Recombinant collagen with good thermal stability

Abstract

The invention relates to recombinant collagen with good thermal stability, the amino acid sequence of the recombinant collagen is shown as SEQ ID NO1, and the recombinant collagen has the advantage of good thermal stability.

Description

Technical Field

[0001] The present invention relates to collagen, in particular to a recombinant collagen with good thermal stability. Background Art

[0002] Collagen is a biopolymer. As the primary component of animal connective tissue, it is also the most abundant and widely distributed functional protein in mammals. Collagen also has a wide range of applications in food, medicine, tissue engineering, and cosmetics. In the food industry, collagen can be used as a food additive to improve the taste and texture of food. In the pharmaceutical field, collagen is used to manufacture medical supplies such as artificial skin and artificial blood vessels. In tissue engineering, collagen is used as a scaffold material for cell culture. In cosmetics, collagen is widely used in skin care products due to its excellent moisturizing and film-forming properties.

[0003] Currently, animal tissues from livestock and poultry are the main way for people to obtain natural collagen and its collagen peptides. However, the key to determining the performance of collagen is the sequence of collagen. The content of proline and hydroxyproline in existing animal-derived collagen is low, but these amino acids can form pyrrolidine rings and hydrogen bonds, thereby affecting the thermal stability of collagen.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention discloses a recombinant collagen with good thermal stability.

[0006] The technical solutions adopted in the present invention are as follows: A recombinant collagen with good thermal stability, the amino acid sequence of the recombinant collagen is shown as SEQ ID NO 1.

[0007] Furthermore, the thermal instability temperature of the recombinant collagen is ≤128°C.

[0008] Furthermore, the recombinant collagen has an instability time of >10 min under the condition of an ambient temperature ≥120°C.

[0009] A method for testing the thermal stability of recombinant collagen with good thermal stability comprises the following steps: Step S1, performing SDS-PAGE on the sample before heating, first heating to 100°C and then heating to 121°C; Step S2, the sample is kept warm at 121°C; Step S3: The sample was cooled from 121° C. to 100° C., and then subjected to SDS-PAGE detection.

[0010] Furthermore, in step S1, the heating time is controlled to be 16 minutes.

[0011] Furthermore, in step S2, the holding time is controlled to 12 minutes. Furthermore, in step S3, the cooling time is controlled to be 15 minutes.

[0012] The beneficial effects of the present invention are as follows: 1. Optimizing the amino acid sequence of recombinant collagen and changing the content and distribution of glycine can enhance the formation of interchain hydrogen bonds, thereby improving the stability of the triple helix structure and improving the thermal stability of recombinant collagen.

[0013] 2. Recombinant collagen is produced through genetic engineering to achieve a high degree of uniformity in its amino acid sequence, thereby reducing the risk of degradation caused by sequence heterogeneity and improving the thermal stability of recombinant collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Flow chart of heat treatment of recombinant collagen.

[0015] Figure 2 Schematic diagram of SDS-PAGE detection of recombinant collagen. DETAILED DESCRIPTION

[0016] The specific embodiments of the present invention will be described below with reference to the accompanying drawings.

[0017] Example 1: A recombinant collagen with good thermal stability, whose amino acid sequence is: gppgpvgppgltgpagepgregspgadgppgrdgaagvkgdrgetgavgapgapgppgspgpagptgkqgdrgeagaqgpmgpsgpagargiqgpqgprgdkgqqgvagerghlgsrgfpgipgpsgppgtkglpgepgpqgpqgpigppgemgpkgppgavgepglpgeagmkgdlgplgtpgeqgligqrg epglegdsgpmgppgpmgltgrpgpvggpgssgakgesgdpgpqgprgvqgppgptgkpgkrgrpgadggrgmpgepgakgdrgfdglpglpgdkghrgergpqgppg ppgddgkpglpglkgergpaglpggpgakgeqgpaglpgkpgltgppgnmgpqgpkgipgshglpgpkgetgpagpagypgakgergspgsdgkpgypgkpgldgpk.

[0018] Example 2: Fermentation expression of a recombinant collagen with good thermal stability. (1) Cultivating expression chassis cells: Platform ID: Bio-52827.

[0019] Specifications: Glycerol / culture.

[0020] Genus name: Corynebacterium Glutamicum.

[0021] Strain name: Corynebacterium glutamicum.

[0022] Other numbers: ACCC16522=ATCC13032.

[0023] Culture medium number: 2.

[0024] Culture temperature: 30℃.

[0025] Cultivation time: 24-48 hours.

[0026] (2) Thermal stability test method of recombinant collagen: like Figure 1 As shown, the thermal stability test method includes the following steps: Step S1: Take the purified recombinant collagen sample and perform SDS-PAGE on the sample before heating. First, heat it to 100°C, then heat it and strictly control the temperature to 121°C for 16 minutes.

[0027] Step S2: Keep the sample at 121° C. for 12 minutes.

[0028] Step S3: Cooling from 121°C to 100°C for 15 min. Finally, SDS-PAGE analysis was performed on the same volume of sample.

[0029] Summarize the SDS-PAGE test results before and after heating the collagen and compare the remaining amount of the target band. The results are as follows: Figure 2 shown.

[0030] Comparative results analysis: Lane 1 on the left is the recombinant collagen sample before heating, and clear bands are visible.

[0031] Lane 2 on the right is the recombinant collagen sample after heating, in which clear bands are visible, and the color of the bands is lighter than that in lane 1.

[0032] This indicates that after high-temperature heating, the bands still remain, which indirectly proves that the recombinant collagen is not completely denatured by heat and has good thermal stability.

[0033] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0034] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A recombinant collagen with good thermal stability, characterized in that: The amino acid sequence of the recombinant collagen is shown in SEQ ID NO 1.

2. The recombinant collagen with good thermal stability according to claim 1, characterized in that: The thermal instability temperature of the recombinant collagen is ≤128°C.

3. The recombinant collagen with good thermal stability according to claim 2, characterized in that: The recombinant collagen has an instability time of more than 10 minutes under the condition of an ambient temperature of ≥120°C.

4. A method for testing the thermal stability of a recombinant collagen with good thermal stability according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step S1, performing SDS-PAGE on the sample before heating, first heating to 100°C and then heating to 121°C; Step S2, the sample is kept warm at 121°C; Step S3: The sample was cooled from 121° C. to 100° C., and then subjected to SDS-PAGE detection.

5. The method for testing the thermal stability of recombinant collagen with good thermal stability according to claim 4, characterized in that: In step S1, the heating time is controlled to be 16 minutes.

6. The method for testing the thermal stability of recombinant collagen with good thermal stability according to claim 4, characterized in that: In step S2, the holding time is controlled to be 12 minutes.

7. The method for testing the thermal stability of recombinant collagen with good thermal stability according to claim 4, characterized in that: In step S3, the cooling time is controlled to be 15 minutes.

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