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Application of wheat resting sulfydryl oxidase in improving stability of emulsion type special medical foods

A technology of sulfhydryl oxidase and sulfhydryl oxidase protein is applied in the fields of genetic engineering and dairy science to achieve the effects of easy amplification, simple purification and improved storage stability

Active Publication Date: 2020-04-03
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, research on QSOX at home and abroad focuses on enzymatic activity, reaction mechanism, and structural characterization, but the application of QSOX as a biological enzyme preparation to improve the stability of emulsion-type special medical food has not been reported yet.

Method used

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  • Application of wheat resting sulfydryl oxidase in improving stability of emulsion type special medical foods
  • Application of wheat resting sulfydryl oxidase in improving stability of emulsion type special medical foods
  • Application of wheat resting sulfydryl oxidase in improving stability of emulsion type special medical foods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Recombinant Wheat Resting Sulfhydryl Oxidase Expression Vector Construction

[0061] The whole gene sequence of wheat 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 28-480 are selected for expression, the corresponding base sequence is 82-1440, and the length is 1359bp, 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.

[0062] Primer F: 5′-GGCC CATATG CTCCGGTCGCTCGGCGTCG-3', wherein the underlined part is the NdeI res...

Embodiment 2

[0065] Example 2 Induced expression and purification of recombinant wheat resting sulfhydryl oxidase (wQSOX)

[0066] 1. Induced expression

[0067] The constructed recombinant plasmid pMAL-c5x-wQSOX 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°C, cultured to OD at 200r / min 600 To about 0.6-0.8, add 0.1-1.0mM IPTG to induce expression for 4-20h, and the induction temperature range is between 16-37°C. Analyzed by SDS-PAGE ( figure 2 ), the recombinant wQSOX 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.

[0068] 2. Affinity purification

[0069] 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 d...

Embodiment 3

[0073] The thiol oxidation activity of embodiment 3 recombinant wQSOX

[0074] The thiol oxidation activity of wQSOX 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 the formation of disulfide bonds catalyzed by wQSOX, 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 wQSOX can be characterized by measuring the change of solution oxygen.

[0075] The enzyme activity assay reaction system was as follows: 10 μmol / L wQSOX was added to 50 mmol / L Tris-HCl (pH 8.0) buffer solution, 10 mmol / L DTT (dithiothreitol) was added to start the reaction, and a liquid-phase oxygen electrode w...

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Abstract

The invention discloses an application of wheat resting sulfydryl oxidase in improving the stability of emulsion type special medical foods, and belongs to the field of genetic engineering and dairy science. The recombinant wheat resting sulfydryl oxidase with sulfydryl oxidation activity is prepared through a prokaryotic expression system, and has the characteristics of being high in expression quantity, simple and convenient to purify, easy to amplify, suitable for industrial application and the like. The invention provides a method for improving the stability of the emulsion type special medical foods by applying the recombinant wheat resting sulfydryl oxidase for the first time, and the recombinant wheat resting sulfydryl oxidase is directly added into the emulsion type special medicalfoods, so that the particle size and the TSI index of emulsion particles can be remarkably reduced, and the storage stability of the emulsion is improved. Therefore, the recombinant wheat resting sulfydryl oxidase can be developed into a biological enzyme preparation for improving the stability of emulsion type special medical foods, and promote the development of the special medical food processing industry in China.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and dairy science, and in particular relates to the application of wheat resting sulfhydryl oxidase in improving the stability of emulsion-type special medical food. Background technique [0002] Malnutrition is a common complication of clinical patients, which seriously affects the life safety and healing cycle of patients. Nutritional therapy is the most effective way to improve the nutritional health of these patients, so it is widely used clinically at home and abroad. Formula food for special medical purposes (referred to as special medical food) is a type of formula food specially processed to meet the needs of enteral nutrition therapy for people with restricted food intake, digestive and absorption disorders, metabolic disorders or specific disease states. Conditions, improving the immunity of patients, reducing complications, and promoting the rapid recovery of patients have remarkabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23L29/00C12N9/02C12N15/52C12N15/70
CPCA23L29/06C12N9/0051C12N15/70
Inventor 刘光张名位魏振承张雁唐小俊邓媛元王智明王佳佳廖娜李萍
Owner SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI
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