CN116694606A - Method for hydrolyzing corn gluten meal prolamin by using keratinase - Google Patents
Method for hydrolyzing corn gluten meal prolamin by using keratinase Download PDFInfo
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- CN116694606A CN116694606A CN202310748011.7A CN202310748011A CN116694606A CN 116694606 A CN116694606 A CN 116694606A CN 202310748011 A CN202310748011 A CN 202310748011A CN 116694606 A CN116694606 A CN 116694606A
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- keratinase
- corn gluten
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- reaction
- prolamin
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- 108010059345 keratinase Proteins 0.000 title claims abstract description 82
- 240000008042 Zea mays Species 0.000 title claims abstract description 57
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 57
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 55
- 235000005822 corn Nutrition 0.000 title claims abstract description 55
- 108010068370 Glutens Proteins 0.000 title claims abstract description 54
- 235000021312 gluten Nutrition 0.000 title claims abstract description 47
- 235000012054 meals Nutrition 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000003301 hydrolyzing effect Effects 0.000 title claims abstract description 11
- 108060006613 prolamin Proteins 0.000 title description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 20
- 230000007062 hydrolysis Effects 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims description 33
- 238000002360 preparation method Methods 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims 2
- 235000009973 maize Nutrition 0.000 claims 2
- 235000018102 proteins Nutrition 0.000 abstract description 30
- 108090000623 proteins and genes Proteins 0.000 abstract description 30
- 102000004169 proteins and genes Human genes 0.000 abstract description 30
- 230000000694 effects Effects 0.000 abstract description 18
- 244000063299 Bacillus subtilis Species 0.000 abstract description 17
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 17
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 abstract description 6
- 229920002494 Zein Polymers 0.000 abstract description 6
- 239000005019 zein Substances 0.000 abstract description 6
- 229940093612 zein Drugs 0.000 abstract description 6
- 108091005658 Basic proteases Proteins 0.000 abstract description 5
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 244000144972 livestock Species 0.000 abstract description 5
- 108091005508 Acid proteases Proteins 0.000 abstract description 4
- 108091005507 Neutral proteases Proteins 0.000 abstract description 4
- 229940116540 protein supplement Drugs 0.000 abstract description 2
- 235000005974 protein supplement Nutrition 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- 230000007935 neutral effect Effects 0.000 abstract 1
- 108091005804 Peptidases Proteins 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 239000004365 Protease Substances 0.000 description 12
- 238000002156 mixing Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 239000007787 solid Substances 0.000 description 9
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 108090000145 Bacillolysin Proteins 0.000 description 3
- 102000011782 Keratins Human genes 0.000 description 3
- 108010076876 Keratins Proteins 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 239000005018 casein Substances 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000021240 caseins Nutrition 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 229920002261 Corn starch Polymers 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- -1 keratin in structure Chemical compound 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000407778 Bacillus subtilis TO-A Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001506304 Kadsura japonica Species 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- GEHJBWKLJVFKPS-UHFFFAOYSA-N bromochloroacetic acid Chemical class OC(=O)C(Cl)Br GEHJBWKLJVFKPS-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- 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
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Abstract
The application discloses a method for hydrolyzing corn gluten prolamin by using keratinase, belonging to the technical field of enzyme engineering. The application hydrolyzes corn gluten prolamine by using keratinase, and adopts 1mL keratinase crude enzyme with enzyme activity of 160000 to react for 3 hours at 60 ℃ to completely hydrolyze insoluble protein in 0.2g corn gluten, and can treat 0.322g corn gluten at most, which is superior to the hydrolysis capability of acid, neutral and alkaline proteases to the corn gluten. The zein adopts keratinase to hydrolyze, so that the generated soluble protein and amino acid are easier to be absorbed by livestock, and the corn gluten meal is better recycled; the keratinase related by the application is produced by fermenting bacillus subtilis, the strain is a food-safe strain, the produced keratinase can be used as an additional protein supplement in a corn gluten hydrolysis system, and the keratinase can be directly added into feed for livestock to use, so that additional economic benefits can be produced.
Description
Technical Field
The application relates to a method for hydrolyzing corn gluten prolamin by using keratinase, belonging to the technical field of enzyme engineering.
Background
Corn gluten meal is a common byproduct of a corn starch processing plant, and specifically refers to waste residue obtained after corn starch is extracted by a wet method, and the main components of the corn gluten meal comprise protein, cellulose, a small amount of starch, fat and the like. Wherein the protein content is about 70% of the solid content, and is a high-quality protein source for feed additives. However, most of proteins in the corn gluten meal are alcohol soluble proteins, are insoluble in water and are difficult to digest by livestock, and the characteristic limits the application range of the corn gluten meal, so that the resource waste is caused.
Prolamine, like keratin in structure, is a disulfide-rich hard protein. The hydrolysis efficiency of the common acid protease, neutral protease and alkaline protease on the market on the zein is poor. Therefore, it is highly desirable to find a method of sufficiently hydrolyzing zein.
Disclosure of Invention
The application provides a method for producing soluble protein and amino acid for feed addition by hydrolyzing corn gluten meal prolamin with keratinase with high hydrolysis efficiency.
The application provides a product of hydrolyzed corn gluten prolamin, which contains keratinase with an amino acid sequence shown as SEQ ID NO. 1.
In one embodiment of the application, the product includes, but is not limited to, a complex enzyme agent, genetically engineered bacteria expressing keratinase.
In one embodiment of the application, the product has at least the following keratinase addition: 100000U/mL.
In one embodiment of the application, the nucleotide sequence encoding the keratinase is shown in SEQ ID NO. 2.
The application provides a hydrolysis method of zein, which comprises the steps of adding keratinase, or an enzyme preparation containing the keratinase, or recombinant cells expressing the keratinase into a reaction system containing corn gluten or zein for hydrolysis reaction.
In one embodiment of the application, the amino acid sequence of the keratinase is shown in SEQ ID NO. 1.
In one embodiment of the application, the nucleotide sequence encoding the keratinase is shown in SEQ ID NO. 2.
In one embodiment of the application, the method is to mix corn gluten meal with keratinase to react, so as to hydrolyze prolamin in the corn gluten meal to obtain soluble protein and amino acid.
In one embodiment of the present application, the amount of the keratinase added to the reaction system is 100000 ～ 200000 U.mL -1 。
In one embodiment of the present application, the amount of the keratinase added to the reaction system is 130000 U.mL -1 。
In one embodiment of the present application, the amount of the keratinase added to the reaction system is 160000 U.mL -1 。
In one embodiment of the present application, the amount of the corn gluten meal added in the reaction system is 0.2 to 0.4 g.multidot.mL -1 。
In one embodiment of the present application, the reaction temperature is 40 to 80 ℃.
In one embodiment of the present application, the reaction temperature is 60 DEG C
In one embodiment of the application, the reaction pH is 7 to 8.
In one embodiment of the application, the reaction time is 1 to 4 hours.
The application also provides a genetically engineered bacterium, which expresses the keratinase.
In one embodiment of the application, the amino acid sequence of the keratinase is shown in SEQ ID NO. 1.
In one embodiment of the application, the nucleotide sequence encoding the keratinase is shown in SEQ ID NO. 2.
In one embodiment of the application, the genetically engineered bacterium is bacillus licheniformis, bacillus subtilis or escherichia coli as an expression host.
In one embodiment of the application, the genetically engineered bacterium is bacillus subtilis as an expression host.
In one embodiment of the application, the recombinant bacillus subtilis expresses a keratinase gene with a pP43NMK plasmid as an expression vector and bacillus subtilis WB600 as a host.
The application also provides a preparation method of the keratinase, which comprises the following steps: inoculating recombinant bacillus subtilis to a fermentation medium for fermentation, and centrifuging fermentation liquor after the fermentation is finished to obtain fermentation supernatant, namely keratinase crude enzyme liquid.
The application also provides application of the method in the aspect of producing soluble protein and amino acid by hydrolyzing corn gluten meal.
The application also provides application of keratinase or an enzyme preparation containing the keratinase or a host cell expressing the keratinase in hydrolyzing corn gluten meal, wherein the amino acid sequence of the keratinase is shown as SEQ ID NO. 1.
In one embodiment of the application, the use is to add keratinase or a complex enzyme containing keratinase or recombinant cells expressing keratinase to a system containing corn gluten meal for hydrolysis.
In one embodiment of the application, the host cell is bacterial or fungal as an expression host.
In one embodiment of the application, the nucleotide sequence encoding the keratinase is shown in SEQ ID NO. 2.
Advantageous effects
(1) According to the application, the corn gluten meal is hydrolyzed by utilizing the crude enzyme liquid of the keratinase, and the hydrolysis of the prolamine meal can be achieved to a larger extent by reacting for 3 hours at 60 ℃, so that more soluble protein is produced compared with the corn gluten meal hydrolyzed by other proteases sold in the market.
(2) The protease mainly used for hydrolyzing the corn gluten meal is alkaline protease at present, the optimal reaction pH of the protease is 10, but the pH of the corn gluten meal is slightly acidic. The zein adopts keratinase to hydrolyze, and the generated soluble protein and amino acid are easier to be absorbed by livestock, so that the corn gluten meal is better recycled.
(3) The keratinase related by the application is produced by fermenting bacillus subtilis, the strain is a food-safe strain, the produced keratinase can be used as an additional protein supplement in a corn gluten hydrolysis system, and the keratinase can be directly added into feed for livestock to use, so that additional economic benefits can be produced.
Drawings
Fig. 1: degree of hydrolysis of keratinase treated corn gluten meal for various times.
Fig. 2: increased amounts of soluble protein in the keratinase treated corn gluten meal for various times.
Fig. 3: measurement of optimal temperature for keratinase treatment of corn gluten meal.
Fig. 4: increased amounts of soluble protein in the corn gluten meal are treated with keratinase and a different commercially available protease.
Detailed Description
The pP43NMK plasmid referred to in the examples below was purchased from the Feng Hui organism; bacillus subtilis (Bacillus subtilis) WB600 referred to in the following examples is described in the patent application publication No. CN 102492645A; the reagents referred to in the examples below were purchased from national pharmaceutical group chemical company, ltd; the BCA protein concentration assay kit referred to in the examples below was purchased from the bi yun biotechnology company; the sulfonated keratins referred to in the examples below were purchased from the chemical industry development company of Boschiza (Shanghai); the acid protease, neutral protease and alkaline protease referred in the following examples were purchased from Shanghai source leaf Biotechnology Inc., under the respective numbers: s10012, S10013, S10154.
Soluble proteins involved in the following examples:
the application adopts BCA kit to detect the soluble protein, and the detected protein concentration comprises all soluble proteins and polypeptides, including keratinase, soluble protein in corn gluten and soluble protein generated after the corn gluten prolamin is hydrolyzed in a reaction system.
The following examples relate to the following media:
LB liquid medium: yeast powder 5 g.L -1 Tryptone 10 g.L -1 、NaCl 10g·L -1 。
LB solid medium: yeast powder 5 g.L -1 Tryptone 10 g.L -1 、NaCl 10g·L -1 20 g.L of agar powder -1 。
Bacillus subtilis fermentation medium: peptone 20 g.L -1 Yeast powder 10 g.L -1 Sucrose 20 g.L -1 、KH 2 PO 4 3g·L -1 、Na 2 HPO 4 6g·L -1 、MgSO 4 0.3g·L -1 。
The detection method involved in the following examples is as follows:
method for measuring keratin hydrolysis activity: 50. Mu.L of a properly diluted fermentation supernatant was taken, 150. Mu.L of a 50mM Gly/NaOH solution as a buffer and 100. Mu.L of 2.5% strength water-soluble keratin (available from Kadsura chemical industry Co., ltd., product code: K0043) as a substrate were added, and after mixing, reacted at 60℃for 20 minutes; the reaction was stopped by adding 200. Mu.L of 4% (w/v) trichloroacetic acid (TCA) and centrifuged at 8000r/min for 3min at room temperature. 200. Mu.L of the supernatant was taken and 1mL of 4% (w/v) Na was added 2 CO 3 And 200. Mu.L of Fu Lin Fen reagent, after mixing uniformly, developing at 50 ℃ for 10min, and measuring the absorbance of the clear liquid at 660nm by using a 0.5cm quartz cuvette; the experimental groups were 3 in parallel, the blank was prepared by adding the reaction terminator TCA prior to the addition of the substrate, and the rest of the procedure was the same. Definition of enzyme activity unit: OD at 60℃and pH 10 660 The amount of enzyme required per increase of 0.001 is one enzyme activity unit (U).
Protease enzyme activity determination method: preheating 1% casein solution in water bath at 40deg.C, adding 0.5mL enzyme solution into preheated 0.5mL casein solution, reacting at 40deg.C for 10min, and adding 1mL 0.4mol.L -1 Trichloroacetic acid (TCA) terminated the reaction. After the completion of the reaction, 1mL of Na was added to 0.2mL of the reaction solution 2 CO 3 Mixing the solutions, adding 0.2mL of Fu Lin Fen reagent, performing color development in water bath at 40deg.C for 20min, and measuring OD 680 . The amount of enzyme required for decomposing casein to 1. Mu.g of tyrosine at 40℃and pH 3.0 for 10min was defined as one enzyme activity unit (U.mL -1 )。
Protein concentration determination method: reference is made to BCA protein concentration assay kit instructions.
The method for calculating the degree of hydrolysis involved in the following examples is:
degree of hydrolysis (%) = (mass of dry corn gluten (g) before reaction-mass of dry matter (g) remaining after reaction)/mass of dry corn gluten (g) before reaction 100%.
The following examples relate to the following media:
LB liquid medium: yeast powder 5 g.L -1 Tryptone 10 g.L -1 、NaCl 10g·L -1 。
Bacillus subtilis fermentation medium: peptone 20 g.L -1 Yeast powder 10 g.L -1 Sucrose 20 g.L -1 、KH2PO4 3g·L -1 、Na2HPO4 6g·L -1 、MgSO4 0.3g·L -1 。
Example 1: preparation of keratinase
Shake flask fermentation, the specific steps are as follows:
(1) Construction of the pP43NMK-Ker recombinant plasmid is described in the text of Chinese patent application publication No. CN108728392A (the nucleotide sequence encoding the keratinase is shown as SEQ ID NO. 2).
(2) Preparation of recombinant bacillus subtilis
The recombinant plasmid pP43NMK-Ker obtained in the step (1) is transformed into bacillus subtilis (Bacillus subtilis) WB600, the transformation product is coated on LB solid medium containing kanamycin resistance, and the bacillus subtilis is cultured for 10 to 12 hours at 37 ℃ to obtain the recombinant bacillus subtilis for producing keratinase KerZ 1: WB600/pP43NMK-Ker.
(3) Preparation of crude enzyme solution
Placing the recombinant bacillus subtilis WB600/pP43NMK-Ker on an LB solid plate, streaking and activating, and selecting single colony to inoculate the strain containing 50 mug.mL -1 The seed solution was prepared by culturing in LB liquid medium of kanamycin at 37℃and 220rpm for 12 hours.
Then the seed solution was inoculated to a seed solution containing 50. Mu.g.multidot.mL in an inoculum size of 5% (v/v) -1 The bacillus subtilis fermentation medium of kanamycin is cultured for 24 hours at 220rpm at 37 ℃ to obtain a fermentation broth.
Centrifuging the fermentation broth at 10000rpm at 4deg.C for 10min to obtain fermentation supernatant, which is crude enzyme liquid of keratinase, and detecting to obtain crude enzyme liquid with enzyme activity of 130000 ～ 160000 U.mL -1 The pH of the crude enzyme solution is 7.0-8.0.
Example 2: determination of the time for hydrolysis of corn gluten meal by keratinase
The method comprises the following specific steps:
(1) Mixing 0.4g corn gluten meal with 1mL deionized water and 1mL enzyme activity of 130000 U.mL -1 After the crude keratinase solution (prepared in example 1) is fully mixed for 5min by vortex shaking, centrifugally sampling 10 microliters to measure the protein concentration in the supernatant, and reacting for 1h, 2h, 3h and 4h respectively at 60 ℃ after being evenly mixed again; the soluble protein content is measured by sampling before and after the reaction.
After the reaction was completed, the reaction was centrifuged at 12000rpm for 5 minutes, the supernatant was discarded, and the precipitate was washed twice with deionized water and dried at 105℃to constant weight, and the mass of the remaining solids (the remaining solids including insoluble prolamin, cellulose, starch, etc. which were not completely hydrolyzed) was measured, and the results are shown in Table 1:
table 1: soluble protein content before and after reaction at different reaction times
The results show that: as can be seen from FIGS. 1 and 2, the maximum hydrolysis level can be achieved in 3 hours, and the soluble protein content increases with the reaction time.
Example 3: optimization of temperature of keratinase hydrolyzed corn gluten meal
The method comprises the following specific steps:
(1) Mixing 0.4g corn gluten meal with 1mL deionized water and 1mL enzyme activity of 130000 U.mL -1 After thoroughly mixing the crude keratinase solution (prepared in example 1) by vortex shaking for 5min, centrifugally sampling 10 microliters to measure the protein concentration in the supernatant, uniformly mixing again, and reacting at 40-80 ℃ for 3h.
(2) Centrifuging at 12000rpm for 5min after the reaction, discarding supernatant, washing precipitate with deionized water twice, oven drying at 105deg.C to constant weight, and measuring the mass of the rest solid (rest solid comprises insoluble prolamin, cellulose, starch, etc. which is not completely hydrolyzed); detecting the hydrolysis degree before and after the reaction at different reaction temperatures; the results are shown in Table 2:
table 2: degree of hydrolysis before and after reaction at different reaction temperatures
As shown in FIG. 3, the optimal reaction temperature of keratinase at 60℃is the optimal temperature at which keratinase hydrolyzes corn gluten meal.
Example 4: comparison of keratinase with commercially available corn gluten meal treated with different proteases
The method comprises the following specific steps:
(1) Mixing 0.2g corn gluten meal with 1mL deionized water and 1mL enzyme activity of 130000 U.mL -1 Crude enzyme solution of keratinase (protease activity prepared in example 1)7000 U.mL -1 ) After thoroughly mixing for 5min by vortex shaking, 10 microlitres of the sample is centrifuged to determine the concentration of the soluble protein in the supernatant, and the mixture is reacted at 60 ℃ for 4h after being evenly mixed again.
(2) After completion of the reaction, the mixture was centrifuged at 12000rpm for 5 minutes, and the concentration of the soluble protein in the supernatant was measured.
(3) The specific steps are the same as the steps (1) to (2), except that the crude keratinase solution is replaced by commercial proteases, and the commercial proteases are respectively prepared as follows: 1mL of acid protease (protease activity 7000 U.mL) -1 ) 1mL neutral protease (protease activity 7000 U.mL) -1 ) 1mL alkaline protease (protease activity 7000 U.mL) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the soluble protein was measured as described above.
The experimental results are shown in table 3:
table 3: soluble protein content before and after the different protease treatment reactions
The results show that: as can be seen from FIG. 4, the hydrolysis of corn gluten meal by keratinase KerZ1 can produce more soluble proteins.
Example 5: determination of ability of keratinase to treat corn gluten meal
The method comprises the following specific steps:
(1) Mixing 0.4g corn gluten meal with 1mL deionized water and 1mL enzyme activity of 130000 U.mL -1 After thoroughly mixing the crude keratinase solution (prepared in example 1) by vortexing for 5min, 10. Mu.l of the supernatant was centrifuged to determine the concentration of soluble protein, and the mixture was reacted at 60℃for 3h after further mixing.
(2) After the reaction was completed, the mixture was centrifuged at 12000rpm for 5 minutes, the supernatant was discarded, and the precipitate was washed twice with deionized water, dried at 105℃to a constant weight, and the mass of the remaining solids was measured.
(3) The specific steps are the same as the steps (1) - (2), and the difference is that:
the crude enzyme liquid of the keratinase is adjusted as follows: 1mL of enzyme activity was 160000 U.mL -1 Keratinase of (a)Crude enzyme solution.
(4) The specific steps are the same as the steps (1) - (2), and the difference is that:
the addition amount of the corn gluten meal is adjusted to be 0.2g.
The degree of hydrolysis was calculated and the results are shown in Table 4.
TABLE 4 keratinase ability to treat corn gluten meal
The results show that: as can be seen from the data in Table 4, increasing the enzyme activity increases the ability of keratinase to treat corn meal.
While the application has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the application as defined in the appended claims.
Claims (10)
1. A product of hydrolyzed corn gluten prolamin contains keratinase with an amino acid sequence shown as SEQ ID NO. 1.
2. The product according to claim 1, wherein the product comprises a complex enzyme agent, a genetically engineered bacterium expressing the keratinase.
3. A method for hydrolyzing corn gluten prolamin is characterized in that the method is to add keratinase with an amino acid sequence shown as SEQ ID NO.1, or an enzyme preparation containing the keratinase, or a host cell expressing the keratinase into a system for preparing corn gluten or corn gluten prolamin for reaction.
4. A method according to claim 3, wherein the nucleotide sequence encoding the keratinase is set forth in SEQ ID No. 2.
5. The method according to claim 4, wherein the amount of the keratinase added to the reaction system is 100000 ～ 200000 U.mL -1 。
6. The method according to claim 5, wherein the concentration of the maize yellow powder in the system is 0.2-0.4 g.ml -1 。
7. The method according to claim 6, wherein the reaction temperature is 40-80 ℃, the pH is 7-8, and the reaction time is 1-4 h.
8. Use of a keratinase or an enzyme preparation containing a keratinase or a host cell expressing a keratinase for hydrolyzing maize yellow meal, characterized in that the amino acid sequence of the keratinase is shown in SEQ ID No. 1.
9. The use according to claim 8, characterized in that the hydrolysis is performed by adding keratinase or a complex enzyme agent containing keratinase or recombinant cells expressing keratinase to a system containing corn gluten meal.
10. The use according to claim 8 or 9, wherein the host cell is bacterial or fungal as an expression host.
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