LU505017B1 - Application of aspergillus cristatus glucose oxidase in improvement of processing quality of flour - Google Patents
Application of aspergillus cristatus glucose oxidase in improvement of processing quality of flour Download PDFInfo
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- LU505017B1 LU505017B1 LU505017A LU505017A LU505017B1 LU 505017 B1 LU505017 B1 LU 505017B1 LU 505017 A LU505017 A LU 505017A LU 505017 A LU505017 A LU 505017A LU 505017 B1 LU505017 B1 LU 505017B1
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- glucose oxidase
- aspergillus cristatus
- flour
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- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 87
- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 80
- 241001205401 Aspergillus cristatus Species 0.000 title claims abstract description 77
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 77
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 77
- 235000013312 flour Nutrition 0.000 title claims abstract description 32
- 230000006872 improvement Effects 0.000 title claims abstract description 14
- 238000012545 processing Methods 0.000 title claims abstract description 11
- 235000008429 bread Nutrition 0.000 claims abstract description 39
- 108010068370 Glutens Proteins 0.000 claims abstract description 21
- 235000021312 gluten Nutrition 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 238000003259 recombinant expression Methods 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 14
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 229940088598 enzyme Drugs 0.000 claims description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 13
- 241000235058 Komagataella pastoris Species 0.000 claims description 12
- 239000013604 expression vector Substances 0.000 claims description 11
- 239000000843 powder Substances 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
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- 238000012258 culturing Methods 0.000 claims description 8
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- 239000002609 medium Substances 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 6
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- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
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- 239000007788 liquid Substances 0.000 claims description 5
- 235000000346 sugar Nutrition 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 235000015112 vegetable and seed oil Nutrition 0.000 claims description 5
- 239000008158 vegetable oil Substances 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 235000012180 bread and bread product Nutrition 0.000 claims description 4
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- 238000012217 deletion Methods 0.000 claims description 2
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- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 8
- 230000000975 bioactive effect Effects 0.000 abstract description 2
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- 235000012794 white bread Nutrition 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
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- 238000002835 absorbance Methods 0.000 description 3
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- 239000013613 expression plasmid Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 235000011844 whole wheat flour Nutrition 0.000 description 3
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 2
- 240000002791 Brassica napus Species 0.000 description 2
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 235000021552 granulated sugar Nutrition 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000011449 brick Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000007986 glycine-NaOH buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 235000011845 white flour Nutrition 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
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Abstract
Disclosed is an application of Aspergillus cristatus glucose oxidase in improvement of processing quality of flour, and belongs to the fields of gene engineering and cereal science. In the present disclosure, a eukaryotic expression method is adopted to obtain a bioactive recombinant Aspergillus cristatus glucose oxidase that has a high expression level, is easy to purify, is easy to amplify, and is suitable for industrial application. The present disclosure provides a method for improving the processing quality of flour by adding Aspergillus cristatus glucose oxidase only, and direct addition of Aspergillus cristatus glucose oxidase may significantly reduce the sulfhydryl content of gluten, strengthen the network structure of gluten, increase the specific volume of bread, and reduce the firmness and chewiness of bread, so Aspergillus cristatus glucose oxidase is a potential novel flour improver.
Description
APPLICATION OF ASPERGILLUS CRISTATUS GLUCOSE OXIDASE IN
IMPROVEMENT OF PROCESSING QUALITY OF FLOUR 17905017
The present disclosure belongs to the fields of gene engineering and cereal science, and specifically, relates to a Pichia pastoris recombinant Aspergillus cristatus glucose oxidase and a preparation method thereof, and an application of the Pichia pastoris recombinant Aspergillus cristatus glucose oxidase in improvement of processing quality of flour.
With the improvement of living standards, people's demand for flour products, such as bread, is gradually increasing, and the demand for high-quality flour is also gradually increasing.
Although there are many varieties of domestic wheat in China, the produced flour generally has the characteristics of "high but not strong gluten, low but not weak gluten”, which is difficult to reach the standard of high-quality flour. Enzyme preparations, as green natural flour improvers, have wide application prospects.
Currently, glucose oxidases are gradually being applied to the bread baking industry. However, varieties and activities of the existing glucose oxidases cannot completely meet the requirements of the flour product processing, and it is urgent to further develop a novel high-quality glucose oxidase. How to provide a glucose oxidase and an excellent production strain is a problem that needs to be solved by those skilled in the art urgently.
Aspergillus cristatus, also referred to as Eurotium cristatum and commonly known as "golden flower", is a predominant probiotic fungus found in Fuzhuan brick tea. Aspergillus cristatus glucose oxidase has typical characteristics of glucose oxidases, is a flavin-dependent protein, and belongs to the glucose/methanol/choline oxidoreductase family. Currently, there are few studies on Aspergillus cristatus glucose oxidase in China and other countries, and there is no report on use of the enzyme as a flour improver in improvement of quality of a flour product.
In order to overcome the shortcomings and deficiencies in the prior art, an objective of the 1 present disclosure is to provide an application of Aspergillus cristatus glucose oxidase in
LU505017 improvement of processing quality of flour.
The objective of the present disclosure is achieved by the following technical solutions.
The present disclosure provides an application of Aspergillus cristatus glucose oxidase in improvement of processing quality of flour.
Specifically, the present disclosure provides an application of Aspergillus cristatus glucose oxidase in improvement of gluten and quality of bread.
The purpose of improving the baking quality of bread may be achieved by adding Aspergillus cristatus glucose oxidase only.
An addition amount of Aspergillus cristatus glucose oxidase to flour is 2-8 ppm (based on the flour), and further, 4-8 ppm (based on the flour), and Aspergillus cristatus glucose oxidase may improve the specific volume of bread and reduce the firmness and chewiness of bread.
An amino acid sequence of Aspergillus cristatus glucose oxidase is represented by SEQ ID
NO. 1. The present disclosure further provides a DNA molecule encoding Aspergillus cristatus glucose oxidase, and a base sequence of the DNA molecule is represented by SEQ ID NO. 2.
Preferably, a polypeptide sequence contains a polypeptide having at least 90% sequence identity with SEQ ID NO. 1, preferably, having at least 95% sequence identity with SEQ ID NO. 1, and most preferably, having 100% sequence identity with SEQ ID NO. 1, or contains a polypeptide derived from SEQ ID NO. 1, which is obtained by modifying SEQ ID NO. 1 through substitution, deletion and/or insertion of one or more amino acids and has more than 90% homology with SEQ ID NO. 1.
Preferably, Aspergillus cristatus glucose oxidase, the flour, sugar, salt, vegetable oil, yeast powder, and water are uniformly stirred, and formed dough is cut, weighed, shaped, fermented, and baked to obtain bread products.
Further, addition amounts of the components are as follows: 90-110 parts by weight of flour, 0.6-1.8 parts by weight of yeast powder, 1-3 parts by weight of salt, 50-70 parts by weight of water, 2
5-10 parts by weight of sugar, and 2-5 parts by weight of vegetable oil.
LU505017
Aspergillus cristatus glucose oxidase may be obtained by fermenting Pichia pastoris transformed with a eukaryotic recombinant expression vector.
The eukaryotic recombinant expression vector is a eukaryotic recombinant expression vector containing Aspergillus cristatus glucose oxidase gene.
Preferably, a starting vector of the eukaryotic recombinant expression vector includes, but is not limited to, pPICZaA.
Preferably, the Pichia pastoris includes, but is not limited to, Pichia pastoris X33.
A Pichia pastoris recombinant expression method for Aspergillus cristatus glucose oxidase includes the following steps: (1) obtaining a codon-optimized Aspergillus cristatus glucose oxidase gene (a sequence is represented by SEQ ID NO. 2) by whole gene synthesis or PCR, joining Aspergillus cristatus glucose oxidase gene to a eukaryotic expression vector, and transforming the obtained eukaryotic recombinant expression vector into Pichia pastoris competent cells to obtain recombinant expression strains ; (2) inoculating the recombinant expression strains constructed in step (1) into a seed medium, culturing by shaking overnight, centrifuging at room temperature, collecting veast ceils, transferring the yeast ceils to a fermentation medium to form a yeast solution with ODeoo of 0.5- 1.0, culturing, adding methanol to the culture medium to 0.5-2.0% v/v every 24 h during culturing, performing solid-liquid separation, and collecting a fermentation supernatant; and (3) collecting the fermentation supernatant obtained in step (2) to obtain a recombinant
Aspergillus cristatus glucose oxidase protein.
In step (2), the recombinant expression strains are cultured by shaking overnight at 25-35°C and 150-250 rpm, and further, at 30°C and 250 rpm; and 3 the cultured yeast cells are centrifuged at room temperature and 2,500-3,000 g for 2-5 min, and further, at 3,000 g for 2 min.
The transferred yeast ceils are cultured at 25-35°C and 150-250 rpm for 24-144 h, and further, at 30°C and 250 rpm for 24-144 h.
Preferably, methanol is added to 1.0% v/v.
Compared with the prior art, the present disclosure has the following advantages and effects.
In the present disclosure, a eukaryotic expression method is adopted to obtain a bioactive recombinant Aspergillus cristatus glucose oxidase. Direct addition of Aspergillus cristatus glucose oxidase may significantly reduce the sulfhydryl content of gluten, strengthen the network structure of gluten, enhance the fermentability of dough, increase the specific volume of bread, and reduce the firmness and chewiness of bread, so Aspergillus cristatus glucose oxidase is a potential novel flour improver.
Fig. 1 shows double digestion results of a recombinant expression plasmid of Example 1, wherein lane M: a molecular weight marker; and lane 1: a double digestion product of the recombinant expression plasmid;
Fig. 2 shows SDS-PAGE analysis results of a recombinant Aspergillus cristatus glucose oxidase of Example 1, wherein lane M: a molecular weight marker; lane 1: the recombinant
Aspergillus cristatus glucose oxidase treated with a reductant B-mercaptoethanol; and lane 2: the recombinant Aspergillus cristatus glucose oxidase without reductant treatment;
Fig. 3 shows enzymatic property analysis results of a recombinant Aspergillus cristatus glucose oxidase of Example 2, wherein A: optimal reaction temperature; B: optimal reaction pH;
C: temperature stability; and D: pH stability; and
Fig. 4 shows influences of a recombinant Aspergillus cristatus glucose oxidase of Example 4 on the specific volume of bread, wherein the experiment is repeated 3 times, and different 4 superscript letters in each column indicate significant differences (p<0.05).
LU505017
The present disclosure will be further described in detail below with reference to examples and drawings, and implementation modes of the present disclosure are not limited thereto.
Test methods without indicating specific experimental conditions in the following examples usually follow the conventional experimental conditions or experimental conditions recommended by manufacturers. Unless otherwise described, reagents and raw materials used herein are all commercially available products or products prepared by recognized methods.
Example 1 Obtaining of Aspergillus cristatus glucose oxidase
A whole gene sequence of Pichia pastoris codon-optimized Aspergillus cristatus glucose oxidase was obtained by whole gene synthesis. An amino acid sequence of Aspergillus cristatus glucose oxidase was represented by SEQ ID NO. 1, and a base sequence encoding Aspergillus cristatus glucose oxidase was represented by SEQ ID NO. 2. Double digestion was performed on the sequence of Aspergillus cristatus glucose oxidase and a vector plasmid pPICZaA by using
EcoRI and Notl, a ligation reaction was performed by using T4 DNA ligase, and a ligation product was transformed into Escherichia coli DH5a competent cells. A single colony was picked, and a plasmid was extracted for electrophoresis detection and stored at -20°C. A target fragment (see Fig. 1) was detected by digestion using EcoRI and Not], the plasmid was transferred to a company for sequencing, and a correctly-sequenced plasmid was a recombinant expression plasmid pPICZoA- cgod of the codon-optimized Aspergillus cristatus glucose oxidase. The correctly-sequenced plasmid was transformed into host cells Pichia pastoris to construct Aspergillus cristatus recombinant expression strains. Aspergillus cristatus recombinant expression strains were inoculated into 10 mL of seed medium, cultured by shaking at 30°C and 250 rpm overnight, and centrifuged at room temperature and 3,000 g for 2 min, yeast cells were collected, transferred to a fermentation medium to form a yeast solution with OD600 of 0.5-1.0, and cultured at 30°C and 250 rpm for 24-144 h, the yeast solution was sampled and methanol was added to the culture medium 1.0% v/v every 24 h during culturing, and after the culturing was completed, solid-liquid separation was performed to obtain a fermentation supernatant. SDS-PAGE analysis results (see
Fig. 2) show that the recombinant strains may induce the expression of the recombinant Aspergillus cristatus glucose oxidase. 5
Example 2 Enzymatic property assay of glucose oxidase HUS050T7 1. Enzyme activity assay 1.25 mL of 0.2 mol/L phosphate buffer (pH=6.0), 1.25 mL of 0.21 mmol/L reduced o-
Dianisidine, 0.3 mL of 18 mg/mL glucose, 100 uL of 60 U/mL horseradish peroxidase solution, and 100 pL of appropriately diluted enzyme solution were thoroughly mixed, and changes in absorbance of the enzyme at 500 nm and 40°C were detected. Enzyme activity was defined as that: the amount of enzyme required for catalyzing 1 pmol of D-glucose to produce D-gluconic acid and H:02 under the foregoing conditions was defined as one unit (U). 2. Enzymatic property assay
The enzyme activity of the recombinant glucose oxidase prepared in Example 2 was measured at different temperatures (20-70°C) and different pH values (3.0-8.0) by the foregoing enzyme activity assay method to determine the optimal reaction temperature and optimal reaction pH of the recombinant glucose oxidase. The enzyme solutions were respectively placed at different temperature (4-80°C) for heat preservation for 1 h, and the residual enzyme activity of the glucose oxidase was measured to determine the temperature stability of the glucose oxidase. The enzyme solutions were respectively placed in buffers with different pH values (3.0-10.0) at room temperature for treatment for 12 h, and the residual enzyme activity of the glucose oxidase was measured to study the pH stability of the glucose oxidase. A 100 mM citrate buffer with a pH value of 3.0-6.0, a 100 mM phosphate buffer with a pH value of 6.0-8.0, a 100 mM Tris-HCI buffer with apH value of 8.0-9.0, and a 100 mM glycine-NaOH buffer with a pH value of 9.0-10.0 were used.
Results show that the optimal temperature of Aspergillus cristatus glucose oxidase is 40°C (see Fig. 3A), and the optimal reaction pH is 6.0 (see Fig. 3B). After being treated at the temperature lower than 60°C for 1 h, the recombinant Aspergillus cristatus glucose oxidase may maintain more than 80% of enzyme activity (see Fig. 3C). After being treated at a pH value of 4.0- 9.0 for 12 h, the glucose oxidase may maintain more than 80% of enzyme activity (see Fig. 3D).
Example 3 Application of recombinant Aspergillus cristatus glucose oxidase in reduction of sulfhydryl content of gluten 6
1. Preparation of gluten
LU505017 100 g of whole-wheat flour, 58 g of purified water, and the recombinant Aspergillus cristatus glucose oxidase were placed into a dough making bowl and stirred to form uniform dough. The formed dough was fermented at 30°C for 30 min, and washed with water to remove starch, and the remaining part was gluten. The gluten was collected, lyophilized, and ground into powder for later use, and gluten without the enzyme was taken as a control. 2. Measurement of sulfhydryl content of gluten 25 mg of gluten was added to 0.5 mL of Tris-glycine protein denaturation buffer (composed of 86 mM tris(hydroxymethyl)aminomethane (Tris), 90 mM glycine, 4 mM ethylene diamine tetraacetic acid, and 8 M urea, pH=8.0), and the mixture was subjected to vortex shaking for 10 min and centrifuged at 15,000 g for 5 min. Then, 100 pL of supernatant was pipetted and added to 150 pL of Tris-glycine protein denaturation buffer and 50 pL of freshly prepared 4 mg/mL 5,5"- dithio-bis-(2-nitrobenzoic acid) solution. 100 pL of mixed solution was pipetted and added to a 96-well plate, and incubated at 30°C for 30 min, and the absorbance of the mixed solution at 412 nm was measured. A buffer with the same volume was taken as a blank control. Reduced glutathione was taken as a standard sample to prepare solutions at a series of standard — concentrations, the absorbance of reduced glutathione was measured by the foregoing method, and a standard curve was drawn.
Results (see Table 1) show that compared with the control group, the free sulfhydryl content of the dough added with the recombinant Aspergillus cristatus glucose oxidase is reduced from 148.79 uM to 116.36 uM, a reduction of 21.80%. In most cases, the strengthening of the network structure of gluten is related to the increase of the number of disulfide bonds between gluten molecules, and the reduction of the sulfhydryl content may indirectly reflect the increase of disulfide bonds in gluten. Therefore, the measurement results of the sulfhydryl content indicate that the addition of the recombinant Aspergillus cristatus glucose oxidase promotes crosslinking of disulfide bonds in gluten, so as to strengthen the network structure of gluten and improve the processing quality of flour.
Table 1 Influences of the recombinant Aspergillus cristatus glucose oxidase on the sulthydryl content of gluten 7
Addition amount of recombinant Aspergillus cristatus Sulfhydryl content (uM) LU505017 glucose oxidase (based on flour)
Control group 148.79+3.78a
Note: the experiment is repeated 3 times, and different superscript letters in each column indicate significant differences (p<0.05).
Example 4 Application of recombinant Aspergillus cristatus glucose oxidase in improvement of quality of white bread 1. Making of bread
A recipe for bread was: 100 parts by weight of flour, 1 part by weight of yeast powder, 1.6 parts by weight of salt, 58 parts by weight of water, 6 parts by weight of sugar, 3 parts by weight of vegetable oil, and the recombinant Aspergillus cristatus glucose oxidase.
A process for making bread was that: the weighed water, white flour, white granulated sugar, edible salt, yeast powder, and glucose oxidase were placed into a dough making bowl and stirred for 18 min to form uniform dough. The formed dough was placed on a balance, cut according to 50 g/piece, kneaded into a round shape, and fermented at 38°C for 60 min, and fermented dough was baked in an oven at 180°C for 10 min to obtain a bread product. The obtained bread was cooled at room temperature for 2 h, and the mass and volume of the bread were measured. The volume of the bread was measured by the rapeseed displacement method, and the specific volume was equal to the volume of the bread / the mass of the bread. The texture of the bread was measured under conditions as follows: the bread was cut according to 2 cm/piece, a 25 mm cylindrical aluminum probe was used, the pre-test speed and the test speed were set as 1 mm/s, the post-test speed was set as 5 mm/s, the compression ratio was 50%, and a time interval between two compressions was 10 s. The firmness and chewiness of the bread were recorded.
Results show that compared with the control group, the specific volume of the white bread added with Aspergillus cristatus glucose oxidase is significantly increased (see Fig. 4). Texture 8 analysis results of the bread show that the firmness and chewiness of the white bread are significantly reduced, and the springiness is significantly improved (see Table 2), which indicates 505017 that the addition of Aspergillus cristatus glucose oxidase may improve the quality of the white bread.
Table 2 Influences of the recombinant Aspergillus cristatus glucose oxidase on texture properties of white bread
Addition amount of recombinant
Aspergillus cristatus glucose Firmness Springiness Chewiness oxidase (based on flour)
Lea [own [ws
Note: the experiment is repeated 3 times, and different superscript letters in each column indicate significant differences (p<0.05).
Example 5 Application of recombinant Aspergillus cristatus glucose oxidase in improvement of quality of whole-wheat bread 1. Making of bread
A recipe for bread was: 100 parts by weight of whole-wheat flour, 1 part by weight of yeast powder, 1.6 parts by weight of salt, 56 parts by weight of water, 6 parts by weight of sugar, 3 parts by weight of vegetable oil, 2.4 parts by weight of milk powder, 8 parts by weight of egg liquid, and the recombinant Aspergillus cristatus glucose oxidase.
A process for making bread was that: the weighed water, whole-wheat flour, white granulated sugar, milk powder, egg liquid, edible salt, yeast powder, and glucose oxidase were placed into a dough making bowl and stirred for 18 min to form uniform dough. The formed dough was placed at 26-30°C for primary fermentation for 30 min, placed on a balance, cut according to 50 g/piece, kneaded into a round shape, and placed at 36-38°C for secondary fermentation for 60 min, and 9 fermented dough was baked in an oven at 180°C for 10 min to obtain a whole-wheat bread product.
The obtained bread was cooled at room temperature for 2 h, and the mass and volume of the bread 505017 were measured. The volume of the bread was measured by the rapeseed displacement method, and the specific volume was equal to the volume of the bread / the mass of the bread. The texture of the bread was measured under conditions as follows: the bread was cut according to 2 cm/piece, a 25 mm cylindrical aluminum probe was used, the pre-test speed and the test speed were set as 1 mm/s, the post-test speed was set as 5 mm/s, the compression ratio was 50%, and a time interval between two compressions was 10 s. The firmness and chewiness of the bread were recorded.
Results (see Table 3) show that compared with the control group, the specific volume and springiness of the whole-wheat bread added with Aspergillus cristatus glucose oxidase are significantly increased, and the firmness and chewiness are significantly reduced, which indicates that the addition of Aspergillus cristatus glucose oxidase may improve the quality of the whole- wheat bread.
Table 3 Influences of the recombinant Aspergillus cristatus glucose oxidase on the quality of whole-wheat bread
Addition amount of recombinant
Aspergillus cristatus | Specific volume Firmness Springiness Chewiness glucose oxidase (based on flour)
C0 [enw | ono [orm | wm
Note: the experiment is repeated 3 times, and different superscript letters in each column indicate significant differences (p<0.05).
The foregoing examples are preferred implementation modes of the present disclosure, and the implementation modes of the present disclosure are not limited to the foregoing examples. Any other changes, modifications, replacements, combinations, and simplifications made without 10 departing from the spirit and principle of the present disclosure shall be equivalent replacement
LU505017 methods and shall fall within the scope of protection of the present disclosure. 11
Claims (10)
1. An application of Aspergillus cristatus glucose oxidase in improvement of processing quality of flour.
2. The application according to claim 1, wherein the application is an application of addition of enzyme preparation mainly containing Aspergillus cristatus glucose oxidase only in improvement of processing quality of flour.
3. The application according to claim 1 or 2, wherein the application is an application of Aspergillus cristatus glucose oxidase in improvement of gluten and quality of bread.
4. The application according to claim 1 or 2, wherein an addition amount of Aspergillus cristatus glucose oxidase to flour is 2-8 ppm (based on the flour), and further, 4-8 ppm (based on the flour).
5. The application according to claim 1 or 2, wherein an amino acid sequence of Aspergillus cristatus glucose oxidase is represented by SEQ ID
NO. 1.
6. The application according to claim 5, wherein a base sequence of a DNA molecule encoding Aspergillus cristatus glucose oxidase is represented by SEQ ID NO. 2, and preferably, a polypeptide sequence contains a polypeptide having at least 90% sequence identity with SEQ ID NO. 1, preferably, having at least 95% sequence identity with SEQ ID NO. 1, and most preferably, having 100% sequence identity with SEQ ID NO. 1, or contains a polypeptide derived from SEQ ID NO. 1, which is obtained by modifying SEQ ID NO. 1 through substitution, deletion and/or insertion of one or more amino acids and has more than 90% homology with SEQ ID NO. 1.
7. The application according to claim 1 or 2, wherein Aspergillus cristatus glucose oxidase, the flour, sugar, salt, vegetable oil, yeast powder, and water are uniformly stirred, and formed dough is cut, weighed, shaped, fermented, and baked to obtain bread products. 12
8. The application according to claim 1 or 2, wherein 17905017 Aspergillus cristatus glucose oxidase is obtained by fermenting Pichia pastoris transformed with a eukaryotic recombinant expression vector, and the eukaryotic recombinant expression vector is a eukaryotic recombinant expression vector containing Aspergillus cristatus glucose oxidase gene.
9. The application according to claim 8, wherein a Pichia pastoris recombinant expression method for Aspergillus cristatus glucose oxidase comprises the following steps: (1) obtaining a codon-optimized Aspergillus cristatus glucose oxidase gene by whole gene synthesis, joining the gene to a eukaryotic expression vector, and transforming the obtained eukaryotic recombinant expression vector into Pichia pastoris competent cells to obtain recombinant expression strains; and (2) inoculating the recombinant expression strains constructed in step (1) into a seed medium, culturing by shaking overnight, centrifuging at room temperature, collecting yeast cells, transferring the yeast cells to a fermentation medium to form a yeast solution with OD600 of 0.5-
1.0, culturing, adding methanol to the culture medium to 0.5-2.0% v/v every 24 h during culturing, performing solid-liquid separation, and collecting a fermentation supernatant to obtain Aspergillus cristatus glucose oxidase protein.
10. The application according to claim 9, wherein in step (2), the recombinant expression strains are cultured by shaking overnight at 25-35°C and 150-250 rpm; in step (2), the yeast cells are centrifuged at room temperature and 2,500-3,000 g for 2-5 min; and in step (2), the yeast cells are cultured at 25-35°C and 150-250 rpm for 24-144 h. 13
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