CN115717148A - Large-scale industrial preparation method of human III-type collagen oligopeptide - Google Patents

Abstract

The invention relates to a large-scale industrial preparation method of human III-type collagen oligopeptide, belonging to the technical field of microbial fermentation. The recombinant collagen has a nucleotide sequence shown as SEQ ID NO.1 and an amino acid sequence shown as SEQ ID NO.2, and the recombinant collagen is used for preparing oligopeptide powder, wherein the molecular weight characteristic distribution of the oligopeptide powder is as follows: oligopeptide with the content of less than 1000 daltons is more than 75%; the ratio of the polypeptide less than 2000 daltons is more than 85%; the ratio of the polypeptide less than 3000 dalton is more than 90%.

Description

Large-scale industrial preparation method of human III-type collagen oligopeptide

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a large-scale industrial preparation method of human type III collagen oligopeptide.

Background

Collagen (collagen) is an extracellular protein, is a fibrous protein twisted into a spiral shape by 3 peptide chains, accounts for more than 30 percent of total proteins of the whole body, has important functions of maintaining normal physiological functions of cells, tissues and organs and repairing damage, and is widely applied to the industries of medicines, health care products and cosmetics. In terms of molecular structure, collagen is rich in amino acids such as glycine (Gly), proline (Pro), hydroxyproline (HYP) and the like required by human bodies.

At present, the main source of collagen is extracted from animal skin tissues, then high-purity and active animal-derived collagen is produced by utilizing an enzymolysis technology, the collagen can be called as a first-generation collagen preparation technology, and along with the development of biotechnology, recombinant collagen is obtained by utilizing a DNA (deoxyribonucleic acid) recombinant technology through a microbial fermentation method, so that the virus hidden trouble existing in the traditional extraction method is solved, and meanwhile, compared with natural collagen, the hydrophilicity and biocompatibility of artificially designed recombinant collagen are obviously improved.

In patent CN 1371919a (a human-like collagen and its production method), the molecular weight of collagen expressed by a prokaryotic expression system is relatively large (357 amino acids in total length), the post-translational processing modification system of the prokaryotic expression system is imperfect, and the presence of expressed protein in the form of inclusion body due to the excessive molecular weight of the protein results in relatively great difficulty in downstream purification and relatively low bioactivity of the expressed product.

The patent CN 103102407A (gene recombinant human collagen) provides a method for expressing humanized gene recombinant human collagen (total length 474 amino acids) by using eukaryotic yeast genetic engineering bacteria, which has a specific affinity purification His tag and two identical segments of human III type collagen. This patent uses two sequences (linked by EFT) identical to partial peptide sequences of native collagen to construct recombinant proteins. Although the His tag can be used for purification by using Ni filler so as to obtain the target protein with higher purity, the His tag can influence the immunogenicity of the protein and the trace Ni ion residue can cause anaphylactic reaction of a human body, thereby influencing the application of the protein in second and third medical devices.

Disclosure of Invention

One of the purposes of the invention is to provide a recombinant collagen, and the nucleotide sequence of the recombinant collagen is shown as SEQ ID NO. 1.

Preferably, the amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 2.

Another object of the present invention is to provide a method for preparing the recombinant collagen, the method comprising the steps of:

(1) Transforming the plasmid containing the recombinant collagen SEQ ID NO.1 sequence into pichia host bacteria;

(2) Seed culture is carried out on the pichia pastoris obtained in the step (1);

(3) Then carrying out fermentation culture;

(4) Collecting fermentation liquor and concentrating to obtain concentrated solution.

Preferably, the plasmid in step (1) is pPIC9K plasmid.

More preferably, the pichia host strain in step (1) is GS115.

The invention also aims to provide a method for preparing oligopeptide powder by using the recombinant collagen, which comprises the following steps: firstly, carrying out enzymolysis on a concentrated solution, then inactivating enzymes at high temperature, then carrying out ultrafiltration concentration, and finally carrying out spray drying to obtain oligopeptide powder.

Preferably, the enzyme used for the enzymatic hydrolysis is subtilisin.

More preferably, the subtilisin is added in an amount of 0.05g/g collagen.

More preferably, the molecular weight characteristic distribution of the oligopeptide powder is: oligopeptide with the content of less than 1000 daltons is more than 75%; the ratio of the polypeptide less than 2000 daltons is more than 85%; the ratio of the polypeptide less than 3000 daltons is more than 90%.

Compared with the prior art, the invention has the following beneficial effects:

(1) The application designs a recombinant human collagen amino acid sequence with small molecular weight and stable structure.

(2) The invention adopts a pichia continuous flow fermentation technology to efficiently express the recombinant human collagen.

Drawings

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

The media used in the following examples are as follows:

YPG:1% Yeast Extract (Yeast Extract), 2% Peptone (Peptone), 2% Glucose) (Glucose).

YNB:3.4g of yeast basic nitrogen source culture medium (without ammonium sulfate) and 10g of ammonium sulfate are fully dissolved in 800m of deionized water together, and finally the volume is determined to 1000ml, filtered and sterilized, and stored at 4 ℃.

BMGY:2% tryptone, 1.34% YNB,1% glycerol (G), 1% yeast extract, 4X 10 ^-6 % biotin, 0.1M K at pH 6.0 ₂ HPO ₄ /KH2PO ₄ And (4) a buffer solution.

PMT1：CuSO4·5H2O 6.0g/L，KI 0.088g/L，MnSO4·H2O 3.0g/L，Na ₂ MoO ₄ ·2H ₂ O 0.2g/L，H ₃ BO ₃ 0.02g/L，CoCl ₂ ·6H ₂ O 0.5g/L，ZnCl ₂ 20.0g/L，FeSO ₄ ·7H ₂ O65.0 g/L, biotin 0.2g/L, concentrated H ₂ SO ₄ 5.0ml。

BSM basal salts medium: 85% H ₃ PO ₄ 26.7ml/L，CaSO ₄ ·2H ₂ O 0.93g/L，K ₂ SO4 18.2g/L MgSO ₄ ·2H ₂ O14.9 g/L, KOH 4.13g/L, glycerol 40g/L, PMT 1.0 ml/L.

Example 1

1. Design of amino acid sequence of human collagen

The invention is designed according to the amino acid sequence of natural human type III collagen, selects peptide segments consisting of 109 Gly-X-Y, and is optimized according to the following conditions: the 109 groups Gly-X-Y are all sequences in human type III collagen alpha 1 (GenBank: KAI 2526100.1) chain and the protein molecular weight is less than 30000 daltons; in the human-like collagen composed of Gly-X-Y tripeptide, glycine (Glycine, abbreviated as Gly or G) is more than 37.0% in amino acid composition, and Proline (Proline, abbreviated as Pro or P) is more than 24.0% in amino acid composition; the theoretical isoelectric point of the peptide section formed by continuous Gly-X-Y is between 6.5 and 7.0.

The DNA sequence SEQ ID NO.1 is as follows:

Ggtggtccaggtagcgacggtaagccaggtccaccaggtagccagggtgaaagcggtcgaccaggtccaccaggtccaagcggtccacgaggtcagccaggtgtcatrggtttcccaggtccaaagggtaacgacggtgcaccaggtaagaacggtgaacgaggtggtccaggtggtccaggtccacagggtccaccaggtaagaacggtgaaaccggtccacagggtccaccaggtccaaccggtccaggtggtgacaagggtgacaccggtccaccaggtccacagggtctccagggtctcccaggtaccggtggtccaccaggtgaaaacggtaagccaggtgaaccaggtccaaagggtgacgcaggtgcaccaggtgcaccaggtggtaagggtgacgcaggtgcaccaggtgaacgaggtccaccaggtctcgcaggtgcaccaggtctccgaggtggtaccggtccaccaggtccagaaggtggtaagggtgcagcaggtccaccaggtccaccaggtgcagcaggtaccccaggtctccagggtatrccaggtgaacgaggtggtctcggtagcccaggtccaaagggtgacaagggtgaaccaggtggtccaggtgcagacggtgtcccaggtaaggacggtccacgaggtccaaccggtccaatcggtccaccaggtccagcaggtcagccaggtgacaagggtgaaggtggtgcaccaggtctcccaggtatygcaggtccacgaggtagcccaggtgaacgaggtgaaaccggtccaccaggtccagcaggtttcccaggtgcaccaggtcagaacggtgaaccaggtggtaagggtgaacgaggtgcaccaggtgaaaagggtgaaggtggtccaccaggtgtcgcaggtccaccaggtggtagcggtccagcaggtccaccaggtccacagggtgtcaagggtgaacgaggtagcccaggtggtcca。

the amino acid sequence SEQ ID NO.2 is as follows:

GGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGTGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGP。

2. preparation of recombinant human III-type collagen

2.1 recombinant human type III collagen Gene

Designing and artificially synthesizing a target gene sequence hCOL (SEQ ID NO. 1) according to the amino acid sequence and pichia pastoris codon preference, connecting the target gene sequence to a plasmid vector pMD19-T simple, and obtaining the plasmid vector through sequencing and identification.

2.2 construction of recombinant human III-type collagen genetically engineered bacteria

Cloning a gene hCOL (the 5 'end is added with Xho I restriction endonuclease cutting site, the 3' end is added with EcoR I restriction endonuclease cutting site) into pPIC9K plasmid, then transferring the plasmid into escherichia coli for large-scale amplification and extracting pPIC9K-hCOL, utilizing restriction endonuclease Sal I to cut enzyme and linearize, then transforming pichia pastoris host strain GS115 (Invitrogen) through an electroporator, screening high-copy transformant through G418, and determining the expression condition through shaking flask fermentation. And (3) detecting the supernatant obtained by the shake flask fermentation through SDS-PAGE electrophoresis, and determining that a position with the theoretical molecular weight of 30.6kDa is provided with an obvious electrophoresis strip to obtain the recombinant human type III collagen genetically engineered bacterium.

3. Fermentation production of recombinant human type III collagen

3.1 seed culture

A fresh single colony on the YPG plate was picked up, inoculated into a 500mL Erlenmeyer flask containing 50mL of BMGY seed medium, and cultured at 30 ℃ at 220r/min until the OD was 6 to 8, and then 50mL of the seed medium was inoculated into a 5L fermentor (containing 1.8L of BSM basic salt medium).

3.2, fermenting and culturing the mixture,

the temperature of the growth stage of the glycerol is controlled at 30 ℃, the pH value is 5.0, and the Dissolved Oxygen (DO) is maintained to be more than 30 percent by adjusting the stirring speed to be between 200 and 800 r/min. When the glycerol in the basic culture medium is exhausted, 50% of glycerol is fed, and the DO is controlled to be 30% -40%, and the glycerol phase feeding time is about 6h. Then glycerol supplementation was stopped.

And (3) entering a methanol induction phase, adding 10g of methanol at one time, monitoring the residual concentration of the methanol in the fermentation liquor by an online methanol electrode to control the methanol feeding rate, and maintaining DO to be more than 30%. If the dissolved oxygen is too low in the later period, the adding speed of the methanol flow is controlled, and the dissolved oxygen is maintained preferentially. Controlling the pH value of the methanol induction stage to be 4.5-5.0 by self-acid production through ammonia water and yeast growth, inducing the temperature to be 20-25 ℃, inducing for 40-50 hours, putting the fermentation tank into a tank, and centrifuging to collect fermentation supernatant.

3.3 concentration by ultrafiltration

And (3) respectively passing the collected fermentation liquor through a 35KD ultrafiltration membrane and a nanofiltration membrane for desalination and concentration.

4. Collagen enzymolysis

Adding the collagen concentrated solution collected in the last step into an enzymolysis tank, adjusting the pH to 7.0-7.5, uniformly stirring, and heating to 55 ℃ in a water bath; adjusting the pH of the protein solution to 8.6 +/-0.2 by using 10mol/L NaOH solution, adding 0.05g/g of liquid subtilisin (Alcalase) to collagen, starting timing enzymolysis for 3.0h, titrating by using 2mol/L NaOH solution in the enzymolysis process, and maintaining the pH at 8.6 +/-0.2.

5. High temperature inactivation

After the enzymolysis reaction is finished, adjusting the pH to 4.0-4.5 by using 3mol/L HCl solution, heating to 90 ℃, maintaining for 30min, cooling to room temperature in water bath, centrifuging for 20min at 4000g and 25 ℃, sieving by a 1200-mesh sieve, and taking supernatant polypeptide solution.

6. Concentrating by ultrafiltration

Passing the supernatant polypeptide solution through a 1000Da ultrafiltration membrane, intercepting polypeptide solution below 1000 Dalton, concentrating the collected peptide solution through a nanofiltration membrane, removing small molecular salt substances, and concentrating the peptide solution to 30% -40%.

7. Spray drying

And (3) rapidly drying the desalted peptide liquid by a spray dryer: after high-pressure homogenization, the solid content of the human III-type collagen polypeptide solution reaches 30-40%, and spray drying can be carried out. Spray drying conditions: the inlet temperature is 125-135 deg.C, the temperature in the tower is 75-80 deg.C, and the exhaust outlet temperature is 75-80 deg.C.

8. Molecular weight detection

Taking 0.1g of collagen oligopeptide powder after spray drying for mass spectrum detection, wherein the detection method comprises the following steps: GB/T22492-2008 appendix A GPC/UV, the specific data are as follows:

the molecular weight distribution is shown in the following table:

TABLE 1

Molecular weight characteristic distribution of human type III collagen oligopeptide: oligopeptide with the content of less than 1000 daltons is more than 75%; the ratio of the polypeptide less than 2000 daltons is more than 85%; the ratio of the polypeptide less than 3000 daltons is more than 90%.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (9)

1. A recombinant collagen, characterized in that the nucleotide sequence of the recombinant collagen is shown as SEQ ID NO. 1.

2. The recombinant collagen according to claim 1, wherein the amino acid sequence of said recombinant collagen is represented by SEQ ID No. 2.

3. A method for preparing recombinant collagen according to claim 1 or 2, comprising the steps of:

(1) Transforming the plasmid containing the recombinant collagen SEQ ID NO.1 sequence into pichia host bacteria;

(2) Seed culture is carried out on the pichia pastoris obtained in the step (1);

(3) Then carrying out fermentation culture;

(4) Collecting fermentation liquor and concentrating to obtain concentrated solution.

4. The method for preparing recombinant collagen according to claim 3, wherein the plasmid in step (1) is pPIC9K plasmid.

5. The method for preparing recombinant collagen according to claim 4, wherein the host Pichia pastoris is GS115 in step (1).

6. A method for preparing oligopeptide powder from the recombinant collagen prepared in claim 3, wherein the method comprises the following steps: firstly, carrying out enzymolysis on a concentrated solution, then inactivating enzymes at high temperature, then carrying out ultrafiltration concentration, and finally carrying out spray drying to obtain oligopeptide powder.

7. The method according to claim 6, wherein the enzyme used for the enzymatic hydrolysis is subtilisin.

8. The method of claim 7, wherein the subtilisin is added in an amount of 0.05g/g collagen.

9. The method according to any one of claims 6 to 8, wherein the molecular weight characteristic distribution of the oligopeptide powder is: the oligopeptide content of less than 1000 daltons is more than 75%; the ratio of the polypeptide less than 2000 daltons is more than 85%; the ratio of the polypeptide less than 3000 daltons is more than 90%.

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