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Application of rice resting sulfydryl oxidase in improvement of flour processing quality

A technology of sulfhydryl oxidase and sulfhydryl oxidase protein, which is applied in the fields of genetic engineering and grain science to achieve the effects of enhancing powder properties, easy purification, and improving baking quality

Active Publication Date: 2021-05-14
SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on QSOX at home and abroad focuses on the aspects of enzymatic activity, reaction mechanism and structural characterization, but there is no report on the application of QSOX as a flour improver to improve the quality of flour products.

Method used

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  • Application of rice resting sulfydryl oxidase in improvement of flour processing quality
  • Application of rice resting sulfydryl oxidase in improvement of flour processing quality
  • Application of rice resting sulfydryl oxidase in improvement of flour processing quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Recombinant Rice Resting Sulfhydryl Oxidase Expression Vector Construction

[0043] The full gene sequence of rice resting sulfhydryl oxidase (SEQ ID NO: 1) was synthesized in Guangzhou Yingzan Biotechnology Co., Ltd., and connected to the cloning vector pGSI. Since the N-terminal has a signal peptide sequence and the C-terminal has a transmembrane sequence, in order not to affect the soluble expression of the protein, amino acids 31-476 are selected for expression, the corresponding base sequence is 91-1428, and the length is 1338bp, the upstream and downstream primers of sequence extension are primer F and primer P respectively. Take 1ng of the recombinant cloning plasmid, add upstream and downstream primers and 2×PfuMix to carry out PCR reaction, after the amplification, perform electrophoresis analysis and recovery and purification on the PCR product.

[0044] Primer F: 5′-GGCC CATATG CGCTCGCTCGGCGGCAGGGA-3', wherein the underlined part is the NdeI restr...

Embodiment 2

[0047] Example 2 Induced Expression and Purification of Recombinant Rice Resting Sulfhydryl Oxidase (rQSOX)

[0048] 1. Induced expression

[0049] The constructed recombinant plasmid pMAL-c5x-rqsox was transformed into Rosetta-gami B (DE3), and 3 single clone colonies were picked and placed in LB liquid medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin , 37 ℃, 200r / min conditions cultured 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. Analyzed by SDS-PAGE ( figure 2 ), the recombinant rQSOX can be expressed in large quantities (lane 2), and the target protein in the broken supernatant (lane 3), indicating that soluble expression has been obtained.

[0050] 2. Affinity purification

[0051] 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 3...

Embodiment 3

[0055] The sulfhydryl oxidation activity of embodiment 3 recombinant rQSOX

[0056] The thiol oxidation activity of rQSOX can be determined by various methods. In this study, a liquid phase oxygen electrode was used to measure the thiol oxidation activity of recombinant protein. The principle is that during rQSOX catalyzing the formation of disulfide bonds, the disulfide bonds at its own catalytic active sites are reduced, and under the action of its prosthetic group FAD, the disulfide bonds at the reduced active sites will be re-oxidized to form disulfide bonds , this process is accompanied by the consumption of solution oxygen, therefore, the thiol oxidation activity of rQSOX can be characterized by measuring the change of solution oxygen.

[0057] The enzyme activity assay reaction system is as follows: 10 μmol / L rQSOX was added to 50 mmol / L Tris-HCl (pH8.0) buffer solution, 10 mmol / L DTT (dithiothreitol) was added to start the reaction, and the oxygen was detected by a liq...

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Abstract

The invention discloses an application of rice resting sulfydryl oxidase in improvement of flour processing quality, and belongs to the field of genetic engineering and cereal science. Through a prokaryotic expression system, the recombinant rice resting sulfydryl oxidase with sulfydryl oxidation activity is prepared, and the recombinant rice resting sulfydryl oxidase has the characteristics of high expression quantity, simplicity and convenience in purification, easiness in amplification, suitability for industrial application and the like. The method for improving the flour processing quality by applying the recombinant rice resting sulfydryl oxidase is provided for the first time, the stabilization time of the flour can be remarkably prolonged, the flour property of the flour can be effectively enhanced and the baking quality of bread can be improved by directly adding the recombinant rice resting sulfydryl oxidase into the flour, and the improvement effect is superior to that of a chemical gluten strengthening agent potassium bromate, the enzyme preparation can be developed into a novel biological flour improving enzyme preparation for replacing potassium bromate, is used for being compounded with other biological improvers, and promotes the development of the flour product processing industry.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and grain science, and in particular relates to the application of rice resting sulfhydryl oxidase in improving the processing quality of flour. 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 bread and other flour products industries in China. 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 protein network, and...

Claims

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Application Information

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IPC IPC(8): A21D2/26A21D13/80C12N9/02C12N15/70C12R1/19
CPCA21D2/26A21D13/80C12N9/0051C12Y108/03002C12N15/70A21D2/00
Inventor 刘光张名位魏振承张雁唐小俊邓媛元王佳佳李萍
Owner SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI
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