Method for improving flour processing quality by applying recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase

A technology of reticulothiol and reductase, applied in the fields of genetic engineering and grain science, can solve the problems of complex additive components and cost, restrict the practical application of wEro1, explore the influence of wEro1, etc., and achieve easy amplification, lower production costs, and simple purification. Effect

Active Publication Date: 2016-08-24
广州英赞生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this patent does not independently explore the influence of wEro1 on bread quality, and the complex addition of components and cost issues seriously restrict the practical application of wEro1

Method used

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  • Method for improving flour processing quality by applying recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase
  • Method for improving flour processing quality by applying recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase
  • Method for improving flour processing quality by applying recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Recombinant wheat endoplasmic reticulum sulfhydryl oxidoreductase expression vector construction

[0038]The wheat endoplasmic reticulum sulfhydryl oxidoreductase gene (SEQ ID NO: 1) was synthesized in Guangzhou Aiji Biotechnology Co., Ltd., and connected to the cloning vector pGSI. Take 1 ng of the recombinant cloned plasmid, react with NdeI and XhoI restriction endonucleases (37°C, 30min), perform agarose gel electrophoresis and purify the wheat endoplasmic reticulum sulfhydryl oxidoreductase gene fragment with sticky ends. 20 μg of the gene fragment was mixed with 5 μg of the plasmid pET-28a(+) digested with the same restriction enzyme, and ligated under the action of T4 DNA ligase at 20° C. for 1 h. After ligation, the product was transformed into Escherichia coli DH5α competent, spread on LB solid medium containing kanamycin and cultured for 12-16 hours. The plasmids were extracted from monoclonal colonies with full shape, and the recombinant plasmids we...

Embodiment 2

[0040] Example 2 Induced expression and purification of recombinant wEro1

[0041] 1. Induced expression of recombinant wEro1

[0042] Transform the constructed recombinant plasmid pET-28a-wero1 into BL21(DE3), pick 3 monoclonal colonies and put them in LB medium containing 50 μg / mL kanamycin, culture at 37°C and 200r / m to OD 600 To about 0.6-0.8, add 0.2-1mM IPTG to induce expression for 8-12 hours, and the induction temperature range is between 16-37°C. Through SDS-PAGE analysis, the recombinant wEro1 obtained soluble expression ( figure 2 ).

[0043] 2. Affinity purification of recombinant wEro1

[0044] The expressed bacteria were collected, resuspended by adding buffer, and the cells of the bacteria were disrupted by a probe-type ultrasonic instrument. The ultrasonic condition was 350w, the duty ratio was 0.4:0.6, and the ultrasonic was 15min. After the sonication was complete, the supernatant was collected after centrifugation at 8000r / m for 20min.

[0045] The su...

Embodiment 3

[0048] The enzymatic properties of embodiment 3 recombinant wEro1

[0049] wEro1 contains a variety of enzymatic properties. In this example, the convenient FAD reducing activity is selected for detection. The principle is that the coenzyme FAD of wEro1 can be reduced to FADH under the action of dithiothreitol (DTT) 2 , the latter has no absorbance at 450nm. Therefore, the FAD reduction activity of wEro1 can be explored by detecting the change of absorbance at 450nm.

[0050] The enzyme activity assay reaction system was as follows: 2 μM wEro1 and 10 μM FAD were added to 50 mM Tris-HCl (pH 8.0) buffer solution, 12.5 mM DTT was added to start the reaction, and the absorbance was measured at 450 nm.

[0051] Result: if Figure 4 As shown, after adding DTT, wEro1 exhibited obvious FAD reducing activity.

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Abstract

The invention discloses a method for improving the flour processing quality by applying recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase, and belongs to the scientific field of genetic engineering and cereals. The method comprises the following steps: protokaryon expresses and purifies the recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase, uniformly stirring the recombined enzyme with flour, sugar, salt (NaCl), plant oil, yeast powder and water to form dough, cutting, weighing, forming and fermenting the formed dough, and baking the dough to obtain a bread finished product. According to the adding level, 0.05 to 0.2 percent (w/w, flour-based) of the recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase is added. The recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase used in the method can obviously improve the flour processing quality, to improve the baking quality such as the specific volume, the height-diameter ratio, the elasticity and the hardness of bread; the improvement effect is equivalent to that of a strong chemical reinforcement agent (azodicarbonamide). Therefore, the recombined wheat endoplasmic reticulum sulfydryl oxidordeuctase can replace the strong chemical reinforcement agent (azodicarbonamide) to be used in the industry of flour product processing.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and grain science, and in particular relates to a method for improving flour processing quality by using recombinant wheat endoplasmic reticulum sulfhydryl oxidoreductase. Background technique [0002] Wheat is one of the three major food crops in the world. The total output of wheat in my country ranks first in the world, accounting for one-sixth of the world's total output. Mainly wheat, suitable for making steamed buns and cakes but not for making bread. However, with the improvement of people's living standards, the consumption demand for bread is increasing year by year, and low-quality medium and low-gluten wheat restricts the development of flour products such as bread in my country. In addition, wheat growing environment, pests and diseases and postharvest treatment lead to uneven processing quality of flour. [0003] The quality of flour processing is mainly determined by the gluten p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A21D8/04A21D13/06
Inventor 胡松青刘光侯轶黄滟波李琳张喜梅
Owner 广州英赞生物科技有限公司
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