Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase

A technology of disulfide bond isomerase and Escherichia coli, which is applied in the fields of genetic engineering, grain chemistry, microbes, and molecular biology, can solve the problems of complex purification and low prokaryotic expression, and achieve improved binding ability and easy mass expression Effect

Active Publication Date: 2013-12-11
广州英赞生物科技有限公司
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Problems solved by technology

[0007] In order to solve the above problems, the present invention achieves a large amount of expression of wPDI through the E. coli expression system, and ob

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  • Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase
  • Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase
  • Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase

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[0028] Example 1

[0029] The method for producing wheat protein disulfide bond isomerase using the E. coli prokaryotic expression system, the specific steps are as follows:

[0030] 1. Construction of wheat protein disulfide isomerase (wPDI) expression vector

[0031] 1. Acquisition of wPDI gene

[0032] Take young wheat leaves that have grown for 8-10 days, grind them into powder in liquid nitrogen, and extract total wheat RNA with an RNA extraction kit (such as figure 1 Shown), the wheat PDI gene was obtained by RT-PCR. According to the sequence analysis of plant PDI on NCBI, PDI has a high degree of conservation. Therefore, primers are designed according to its conserved regions. The primer sequences used in RT-PCR are: 5'-ATGGCGATCTGCAAGGTCTGG-3', 5'-TCAGAGCTCGTCCTTCAGAGGC-3', After the wheat PDI gene is cut and recovered, the A tail is added and connected to PMD-19T, which is sent to BGI for gene sequencing.

[0033] The sequencing results showed that the size of the cloned PDI ...

Example Embodiment

[0050] Example 3

[0051] Effect of reorganized wPDI on the quality and function of flour processing

[0052] Add 8.9ml of water / BSA solution / protein solution for every 8g of flour (the protein addition amount is: 1mg protein / g flour, the experimental group with BSA added are all controls) for kneading, kneading for 5 minutes, and preparing it to have a side length of about 1cm. A rectangular parallelepiped with a height of about 1.3 cm is placed on the texture analyzer for measurement. 5 samples are made for each group of experiments, and the average value of the 5 measured data is taken. The texture analyzer probe is P / 36R, the pre-test speed is 1.0mm / s, the test speed is 2.0mm / s, the post-test speed is 2.0mm / s, the compression ratio is 50%, and the interval between two runs is 10s.

[0053] Experimental results: Compare the texture data of blank / BSA / wPDI during the mixing process (see Table 1), and the experimental group added wDPI during the mixing process, in the dough hardness...

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Abstract

The invention discloses wheat protein disulfide isomerase produced by utilizing an escherichia coli prokaryotic expression system, as well as a method and application of the wheat protein disulfide isomerase. According to the technical scheme, the method comprises the following steps: extracting wheat total RNA (ribonucleic acid) from wheat spires, and obtaining wPDI (wheat protein disulfide isomerase) gene through RT-PCR (reverse transcription-polymerase chain reaction); connecting a target gene to a carrier PET-30b (polyethylene glycol terephthalate-30b) to construct a recombinant plasmid; transferring the recombinant plasmid into a competent cell of escherichia coli BL21, performing inducible expression in an LB culture medium to culture the recombinant escherichia coli, and centrifugally collecting a bacterial cell; crushing the bacterial cell by using an ultrasonic method, centrifuging and collecting supernate; preparing wPDI through once Ni-sepharose affinity chromatography. The wPDI massive expression and simple purification process can be realized through the escherichia coli expression system; the prepared wPDI plays an excellent effect in improving the wheat processing quality in a flour processing quality experiment, and can be used in flour processing.

Description

technical field [0001] The invention designs a method for producing wheat protein disulfide bond isomerase using an Escherichia coli prokaryotic expression system and its application in flour processing, belonging to the fields of microorganisms, molecular biology, genetic engineering and grain chemistry. Background technique [0002] Wheat is an important food crop, providing the main food for nearly 40% of the world's population. my country's wheat production accounts for one-sixth of the world's total production. Wheat flour not only has good nutritional function, but also can be processed into flour products with different needs, good palatability and beautiful appearance. The network structure formed by glutenin in the dough endows wheat flour with processing quality, and the disulfide bonds between glutenin subunits are the basis for the formation of the network structure. Therefore, the processing quality of flour is related to the number of disulfide bonds in gluteni...

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Application Information

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IPC IPC(8): C12N9/90C12N15/70A21D8/04C12R1/19
Inventor 胡松青张婷婷侯轶刘光毅李琳
Owner 广州英赞生物科技有限公司
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