CN106978406B - Short peptide self-assembled hydrogel with esterase activity and application thereof - Google Patents

Short peptide self-assembled hydrogel with esterase activity and application thereof Download PDF

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CN106978406B
CN106978406B CN201710225453.8A CN201710225453A CN106978406B CN 106978406 B CN106978406 B CN 106978406B CN 201710225453 A CN201710225453 A CN 201710225453A CN 106978406 B CN106978406 B CN 106978406B
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刘燕飞
张玲
卫巍
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Affiliated Hospital of Zunyi Medical University
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Abstract

The invention relates to a short peptide self-assembly hydrogel with esterase activity, which can promote the self-assembly of short peptides through the action of ions in PBS buffer solution so as to form colorless and transparent hydrogel. The hydrogel can simulate natural enzyme to catalyze 2,4-dinitrophenyl ester and p-nitrophenyl ester with short-chain fatty acid. After the catalytic reaction is completed, the hydrogel can be washed and recovered by a buffer solution and reused. Compared with the traditional acid-base catalysis, the catalytic reaction has mild conditions, can be carried out at neutral pH, normal temperature and normal pressure, and is easy to recycle. The self-assembled short peptide is easy to synthesize, simple in component and structure, easy in substrate purification and capable of simulating the catalytic process of enzyme, and provides a novel research tool for researching the catalytic process of enzyme, enzyme and substrate identification and the like.

Description

Short peptide self-assembled hydrogel with esterase activity and application thereof
Technical Field
The invention belongs to the field of nano biomaterials, and particularly relates to novel short peptide self-assembled hydrogel with esterase activity and application of the short peptide self-assembled hydrogel in catalyzing 2,4-dinitrophenyl ester and p-nitrophenyl ester by simulated esterase.
Background
The self-assembly short peptide is a novel nano biological material, takes amino acid as a raw material, and can be self-assembled into nano-scale fibers and even form hydrogel in response to specific environmental changes. Since the 90 s of the 20 th century, the self-assembled short peptide system has been widely used in three-dimensional cell culture, drug release, and tissue engineering due to its excellent biological and physicochemical properties. However, the design of new self-assembled short peptides and the expansion of the application range of self-assembled short peptides are still the target of the technical workers.
2,4-dinitrophenyl ester (2, 4-dinitrophenyl acetate) and p-nitrophenyl ester (p-nitrophenylester) and hydrolysis products thereof, 2, 4-dinitrophenol, nitrophenol and organic acids, are important raw materials or intermediates widely used in the pesticide, medicine and dye industries. Currently, the hydrolysis of 2,4-dinitrophenyl ester and p-nitrophenyl ester is mainly catalyzed by acid and base, enzyme and the like. The acid-base catalysis method is mainly carried out under the conditions of strong acid or strong base and high-temperature heating. Whereas enzymatic hydrolysis is carried out under mild conditions at room temperature, but requires the presence of large amounts of enzyme. This method is extremely costly, since the enzyme is volatile and difficult to purify. Therefore, it is necessary to find a novel catalytic material with stable reaction structure and mild reaction conditions.
Disclosure of Invention
The invention aims to design a novel self-assembly short peptide by using a nano short peptide self-assembly technology, wherein the short peptide can form hydrogel through self-assembly and simulate esterase in the nature to effectively catalyze esters with short fatty acid chains such as 2,4-dinitrophenyl ester, p-nitrophenyl ester and the like to hydrolyze.
A self-assembled hydrogel of short peptide with esterase activity is prepared by connecting 1-3 histidines with C end of RADA16 directly through amido bond; two glycines are used as a spacer between histidine and RADA16 to ensure that histidine is not sterically affected by the RADA16 peptide chain; the N end of the short peptide is acetylated and protected, while the C end is not protected and is still free-COOH;
the steps of adopting the short peptide self-assembly hydrogel to catalyze ester hydrolysis are as follows: firstly, preparing self-assembly short peptide; then carrying out hydrogel preparation and enzymatic reaction; and finally, recovering the reacted hydrogel.
The working concentration of the self-assembly short peptide is 1% (10 mg/mL) to 2% (20 mg/mL).
The hydrogel formation was promoted using 2 volumes of 50 mM PBS buffer, pH7.4, with a 30 min buffer incubation time.
Suitable esters for the catalytic reaction are 2, 4-dinitrobenzyl, 2, 4-dinitrophenethyl, p-nitrophenylmethyl and p-nitrophenylethyl.
When the catalyst is used for catalyzing the hydrolysis of the p-nitrophenyl ester, the catalytic speed of the p-nitrophenyl methyl ester and the p-nitrophenyl ethyl ester with shorter fatty acid chains is higher than that of the p-nitrophenyl ester with longer fatty acid chains.
The buffer solution for catalytic reaction is PBS buffer solution, HEPES buffer solution and acetic acid buffer solution with the pH value of 4-10.
The hydrogel after the catalytic reaction is slowly washed by 50 mM PBS buffer solution with pH7.4, and the washed hydrogel can be reused for 3-5 times.
The self-assembled short peptide of the invention is composed of a catalytic histidine region, RADA16-
Figure 899700DEST_PATH_IMAGE001
A self-assembly region and a connecting region, the catalytic region is located at the carboxyl segment of the short peptide, and RADA16-
Figure 754524DEST_PATH_IMAGE001
The self-assembly short peptide sequences are three, the sequence difference is that the number of catalytic region amino acids is different, the amino acid sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in the sequence table, which are abbreviated as RADA16-H, RADA16-HH and RADA16-HHH, the molecular model diagram of the self-assembly short peptide is shown as FIG. 1, and the molecular weights are 1965.05 g/mol, 2102.19 g/mol and 2239.33 g/mol respectively.
Experiments show that the self-assembled short peptide can form β -folded conformation in neutral pH salt solution and self-assemble to form nano-fiber, finally form hydrogel, and RADA16-
Figure 534261DEST_PATH_IMAGE001
The self-assembly region sequence mutation short peptide RAGA16-HH (the contrast group short peptide, the amino acid sequence of which is shown in SEQ ID NO.4 in the sequence table) adopts random coil conformation, and can not form nano fiber and hydrogel.
Experiments show that RADA16-H, RADA16-HH and RADA16-HHH can effectively catalyze hydrolysis of 2,4-dinitrophenyl ester (DNPA) under mild conditions (normal temperature and pressure, pH 7.4), and the catalytic kinetic curves of the RADA16-H, RADA16-HH and the RADA16-HHH accord with Michaelis-Menten (Michaelis-Menten), which means that RADA16-H, RADA16-HH and RADA16-HHH can simulate the catalysis of substrates by esterase existing in nature, and K iscat/KuncatAround 100. The control short peptide, RAGA16-HH, did not have this catalytic property.
Experiments show that RADA16-H, RADA16-HH and RADA16-HHH can catalyze nitre under mild conditions (normal temperature and pressure, pH 7.4)Hydrolysis of phenyl ester. The catalytic kinetics curves of RADA16-H, RADA16-HH and RADA16-HHH vs pNPC2 and pNPC4 also conform to the Mie's equation, Kcat/KuncatBetween 180 and 390 at about 100. And p-nitroacetoacetate having a short fatty acid chain length (pNitrophenyl acetate, pNPA or pNPC 2) and p-nitrobenzoate ester(s) ((II)pNitrophenyl butyrate, pNPC 4) at a significantly higher catalytic rate than p-nitrophenylhexanoate (p-nitrophenylhexanoate) which is longer than the fatty acid chainpNitrophenyl caprate, pNPC 6), p-Nitrophenyl octanoate (p-Nitrophenyl octanoate)p-Nitrophenyl octanoate, pNPC 8), p-nitrophenyldecanoate: (p-Nitrophenyl decanoate)pNitrophenyl captate, pNPC 10), p-nitrophenyllaurate (p-Nitrophenyl laurate)p-Nitrophenyl laurate, pNPC 12), p-nitrophenylmyristate: (p-Nitrophenyl myristate)p-Nitrophenyl myrisitate, pNPC 14), this substrate specificity and esterases in nature (which can usually only recognize p-Nitrophenyl esters catalyzing short fatty acid chains with 2 or 3 carbon chains, such as α -chymotrypsin).
Experiments show that the hydrophobic effect between the hydrophobic surface of β -folded sheet formed by self-assembly short peptide fiber and the substrate plays an important role in the substrate recognition process of the short peptides RADA16-H, RADA16-HH and RADA 16-HHH.
Experiments show that the hydrogel formed by the RADA16-H, RADA16-HH and the RADA16-HHH can be repeatedly used, and the activity is not obviously reduced after three times of catalysis.
The invention has the following beneficial effects:
provides a novel self-assembly short peptide, increases the short peptide types of the self-assembly short peptide series, and expands the application range of the self-assembly short peptide.
Provides a novel catalytic material with stable structure, repeated use and mild reaction conditions (normal temperature and pressure and neutral pH), and has obvious social and economic effects in bioengineering, pesticide, medicine and dye industries.
The self-assembled short peptide is easy to synthesize, simple in component and structure, easy in substrate purification and capable of simulating the catalytic process of enzyme, and provides a novel research tool for researching the catalytic process of enzyme, enzyme and substrate identification and the like.
Drawings
FIG. 1 is a High Performance Liquid Chromatography (HPLC) chart of purified self-assembled short peptide RADA16-H prepared by the method described in example 1.
FIG. 2 is a High Performance Liquid Chromatography (HPLC) chart of the purified self-assembled short peptide RADA16-HH prepared in example 2.
FIG. 3 is a High Performance Liquid Chromatography (HPLC) chart of purified self-assembled short peptide RADA16-HHH prepared by the method described in example 3.
FIG. 4 is a High Performance Liquid Chromatography (HPLC) chart of the purified control short peptide RAGA16-HH prepared in accordance with the method described in example 4.
FIG. 5 is a mass spectrum of the self-assembled short peptide RADA16-H prepared by the method described in example 1.
FIG. 6 is a mass spectrum of the self-assembled short peptide RADA16-HH prepared according to the method described in example 2.
FIG. 7 is a mass spectrum of the self-assembled short peptide RADA16-HHH prepared by the method described in example 3.
FIG. 8 is a three-dimensional molecular model of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH of the present invention.
FIG. 9 is a schematic diagram of the catalytic reaction of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH of the present invention.
FIG. 10 is a Circular Dichroism (CD) plot of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH and the control short peptide RAGA16-HH according to the present invention.
FIG. 11 is an Atomic Force Microscope (AFM) image of the self-assembling short peptides RADA16-H (FIG. a), RADA16-HH (FIG. b) and RADA16-HHH (FIG. c) of the present invention and the control short peptide RAGA16-HH (FIG. d).
FIG. 12 is a rheological diagram of a hydrogel formed by the self-assembled short peptide RADA16-H of the present invention exposed to phosphate buffers of different ion concentrations.
FIG. 13 is an HPLC chromatogram of 2, 4-dinitrophenol formed from the catalysis of 2, 4-dinitrophenylacetic acid (DNPA) by a hydrogel formed from the self-assembled short peptide RADA 16-H. FIG. a shows the reaction time before, and FIG. b shows the reaction time after 2 hours.
FIG. 14 is a kinetic plot of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH catalyzed DNPA.
FIG. 15 is a plot of control short peptide RAGA16-HH and imidazolyl-catalyzed DNPA.
FIG. 16 is a kinetic profile of self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH catalyzed p-nitrophenylacetate (pNPA).
FIG. 17 is a graph of control short peptides RAGA16-HH and imidazolyl-catalyzed pNPA.
FIG. 18 is a graph comparing the maximal catalytic rates of self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH versus p-nitrophenylate.
Detailed Description
Example 1: synthesis of self-assembled short peptide RADA16-H
1. Material
Fmoc amino acids were purchased from Sigma Aldrich, USA; PyBOP, Boc-His-Merrifield resin, piperidine, lutidine were purchased from Merck, USA; 1-Hydroxybenzotriazole (HOBT) was purchased from Sichuan Shengxin biopharmaceutical; dichloromethane (DCM) was purchased from shanghai biochemical reagents, china medicine (group); n-methylmorpholine (NMM), Dimethylamide (DMF) and trifluoroacetic acid (TFA) were purchased from Doudao-Hei biochemistry, Inc.
2. Preparation method
(1) Weighing 100 mg of Boc-His-Merrifield resin into a sand core filtration reactor, adding DCM, soaking and washing, adding 10% TFA5 ml, incubating at room temperature for 2 hours, adding DCM, soaking and washing for 3 times after the reaction is finished, neutralizing with 5% triethylamine 5 ml, washing for 5 times with DCM, and then putting into the reactor for subsequent reaction.
(2) 100 mg of His-Merrifield resin was weighed and put into a reactor of 431A polypeptide synthesizer, applied biosystems, USA, and PYBOP and HOBT were added to the reactor at the same time in the same molar amount. Fmoc-Gly-OH (89.2 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg). One reaction period was 40 minutes.
Adding the following components in each step of reaction simultaneously:
PyBOP：1560.9 mg
HOBT+H20 ：6000 mL，0.50 [mmoL/mL DMF]
NMM：324 mL，1 [mmoL/mL DMF]
(3) after the synthesis is finished, removing the Fmoc-protecting group, washing the resin with methanol for 4 times, performing suction filtration to dry, then performing vacuum drying for 12 hours, adding TFA to perform cleavage for 2 hours, performing suction filtration again, and collecting filtrate. Ice-cold diethyl ether was added to the filtrate to precipitate and precipitate the polypeptide. Centrifuging, collecting the precipitate, and vacuum drying to obtain crude polypeptide product. The amino acid sequence is shown as SEQ ID NO.1 in the sequence table.
Example 2: synthesis of self-assembled short peptide RADA16-HH
1. Material
Fmoc amino acids were purchased from Sigma Aldrich, USA; PyBOP, Boc-His-Merrifield resin, piperidine, lutidine were purchased from Merck, USA; 1-Hydroxybenzotriazole (HOBT) was purchased from Sichuan Shengxin biopharmaceutical; dichloromethane (DCM) was purchased from shanghai biochemical reagents, china medicine (group); n-methylmorpholine (NMM), Dimethylamide (DMF) and trifluoroacetic acid (TFA) were purchased from Doudao-Hei biochemistry, Inc.
2. Preparation method
(1) Weighing 100 mg of Boc-His-Merrifield resin into a sand core filtration reactor, adding DCM, soaking and washing, adding 10% TFA5 ml, incubating at room temperature for 2 hours, adding DCM, soaking and washing for 3 times after the reaction is finished, neutralizing with 5% triethylamine 5 ml, washing for 5 times with DCM, and then putting into the reactor for subsequent reaction.
(2) 100 mg of His-Merrifield resin was weighed and put into a reactor of 431A polypeptide synthesizer, applied biosystems, USA, and PYBOP and HOBT were added to the reactor at the same time in the same molar amount. Then Fmoc-His (Trt) -OH (185.93 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg) were added in this order after the reaction was completed, Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg). One reaction period was 40 minutes.
Adding the following components in each step of reaction simultaneously:
PyBOP：1560.9mg
HOBT+H20 ：6000 mL，0.50 [mmoL/mL DMF]
NMM：324 mL，1 [mmoL/mL DMF]
(3) after the synthesis is finished, removing the Fmoc-protecting group, washing the resin with methanol for 4 times, performing suction filtration to dry, then performing vacuum drying for 12 hours, adding TFA to perform cleavage for 2 hours, performing suction filtration again, and collecting filtrate. Ice-cold diethyl ether was added to the filtrate to precipitate and precipitate the polypeptide. Centrifuging, collecting the precipitate, and vacuum drying to obtain crude polypeptide product. The amino acid sequence is shown as SEQ ID NO.2 in the sequence table.
Example 3: synthesis of self-assembled short peptide RADA16-HHH
1. Material
Fmoc amino acids were purchased from Sigma Aldrich, USA; PyBOP, Boc-His-Merrifield resin, piperidine, lutidine were purchased from Merck, USA; 1-Hydroxybenzotriazole (HOBT) was purchased from Sichuan Shengxin biopharmaceutical; dichloromethane (DCM) was purchased from shanghai biochemical reagents, china medicine (group); n-methylmorpholine (NMM), Dimethylamide (DMF) and trifluoroacetic acid (TFA) were purchased from Doudao-Hei biochemistry, Inc.
2. Preparation method
(1) Weighing 100 mg of Boc-His-Merrifield resin into a sand core filtration reactor, adding DCM, soaking and washing, adding 10% TFA5 ml, incubating at room temperature for 2 hours, adding DCM, soaking and washing for 3 times after the reaction is finished, neutralizing with 5% triethylamine 5 ml, washing for 5 times with DCM, and then putting into the reactor for subsequent reaction.
(2) 100 mg of His-Merrifield resin was weighed and put into a reactor of 431A polypeptide synthesizer, applied biosystems, USA, and PYBOP and HOBT were added to the reactor at the same time in the same molar amount. Then Fmoc-His (Trt) -OH (185.93 mg), Fmoc-His (Trt) -OH (185.93 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-OH (59f) -OH (194.64 mg) were added in this order after the reaction was completed, Fmoc-Ala-OH (93.41 mg), Fmoc-Asp (OtBu) -OH (123.44 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg). One reaction period was 40 minutes.
Adding the following components in each step of reaction simultaneously:
PyBOP：1560.9mg
HOBT+H20 ：6000 mL，0.50 [mmoL/mL DMF]
NMM：324 mL，1 [mmoL/mL DMF]
(3) after the synthesis is finished, removing the Fmoc-protecting group, washing the resin with methanol for 4 times, performing suction filtration to dry, then performing vacuum drying for 12 hours, adding TFA to perform cleavage for 2 hours, performing suction filtration again, and collecting filtrate. Ice-cold diethyl ether was added to the filtrate to precipitate and precipitate the polypeptide. Centrifuging, collecting the precipitate, and vacuum drying to obtain crude polypeptide product. The amino acid sequence is shown as SEQ ID NO.3 in the sequence table.
Example 4: synthesis of control short peptide RAGA16-HH
1. Material
Fmoc amino acids were purchased from Sigma Aldrich, USA; PyBOP, Boc-His-Merrifield resin, piperidine, lutidine were purchased from Merck, USA; 1-Hydroxybenzotriazole (HOBT) was purchased from Sichuan Shengxin biopharmaceutical; dichloromethane (DCM) was purchased from shanghai biochemical reagents, china medicine (group); n-methylmorpholine (NMM), Dimethylamide (DMF) and trifluoroacetic acid (TFA) were purchased from Doudao-Hei biochemistry, Inc.
2. Preparation method
(1) Weighing 100 mg of Boc-His-Merrifield resin into a sand core filtration reactor, adding DCM, soaking and washing, adding 10% TFA5 ml, incubating at room temperature for 2 hours, adding DCM, soaking and washing for 3 times after the reaction is finished, neutralizing with 5% triethylamine 5 ml, washing for 5 times with DCM, and then putting into the reactor for subsequent reaction.
(2) 100 mg of His-Merrifield resin was weighed and put into a reactor of 431A polypeptide synthesizer, applied biosystems, USA, and PYBOP and HOBT were added to the reactor at the same time in the same molar amount. Then Fmoc-His (Trt) -OH (185.93 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (Pb 194.64 mg), Fmoc-Ala-OH (93.41 mg) were added in this order after the reaction was completed, Fmoc-Gly-OH (89.2 mg), Fmoc-Ala-OH (93.41 mg), Fmoc-Arg (Pbf) -OH (194.64 mg). One reaction period was 40 minutes.
Adding the following components in each step of reaction simultaneously:
PyBOP：1560.9mg
HOBT+H20 ：6000 mL，0.50 [mmoL/mL DMF]
NMM：324 mL，1 [mmoL/mL DMF]
(3) after the synthesis is finished, removing the Fmoc-protecting group, washing the resin with methanol for 4 times, performing suction filtration to dry, then performing vacuum drying for 12 hours, adding TFA to perform cleavage for 2 hours, performing suction filtration again, and collecting filtrate. Ice-cold diethyl ether was added to the filtrate to precipitate and precipitate the polypeptide. Centrifuging, collecting the precipitate, and vacuum drying to obtain crude polypeptide product. The amino acid sequence is shown as SEQ ID NO.4 in the sequence table.
Example 5: purification of self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH and control short peptide RAGA16-HH
The crude short peptides prepared in examples 1, 2, 3 and 4 were purified by High Performance Liquid Chromatography (HPLC) and tested, and the purities were 97.52%, 92.05%, 91.40% and 94.55%, respectively, with the results shown in fig. 1-4.
The short peptides RADA16-H, RADA16-HH and RADA16-HHH prepared in examples 1, 2 and 3 respectively are detected by mass spectrometry, the molecular weights of the short peptides RADA16-H, RADA16-HH and RADA16-HHH are respectively 1964.4, 2101.1 and 2238.7, and the molecular weights are matched with respective theoretical molecular weights (1965.02, 2102.16 and 2239.30), which shows that the synthesized short peptides are designed self-assembly short peptides, and the results are shown in FIGS. 5-7.
Example 6: three-dimensional molecular model drawing of self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH
A three-dimensional molecular model schematic diagram is drawn by Molsoft. ICM-pro software on the basis of the principle of energy minimization in aqueous solution for the self-assembled short peptides RADA16-H, RADA16-HH and RADA 16-HHH. The three-dimensional dimensions of the molecular model were approximately 7 nm long, 1.2 nm high, 04 nm wide, as shown in FIG. 8.
Example 7: circular Dichroism (CD) detection of self-assembled short peptide molecules
The CD spectra were collected using an AVIV400 spectrometer (Aviv Biomedical, Inc.) at 20 ℃ using a quartz cuvette with a 2 mm optical path. The wavelength ranges from 190 nm to 260 nm with a step size of 1 nm. All data areCorrected by subtracting the background value to average residue molar ellipticity [ theta ]]Is expressed in units of [ deg. cm2·dmol-1]. All short peptide samples were at a concentration of 0.075 mM.
The detection results are shown in FIG. 10, and the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH of the invention respectively show positive peaks and negative peaks at 193 nm and 216-218 nm, which indicates that the short peptides in aqueous solution show a typical β -folded secondary structure, while the control short peptide RAGA16-HH shows a random coil structure.
Example 8: atomic Force Microscope (AFM) detection of short peptide self-assembly micro morphology
All short peptide solutions were diluted to 0.1 mM before image scanning. 5L of the oligopeptide solution and the freshly peeled mica sheet were each taken, left to stand for 15 s, and then the mica surface was rinsed with 400L of Milli-Q deionized water, and after the sample had dried naturally, it was scanned in a tapping mode by an atomic force microscope (SPA 400, SII Nanotechnology, Inc.) at room temperature. The scan parameters were as follows: the vibration frequency is 124 kHz, the integral gain coefficient is 0.2-0.4, the proportional gain is 0.02-0.05, the scanning speed is 1 Hz, and the resolution is 512 multiplied by 512 pixels.
As shown in FIG. 11, the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH of the present invention can self-assemble to form fibers with a height of 1.2-1.5 nm and a length of several hundred nanometers to several micrometers, while the control short peptide RADA16-HH has no self-assembly ability and can only form particle aggregates with a diameter of 40-200 nm.
Example 9: rheological property detection of self-assembled short peptide RADA16-H hydrogel
Rheological properties of the self-assembled short peptide RADA16-H hydrogel were measured using a rotational rheometer (HAAKE RheostassI), and the lamina system was as follows: cone diameter 2 cm, angle 1 °, truncation 51 m. Before detection, 100L of a solution of the short peptide RADA16-H with a concentration of 5 mM was placed on a plate, 100L of phosphate buffers (pH 7.4) with a concentration of 10 mM and 50 mM, respectively, were added dropwise around the short peptide solution, excess solution was removed after 30 min, leaving only the hydrogel on the vertebral plate, and a time sweep was performed at a fixed shear stress of 1Pa and a fixed frequency of 6 rad/s for 30 min. The temperature was controlled at 25 ℃ by a circulating water bath (HAAKE phoenix II).
As shown in FIG. 12, when RADA16-H molecules were exposed to an ionic solution (e.g., PBS), the storage modulus G' was much greater than the dissipation modulus G ″, indicating that the material was capable of forming a hydrogel under the action of ions and that the gel strength was proportional to the ionic strength over a certain range of ionic strengths.
Example 10: catalysis of 2, 4-dinitrophenylacetic acid (2, 4-dinitrophenyl acetate, DNPA) by short-peptide RADA16-H hydrogel
A suitable amount of RADA16-H short peptide was weighed out and dissolved in sterilized ultrapure water to a concentration of 5 mM. Ultrasonically treating for 15min by an ultrasonic cleaner, and standing overnight at room temperature; 100L of the short peptide solution was transferred to the wells of a 24-well plate, 200L of PBS buffer (pH 7.4) was carefully added, and incubated at room temperature for at least 30 minutes to form a colorless transparent hydrogel. Carefully aspirate off the PBS buffer and wash with HEPES buffer (pH 7.4); after adding 500L of HEPES buffer (pH 7.4) containing 0.01 mmoL of 2, 4-dinitrophenylacetic acid (1% acetonitrile in the buffer) and reacting at 25 ℃ for 2 hours, the upper reaction solution was taken out and subjected to HPLC analysis, and as shown in FIG. 13, >99% of DNPA was converted into 2, 4-dinitrophenol and acetic acid. The RADA16-H short peptide hydrogel can be reused at least three times after being washed by buffer solution, and the catalytic activity of the RADA16-H short peptide hydrogel is not obviously reduced.
Example 11: catalytic kinetic study of self-assembling short peptides RADA16-H, RADA16-HH and RADA16-HHH on DNPA
(1) In the experiment, an ultraviolet spectrophotometer is adopted to detect the absorption of 400 nm to track the generation of the product 2, 4-dinitrophenol. The self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH and the control short peptides RADA16-HH and imidazolyl were diluted to 2.5X 10 with HEPES buffer (pH 7.4)-5mol/L, then adding substrate DNPA with different concentrations into the short peptide solution to ensure that the concentration of DNPA is 2.5 multiplied by 10 respectively-5mol/L、5×10-5mol/L、10-4mol/L、2×10-4mol/L、4×10-4mol/L 、8×10-4mol/L and 1.6X 10-3mol/L. All reactions were carried out at 25 ℃ and the reaction system contained 1% acetonitrile；
(2) Ultraviolet spectrophotometer data was collected at 400 nm wavelength and converted to the concentration of the product 2, 4-dinitrophenol according to Beer-Lambert's law. Wherein the apparent reaction rate (V)obs) It is necessary to subtract the background hydrolysis rate (V) in the absence of any catalystuncat) I.e. true reaction rate Vnet=Vobs-Vuncat. First order reaction constant KcatFrom Kcat= Vmax/[E]Calculated, E is the short peptide concentration. Second order reaction constant K2Calculated from the reaction rates of the short peptides RAGA16-HH and imidazolyl and the ratio of the concentrations of RAGA16-HH and imidazolyl. The results are shown in FIG. 14 and Table 1, and the catalytic kinetic curves V of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH versus DNPAnet/[S]The enzyme conforms to the Michaelis equation and shows that the Michaelis constant K is the enzymatic reactionmIs 7.90X 10-4-1.11×10-3mol/L, close to that of esterases in nature (e.g. α -chymotrypsin), KcatIs 4.67X 10-3s-1-1.03×10-2s-1，Kcat/KuncatTo achieve
Figure 116421DEST_PATH_IMAGE002
100, respectively; as shown in FIG. 15, the reaction rates for the control short peptide, RAGA16-HH and imidazolyl were proportional to the substrate concentration and did not conform to the Michaelis equation.
Figure 930793DEST_PATH_IMAGE003
Example 12: self-assembling short peptides RADA16-H, RADA16-HH and RADA16-HHH p-nitroacetoacetate (R) ((R))pCatalytic kinetics study of Nitrophenyl acetate, pNPA)
(1) In the experiment, an ultraviolet spectrophotometer is adopted to detect the absorption of 400 nm to track the generation of the product p-nitrophenol. The self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH and the control short peptides RADA16-HH and imidazolyl were diluted to 10X 10 with HEPES buffer (pH 7.4)-4mol/L, then adding different concentrations of substrate pNPA into the short peptide solution to ensure that the concentrations of the pNPA are 2 respectively.5×10-5mol/L、5×10-5mol/L、10-4mol/L、2×10-4mol/L、4×10-4mol/L 、8×10-4mol/L and 1.6X 10-3mol/L. All reactions were carried out at 25 ℃, with the reaction system containing 1% acetonitrile;
(2) ultraviolet spectrophotometer data were collected at 400 nm wavelength and converted to the concentration of the product p-nitrophenol according to Beer-Lambert law. Wherein the apparent reaction rate (V)obs) It is necessary to subtract the background hydrolysis rate (V) in the absence of any catalystuncat) I.e. true reaction rate Vnet=Vobs-Vuncat. First order reaction constant KcatFrom Kcat= Vmax/[E]Calculated, E is the short peptide concentration. Second order reaction constant K2Calculated from the reaction rates of the short peptides RAGA16-HH and imidazolyl and the ratio of the concentrations of RAGA16-HH and imidazolyl. The results are shown in FIG. 16 and Table 1, and the catalytic kinetic curves V of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH versus pNPAnet/[S]The enzyme conforms to the Michaelis equation and shows that the Michaelis constant K is the enzymatic reactionmIs 8.7 multiplied by 10-4-1.6×10-3mol/L，KcatIs 5.3X 10-4s-1-1.2×10-3s-1，Kcat/KuncatIs 180
Figure 690939DEST_PATH_IMAGE002
390; as shown in FIG. 17, the reaction rates for the control short peptide, RAGA16-HH and imidazolyl were proportional to the substrate concentration and did not conform to the Michaelis equation.
Figure 957972DEST_PATH_IMAGE005
Example 13: self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH catalytic rate study on p-nitrobenzoate
Using the method described in example 12, the reaction substrates were respectively replaced with p-nitrobenzoic butyrate (p-nitrobenzoic butyrate) having different lengths of fatty acid chainspNitrophenyl butyrate, pNPC 4), p-nitrophenylhexanoate ((II) and (III) in the presence of a catalystp-NitrophenylcaproatepNPC 6), p-nitrophenyl octanoate (p-nitrophenyl octanoate)p-Nitrophenyl octanoate, pNPC 8), p-nitrophenyldecanoate: (p-Nitrophenyl decanoate)pNitrophenyl captate, pNPC 10), p-nitrophenyllaurate (p-Nitrophenyl laurate)pNitrophenyl laurate, pNPC 12) and p-nitrophenylmyristate ((P-Nitrophenyl-L-myristate)p-Nitrophenyl myrisate, pNPC 14). The maximum reaction rates are shown in FIG. 18, which shows that the catalytic rates of the self-assembled short peptides RADA16-H, RADA16-HH and RADA16-HHH to p-nitrophenyl ester with shorter fatty acid chain are obviously higher than those of p-nitrophenyl ester with longer fatty acid chain, and the substrate specificity is similar to that of esterase in nature (which can only recognize p-nitrophenyl ester catalyzing short fatty acid chain with 2 or 3 carbon chains generally).
Zunyi medical college affiliated hospital
SEQ ID NO.1
[CH3CO]-Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala AspAla Gly Gly His-[COOH]
SEQ ID NO.2
[CH3CO]-Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala AspAla Gly Gly His His-[COOH]
SEQ ID NO.3
[CH3CO]-Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala AspAla Gly Gly His His His-[COOH]
SEQ ID NO.4
[CH3CO]-Arg Ala Gly Ala Arg Ala Gly Ala Arg Ala Gly AlaArg Ala GlyAla Gly Gly His His-[COOH]

Claims (2)

1. A self-assembled hydrogel of short peptides with esterase activity, which is characterized in that: the amino acid sequence of the short peptide self-assembly hydrogel with esterase activity is SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
2. Use of a self-assembled hydrogel of short peptide with esterase activity according to claim 1 in simulating esterase catalysis of 2,4-dinitrophenyl ester and p-nitrophenyl ester.
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