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A strain of Bacillus amyloliquefaciens efficiently secreting and expressing foreign proteins and its application

A technology for the secretion and expression of amyloliquefaciens, which is applied in the biological field and can solve the problems of low transformation rate of Bacillus amyloliquefaciens

Active Publication Date: 2021-04-09
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The conversion rate of wild-type Bacillus amyloliquefaciens is low, and due to the limitation of the modification system in the bacteria, at present, there are no reports on the screening of Bacillus amyloliquefaciens and the patent for its expression system modification

Method used

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  • A strain of Bacillus amyloliquefaciens efficiently secreting and expressing foreign proteins and its application
  • A strain of Bacillus amyloliquefaciens efficiently secreting and expressing foreign proteins and its application
  • A strain of Bacillus amyloliquefaciens efficiently secreting and expressing foreign proteins and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Screening and Identification of Neutral Protease High Yield Strain

[0030] 1. Primary screening of strains

[0031] Collect soil samples from Yishui County Food Industry Park, and grind the collected soil samples with a mortar; take 5g of ground samples and put them into a large triangular bottle filled with 95ml of sterile water to fully shake to obtain a soil suspension ; The soil suspension is gradiently diluted to obtain soil suspensions of different concentrations; the selected concentration is 10 -6 、10 -7 and 10 -8 Take 100 μl of the soil suspension and add it to the SX medium plate, spread it evenly, and culture it in a 37°C incubator for 1-3 days; select a large single colony with a transparent circle and streak it on the SX medium plate again, Carry out secondary confirmation and isolation and purification, culture at 37°C for 1-3 days, obtain a single colony after isolation and purification, take a single colony and inoculate it in a test tube c...

Embodiment 2

[0046] The mutagenesis of embodiment 2 bacterial strains and the screening of non-spore-producing and enzyme-activity-level-improved bacterial strains

[0047] 1. Preparation of Bacterial Suspension

[0048] Take the frozen strain LKT-10261, streak it on the solid plate of SX medium, culture at 37°C for 1-3 days, use an inoculation needle to take a single colony with a relatively large transparent circle on the plate and inoculate it into a medium containing 100mL of SX liquid medium In a conical flask, place in a shaker at 37°C, 200rpm, culture for 1.5 days, draw 40mL of the above seed solution into a centrifuge tube, centrifuge at 8000rpm for 5min, discard the supernatant, and wash the bacteria with 30mL of normal saline. Centrifuge twice under the same conditions, and resuspend the precipitated bacteria with 10 mL of sterile water to obtain a bacterial suspension, the concentration of which is 10 7 A / mL or so.

[0049] 2. Normal temperature and pressure plasma mutagenesis...

Embodiment 3

[0056] Example 3 Verification of whether the three mutagenized strains can carry out molecular genetic manipulation

[0057] Some scholars (Connaughton J F, et al. Gene Analysis Techniques, 1988, 5(6): 116-124.) proposed that the restriction modification system of Bacillus amyloliquefaciens is mainly the R.BamHI / M.BamHI system. The DNA of the kylated GGATCC fragment could hardly transform Bacillus amyloliquefaciens. And there is a BamHI site on the plasmid pKS1, see appendix figure 2 Therefore, if the restriction modification system of the strain is not destroyed by mutagenesis, the plasmid pKS1 will be degraded by the endogenous BamHI restriction endonuclease after transformation into Bacillus amyloliquefaciens, otherwise the transformant will grow on the resistance plate and Shows resistance to kana and erythromycin on the plasmid. Therefore, the three mutagenized strains ZD-10916, ZD-708 and ZD-6628 screened above were used to transform the plasmid pKS1 to verify whether...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a strain of Bacillus amyloliquefaciens capable of integrating and efficiently secreting and expressing foreign proteins and its application. The strain is specifically Bacillus amyloliquefaciens ZD‑708 (ΔGAΔEGL), which is based on a strain of Bacillus amyloliquefaciens LKT‑10261 that can efficiently produce neutral protease, and the ZD‑708 strain is obtained after ARTP mutagenesis. The ‑708 strain is obtained by knocking out the glucoamylase gene GA and the cellulase gene EGL, and its restriction modification system has failed to cut the pKS1 plasmid. The activity of the neutral protease obtained from the fermentation culture of the strain is 20200-21930U / ml, and the protein content in the supernatant is 95-100mg / mL. The expressed protease activity level is high, which greatly reduces the production cost and broadens the application of the enzyme field.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of Bacillus amyloliquefaciens capable of integrating and efficiently secreting and expressing foreign proteins and its application. Background technique: [0002] With the development of biotechnology, in order to reduce production costs, expand the conditions and scope of action, and meet the needs of diversified products in the market, it is necessary to develop or optimize multiple strains for different products, with high enzyme production activity, good production stability, and strong adaptability. production strains. Since the development of multiple new strains takes a long time, and the enzyme products of multiple varieties are often produced by the same type of strains, therefore, using the same type of expression system strains to produce multiple different enzyme products will make the production The speed of research and development is accelerated,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/54C12R1/07
CPCC12N9/2428C12N9/2437C12N9/54C12Y302/01004C12N1/205C12R2001/07
Inventor 刘文龙王兴吉郭庆文王克芬王金余钱娟娟佟新伟
Owner SHANDONG LONGKETE ENZYME PREPARATION
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