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Signal peptide for optimizing high-level secretory expression of keratinase Ker and application of signal peptide

A technology for keratinase and secretion expression, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low extracellular expression of keratinase and is difficult to meet industrial needs, and achieves the effect of improving secretion efficiency and facilitating industrial application.

Active Publication Date: 2017-09-26
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the main problem in the industrial production and application of keratinase is that the extracellular expression of keratinase is low, which is difficult to meet the needs of industry

Method used

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  • Signal peptide for optimizing high-level secretory expression of keratinase Ker and application of signal peptide
  • Signal peptide for optimizing high-level secretory expression of keratinase Ker and application of signal peptide
  • Signal peptide for optimizing high-level secretory expression of keratinase Ker and application of signal peptide

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Experimental program
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Effect test

Embodiment 1

[0028] The preliminary screening of embodiment 1 signal peptide

[0029] (1) Synthesis of gene ker

[0030] According to the NCBI accession number JX504681 of keratinase Ker derived from Bacillus licheniformis BBE11-1, its gene sequence was searched and submitted to Shanghai Boyi Biotechnology Co., Ltd. for the whole gene synthesis of keratinase Ker.

[0031] (2) Construction of expression vector pSTOP1622-ker

[0032] Design PCR primers P1 and P2 (Table 1) according to the gene sequence of Ker, use the synthetic gene ker as a template, and use P1 and P2 as primers to perform PCR amplification to obtain the amplified product ker (signal peptide, propeptide and Nucleotide sequence of mature enzyme); PCR amplification conditions are: 98°C 5min; 98°C 20sec, 60°C 40sec, 74°C 2min, 30 cycles; 74°C, 10min; Restriction digestion, connection to vector pSTOP1622, construction of recombinant vector pSTOP1622-ker.

[0033] Table 1 Primers required for vector construction

[0034] ...

Embodiment 2

[0053] Optimization of embodiment 2 signal peptide

[0054] (1) Gene synthesis of signal peptide mutants

[0055] The preliminary screening results of signal peptides showed that signal peptides YfkN, Bpr, Mpr, PhoB, WapA, AbnA can effectively improve the secretion efficiency of keratinase Ker. According to the structural characteristics of the signal peptides, the amino acid sequences and nucleotide sequences of the above six signal peptides were partitioned, that is, N-terminal, H-segment, and C-terminal, as shown in Tables 4-9. The three regions of these six signal peptides were recombined, and a total of 216 (6×6×6) signal peptide mutants were obtained ( image 3 ). According to the gene sequences of these six signal peptides, the nucleotide sequence "5'-ACAATGGTCCAAACTAGT+the nucleotide sequence of the signal peptide mutant+GCTCAGCCGGCGAAAA-3'" was designed and synthesized by Shanghai Boyi Biotechnology Co., Ltd.

[0056] Table 4 Signal peptide YfkN

[0057]

[005...

Embodiment 3

[0074] Example 3 Verification of high secretion capacity keratinase production strain

[0075] The batch-fed fermentation method was used to perform fermentation verification on the recombinant Bacillus subtilis WB600 / pSTOP1622-24-kerds constructed in Example 2. The seed culture conditions of recombinant bacteria Bacillus subtilis WB600 / pSTOP1622-24-kerds and Bacillus subtilis WB600 / pSTOP1622-ker are as follows: LB liquid medium is used for cultivation in a 500mL Erlenmeyer flask, wherein the liquid volume of the medium is 50mL, and the cultivation temperature The temperature is 37°C, the rotational speed is 200rpm, and the incubation time is 10h. The batch-fed fermentation conditions of the recombinant bacteria are as follows: B. subtilis WB600 batch fermentation medium is used to inoculate the seeds in a 3L automatic fermenter, the stirring speed is 400rpm, the culture temperature is 37°C, and the ventilation rate is 1.5vvm. Use ammonia water to control the pH not lower tha...

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Abstract

The invention discloses a signal peptide for optimizing high-level secretory expression of keratinase Ker and application of the signal peptide and belongs to the fields of genetic engineering and enzyme engineering. The signal peptide is characterized in that 46 secretory signal peptides from bacillus subtilis are preliminarily screened and 6 secretory signal peptides capable of obviously improving the secretion volume of the keratinase in the 46 secretory signal peptides are directionally transformed, so the signal peptide for optimizing the high-level secretory expression of keratinase is obtained. An N terminal of the signal peptide is provided with first nine amino acid residues of a signal peptide AbnA; an H terminal of the signal peptide is provided with a hydrophobic sequence of a signal peptide YfkN, and a C terminal of the signal peptide is provided with last four amino acid residues of a signal peptide PhoB. The signal peptide disclosed by the invention is fused at the N terminal of the keratinase Ker, so the secretion efficiency of the keratinase of recombinant bacillus subtilis is remarkably improved, and the enzyme activity of extracellular keratinase of the bacillus subtilis is improved by 3.39 times. The extracellular keratinase producing capability of a transformed strain is remarkably improved, the industrial application is better facilitated, the production cost can be reduced, and the production efficiency is improved.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, in particular to a signal peptide for optimizing the efficient secretion and expression of keratinase Ker and its application. Background technique [0002] Keratin is a kind of insoluble hard protein, which widely exists in the tissues of organisms and is the main component of feathers, hair, wool, nails, horns, hooves and scales. [0003] As a by-product of the poultry industry, keratin is a considerable renewable resource. Due to the special structure of keratin, keratin is difficult to be degraded by general proteases such as papain, pepsin and trypsin. The traditional keratinase treatment process is mainly high-temperature heating acid-base hydrolysis, which will not only pollute the environment, but also destroy some amino acids, thus affecting the utilization rate of keratin. Using biotransformation methods such as using keratinase to degrade feathers, hair, and...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N1/21C12N15/70C12N9/54C12R1/19
CPCC07K14/00C12N9/54
Inventor 曾静袁林郭建军郭浩王林庚
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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