阅读说明：本技术 水稻静息巯基氧化酶在改善面粉加工品质中的应用 (Application of rice resting sulfhydryl oxidase in improving flour processing quality ) 是由 刘光 张名位 魏振承 张雁 唐小俊 邓媛元 王佳佳 李萍 于 2019-10-28 设计创作，主要内容包括：本发明公开一种水稻静息巯基氧化酶在改善面粉加工品质中的应用,属于基因工程和谷物科学领域。本发明通过原核表达系统,制备了具有巯基氧化活性的重组水稻静息巯基氧化酶,具有表达量高,纯化简便,易于放大,适合工业化应用等特点。本发明首次提供了一种应用重组水稻静息巯基氧化酶改善面粉加工品质的方法,直接添加该重组酶到面粉中能够显著延长面粉的稳定时间,有效增强面粉粉质特性,改善面包的烘焙品质,其改善效果优于化学强筋剂溴酸钾,因此,可发展成为替代溴酸钾的新型生物面粉改良酶制剂,用于与其它生物改良剂的复配,促进面制品加工行业的发展。(The invention discloses application of resting thiol oxidase of rice in improving the processing quality of flour, belonging to the fields of genetic engineering and grain science. The recombinant rice resting thiol oxidase with thiol oxidation activity is prepared by a prokaryotic expression system, and has the characteristics of high expression quantity, simple and convenient purification, easy amplification, suitability for industrial application and the like. The invention provides a method for improving the processing quality of flour by applying recombinant rice resting sulfhydryl oxidase for the first time, the recombinase is directly added into the flour, the stability time of the flour can be obviously prolonged, the flour quality characteristic is effectively enhanced, the baking quality of bread is improved, and the improvement effect is better than that of a chemical strengthening agent potassium bromate, so that the recombinase can be developed into a novel biological flour improving enzyme preparation for replacing potassium bromate, is used for compounding with other biological modifiers, and promotes the development of the flour product processing industry.)

1. The application of rice resting sulfhydryl oxidase in improving flour processing quality.

2. Use according to claim 1, characterized in that:

the application of an enzyme preparation mainly containing rice resting sulfhydryl oxidase in improving flour processing quality is separately added.

3. Use according to claim 1 or 2, characterized in that:

the rice resting sulfhydryl oxidase is applied to improving the quality of dough and bread.

4. Use according to claim 1 or 2, characterized in that:

the addition level of the rice resting sulfhydryl oxidase in the flour is 0.05-1.0% w/w of the flour base.

5. Use according to claim 1 or 2, characterized in that:

the amino acid sequence of the rice resting thiol oxidase is shown as SEQ ID NO: 2, or, as shown in SEQ ID NO: 2 from amino acid 31 to amino acid 476.

6. A method for improving flour processing quality by applying recombinant rice resting sulfhydryl oxidase is characterized in that: uniformly stirring the recombinant rice resting sulfhydryl oxidase, flour, sugar, salt, vegetable oil, yeast powder and water, cutting, weighing, forming and proofing the formed dough, and baking to obtain a bread finished product;

the addition level of the recombinant rice resting sulfhydryl oxidase is 0.05-1.0% w/w of flour base.

7. The method of claim 6, wherein:

the amino acid sequence of the rice resting thiol oxidase is shown as SEQ ID NO: 2, or, as shown in SEQ ID NO: 2 from amino acid 31 to amino acid 476.

8. The method according to claim 6 or 7, characterized in that:

the preparation method of the recombinant rice resting sulfhydryl oxidase adopts an escherichia coli expression system to express, and adopts a column chromatography method to purify recombinant rQSOX protein; according to rQSOX: FAD is 1: adding FAD into the purified recombinant rQSOX protein solution at a molar ratio of 1-5 to obtain the recombinant rice resting thiol oxidase with thiol oxidation activity.

9. The method of claim 8, wherein:

the starting expression vector adopted in the escherichia coli expression system is pMAL-c5x, and the starting expression strain is escherichia coli Rosetta-gami B (DE 3).

10. The method of claim 9, wherein:

the preparation method of the recombinant rice resting sulfhydryl oxidase comprises the following steps:

(1) synthesizing a whole gene sequence of the rice resting sulfhydryl oxidase by using a whole gene synthesis technology, and performing PCR amplification by using a primer F and a primer P; connecting the purified target gene fragment to a vector plasmid pMAL-c5x, wherein the restriction enzyme sites are NdeI and EcoRI, and constructing a rice resting sulfhydryl oxidase recombinant expression plasmid pMAL-c5 x-rqsox;

and (3) primer F: 5' -GGCCCATATGCGCTCGCTCGGCGGCAGGGA-3', wherein the underlined section is the NdeI restriction site;

primer P: 5' -GGCCGAATTCGCTTGCAGCATTAGAAATCGATG-3'; wherein the underlined part is an EcoRI restriction site;

(2) transferring the recombinant expression plasmid pMAL-c5x-rqsox into escherichia coli Rosetta-gami B (DE3) competent cells to obtain recombinant expression bacteria; inoculating the recombinant expression bacteria to LB liquid culture medium to culture to OD₆₀₀When the temperature reaches 0.6-0.8 ℃, adding 0.2-1.0 mM IPTG (isopropyl thiogalactoside) to induce the recombinant protein to express for 8-12 hours at the temperature of 16-37 ℃, and collecting thalli;

(3) after the collected thalli are subjected to ice bath ultrasonic crushing, centrifuging and collecting supernatant, and purifying recombinant rice resting thiol-based oxidase protein by using Ni-NTA;

(4) according to rQSOX: FAD is 1: adding FAD into a purified recombinant rice resting thiol-based oxidase protein solution at a molar ratio of 1-5, incubating overnight at a low temperature, and removing redundant FAD through dialysis and desalination treatment to obtain the recombinant rice resting thiol-based oxidase with thiol oxidation activity.

Technical Field

The invention belongs to the fields of genetic engineering and grain science, and particularly relates to application of resting sulfhydryl oxidase of rice in improving the processing quality of flour.

Background

Wheat is one of three major food crops in the world, the total yield of wheat in China is at the head of the world and accounts for one sixth of the total yield of the world, but is limited by varieties, climate, soil and the like, and the wheat produced in China mostly takes middle-low gluten wheat as the main part, so that the wheat is suitable for making steamed bread and cakes but not suitable for making bread. However, with the improvement of living standard of people, the consumption demand of bread rises year by year, and the low-quality medium-low gluten wheat restricts the development of bread products such as bread in China. In addition, wheat growth environment, pest and disease damage and post-harvest treatment cause uneven processing quality of flour.

The quality of flour processing is mainly determined by the gluten protein network, and intermolecular disulfide bonds are the basis for forming the network structure. In recent years, the flour product processing industry has improved the processing quality of flour products by adding flour improving agents that promote the formation of intermolecular disulfide bonds. Cheap and efficient chemical flour modifiers such as potassium bromate, azodicarbonamide and the like are widely applied to the flour product processing industry for a long time. However, the food safety implications of "chemical" modifiers have been increasingly appreciated and demonstrated, as bromate has been found to be carcinogenic and genotoxic. Therefore, the search for novel flour improving agents becomes a development direction in the flour product processing field, and safe, efficient and green biological enzyme preparations meet the development trend.

Rice resting sulfhydryl oxidase (rQSOX) is sulfhydryl oxidase containing prosthetic group FAD, and can directly catalyze the formation of disulfide bond, and the reduced disulfide bond can be reoxidized under the action of prosthetic group FAD, thereby realizing the continuous exertion of sulfhydryl oxidation activity. Furthermore, QSOX belongs to a family member of sulfhydryl oxidases, which are Generally Recognized as GRAS (Generally Recognized as Safe) by the Food and Drug Administration (FDA), and therefore have the potential to be developed as a Safe and efficient flour improver. At present, research on QSOX at home and abroad focuses on the aspects of enzymatic activity, reaction mechanism, structural characterization and the like, but the application of QSOX as a flour improver to the quality improvement of flour products is not reported.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the application of the rice resting sulfhydryl oxidase in improving the flour processing quality.

The recombinant rice resting sulfhydryl oxidase with sulfhydryl oxidation activity is obtained by constructing an optimal expression system. The flour quality improvement test result shows that the recombinant rice resting sulfhydryl oxidase can obviously prolong the flour stabilizing time and improve the bread baking quality.

The invention also aims to provide a method for improving the processing quality of flour by using the recombinant rice resting sulfhydryl oxidase. The method creates a new way for improving the flour products in China.

The purpose of the invention is realized by the following technical scheme:

the invention provides application of rice resting sulfhydryl oxidase in improving flour processing quality, in particular application of independently adding an enzyme preparation mainly comprising the rice resting sulfhydryl oxidase in improving flour processing quality.

In particular to application of rice resting sulfhydryl oxidase in improving the quality of dough and bread.

The aims of enhancing flour quality and improving bread baking quality can be achieved only by adding the rice resting sulfhydryl oxidase into flour.

The addition level of the rice resting sulfhydryl oxidase in the flour is 0.05-1.0% (w/w, flour base), further 0.2-0.5% (w/w), and further 0.5% (w/w); can increase the stabilization time and the quality number of the flour, reduce the weakening degree of the flour and reduce the hardness and the viscosity of the bread.

The amino acid sequence of the rice resting thiol oxidase is shown as SEQ ID NO: 2, or, as shown in SEQ ID NO: 2 at amino acids 31-476; the nucleotide sequence of the gene is shown as SEQ ID NO: 1, or, as shown in SEQ ID NO: 1, from base 91 to base 1428.

A method for improving flour processing quality by using recombinant rice resting sulfhydryl oxidase comprises the steps of uniformly stirring the recombinant rice resting sulfhydryl oxidase, flour, sugar, salt (NaCl), vegetable oil, yeast powder and water, cutting, weighing, forming, fermenting and baking the formed dough to obtain a finished bread product. Wherein the addition amount of each component is as follows: 90-110 parts of flour, 0.6-1.8 parts of yeast powder, 1-3 parts of salt, 50-70 parts of water, 5-10 parts of sugar and 2-5 parts of vegetable oil.

The addition level of the recombinant rice resting sulfhydryl oxidase is 0.05-1.0% (w/w, flour base), further 0.2-0.5% (w/w), and further 0.5% (w/w).

The method for improving the flour processing quality by applying the recombinant rice resting sulfhydryl oxidase comprises the following specific steps:

(1) materials: 90-110 parts of flour, 0.6-1.8 parts of yeast powder, 1-3 parts of salt, 50-70 parts of water, 5-10 parts of sugar, 2-5 parts of vegetable oil and 0.05-1.0% (w/w, flour base) of recombinant rice resting sulfhydryl oxidase;

(2) kneading: mixing the materials, and stirring at the speed of 150-180 r/min for 10-15 min to form uniform dough;

(3) molding: dividing the well kneaded dough into 40-60 parts by weight per dough, rounding, forming and dishing;

(4) fermentation: placing the fermentation plate filled with the dough into a fermentation box for fermentation, wherein the temperature is controlled to be 30-35 ℃, the relative humidity is 75-85%, and the fermentation time is 60-80 min;

(5) baking: and baking the proofed dough for 10-15 min at the temperature of 180-200 ℃ in an oven to obtain a finished bread product.

The preparation method of the recombinant rice resting sulfhydryl oxidase adopts an escherichia coli expression system to express, and adopts a column chromatography method to purify recombinant rQSOX protein; according to rQSOX: FAD is 1: adding FAD into the purified recombinant rQSOX protein solution at a molar ratio of 1-5 to obtain the recombinant rice resting thiol oxidase with thiol oxidation activity.

The starting expression vector adopted in the escherichia coli expression system is pMAL-c5x, and the starting expression strain is escherichia coli Rosetta-gami B (DE 3).

The method comprises the following steps:

(1) synthesizing a whole gene sequence (SEQID NO: 1) of the rice resting sulfhydryl oxidase by using a whole gene synthesis technology, and obtaining a 91-1428 base fragment by PCR amplification, wherein the used amplification primers are a primer F and a primer P respectively; connecting the purified target gene fragment to a vector plasmid pMAL-c5x, wherein the restriction enzyme sites are NdeI and EcoRI, and constructing a rice resting sulfhydryl oxidase recombinant expression plasmid pMAL-c5 x-rqsox;

and (3) primer F: 5' -GGCCCATATGCGCTCGCTCGGCGGCAGGGA-3', wherein the underlined section is the NdeI restriction site;

primer P: 5' -GGCCGAATTCGCTTGCAGCATTAGAAATCGATG-3'; wherein the underlined part is an EcoRI restriction site;

(2) transferring the recombinant expression plasmid pMAL-c5x-rqsox into escherichia coli Rosetta-gami B (DE3) competent cells to obtain recombinant expression bacteria; inoculating the recombinant expression bacteria to LB liquid culture medium to culture to OD₆₀₀When the temperature reaches 0.6-0.8 ℃, adding 0.2-1.0 mM IPTG (isopropyl thiogalactoside) to induce the recombinant protein to express for 8-12 hours at the temperature of 16-37 ℃, and collecting thalli;

(3) after the collected thalli are subjected to ice bath ultrasonic crushing, centrifuging and collecting supernatant, and purifying recombinant rice resting thiol-based oxidase protein by using Ni-NTA;

(4) according to rQSOX: FAD is 1: adding FAD into a purified recombinant rice resting thiol-based oxidase protein solution at a molar ratio of 1-5, incubating overnight at a low temperature, and removing redundant FAD through dialysis and desalination treatment to obtain the recombinant rice resting thiol-based oxidase with thiol oxidation activity. After the recombinant protein is concentrated, the recombinant protein is frozen and stored at the temperature of minus 20 ℃ for standby.

Preferably, the recombinant rice resting thiol oxidase is added into bread according to the proportion of 0.5% (w/w, flour base), and the recombinant rice resting thiol oxidase is found to be superior to a chemical modifier potassium bromate in the aspect of improving the bread quality effect through bread texture analysis.

The mechanism of the invention is as follows: the invention prepares the recombinant rice resting sulfhydryl oxidase with sulfhydryl oxidation activity by a prokaryotic expression system. The recombined rice resting sulfhydryl oxidase is directly added into the flour, so that the stability time of the flour can be obviously prolonged, and the baking quality of the bread can be improved. The quality improving effect of the recombined rice resting sulfhydryl oxidase on flour is superior to that of a chemical modifier potassium bromate, so that the recombined rice resting sulfhydryl oxidase can be developed into a novel biological flour improving enzyme preparation which is used for compounding with other biological modifiers to promote the development of the flour product processing industry.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a method for improving the processing quality of flour by applying recombinant rice resting sulfhydryl oxidase for the first time. The recombinase can effectively enhance the flour quality characteristic and improve the baking quality of bread, the improvement effect of the recombinase is superior to that of the chemical strengthening agent potassium bromate, and the recombinase can be developed into a novel biological flour improver for replacing the potassium bromate and used for the flour product processing industry.

(2) The invention provides a recombinant rice resting sulfhydryl oxidase which is prepared by adopting prokaryotic expression and has sulfhydryl oxidation activity, and has the characteristics of high expression quantity, simple and convenient purification, easy amplification, suitability for industrial application and the like.

Drawings

FIG. 1 shows colony PCR identification of recombinant bacteria; wherein, lane M: DNA Marker D5000; lanes 1-6: the monoclonal bacteria were picked.

FIG. 2 is an expression and solubility analysis of recombinant rQSOX; wherein, lane M: a molecular weight Marker; lane 1: non-induced bacteria liquid; lane 2: inducing to obtain a bacterial liquid; lane 3: cell disruption solution supernatant; the recombinant rQSOX protein is underlined.

FIG. 3 is an SDS-PAGE analysis of recombinant rQSOX after affinity chromatography; wherein, lane M: a molecular weight Marker; lane 1: cell disruption supernatant; lane 2: target protein after Ni-NTA affinity chromatography; lane 3: and (5) desalting the target protein.

FIG. 4 is a graph of the thiol oxidation activity of recombinant rQSOX measured using an oxygen electrode.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

For process parameters not specifically noted, reference may be made to conventional techniques. The flour used in the examples of the present invention was purchased from the grain market; restriction enzymes NdeI and EcoRI were purchased from Thermo Fisher scientific; the pMAL-c5x plasmid was purchased from Nelumbo technologies, USA; coli DH5 α and Rosetta-gami B (DE3) competent cells were purchased from Novagen; the whole gene sequence synthesis was performed by Hispanic Biotech, Inc., Guangzhou.

Example 1 construction of recombinant Rice resting thiol oxidase expression vector

The rice resting thiol oxidase complete gene sequence (SEQ ID NO: 1) was synthesized in Hispanic Biotech, Inc., Guangzhou and ligated to the cloning vector pGSI. As the N-terminal is provided with a signal peptide sequence and the C-terminal is provided with a transmembrane sequence, in order to not influence the soluble expression of the protein, 31-476 amino acids are selected for expression, the corresponding base sequence is 91-1428, the length is 1338bp, and the upstream primer and the downstream primer of the sequence extension are respectively a primer F and a primer P. Taking 1ng of recombinant clone plasmid, adding upstream and downstream primers and 2 XPfumix to perform PCR reaction, and after the amplification is finished, performing electrophoretic analysis and recovery and purification treatment on a PCR product.

And (3) primer F: 5' -GGCCCATATGCGCTCGCTCGGCGGCAGGGA-3', wherein the underlined section is the NdeI restriction site;

primer P: 5' -GGCCGAATTCGCTTGCAGCATTAGAAATCGATG-3'; wherein the underlined part is an EcoRI restriction site;

and carrying out double enzyme digestion on the recovered extended fragment by NdeI and EcoRI restriction enzymes, and after the enzyme digestion reaction (37 ℃ and 30min) is finished, purifying and recovering the rice resting sulfhydryl oxidase gene fragment with the sticky end. Mu.g of the gene fragment were mixed with 5. mu.g of plasmid pMAL-c5x digested with the same restriction enzymes and ligated at 20 ℃ for 1h with T4DNA ligase. The product after connection is transferred into escherichia coli DH5 alpha competent cells, and the cells are spread on LB solid culture medium containing kanamycin to be cultured for 12-16 h. And selecting the monoclonal colony with plump shape for colony PCR identification, wherein the positive clone bacterial group has obvious bands. As can be seen from FIG. 1, the specific bands are amplified from the selected 6 monoclonals except the 5 th monoclonals, and the band size is about 1350bp and is close to the theoretical size of the target gene. Therefore, the results show that pMAL-c5x-rqsox recombinant expression vector was successfully constructed.

Example 2 inducible expression and purification of recombinant Rice resting thiol oxidase (rQSOX)

1. Inducible expression

The constructed recombinant plasmid pMAL-c5x-rqsox is transformed into Rosetta-gami B (DE3), 3 monoclonal bacteria are selected and dropped into LB liquid culture medium containing 50 ug/mL kanamycin and 100 ug/mL ampicillin, and cultured at 37 ℃ and 200r/min until OD is reached₆₀₀When the temperature is about 0.6-0.8, 0.2-1 mM IPTG is added to induce expression for 8-12 h, and the induction temperature range is 16-37 ℃. The recombinant rQSOX was able to be expressed in large amounts (lane 2) by SDS-PAGE analysis (FIG. 2), and the desired protein in the supernatant was disrupted (lane 3), indicating that soluble expression was obtained.

2. Affinity purification

And collecting the expressed thallus, adding a buffer solution for resuspension, and crushing thallus cells by using a probe type ultrasonic instrument under the ultrasonic condition of 350w and the duty ratio of 0.4:0.6 for 15min by ultrasonic. After the ultrasonic treatment is completed, the supernatant is collected after 8000r/m centrifugation for 20 min.

The supernatant was slowly injected into a Ni-NTA affinity column, the target protein was purified by linear elution, the fractions of each effluent peak were collected, and the purity of the recombinant protein was checked by SDS-PAGE (fig. 3).

The protein solutions of interest were pooled according to rQSOX: FAD is 1: adding FAD in a molar ratio of 1-5, processing at 4 ℃ overnight, desalting, concentrating, and freezing at-20 ℃ for later use.

The results show that the recombinant rQSOX obtains soluble expression and obtains high-purity target protein through one-step affinity chromatography.

Example 3 thiol Oxidation Activity of recombinant rQSOX

The thiol oxidation activity of rQSOX can be measured in a number of ways, and this study used liquid phase oxygen electrodes to measure the thiol oxidation activity of recombinant proteins. The principle is that during the formation of rQSOX catalytic disulfide bonds, the disulfide bonds of the catalytic active sites of the rQSOX catalytic disulfide bonds are reduced, and under the action of the prosthetic group FAD, the reduced disulfide bonds of the active sites are reoxidized to form disulfide bonds, and the consumption of solution oxygen is accompanied in the process, so that the rQSOX sulfhydryl oxidation activity can be represented by measuring the change of the solution oxygen.

The enzyme activity determination reaction system is as follows: mu. mol/L rQSOX was added to 50mmol/L Tris-HCl (pH8.0) buffer solution, 10mmol/L DTT (dithiothreitol) was added to start the reaction, and the change in oxygen concentration was detected using a liquid-phase oxygen electrode.

The results are shown in fig. 4, where DTT or rQSOX alone had little effect on solution oxygen, but the solution oxygen concentration decreased significantly after DTT and rQSOX were added simultaneously, indicating that rQSOX had significant thiol oxidation activity.

Example 4 Effect of recombinant rQSOX on flour quality characteristics

And (3) adopting a trace powder instrument to characterize the influence of the recombinant rQSOX on the characteristics of the flour. Adding 4g of flour into a powder bowl, premixing at 63r/min for 1min, adding 57% of water, and continuously stirring at the same speed for 15min to obtain the powder quality parameters. Negative control group without any substance, positive control group with potassium bromate (20. mu.g/g) and experimental group with recombinant rQSOX protein (0.2%, w/w, flour base).

As a result: compared with a negative control, after the potassium bromate and the rQSOX are added, the flour quality parameter changes obviously, and the dough stabilizing time and the dough quality number are improved obviously. However, the addition of recombinant rQSOX had a greater effect on flour stability time and mass number than the addition of the potassium bromate group (Table 1), indicating that recombinant rQSOX is superior to the "chemical" modifier potassium bromate in improving flour quality characteristics.

TABLE 1 comparison of the Effect of recombinant rQSOX and Potassium bromate on flour quality parameters

	Stabilization time (min)	Degree of weakness (FU)	Mass number
				Control	0.73±0.06b	178±6a	34±1.3b
Potassium bromate	1.03±0.06a	149±0b	43±2.1a
				rQSOX	1.16±0.05a	138±3c	46±1.7a

Note: potassium bromate addition level was 20. mu.g/g, recombinant rQSOX addition level was 0.2% (w/w, flour based). Different letters in the same column indicate significant differences (P < 0.05).

Example 5rQSOX method to improve bread baking quality

The method for improving bread baking quality by using the recombinant rice resting sulfhydryl oxidase is characterized by adopting the following process flow:

(1) materials: 100 parts of flour, 1.5 parts of yeast powder (Angel high-activity dry yeast sold in the market), 2 parts of salt, 60 parts of water, 7 parts of sugar, 4 parts of vegetable oil (Jinlongyu mixed oil sold in the market) and 0.5 percent (w/w, flour base) of recombinant rice resting sulfhydryl oxidase.

(2) Kneading: adding the above materials into dough kneading bowl, and stirring at 180r/min for 15min to form uniform dough.

(3) Molding: and placing the kneaded dough on a balance, dividing into 50 parts by weight per piece, rounding on a bread forming machine, forming and dishing.

(4) Fermentation: placing the dough-filled proving tray in a proving box for proving, wherein the temperature is controlled at 32 ℃, the relative humidity is 80%, and the fermentation time is 70 min.

(5) Baking: and (3) baking the proofed dough in an oven for 12min, wherein the oven temperature is 190 ℃, and obtaining a finished bread product.

And cooling the prepared bread for 2 hours at room temperature, and then carrying out bread texture quality analysis. Vertically cutting the bread into 2 cm/block, and determining the bread texture: the speed of the p/25 probe before and after measurement is 1mm/s, the speed after measurement is 5mm/s, the compression ratio is 50 percent, and the pressing interval is 10 s. No substance was added as a negative control, and potassium bromate (20. mu.g/g) was added as a positive control.

The bread texture result shows that the addition of potassium bromate or recombinant rQSOX can obviously reduce the hardness and viscosity of the bread and improve the elasticity of the bread, and both can improve the quality of the bread. Furthermore, there was no significance of differences between the breads to which recombinant rQSOX and potassium bromate were added (Table 2). Thus, the texture results further indicate that recombinant rQSOX can replace potassium bromate, exerting comparable bread quality improving ability.

TABLE 2 recombinant rQSOX and Potassium bromate impact on bread baking quality

	Hardness of	Viscosity	Elasticity
				Control	715±12a	418±16a	0.92±0.04b
Potassium bromate	620±23b	375±12b	0.95±0.09a
				rQSOX	580±17c	346±11c	0.96±0.11a

Note: potassium bromate addition levels were 20. mu.g/g, recombinant rQSOX addition levels were 0.5% (w/w, flour based). Different letters in the same column indicate significant differences (P < 0.05).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Bombycis of Guangdong province academy of agricultural sciences and institute of agricultural product processing

Application of rice resting sulfhydryl oxidase in improving flour processing quality

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1542

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of rice resting sulfhydryl oxidase

<400> 1

atggctgcgg cggcggtggc gcgccgcgtc gtcctggtgc tcgtcctcgc cgccgcctcc 60

ctcgccgcgg cgccgcgcgg ggcggccgct cgctcgctcg gcggcaggga gggccccggg 120

gaggtcgacg ccgacgccgc cgtcgacctc aacgccacca acttcgacgc cttcctcaag 180

gcctcgctgg agccctgggc cgtcgtcgag ttcttcgccc actggtgtcc agcttgcaga 240

aactacaagc ctcattatga gaaggttgca aaactattca atggtcggga cgctgcacat 300

ccagggttaa tactgatggc tagggttgat tgtgcatcaa aggtgaacat cgatctttgc 360

aatagattct cagttgacca ttaccctttc ctactttggg gtccaccaac aaaatttgct 420

tctgctaagt gggatcccaa gcaagagaat aatgaaataa agttaattga tgatggaaga 480

acagcagaac gtttactgaa gtggataaat aatcagatga aaagctcttt cagtttagaa 540

gacaaaaagt acgagaatga aaatatgctt ccaaagaatg cttcggatcc tgagcagatt 600

gttcaagcaa tttacgatgt tgaggaagca acagctcaag cgttacagat aattttggag 660

cgcaagacga tcaaaccaaa gaatcgtgat tcgctcatca gatttttaca aattttggtg 720

gctcgtcacc catccaagag gtgccgaagg ggatctgctg agctacttat taacttcgat 780

gatcactggt catcgaatct gtcgttaagt tcacaagagg gttctaaatt gttggaaagt 840

gttgcggaag agaaccactg gatctgtgga aaagaggtgc cacgtggata ttggctgttc 900

tgccgcggca gtaaaagtga aacaagagga tttagctgtg gtctatgggt tttgatgcat 960

tcactaaccg tccgaattgg ggatggagag agccagtcaa cctttacatc aatatgtgat 1020

tttattcaca acttcttcat ctgtgaggaa tgccgcaagc acttttatga aatgtgttca 1080

agcgtgtcag cccccttcag aactgctcgt gagctcagtc tctggttatg gagcacacat 1140

aacaaagtca atatgagatt gatgaaagaa gaaaaggata tgggaactgg tgatcccttg 1200

tttccgaagg ttacctggcc tccaaatcag ctctgcccat cttgctaccg ctcaagcaag 1260

gtcaccgatg gagctgtgga ctggaacgag gatgcagtgt atcaattctt ggttaactac 1320

tatggaaaga agcttgtatc atcctacaag gagacctaca tggagtctct tcagcaacaa 1380

gagaagaaga tagtttcaga ggattcatcg atttctaatg ctgcaagcgt cccaattgga 1440

gctgccctgg gtgttgcgat tgccagctgt acatttgggg cgctggcttg cttctggagg 1500

gcccagcaga agaacagaaa gcaaagaaag aactggaact ga 1542

<210> 2

<211> 513

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of rice resting thiol oxidase

<400> 2

Met Ala Ala Ala Ala Val Ala Arg Arg Val Val Leu Val Leu Val Leu

1 5 10 15

Ala Ala Ala Ser Leu Ala Ala Ala Pro Arg Gly Ala Ala Ala Arg Ser

20 25 30

Leu Gly Gly Arg Glu Gly Pro Gly Glu Val Asp Ala Asp Ala Ala Val

35 40 45

Asp Leu Asn Ala Thr Asn Phe Asp Ala Phe Leu Lys Ala Ser Leu Glu

50 55 60

Pro Trp Ala Val Val Glu Phe Phe Ala His Trp Cys Pro Ala Cys Arg

65 70 75 80

Asn Tyr Lys Pro His Tyr Glu Lys Val Ala Lys Leu Phe Asn Gly Arg

85 90 95

Asp Ala Ala His Pro Gly Leu Ile Leu Met Ala Arg Val Asp Cys Ala

100 105 110

Ser Lys Val Asn Ile Asp Leu Cys Asn Arg Phe Ser Val Asp His Tyr

115 120 125

Pro Phe Leu Leu Trp Gly Pro Pro Thr Lys Phe Ala Ser Ala Lys Trp

130 135 140

Asp Pro Lys Gln Glu Asn Asn Glu Ile Lys Leu Ile Asp Asp Gly Arg

145 150 155 160

Thr Ala Glu Arg Leu Leu Lys Trp Ile Asn Asn Gln Met Lys Ser Ser

165 170 175

Phe Ser Leu Glu Asp Lys Lys Tyr Glu Asn Glu Asn Met Leu Pro Lys

180 185 190

Asn Ala Ser Asp Pro Glu Gln Ile Val Gln Ala Ile Tyr Asp Val Glu

195 200 205

Glu Ala Thr Ala Gln Ala Leu Gln Ile Ile Leu Glu Arg Lys Thr Ile

210 215 220

Lys Pro Lys Asn Arg Asp Ser Leu Ile Arg Phe Leu Gln Ile Leu Val

225 230 235 240

Ala Arg His Pro Ser Lys Arg Cys Arg Arg Gly Ser Ala Glu Leu Leu

245 250 255

Ile Asn Phe Asp Asp His Trp Ser Ser Asn Leu Ser Leu Ser Ser Gln

260 265 270

Glu Gly Ser Lys Leu Leu Glu Ser Val Ala Glu Glu Asn His Trp Ile

275 280 285

Cys Gly Lys Glu Val Pro Arg Gly Tyr Trp Leu Phe Cys Arg Gly Ser

290 295 300

Lys Ser Glu Thr Arg Gly Phe Ser Cys Gly Leu Trp Val Leu Met His

305 310 315 320

Ser Leu Thr Val Arg Ile Gly Asp Gly Glu Ser Gln Ser Thr Phe Thr

325 330 335

Ser Ile Cys Asp Phe Ile His Asn Phe Phe Ile Cys Glu Glu Cys Arg

340 345 350

Lys His Phe Tyr Glu Met Cys Ser Ser Val Ser Ala Pro Phe Arg Thr

355 360 365

Ala Arg Glu Leu Ser Leu Trp Leu Trp Ser Thr His Asn Lys Val Asn

370 375 380

Met Arg Leu Met Lys Glu Glu Lys Asp Met Gly Thr Gly Asp Pro Leu

385 390 395 400

Phe Pro Lys Val Thr Trp Pro Pro Asn Gln Leu Cys Pro Ser Cys Tyr

405 410 415

Arg Ser Ser Lys Val Thr Asp Gly Ala Val Asp Trp Asn Glu Asp Ala

420 425 430

Val Tyr Gln Phe Leu Val Asn Tyr Tyr Gly Lys Lys Leu Val Ser Ser

435 440 445

Tyr Lys Glu Thr Tyr Met Glu Ser Leu Gln Gln Gln Glu Lys Lys Ile

450 455 460

Val Ser Glu Asp Ser Ser Ile Ser Asn Ala Ala Ser Val Pro Ile Gly

465 470 475 480

Ala Ala Leu Gly Val Ala Ile Ala Ser Cys Thr Phe Gly Ala Leu Ala

485 490 495

Cys Phe Trp Arg Ala Gln Gln Lys Asn Arg Lys Gln Arg Lys Asn Trp

500 505 510

Asn

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer F

<400> 3

ggcccatatg cgctcgctcg gcggcaggga 30

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer P

<400> 4

ggccgaattc gcttgcagca ttagaaatcg atg 33