CN117820433B

CN117820433B - A sea cucumber-derived polypeptide with moisturizing function

Info

Publication number: CN117820433B
Application number: CN202311853771.0A
Authority: CN
Inventors: 吴晓军; 杨凯声
Original assignee: Xi'an Huipu Biotechnology Co ltd
Current assignee: Xi'an Huipu Biotechnology Co ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2025-01-28
Anticipated expiration: 2043-12-29
Also published as: CN117820433A

Abstract

本发明涉及一种来源自海参的具有保湿功能的氨基酸序列，所述氨基酸序列为TIGLEVEPSDTIENGK，本发明还涉及一种来源自海参的具有保湿功能的多肽，所述多肽的氨基酸序列为(TIGLEVEPSDTIENGK)_n，n为1～5的正整数。本申请海参中提取出由重复的短氨基酸序列构成的具有高保湿性能的多肽，其亲水性使其具有优秀的保湿性，并且由于其来自于天然海参组分，在进一步制作保湿剂及含该保湿剂的化妆品等产品时不会对使用者的皮肤造成刺激或副作用。The present invention relates to an amino acid sequence with moisturizing function derived from sea cucumber, the amino acid sequence is TIGLEVEPSDTIENGK, and the present invention also relates to a polypeptide with moisturizing function derived from sea cucumber, the amino acid sequence of the polypeptide is (TIGLEVEPSDTIENGK) _n , n is a positive integer of 1 to 5. The present application extracts a polypeptide with high moisturizing performance composed of a repeated short amino acid sequence from sea cucumber, and its hydrophilicity makes it have excellent moisturizing property, and because it comes from natural sea cucumber components, it will not cause irritation or side effects to the user's skin when further preparing moisturizing agents and cosmetics containing the moisturizing agents.

Description

Sea cucumber-derived polypeptide with moisturizing function

Technical Field

The invention relates to the field of biology, and mainly relates to a polypeptide and application thereof in preparation of moisturizers or cosmetics.

Background

The ocean is a life source of everything on earth, more than 80% of living organisms in the ocean are biological resources, and the ecological environment of marine organisms and land organisms are greatly different. Marine organisms have extremely rich biodiversity and functional diversity, and due to the special living environment, unique genetic metabolism mechanisms and chemical defense mechanisms are formed in the evolution process of adapting to the environment, so that the marine organisms are the main sources of new structures or new active natural products at present, and the marine organisms attract the attention of a plurality of researchers due to the potential bioactivity.

Sea cucumber is a generic term for animals of the class Stichopus japonicus of the phylum Echinodermata. Sea cucumbers are cylindrical, but the thickness, shape and size vary greatly according to the variety. The common large-scale edible sea cucumbers are in a thicker cylindrical shape, wart feet are arranged on the back surfaces, and tubular feet are arranged on the ventral surfaces. Sea cucumber has flexible body wall, rich connective tissue and thickness varying with the kind. Because of its cucumber-like appearance, the english name is "sea cucumber". The sea cucumber has 900 kinds in the world and 140 kinds in China. Since ancient times, sea cucumbers are rare and excellent products for nourishing and strengthening body and good medicines for preventing and treating some diseases, and records are mostly in the medical literature of the past generation in China. Sea cucumber has sweet and warm taste, has the effects of tonifying kidney, replenishing essence, nourishing blood, moistening dryness and prolonging life, and can be compared with human body. Sea cucumber is one of the potential marine organisms that have beneficial effects on human health. Recent pharmacological studies show that sea cucumber peptide has various bioactive functions such as antioxidation, anti-inflammatory, antifatigue, anti-tumor, immunoregulation, blood pressure reduction and the like as a marine bioactive peptide. Protein is the main nutrition component of sea cucumber, and is difficult to digest and absorb because of containing more collagen, and previous researches report that the effective utilization rate of the sea cucumber protein is only about 20%. The sea cucumber polypeptide is an active peptide with special physiological functions, which is prepared from sea cucumber through enzymolysis, and is a small molecular peptide or polypeptide consisting of 2-12 amino acids. Such as neuropeptides, glycopeptides, and antimicrobial peptides. Sea cucumber polypeptide has effects of resisting aging, enhancing immunity, scavenging oxygen free radicals, improving erythrocyte SOD activity, and relieving fatigue. The polypeptide has excellent properties of solubility, low viscosity, strong thermal stability and the like, and is easy to be absorbed by human bodies.

Disclosure of Invention

Sea cucumber polypeptide has many physiological functions, but in the prior art, the research is mainly conducted on the oxidation resistance and the inflammation resistance of sea cucumber polypeptide, and the research on other functions is less. The application aims to provide an active polypeptide with a moisturizing function, which is derived from sea cucumbers.

In particular, the invention relates to the following:

1. An amino acid sequence with a moisturizing function, wherein the amino acid sequence is Seq id No.1 (TIGLEVEPSDTIENGK).

2. A polypeptide with a moisturizing function, wherein the polypeptide comprises an amino acid sequence (TIGLEVEPSDTIENGK) _n, and n is a positive integer of 1-5.

3. The polypeptide of claim 2, which has the amino acid sequence TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK (Seq ID No. 5).

4. The amino acid sequence according to claim 1 or the polypeptide according to any one of claims 2 to 4, which is of sea cucumber origin.

5. A nucleic acid sequence encoding the amino acid sequence of claim 1 or the polypeptide of any one of claims 2-4.

6. A method of producing a polypeptide, wherein the polypeptide is a polypeptide according to any one of claims 2 to 4.

7. The method of claim 6, wherein comprising:

The polypeptide is obtained by expression and purification of pichia pastoris, wherein the pichia pastoris carries an expression vector containing the nucleic acid sequence of claim 6.

8. The method of claim 7, wherein the pichia pastoris is pichia pastoris GS115, preferably the expression vector is pPIC9K.

9. Use of an amino acid sequence according to claim 1 or a polypeptide according to any one of claims 2 to 3 or a polypeptide prepared according to the method of any one of claims 6 to 8 in the preparation of a humectant.

10. Use of the amino acid sequence of claim 1 or the polypeptide of any one of claims 2 to 3 or the polypeptide prepared according to the method of any one of claims 6 to 8 in the preparation of a cosmetic.

11. A humectant comprising the amino acid sequence of claim 1 or the polypeptide of any one of claims 2 to 3 or the polypeptide prepared according to the method of any one of claims 6 to 8.

12. A cosmetic comprising an amino acid sequence according to claim 1 or a polypeptide according to any one of claims 2 to 3 or a polypeptide prepared according to the method of any one of claims 6 to 8.

Effects of the invention

The polypeptide with high moisturizing performance, which is formed by repeated short amino acid sequences, is extracted from the sea cucumber, the hydrophilicity of the polypeptide enables the polypeptide to have excellent moisturizing performance, and the polypeptide is derived from natural sea cucumber components, so that the polypeptide can not cause irritation or side effect on the skin of a user when the moisturizing agent and cosmetics containing the moisturizing agent are further prepared.

Drawings

Mass spectrum of sea cucumber polypeptide in example 1 shown in fig. 1.

FIG. 2 shows a mass spectrum of sea cucumber polypeptide obtained by mass spectrometry in example 1.

FIG. 3 shows the net charge of the sea cucumber polypeptide sequence with moisturizing function of example 2.

FIG. 4 shows the results of the measurement of the moisturizing ability of the sea cucumber polypeptide sequences having the moisturizing ability of example 3 and comparative example 1.

Detailed Description

The present application is described in further detail below in conjunction with the detailed description of the application, examples are given to provide a better understanding of the present application and to fully convey the scope of the application to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description proceeds with reference to the general principles of the description. The scope of the application is defined by the appended claims.

In the following amino acid sequences and polypeptide sequences, I, P, V, Q, E, S, T, W and like capital letters each represent an amino acid or an amino acid residue thereof, and the correspondence between each capital letter and an amino acid is shown in Table 1.

Table 1 amino acids and their corresponding capital letters

Chinese name	Letter abbreviations	Chinese name	Letter abbreviations
				Glycine (Gly)	G	Serine (serine)	S
Alanine (Ala)	A	Threonine (Thr)	T
				Valine (valine)	V	Cysteine (S)	C
Leucine (leucine)	L	Asparagine derivatives	N
				Isoleucine (Ile)	I	Glutamine	Q
Proline (proline)	P	Aspartic acid	D
				Phenylalanine (Phe)	F	Glutamic acid	E
Tryptophan	W	Lysine	K
				Methionine (methionine)	M	Arginine (Arg)	R
Tyrosine	Y	Histidine	H

The present application provides an amino acid sequence having a moisturizing function, and in particular, the amino acid sequence provided by the present application is rich in a large amount of free amino groups and carboxyl groups therethrough so as to have excellent hydrophilicity.

In a specific embodiment, the amino acid sequence is Seq ID No.1 (TIGLEVEPSDTIENGK).

The application also provides a polypeptide with a moisturizing function, wherein the polypeptide comprises an amino acid sequence (TIGLEVEPSDTIENGK) _n, n is a positive integer of 1-5, for example, 1,2,3,4,5.

When n=1, the amino acid sequence of the polypeptide is:

TIGLEVEPSDTIENGK(Seq ID No.1)。

when n=2, the amino acid sequence of the polypeptide is:

TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK(Seq ID No.2)。

when n=3

TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK(Seq ID No.3)。

When n=4

TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK(Seq ID No.4)。

When n=5

TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK(Seq ID No.5)。

In a specific embodiment, the amino acid sequence of the polypeptide is TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK (Seq ID No. 5).

In a specific embodiment, the amino acid sequence or polypeptide is of sea cucumber origin. In some embodiments, the amino acid sequence or polypeptide is obtained by hydrolyzing sea cucumber by enzymatic hydrolysis.

In an embodiment of the application, a nucleic acid sequence encoding said amino acid sequence or said polypeptide is also included.

In some embodiments, the amino acid sequence or polypeptide may be produced by chemical synthesis. In some embodiments, the polypeptide may be produced by biosynthesis. In some embodiments, the polypeptide may be extracted from food or obtained via enzymatic hydrolysis.

In a specific embodiment, the polypeptide is obtained by expression and purification of recombinant pichia pastoris, which contains the nucleic acid sequence described above.

Pichia pastoris is a single-cell fungus belonging to the family Saccharomyces, the Pichia pastoris is fermented under anaerobic conditions, the cell morphology of the Pichia pastoris is usually elliptic or spherical, the diameter of the Pichia pastoris is 2-4 microns, and candida is sometimes formed. The optimal growth temperature of the pichia pastoris is 28-30 ℃, but the pichia pastoris can grow at 37 ℃. The pichia pastoris has an AOX1 gene, the AOX1 gene is a methanol utilization gene of the pichia pastoris, the AOX1 gene promoter is one of the most stringent promoters of the most emphasized control mechanism at present, meanwhile, the constructed pichia pastoris can be stably integrated in a single copy or multiple copies at a specific site of a genome of the pichia pastoris, the strain of the pichia pastoris is easy to carry out high-density fermentation, the expression quantity of exogenous proteins is high, peroxisomes exist in the pichia pastoris, the expressed proteins are stored in the pichia pastoris, the degradation of protease can be avoided, and the toxic effect on cells is reduced.

In a specific embodiment, the pichia is pichia pastoris GS115.

The Pichia pastoris GS115 strain is a yeast commonly used in biotechnology and protein expression systems, has an AOX1 gene, can normally express the AOX1 gene to generate enzyme for converting methanol into formaldehyde, and belongs to normal yeast for utilizing methanol.

In a specific embodiment, the expression vector is pPIC9K.

The pPIC9K plasmid is a plasmid for use in an expression system for E.coli (ESCHERICHIA COLI), which is commonly used for expressing foreign genes in E.coli. The selection marker is included in pPIC9K, primarily to screen and maintain the presence of the plasmid in the pichia medium. A selectable marker is a gene or DNA fragment that allows only cells carrying a particular plasmid to grow and reproduce. pPIC9K typically contains multiple cloning sites that allow researchers to insert exogenous genes of interest into it. These sites are typically located around some restriction sites to facilitate insertion of the DNA fragment.

In a specific embodiment, the method for preparing the polypeptide comprises the steps of carrying out codon optimization on the amino acid sequence or the polypeptide to obtain a nucleic acid sequence for encoding the amino acid sequence or the polypeptide sequence, connecting the nucleic acid sequence with a vector pPIC9K to obtain a recombinant vector, introducing the recombinant vector into Pichia pastoris GS115 by electric shock to obtain recombinant Pichia pastoris, and carrying out induced fermentation on the recombinant Pichia pastoris to obtain the polypeptide.

In a specific embodiment, the induction culture is an induction culture with methanol.

In one embodiment, the fermentation medium used for the induction culture comprises BMGY medium, wherein the BMMY medium comprises 20g/L peptone, 10g/L yeast extract, 13.4g/L YNB, 10g/L glycerol, 0.1M KH2P04/K2HP04 buffer solution with pH=6.0, and the BMMY medium comprises 20g/L peptone, 10g/L yeast extract, 13.4g/LYNB, 10g/L methanol and 0.1 MPH=6.0 KH2P04/K2HP04 buffer solution.

In one embodiment, recombinant Pichia pastoris GS115/pPIC9K is activated and transferred to 30mL BMGY medium, cultured at 220rpm for 24h at 30℃and transferred to a sterile centrifuge tube, centrifuged at 5000r/min for 5min and washed twice with sterile water. The resulting strain was resuspended in 30mL of BMMY medium, and cultured at 220rpm for 4d at 28℃and induced by adding methanol once every 24 hours to a final concentration of 1% (v/v). Centrifuging at 8000r/min for 5min after induction, and collecting supernatant to obtain crude extract.

In one embodiment, the crude enzyme solution is further purified after it is obtained.

In one embodiment, the purification is performed using a Ni column.

Further, embodiments of the application also provide for the use of the amino acid sequence or the polypeptide in the preparation of a humectant.

In some embodiments also provided is the use of the amino acid sequence or the polypeptide in the preparation of a cosmetic.

The application also provides a humectant comprising an amino acid sequence as described above or a polypeptide as described above. In some embodiments, the moisturizing agent comprises an amino acid sequence as described above or a polypeptide having a moisturizing function as described in the former, two or more.

The application also provides a cosmetic comprising an amino acid sequence as defined above or a polypeptide as defined above.

The sea cucumber polypeptide with high moisturizing performance, which is formed by repeated short amino acid sequences, is extracted from sea cucumber, the hydrophilicity of the polypeptide enables the polypeptide to have excellent moisturizing performance, the moisturizing rate of the sea cucumber polypeptide is obviously higher than that of glycerol under the same condition, and the moisturizing rate of the sea cucumber polypeptide can reach 90.9% after the sea cucumber polypeptide is placed for 8 hours under the environment that the humidity of 22 ℃ is 40%. And because the natural sea cucumber component is derived from the natural sea cucumber component, the skin care product can not cause irritation or side effect to users when the skin care product and the cosmetic containing the skin care product are further prepared.

Examples

EXAMPLE 1 extraction of sea cucumber polypeptide

Fresh sea cucumber, removing mouth, viscera and inner wall membrane, cleaning, pulverizing, adding 6 times of ultrapure water, adjusting pH to 7.0, adding 1% neutral protease, performing enzymolysis at 27deg.C for 8 hr, heating hydrolysate at 100deg.C for 40 min, inactivating enzyme, cooling to room temperature to obtain sea cucumber enzymolysis solution, adding 95% ethanol to sea cucumber enzymolysis solution until ethanol volume fraction is 80%, standing for 12 hr, centrifuging with centrifuge at 10000rpm for 30min, recovering ethanol of supernatant, decolorizing and removing fishy smell to the mixture of sea cucumber polypeptide by anion resin to obtain sea cucumber polypeptide extract. Desalination treatment was performed at 4 ℃ using dialysis bags. And (3) placing the extracting solution in liquid nitrogen for quick freezing and drying to obtain the sea cucumber polypeptide freeze-dried powder.

The determination of the molecular weight range of sea cucumber polypeptide comprises dissolving the sea cucumber polypeptide lyophilized powder in 50% acetonitrile (0.1% TFA), centrifuging at 10000rpm with a centrifuge for 5 min. 10mg/mL of 10mg/mL CHCA was prepared prior to the experiment as a 70% acetonitrile (0.1% TFA) matrix solution. In the experiment, 1. Mu.L each of the sample and the matrix solution was mixed at a ratio of 1:1 as a Matrix Assisted Laser Desorption Ionization (MALDI) target, and a MALDI-TOF mass spectrum was obtained by an AXIMA-CFP plus (KRATOS Analytical, shimadzu Group Company) mass spectrometer equipped with a nitrogen laser (337.1 nm). In the positive ion linear mode, mass spectrum is obtained by transmitting more than 100 times on average under the acceleration voltage of 20 kV. External standard calibration with cytochrome C was performed at m/z66256.00 and 12328.00 with a mass measurement accuracy of about 50ppm.

Example 2 screening for polypeptide sequences with moisturizing Functions

The sea cucumber polypeptide freeze-dried powder prepared in the example 1 is taken and dissolved in 1% formic acid solution. Analysis was performed using an orbital ion trap mass spectrometer (Thermo, america). The flow rate was 0.300. Mu.l/min, the analytical column was ACCLAIM PEPMAP RSLC column (75. Mu.m ID,250mm length, C18) and mobile phase A was 0.1% formic acid-water, mobile phase B was 80% acetonitrile and 0.1% formic acid. Gradient elution conditions: 0-5min,3% B, 5-80min,22% B, 80-92min,35% B, 92-103min,90% B, 103-109min,90% B, 109-110min,3% B. The scanning range is 600-10000m/z, and the resolution is 60000.

Polypeptide sequences with moisturizing function are screened, namely, the freeze-dried powder of sea cucumber is subjected to Orbitrap-MS analysis, and corresponding ncbi sea cucumber protein databases are searched to obtain 327 peptide spectrums, 74 proteins, 24 proteomes and 197 peptides in total. 98.98% of the polypeptide has a theoretical molecular weight of 800Da to 2000Da. MS/MS spectra were searched using software byonic (version 3.2.0) according to the selected sea cucumber protein database described above. GO analysis was performed on 74 proteins (Gene Ontology (GO) analysis was performed by the GO tool provided by the UniProt community (http:// www.uniprot.org /). Wherein 64 IDs are expressed, 8 molecular functions are obtained, 11 biological processes are involved, the sea cucumber polypeptide sequences with moisturizing function are screened to be TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK (Seq ID No. 5) in 5 cell components.

The following example uses the polypeptide of sequence TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK (Seq ID No. 5) obtained in example 2 as a test sample.

EXAMPLE 3 determination of the moisturizing Properties of sea cucumber peptide

1G of the polypeptide obtained in example 2 (accurate to 0.0001 g) was weighed using a weighing bottle as a carrier, the mass of the weighing bottle and the mass of the sample were recorded, the sample was placed in an environment with a temperature of 22.+ -. 2 ℃ and a humidity of 40.+ -. 5%, the data were weighed and recorded every time a period, the moisture retention rate was calculated, and a change curve was drawn.

The formula for calculating the moisture retention rate is as follows:

Moisture retention (%) = (weight of sample after placement/weight of sample before placement) ×100%.

Comparative example 1

This comparative example differs from example 2 only in that a 5% aqueous glycerol solution was used.

Analysis of experimental results:

the statistics of the test results of 1h, 2h, 4h and 8h are shown in Table 1.

TABLE 1

Time/h	Example 1 moisture retention (%)	Comparative example 1 moisture retention (%)
			1	98.9%	98.4%
2	96.7%	96.1%
			4	93.6%	93.4%
8	90.9%	90.2%

Example 4 human safety test of sea cucumber polypeptide

Subjects meeting 40 criteria were screened by reference to cosmetic safety Specification (2015 edition) using the sequence TIGLEVEPSDTIENGK obtained in example 2

TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK

TIGLEVEPSDTIENGK (Seq ID No. 5). The polypeptide of (2) is used as an experimental sample to carry out human body patch experiments. The test results are shown in Table 2.

0 Pole	Mild itching response	Mild erythema
			38 People	1 Person	1 Person

The results showed that 38 out of 40 subjects were rated 0, meaning no response, 1 subject exhibited a mild itching response, and 1 subject had mild erythema.

According to the inspection results, glycerol has excellent moisturizing performance, and the moisturizing rates of the sea cucumber polypeptide sequences 1h, 2h, 4h and 8h obtained in the example 2 are respectively 98.9%, 96.7%, 93.6% and 90.9%, which are all superior to the 5% glycerol aqueous solution used in the comparative example 1, so that the moisturizing performance of the sea cucumber polypeptide sequences obtained in the example 2 is obviously superior to that of the 5% glycerol aqueous solution under the same conditions. The sea cucumber polypeptide sequence obtained in the example 2 is derived from natural sea cucumber components, and can be used for replacing glycerol components without causing irritation to skin of a user when further preparing cosmetics.

EXAMPLE 5 preparation of sea cucumber polypeptide by recombinant vector

The culture medium involved in this example is as follows:

The BMGY liquid culture medium comprises 20g/L peptone, 10g/L yeast extract, 13.4g/L YNB, 10g/L glycerol and 0.1M KH2PO4/K2HPO4 buffer solution with pH=6.0.

BMMY liquid culture medium comprises 20g/L peptone, 10g/L yeast extract, 13.4g/L YNB, 10g/L methanol, and 0.1M KH2PO4/K2HPO4 buffer solution with pH=6.0.

The YPD solid culture medium comprises 20g/L peptone, 10g/L yeast extract, 20g/L glucose and 20g/L agar powder.

BSM liquid medium comprises 85% H3PO426.7mL/L, caSO42H2O0.93g/L, K2SO418.2g/L, mgSO H2O 14.9g/L, KOH.13 g/L, glycerol 40g/L, PTM 1.0 mL/L.

LB liquid culture medium, which comprises 10g/L peptone, 5g/L yeast extract and 10g/LNaCl.

The operation of the electric shock method according to the present embodiment is as follows:

competent cells were mixed with linearized DNA and then transferred to an ice-chilled cuvette, which was tapped to ensure that the cells were sinking to the bottom of the cup.

(2) And electric shock is carried out on the electric shock cup.

(3) Immediately after shock, 1mL ice-cold sorbitol was added to the cup and transferred to a 1.5mL centrifuge tube.

(4) And placing the centrifuge tube at 28 ℃ for static culture for 1-2 h.

(5) Mu.L of the cell fluid was uniformly spread on the MD plate.

(6) Plates were incubated upside down at 28℃for 3-10 d until colonies appeared.

(7) The selection was performed on YPD medium containing G418 at a concentration of 0.5G/L, 2G/L, 4G/L, 6G/L, respectively.

5.1 Preparation of recombinant vectors

Gene synthesis nucleotide sequences such as

ACTATTGGTTTGGAAGTTGAACCATCTGATACTATTGAAAACGGTAAG

ACTATTGGTTTGGAAGTTGAACCATCTGATACTATTGAAAACGGTAAGCATCATCATCATCATCAT (Seq ID No. 6) and the vector pPIC9K are constructed by adopting a one-step cloning method to obtain a recombinant vector.

5.2 Preparation of recombinant Pichia pastoris

Converting the prepared recombinant plasmid into Pichia pastoris GS115 by adopting an electric shock method, and preparing the recombinant Pichia pastoris;

5.3 production of sea cucumber polypeptide by shake flask fermentation of recombinant strains

Culturing the prepared recombinant Pichia pastoris in YPD solid culture medium at 30 ℃ in an incubator for 3d to obtain single colony, inoculating the single colony into 30mL BMGY liquid culture medium, culturing at 220rpm for 24h at 30 ℃ to obtain fermentation broth, placing the fermentation broth in a sterilizing centrifuge tube, centrifuging at 5000r/min for 5min, taking precipitate, washing with sterilizing water twice to obtain thalli, suspending the thalli in 30mL BMMY liquid culture medium at 220rpm, culturing at 28 ℃ for 4d, adding methanol once every 24h for induction, and adding the methanol to the final concentration of 1% (v/v) each time. Centrifuging at 8000r/min for 5min after induction, and collecting supernatant to obtain crude enzyme solution.

The crude enzyme solution was subjected to SDS polyacrylamide gel electrophoresis.

Detection of sea cucumber peptide content:

The content of sea cucumber peptide is determined by high performance liquid chromatography, namely Waters, agilent C18 column, methanol with mobile phase of 10:90 and formic acid with 0.1%, and flow rate of 1mL/min.

According to the embodiment, the recombinant strain capable of heterologously expressing the sea cucumber peptide is obtained by construction, and after the sea cucumber peptide is produced by fermenting the recombinant strain, the single product of the sea cucumber peptide is rapidly and massively produced.

The above description is only of the preferred embodiments of the present application, and is not intended to limit the present application in any way. Any person skilled in the art may make variations or modifications to the equivalent embodiments using the teachings disclosed above. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A polypeptide having a moisturizing function, wherein the amino acid sequence of the polypeptide is TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK TIGLEVEPSDTIENGK (Seq ID No. 5).

2. The polypeptide of claim 1, which is of sea cucumber origin.

3. A nucleic acid sequence encoding the polypeptide of claim 1.

4. A method of producing a polypeptide, wherein the polypeptide is the polypeptide of claim 1.

5. The method of claim 4, wherein comprising:

the polypeptide is obtained by expression and purification of pichia pastoris, wherein the pichia pastoris carries an expression vector containing the nucleic acid sequence of claim 4.

6. The method of claim 5, wherein the pichia pastoris is pichia pastoris GS115, preferably the expression vector is pPIC9K.

7. Use of the polypeptide of claim 1 or the polypeptide prepared according to the method of any one of claims 4-6 in the preparation of a humectant.

8. Use of the polypeptide of claim 1 or the polypeptide prepared according to the method of any one of claims 4-6 in the preparation of a cosmetic.

9. A humectant comprising the polypeptide of claim 1 or a polypeptide prepared according to the method of any one of claims 4-6.

10. A cosmetic comprising the polypeptide of claim 1 or the polypeptide prepared according to the method of any one of claims 4-6.