieting transaminases ATA-117 to substrate, shortens substrate conversion
The time of rate, its substrate conversion efficiency highest can improve 30% or so within the identical reaction time, reach highest substrate conversion efficiency
Time self assembly catalytic nanometer line Sup35-ATA-117 shortened compared to resolvase ATA-117 more than 4 times.
Brief description of the drawings
Fusion protein S up35-MPH (Sup35-ATA-117) expression and nano wire zymoprotein in Fig. 1 embodiment of the present invention
Self-assembled structures schematic diagram.
Sup35-MPH multienzyme complexes and Sup35-ATA-117 multienzyme complexes SDS-PAGE coagulate in Fig. 2 embodiment of the present invention
Gel electrophoresis detection figure.
The electron microscope for the Sup35-MPH nano wires being self-assembly of in Fig. 3 A. embodiment of the present invention.
The electron microscope for the Sup35-ATA-117 nano wires being self-assembly of in Fig. 3 B. embodiment of the present invention.
In Fig. 4 A. embodiment of the present invention in the Sup35-MPH reaction process of resolvase MPH and nano wire state, product
OD405Value changes with time condition diagram.
The zymoprotein Sup35-MPH dynamics of resolvase MPH and nano wire state compares knot in Fig. 4 B. embodiment of the present invention
Fruit is schemed.
The resolvase ATA-117 and zymoprotein Sup35-ATA-117 of nano wire state catalysis in Fig. 5 embodiment of the present invention
Ability comparative result figure.
Embodiment
Explanation：In the specific embodiment of the invention, embodiment abridge " MPH ", " ATA-117 ", " Sup35-MPH ",
" Sup35-ATA-117 " case-insensitive, can represent fusion, fusion protein or self-assembled nanowires, specific representative
Implication according to context understanding.
" Sup35-MPH " can represent Yeast prion protein Sup35 and MPH and be directly connected to, and can also represent Yeast prion protein
Sup35 is connected with MPH by being connected peptide or street corner albumen, and specific representative implication is according to context understanding.
" Sup35-ATA-117 " can represent Yeast prion protein Sup35 and ATA-117 and be directly connected to, and can also represent yeast
PrPC Sup35 is connected with ATA-117 by being connected peptide or adaptor protein, and specific representative implication is according to context understanding.
Connection peptide in the specific embodiment of the invention, can be replaced with adaptor protein as needed.For example, leucine zipper
Deng.
With reference to specific embodiment, by taking Sup35-MPH/Sup35-ATA-117 as an example, multienzyme complex to the present invention,
Self assembly catalytic nanometer line and preparation method thereof is further elaborated.
The preparation of embodiment 1Sup35-MPH multienzyme complexes
1st, Sup35-MPH multienzyme complexes are cloned
By molecular cloning by Yeast prion protein self-assembled structures domain (the 1-61 amino acid of Sup35, abbreviation Sup35,
Particular sequence is referring to shown in SEQ ID NO.1) and Methyl Parathion Hydrolase (the 36-331 amino acid of MPH, particular sequence
Referring to shown in SEQ ID NO.2) formed and melted by flexible peptide linker (particular sequence is referring to shown in SEQ ID NO.3) fusion connection
Hop protein Sup35-MPH, then merges His labels in its N-terminal.It is control with single Methyl Parathion Hydrolase (MPH).
Comprise the following steps that：
1) using plasmid pET28a as template, PCR amplification is carried out using primer MPH-Primer-1, is obtained containing primer MPH-
The plasmid pET28a of Primer-1 sequences；
2) using MPH genetic fragments as template, PCR amplification is carried out using primer MPH-Primer-3, obtains N-terminal band His-
The MPH genetic fragments of tag；
3) with C-terminal band His existing for this laboratorytagPlasmid pET28a-Sup351-61–Linker–MPH-Histag(tool
Body can be found in document：Men Dong, multifunctional nano line and the ultrasensitive biological sensing based on PrPC self assembly, doctor, which graduates, to be discussed
Text) it is template, PCR amplification is carried out using MPH-Primer-2, obtains Sup35 of the N-terminal with His-tag1-61- MPH fragments；
4) by step 1) with 2) in obtained genetic fragment carry out homologous recombination, you can obtain expression vector pET28a-
His-MPH；
5) and step 1) with 3) in obtained genetic fragment do homologous recombination reaction, you can obtain expression vector pET28a-
Histag-Sup351-61-Linker-MPH。
2nd, the expression of Sup35-MPH multienzyme complexes, purifying
Respectively by expression vector pET28a-His-MPH and pET28a-Histag-Sup351-61- Linker-MPH is transformed into
E. coli expression strains BL21, kalamycin resistance tablet picking positive colony.By the positive colony E.coli BL21 of picking
Re-activation is to kalamycin resistance LB culture mediums, 37 DEG C, 200rpm shaken cultivations to exponential phase (OD values are about 0.5).
Added into culture work final concentration of 1mM IPTG and work in concentration be 50 μM (final concentration of Kan is 50 μ g/ml)
Kanamycins, 25 DEG C, when 120rpm shaken cultivations inducible protein expression 8 is small.Thalline is collected by centrifugation 5 minutes in 8000rpm, and ultrasound is broken
Broken thalline, 10000 × g are centrifuged 30 minutes and are removed cell fragment, take supernatant Ni affinitive layer purification target proteins, that is, purified
MPH and Sup35-MPH (as shown in Figure 1).MPH is free Methyl Parathion Hydrolase albumen, as control.
Those skilled in the art know, design parameter in the expression and purification of albumen in the above-mentioned embodiment of the present invention
(such as concentration, time, temperature etc.) numerical value is not intended to limit the invention, and those skilled in the art can make according to actual demand
Adjustment.
3rd, the controllable preparation of self assembly catalytic nanometer line Sup35-MPH
(1) preparation of Sup35-MPH seeds：A part of multienzyme complex Sup35-MPH is placed in 4 DEG C as monomeric protein to incubate
Educate after a week, by shearing force caused by ultrasound, long nano wire is interrupted and prepares seed as nano wire fragment, the seed
The fusion protein that fusion, which can quickly be induced, self-assembled structures domain is assembled in its end.
(2) preparation of Sup35-MPH nano wires：The Sup35-MPH seeds that will be prepared, with another part Sup35-
MPH multienzyme complexes are incubated 8 hours in 4 DEG C, bring it about the quick assembling of Seed inducement according to certain mixed in molar ratio
(as shown in Figure 1).Sup35-MPH seeds and the molar ratio of Sup35-MPH multienzyme complexes can be any ratio, be selected in the present embodiment
1:4 ratio.It is control with free Methyl Parathion Hydrolase albumen (MPH).
The detection of embodiment 2Sup35-MPH multienzyme complexes and nano wire
SDS-PAGE electrophoresis is carried out to MPH made from embodiment 1 and Sup35-MPH, coomassie is carried out to obtained gel
Light blue dyes, and the results are shown in Figure 2 for it.As shown in Figure 2, between 30KDa and 40KDa, 40KDa nearby has clear obvious bar
Band, it corresponds respectively to free Methyl Parathion Hydrolase albumen (MPH) and Sup35-MPH multienzyme complexes.Electrophoresis showed without
Obvious foreign protein band, illustrates that the Methyl Parathion Hydrolase albumen (MPH) of the preparation of embodiment 1 and Sup35-MPH enzymes are compound
The purity and expression quantity of thing are all very high.
Electronic Speculum detection, its result such as Fig. 3 A institutes are carried out to self assembly catalytic nanometer line Sup35-MPH prepared by embodiment 1
Show.From Fig. 3 A, the nano wire concentration and distribution of lengths being self-assembly of are uniform.
The preparation of embodiment 3Sup35-ATA-117 multienzyme complexes
1st, Sup35-ATA-117 multienzyme complexes are cloned
By molecular cloning by Yeast prion protein self-assembled structures domain (the 1-61 amino acid of Sup35, abbreviation Sup35,
Particular sequence is referring to shown in SEQ ID NO.1) (ATA-117, particular sequence is referring to SEQ ID NO.4 with Xi Talieting transaminases
It is shown) pass through flexible peptide linker (specific sequence is referring to shown in SEQ ID NO.3) fusion connection formation fusion protein S up35-
ATA-117, then merges His labels in its N-terminal.It is control with single Xi Talieting transaminases (ATA-117).Specific step
It is rapid as follows：
1) using plasmid pET28a as template, PCR amplification is carried out using primer ATA-Primer-1, is obtained containing primer ATA-
The pET28a carriers of Primer-1 sequences；
2) using plasmid PUC57-ATA-117 as template, PCR amplification is carried out using primer ATA-Primer-2, obtains N-terminal band
The His of His-tagtag- ATA-117 genetic fragments；
3) with plasmid pET28a-Histag-Sup351-61- Linker-MPH is template, using primer ATA-Primer-3 into
Row PCR amplification, obtains His of the N-terminal with His-tagtag-Sup351-61- Linker genetic fragments；
4) using PUC57-ATA-117 as template, PCR reactions is carried out using primer ATA-Primer-4, obtain Linker-
ATA-117 genetic fragments；
5) with the His obtained by step 3)tag-Sup351-61Linker-ATA-117 pieces obtained by-Linker fragments and step 4)
Section is template, carries out PCR amplification using primer ATA-Primer-5, amplification obtains Sup351-61- Linker-ATA-117 genes
Fragment；
6) by step 1) with 2) in obtained genetic fragment carry out homologous recombination, you can obtain expression vector pET28a-
Histag-ATA-117；
7) by step 1) with 5) in obtained genetic fragment carry out homologous recombination, you can obtain expression vector pET28a-
Histag-Sup351-61-ATA-117。
2nd, the expression of Sup35-ATA-117 multienzyme complexes, purifying
Respectively by expression vector pET28a-Histag-ATA-117、pET28a-Histag-Sup351-61- ATA-117 is converted
Enter E. coli expression strains BL21, kalamycin resistance tablet picking positive colony.By the positive colony E.coli of picking
BL21 re-activations are to kalamycin resistance LB culture mediums, 37 DEG C, (OD values are about 200rpm shaken cultivations to exponential phase
0.5).Added into culture work final concentration of 1mM IPTG and work in concentration be 50 μM kanamycins, 25 DEG C,
When 120rpm shaken cultivations inducible protein expression 8 is small.Thalline is collected by centrifugation 5 minutes in 8000rpm, ultrasonication thalline, 10000 ×
G centrifuge 30 minutes remove cell fragment, take supernatant Ni affinitive layer purification target proteins, that is, obtain purifying ATA-117 and
Sup35-ATA-117 (as shown in Figure 1).ATA-117 is free Xi Talieting apotransminases, as control.
Those skilled in the art know, design parameter in the expression and purification of albumen in the above-mentioned embodiment of the present invention
(such as concentration, time, temperature etc.) numerical value is not intended to limit the invention, and those skilled in the art can make according to actual demand
Adjustment.
3rd, the controllable preparation of self assembly catalytic nanometer line Sup35-ATA-117
(1) preparation of Sup35-ATA-117 seeds：Using a part of multienzyme complex Sup35-ATA-117 as monomeric protein
It is placed in 4 DEG C to be incubated after a week, by shearing force caused by ultrasound, long nano wire is interrupted and prepares kind as nano wire fragment
Son, the fusion protein which can quickly induce fusion and have self-assembled structures domain are assembled in its end.
(2) preparation of Sup35-ATA-117 nano wires：The Sup35-ATA-117 seeds that will be prepared, with another part
Sup35-ATA-117 multienzyme complexes are incubated 8 hours in 4 DEG C, bring it about Seed inducement according to certain mixed in molar ratio
Quick assembling (as shown in Figure 1).Sup35-ATA-117 seeds and the molar ratio of Sup35-ATA-117 multienzyme complexes can be any
Than selecting 1 in the present embodiment:6 ratio.It is control with free Xi Talieting apotransminases (ATA-117).
The detection of embodiment 4Sup35-ATA-117 multienzyme complexes and nano wire
SDS-PAGE electrophoresis is carried out to ATA-117 made from embodiment 3 and Sup35-ATA-117, to obtained gel into
Row coomassie brilliant blue staining, the results are shown in Figure 2 for it.As shown in Figure 2, there have near 40KDa, between 50KDa and 60KDa to be clear
Obvious band, it corresponds respectively to free Xi Talieting apotransminases (ATA-117) and Sup35-ATA-117 enzymes are compound
Thing.Electrophoresis showed without obvious foreign protein band, illustrate Xi Talieting apotransminases (ATA-117) prepared by embodiment 3 and
The purity and expression quantity of Sup35-ATA-117 multienzyme complexes are very high.
Electronic Speculum detection, its result such as Fig. 3 B are carried out to self assembly catalytic nanometer line Sup35-ATA-117 prepared by embodiment 3
It is shown.From Fig. 3 B, the distribution of the nano wire concentration and length that are self-assembly of is all very uniform.
Respectively with self assembly catalytic nanometer the line Sup35-MPH and Sup35-ATA- prepared by embodiment 1 and embodiment 3
117 carry out enzyme kinetics and enzyme stability analysis and research.
5 self assembly catalytic nanometer line Sup35-MPH enzyme kinetics of embodiment and stability study
Self assembly catalytic nanometer line Sup35-MPH (being denoted as SMPH) prepared by embodiment 1 can be catalyzed substrate such as methyl to sulphur
Phosphorus reacts, its product is yellow, and has light absorbs in wavelength for 405nm, and the generation of its product is detected using microplate reader
And the situation of change of product amount, to analyze the zymetology aerodynamic and stability change feelings of self assembly catalytic nanometer line Sup35-MPH
Condition, experimental result such as Fig. 4 A and Fig. 4 B.Using free MPH as control (being denoted as MPH).
As can be seen that the reaction rate of resolvase MPH is well below nano wire state enzyme Sup35 from Fig. 4 A1-61-MPH
That is SMPH, SMPH reach the equalization point of reaction within a very short time.
Fig. 4 B the picture left above is Michaelis constant test result figure, as can be seen that nano wire state enzyme from Fig. 4 B the picture left above
The Michaelis constant K of SMPHmReduce 5.4 times compared with resolvase MPH, i.e. nano wire state enzyme SMPH is more easy to and Binding Capacity, its with
The affinity of substrate is remarkably reinforced.
Fig. 4 B top right plots are catalytic constant test result figure, as can be seen that nano wire state enzyme from Fig. 4 B top right plots
The catalytic constant K of SMPHcat1 times, i.e., under concentration of substrate saturation state is improved compared with resolvase MPH, nano wire state enzyme SMPH is urged
The speed for changing same reaction is significantly larger than resolvase MPH.
Fig. 4 B lower-lefts figure is maximum catalytic rate test result figure, from Fig. 4 B lower-lefts it can be seen from the figure that, in the same terms
Under, the maximum rate V of nano wire state enzyme SMPHmax26.5 times are improved compared with resolvase MPH.
Fig. 4 B bottom-right graphs are enzymatic activity test result figure, as can be seen that nano wire state enzyme SMPH from Fig. 4 B bottom-right graphs
Rate activity (Enzyme specific activity) improve 4.8 times, i.e. every milligram of nano wire state enzyme compared with resolvase MPH
Contained enzyme activity unit number is significantly larger than the enzyme activity unit number contained by every milligram of resolvase MPH in SMPH albumen.
In conclusion self assembly catalytic nanometer line Sup35-MPH is equal compared to its catalysis characteristics of free MPH and stability
Significantly improve.
6 self assembly catalytic nanometer line Sup35-MPH enzyme kinetics of embodiment and stability study
Self assembly catalytic nanometer line Sup35-ATA-117 prepared by embodiment 3 can be catalyzed 4 (Xi Ta of Xi Talieting intermediates
Row spit of fland precursor ketone) change into Xi Talieting.Using the generation of HPLC separation detections Xi Talieting and the situation of change of product amount, with
Analyze the catalytic capability of self assembly catalytic nanometer line Sup35-ATA-117, experimental result such as Fig. 5.Using free ATA-117 enzymes as pair
According to.
From figure 5 it can be seen that the substrate conversion efficiency of self assembly catalytic nanometer line Sup35-ATA-117 is apparently higher than free
The substrate conversion efficiency of enzyme ATA-117.Its substrate conversion efficiency highest can improve 30% or so within the identical reaction time, reach most
The time self assembly catalytic nanometer line Sup35-ATA-117 of high substrate conversion efficiency shortens 4 times compared to resolvase ATA-117
It is more.
In conclusion fibroblast cells (such as Sup35) can improve enzyme molecule catalysis partnership can and stability.Into
Fibrin is assembled into protein nano structure using the characteristic of its own, by enzyme molecule high density, is illustrated in albumen to array
The surface of nanostructured, forms the nanostructured with catalytic capability.The nanostructured simulates the natural of desmoenzyme molecule
Compartmentalization state, so that in the case where not being changed in itself to enzyme molecule, improves the catalytic activity and stability of enzyme.
Method provided by the invention or application can as the directed evolution technologies (directed evolution) of enzyme molecule,
Abbreviation directed enzyme evolution.Enzyme molecule ensure that adaptability of the enzyme to any change of environment during natural evolution, but certainly
So evolve both without specific direction, also without specific target, it is spontaneous in the breeding of whole biology and survival processes
Carry out.The natural evolution of enzyme is primarily not the continuous improvement for the vigor and stability for showing as some enzyme molecule, but is to give birth to
The adaptability of thing entirety, the enhancing of ability of regulation and control, therefore, usually only requires enzyme in vivo to specific biological function
There is specificity.
The evolutionary process that directed evolution technologies can enable generation very long in nature is simulated in the lab, is made one
Class according to the wish of oneself and can need to transform enzyme molecule, or even design in nature original not existing brand-new enzyme point
Sub (brand-new protein).Compared with natural evolution, the orthogenesis process of enzyme molecule is entirely in people's lower progress in order to control, is made
Enzyme molecule towards it is intended that specific objective evolve.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Within god and principle, any modification for being made, equivalent substitution etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Wuhan Virology Institute,Chinan academy of Sciences
<120>Multienzyme complex and self assembly catalytic nanometer line
<130> 2017
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 61
<212> PRT
<213>Yeast prion protein (prion)
<400> 1
Met Ser Asp Ser Asn Gln Gly Asn Asn Gln Gln Asn Tyr Gln Gln Tyr
1 5 10 15
Ser Gln Asn Gly Asn Gln Gln Gln Gly Asn Asn Arg Tyr Gln Gly Tyr
20 25 30
Gln Ala Tyr Asn Ala Gln Ala Gln Pro Ala Gly Gly Tyr Tyr Gln Asn
35 40 45
Tyr Gln Gly Tyr Ser Gly Tyr Gln Gln Gly Gly Tyr Gln
50 55 60
<210> 2
<211> 296
<212> PRT
<213>Methyl Parathion Hydrolase (Pseudomonas sp.)
<400> 2
Ala Ala Pro Gln Val Arg Thr Ser Ala Pro Gly Tyr Tyr Arg Met Leu
1 5 10 15
Leu Gly Asp Phe Glu Ile Thr Ala Leu Ser Asp Gly Thr Val Ala Leu
20 25 30
Pro Val Asp Lys Arg Leu Asn Gln Pro Ala Pro Lys Thr Gln Ser Ala
35 40 45
Leu Ala Lys Ser Phe Gln Lys Ala Pro Leu Glu Thr Ser Val Thr Gly
50 55 60
Tyr Leu Val Asn Thr Gly Ser Lys Leu Val Leu Val Asp Thr Gly Ala
65 70 75 80
Ala Gly Leu Phe Gly Pro Thr Leu Gly Arg Leu Ala Ala Asn Leu Lys
85 90 95
Ala Ala Gly Tyr Gln Pro Glu Gln Val Asp Glu Ile Tyr Ile Thr His
100 105 110
Met His Pro Asp His Val Gly Gly Leu Met Val Gly Glu Gln Leu Ala
115 120 125
Phe Pro Asn Ala Val Val Arg Ala Asp Gln Lys Glu Ala Asp Phe Trp
130 135 140
Leu Ser Gln Thr Asn Leu Asp Lys Ala Pro Asp Asp Glu Ser Lys Gly
145 150 155 160
Phe Phe Lys Gly Ala Met Ala Ser Leu Asn Pro Tyr Val Lys Ala Gly
165 170 175
Lys Phe Lys Pro Phe Ser Gly Asn Thr Asp Leu Val Pro Gly Ile Lys
180 185 190
Ala Leu Ala Ser His Gly His Thr Pro Gly His Thr Thr Tyr Val Val
195 200 205
Glu Ser Gln Gly Gln Lys Leu Ala Leu Leu Gly Asp Leu Ile Leu Val
210 215 220
Ala Ala Val Gln Phe Asp Asp Pro Ser Val Thr Thr Gln Leu Asp Ser
225 230 235 240
Asp Ser Lys Ser Val Ala Val Glu Arg Lys Lys Ala Phe Ala Asp Ala
245 250 255
Ala Lys Gly Gly Tyr Leu Ile Ala Ala Ser His Leu Ser Phe Pro Gly
260 265 270
Ile Gly His Ile Arg Ala Glu Gly Lys Gly Tyr Arg Phe Val Pro Val
275 280 285
Asn Tyr Ser Val Val Asn Pro Lys
290 295
<210> 3
<211> 5
<212> PRT
<213>Artificial sequence (Artificial Sequence)
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Gly Gly Gly Gly Ser
1 5
<210> 4
<211> 330
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Met Ala Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Phe Tyr Asn Ser Asp
50 55 60
Ala Thr Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Gly Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Met Val Thr Val Thr Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Leu Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Leu Pro Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Pro Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu
260 265 270
Tyr Asp Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
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Pro Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330
<210> 6
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tgagatccgg ctgctaac 18
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<213>Artificial sequence (Artificial Sequence)
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gtgatgatga tgatgatggc tgctgcccat gg 32
<210> 7
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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catcatcatc atcacatgtc ggattcaaac caaggc 36
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<213>Artificial sequence (Artificial Sequence)
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<213>Artificial sequence (Artificial Sequence)
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<213>Artificial sequence (Artificial Sequence)
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<213>Artificial sequence (Artificial Sequence)
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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catcatcatc atcacatgtc ggattcaaac caaggc 36
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<213>Artificial sequence (Artificial Sequence)
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<213>Artificial sequence (Artificial Sequence)
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agcagccgga tctcactatt agtactgcac aggcgtaagc 40
<210> 19
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
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catcatcatc atcacatgtc ggattcaaac caaggc 36
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Claims (12)
1. application of the fibroblast cells in enhancing enzymatic activity and/or enzyme stability.
2. application as claimed in claim 1, the fibroblast cells and the enzyme form multienzyme complex, the enzyme directly or
Be connected to the fibroblast cells in succession, it is preferable that the enzyme by connect peptide and/or street corner albumen be connected to it is described into fiber egg
In vain.
What 3. application as claimed in claim 1 or 2, the fibroblast cells, or the fibroblast cells were formed with the enzyme
Multienzyme complex is self-assembly of fiber-like nanostructures, and the fibroblast cells are Yeast prion protein or amyloid, preferably
Ground, the fibroblast cells are Yeast prion protein Sup35；And optionally, the enzyme is selected from oxidoreducing enzyme, transferase, water
Solve enzyme, lyase, isomerase or synzyme.
4. application as claimed in claim 3, the enzyme are selected from glucose oxidase, amylase, pepsin, alkaline phosphatase
Enzyme, cellulase, dextranase, lactase, lipase, esterase, Methyl Parathion Hydrolase or Xi Talieting transaminases, preferably
Ground, the enzyme are Methyl Parathion Hydrolase or Xi Talieting transaminases.
5. in a kind of application as any one of claim 2-4, the preparation method of the multienzyme complex include or by
Following steps form：
By molecular cloning, by fibroblast cells merged with enzyme molecule or by fibroblast cells, connection peptide (or street corner albumen),
Enzyme molecule merges to form antigen-4 fusion protein gene, and the antigen-4 fusion protein gene is expressed.
6. self assembly catalytic nanometer line, including prepared by multienzyme complex any one of claim 2-4 or claim 5
Multienzyme complex, wherein, more than 50% fibroblast cells direct or indirect connection has enzyme in the multienzyme complex, it is preferable that
60% or 70% or 80% or 90% or more than 95% fibroblast cells direct or indirect connection has enzyme.
7. self assembly catalytic nanometer line as claimed in claim 6, the fibroblast cells are self-assembly of fibrous nano knot
Structure.
8. the preparation method of the self assembly catalytic nanometer line described in claim 6 or 7, includes the following steps：
Mix what is formed by a part of multienzyme complex or by the part multienzyme complex and the compound fibroblast cells of no enzyme
Mixture is incubated as monomer, forms nano wire, nano wire is broken for nano wire fragment by ultrasound, prepares nano wire kind
Son；
By the nano wire seed prepared and another part multienzyme complex or the part multienzyme complex with without enzyme it is compound into
Fibrin mixes the mixture to be formed mixing and is incubated, and carries out Seed inducement and is self-assembly of self assembly catalytic nanometer line,
The wherein described multienzyme complex is obtained by multienzyme complex or claim 5 any one of claim 2-4
Multienzyme complex.
9. made from a kind of multienzyme complex as any one of claim 2-4 or preparation method as claimed in claim 5
Multienzyme complex or self assembly catalytic nanometer line as claimed in claims 6 or 7 or preparation method as claimed in claim 8 are made
Application of the self assembly catalytic nanometer line as catalyst.
10. fibroblast cells are being prepared for strengthening in the self assembly catalytic nanometer line of enzyme catalysis and/or enzyme stability
Using, it is preferable that the self assembly catalytic nanometer line as claimed in claims 6 or 7 or as prepared by claim 8 from group
Fill catalytic nanometer line.
11. application of the fiber-like nanostructures in enhancing enzymatic activity and/or enzyme stability.
12. a kind of method for increasing enzymatic activity and/or stability, it includes：
(1) enzyme is directly or indirectly connected to fibroblast cells and obtains multienzyme complex；
(2) multienzyme complex obtained in (1) is self-assembled into nano wire, so that the enzyme is illustrated in self-assembled nanowires
On.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711099649.3A CN108004226A (en) | 2017-11-09 | 2017-11-09 | Multienzyme complex and self assembly catalytic nanometer line |
| PCT/CN2018/114833 WO2019091454A1 (en) | 2017-11-09 | 2018-11-09 | Enzyme complex and self-assembly catalytic nanowires |
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| CN201711099649.3A CN108004226A (en) | 2017-11-09 | 2017-11-09 | Multienzyme complex and self assembly catalytic nanometer line |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116217660A (en) * | 2022-10-31 | 2023-06-06 | 潍坊医学院 | Matrix metalloproteinase-2 responsive small molecule peptide and application thereof |
-
2017
- 2017-11-09 CN CN201711099649.3A patent/CN108004226A/en active Pending
-
2018
- 2018-11-09 WO PCT/CN2018/114833 patent/WO2019091454A1/en active Application Filing
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| Title |
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| LENG Y等: "Integration of a fluorescent molecular biosensor into self-assembled protein nanowires: a large sensitivity enhancement", 《ANGEW CHEM INT ED ENGL.》 * |
| MEN D等: "Seeding-induced self-assembling protein nanowires dramatically increase the sensitivity of immunoassays", 《NANO LETT.》 * |
| 冷艳: "甲基对硫磷水解酶高活筛选及荧光纳米分子生物传感器的研究", 《中国博士学问论文全文数据库（基础科学辑）》 * |
| 印文 等: "酿酒酵母Sup35 朊蛋白结构域的自组装机理及其应用的研究进展", 《生物工程学报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116217660A (en) * | 2022-10-31 | 2023-06-06 | 潍坊医学院 | Matrix metalloproteinase-2 responsive small molecule peptide and application thereof |
| CN116217660B (en) * | 2022-10-31 | 2024-01-23 | 潍坊医学院 | Matrix metalloproteinase-2 responsive small molecule peptide and application thereof |
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| WO2019091454A1 (en) | 2019-05-16 |
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