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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

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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 obtains a relatively high-purity enzyme through a column chromatography, which overcomes the problems of low prokaryotic expression and complicated purification.

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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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Embodiment 1

[0029] The method for producing wheat protein disulfide bond isomerase by using Escherichia 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 wheat young leaves that have grown for 8-10 days, grind them into powder in liquid nitrogen, and extract wheat total 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, it shows that PDI has a high degree of conservation, so primers were designed according to its conserved region. The primer sequence used in RT-PCR is: 5'-ATGGCGATCTGCAAGGTCTGG-3', 5'-TCAGAGCTCGTCCTTCAGAGGC-3', After the obtained wheat PDI gene was recovered by gel cutting, A tail was added, connected to PMD-19T, and sent to BGI for sequencing.

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

Embodiment 3

[0051] Effects of Recombinant wPDI on Flour Processing Quality Function

[0052] For every 8g of flour, add 8.9ml of water / BSA solution / protein solution (the amount of protein added is: 1mg protein / g flour, and the experimental group with BSA added is the control) for kneading. The kneading time is 5min, and the side length is about 1cm. A cuboid with a height of about 1.3 cm is placed on a texture analyzer for measurement. Five samples are made for each group of experiments, and the average value of the five 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 times is 10s.

[0053] Experimental results: compare the texture data of blank / BSA / wPDI during the dough mixing process (see Table 1), and the experimental group with wDPI added during the dough mixing process, the dough hardness, elasticity, cohesion, stickiness, c...

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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...

Claims

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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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