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Promoter for heterologous expression of keratinase, and application thereof

A technology of keratinase and promoter, which is applied in the field of promoters for heterologous expression of keratinase, to achieve the effects of long cycle, reduced workload and high expression efficiency

Active Publication Date: 2018-12-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high-efficiency expression of keratinase through promoter modification has not been reported in any literature, which is also of great significance for the deep and wide application of keratinase

Method used

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  • Promoter for heterologous expression of keratinase, and application thereof
  • Promoter for heterologous expression of keratinase, and application thereof
  • Promoter for heterologous expression of keratinase, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Promoter Molecular Modification Improves Keratinase Activity and Expression

[0033] The nucleotide sequence of the promoter is amplified from the Bacillus genome and modified by mutation. In this study, the promoters selected and modified are all constitutive promoters or self-inducible promoters without adding inducers, which are trnQ, sigX, yolA, wapA, gapB, cdd, veg, mpr, nprE, The base sequences of the promoters of aprE, epr, bpr, nprB, pst, gsiB, and srfA genes are shown in SEQ ID NO: 1-SEQ ID NO: 16. According to the promoter sequences of these genes, the primers are designed in Table 1.

[0034] Table 1. Promoter primer design

[0035]

[0036] In the research on the screening of promoters for high-efficiency expression of keratinase, a promoter screening system was constructed in the Bacillus subtilis expression host. The promoter can be inserted into the coding sequence of the target gene by enzyme digestion and ligation, such as keratinase reco...

Embodiment 2

[0038] Embodiment 2: PaprE promoter transforms recombinant bacteria shaking flask fermentation situation

[0039] Nutritional components such as glycerol, maltose, sucrose, beef extract and corn steep liquor were added to the TB medium in an amount of 20 g / L, and the enzyme production levels in different culture medium were detected after inoculation of PaprE promoter-transformed recombinant bacteria. Depend on Figure 4 It can be seen that when the fermentation lasted for 30 hours, the keratinase production in the TB medium was the highest, while the keratinase production in the experimental group added nutrients at the same period was lower, which may be caused by the prolonged growth period of the recombinant strain. When fermented to 48h, compared with the control TB medium, all the experimental groups added with nutritional components showed relatively higher keratinase production. The enzyme activity reaches above 4000U / mL.

Embodiment 3

[0040] Embodiment 3: PaprE promoter transformation recombinant bacteria 5L fermentation tank fermentation situation

[0041] The situation of enzyme production by PaprE promoter-transformed recombinant bacteria in 5L tank was further investigated. During the fermentation process, after inoculation with 5% inoculum, the pH value in the fermentation broth first gradually decreased, and after about 18 hours of fermentation, the pH value began to gradually increase, accompanied by the generation of a large amount of recombinant keratinase. The cell density in the fermentation broth increases rapidly in the early stage. When the fermentation reaches 30h, the OD 600 The maximum reached was 32.2, about 2.2 times that of shake flask culture. The keratinase enzyme activity in the fermentation broth reached the highest value when it was fermented to 36h, which was 7176U / mL ( Figure 5 ), approximately 1.6 times the shake flask level. The yield of recombinant keratinase obtained in th...

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Abstract

The invention discloses a promoter for heterologous expression of keratinase, and an application thereof, and belongs to the field of industrial biotechnology. Keratinase recombinant bacteria carrying16 different promoter sequences are successfully constructed, 7 promoter sequences can increase the expression level of keratinase, and the enzyme activity level of a ParpE promoter in transforming the recombinant bacteria is highest, reaches 2605 U / mL, and is 20 times higher than that of control bacteria. The keratinase activity in a fermentation solution in a 5L fermenter reaches up to 7176 U / mL, and is the highest level of recombinant keratinase expression reported in the literature, so the promoter can well serve in practical applications. An effective strategy is provided for high-efficiency expression of keratinase and enzyme production researches. Traditional genetic engineering transformation usually requires a high-throughput screening method and large-scale library screening, solarge workload, long cycle and high cost exist; compared with the traditional method, the reconstruction method in the invention has the advantages of great reduction of the workload, and improvementof the expression efficiency.

Description

technical field [0001] The invention relates to a promoter for heterologous expression of keratinase and its application, belonging to the field of industrial biotechnology. Background technique [0002] Keratin is an insoluble structural protein with high stability due to a high degree of crosslinking by disulfide and hydrogen bonds. As a by-product of the development process of animal husbandry, leather industry and textile industry, a large amount of hair is discarded in nature every year, causing huge pollution to the environment. At present, hair is mainly degraded through physical and chemical methods such as high temperature, high pressure, acid, alkali, oxidation, etc., but these methods will deplete useful amino acids in keratin, and have low efficiency, high energy consumption, and large pollution. Keratinases open complex disulfide bonds in keratin through disulfide bond reduction, and then degrade keratin through proteolysis. Degrading keratin with keratinase i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/75C12N1/21C12N9/50
CPCC12N15/75C07K14/32C12N9/50
Inventor 史劲松龚劲松陶丽妍许正宏苏畅
Owner JIANGNAN UNIV
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