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Polypeptide having serrapeptase activity and preparation method thereof

A serrapeptase and activity technology, applied in the field of polypeptides with serrapeptase activity, can solve the problems of limited market promotion and application, toxicity of serrapeptase, limited research materials, etc., and achieve great application prospects and commercial Value, the effect of shortening the strain culture period and reducing the production cost

Inactive Publication Date: 2019-02-01
NINGBO XINUOYA MARINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] But the production of serrapeptase mainly adopts to extract from the fermented liquid of natural bacterial strain Serratia E15 bacterial classification, and this technology is owing to Serratia latent research data is limited, and natural bacterial strain fermentation is unstable, and serrapeptase is for bacteria Due to the toxicity of the body itself, the process has not been able to achieve greater breakthroughs
Ultimately, serrapeptase is expensive to produce, and product marketing and applications are limited

Method used

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  • Polypeptide having serrapeptase activity and preparation method thereof
  • Polypeptide having serrapeptase activity and preparation method thereof
  • Polypeptide having serrapeptase activity and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] The target gene was obtained by PCR amplification by designing homologous primers. Wherein the forward primer is SerF: ccggaattc gccgcgacaacc (SEQ ID NO: 1), and the reverse primer is: tgctctaga ttacacgataaagtc (SEQ ID NO: 2). PCR amplification was performed using the genome extracted from Serratia marcescens CICC 23703 as a template. The PCR system and program follow the procedure below

[0022] PCR reaction system:

[0023]

[0024]

[0025] PCR reaction program:

[0026]

[0027] The DNA sequence of the polypeptide with serrapeptase activity in the target gene has been obtained by PCR amplification, as shown in SEQ ID NO: 3. According to the SEQ ID NO: 3, translation is carried out according to the central principle, and the polypeptide with serrapeptase activity can be obtained. The amino acid sequence of the active polypeptide is shown in SEQ ID NO:4.

[0028] Digest the target gene according to the restriction endonuclease sites involved in the primer...

Embodiment 2

[0030] After a single colony grows, pick a single colony and inoculate it into LB liquid medium, the culture condition is 37°C, and the rotation speed of the culture shaker is 150-250rpm / min. When the bacterium concentration grows to OD (measured at 600nm) between 0.5-1, add IPTG for induction, the final concentration of IPTG induction is 0.1-1mmol / L, and the induction time is 72h. 5 different Bacillus strains, each of which screened 5 highly active expression strains, the results are shown in Table 1 below:

[0031]

[0032]

[0033] It can be seen that the WB800 series bacillus has the highest activity, with an average higher than 2000SpU / g. Select the strain with the highest activity (WB6004, WB800N4, BS1682, CMCC02, SCK603) from 5 different strains, and perform SDS-PAGE electrophoresis. The results are shown in Figure 5 , SDS-PAGE showed that the molecular weight of the polypeptide with serrapeptase activity was about 50kD.

Embodiment 3

[0035] In each bacillus, the bacterial strain with the highest activity was screened out, and lactose (final concentration 1%), isopropylthiogalactopyranoside (final concentration 0.5mmol / L) or other lactose analogs (O-nitrogalactoside) glycoside (ONPG)) for inducible expression. The culture conditions and induction expression conditions are the same as in Example 2. Lactose (final concentration 1%) test results see figure 2 , see the experimental results of isopropylthiogalactoside (final concentration 0.5mmol / L) image 3 , O-nitrogalactoside (ONPG) experimental results see Figure 4 . After comparative analysis, it was found that after 72 hours of induction, the WB800N4 strain induced by IPTG had the highest activity.

[0036] It can be seen from the examples that the present invention can obtain a polypeptide with high activity of serrapeptase through genetic engineering, and the culture period is short, which is beneficial to the popularization of the production metho...

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Abstract

The invention provides a polypeptide having serrapeptase activity. The polypeptide has the gene sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4. A serrapeptase gene is cloned from serratia, the gene is cloned into a pHT43 vector by a genetic engineering technology to construct an expression vector, and a constructed expression plasmid is led into different expressionstrains through conversion; through culture and induced expression, the strain can express the polypeptide having high serrapeptase activity. The polypeptide having high serrapeptase activity is expressed in bacillus by the gene cloning technology. The strain culture cycle is shortened, the production cost is reduced, the product purity is higher, and the extraction process is simpler. The polypeptide has great application prospect and commercial value in the biomedical technical field.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a polypeptide having serrapeptase activity and a preparation method thereof. Background technique [0002] Serrapeptase is a protease synthesized by the enterobacteria called Serratia E15 (Serratia E15). This bacterium can be found in the gut of silkworms. There are many types of Serratia, but most of them except E15 are pathogenic. Serrapeptase has a strong effect of dissolving fibrin lumps, eliminating viscous purulent sputum, and purifying inflammatory lesions. It is widely used clinically for difficult sputum discharge caused by respiratory diseases. Serrapeptase has the functions of reducing phlegm, reducing inflammation, and eliminating edema or swelling. Serrapeptase has been used as an alternative medicine in many countries (such as Japan) and has a long history of application. Recent studies have also provided more evidence of its effectiveness in treating pain a...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/56C12N1/21C12N15/74C12R1/07
CPCC12N9/52C12N15/74
Inventor 田健陈豪曹阳诸辉
Owner NINGBO XINUOYA MARINE BIOTECH CO LTD
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