CN108179143B - Magnetic graphene oxide immobilized enzyme and application thereof in wet corn soaking - Google Patents

Magnetic graphene oxide immobilized enzyme and application thereof in wet corn soaking Download PDF

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CN108179143B
CN108179143B CN201810239162.9A CN201810239162A CN108179143B CN 108179143 B CN108179143 B CN 108179143B CN 201810239162 A CN201810239162 A CN 201810239162A CN 108179143 B CN108179143 B CN 108179143B
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graphene oxide
immobilized enzyme
ferrous
ferric
salt
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CN108179143A (en
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刘庆艾
史建国
马耀宏
杨俊慧
孟庆军
王丙莲
马恒
李秋顺
公维丽
蔡雷
郑岚
高广恒
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Shandong Keqiao Biotechnology Co.,Ltd.
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Biology Institute of Shandong Academy of Sciences
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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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00—Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/04—Extraction or purification
    • C08B30/042—Extraction or purification from cereals or grains
    • C08B30/044—Extraction or purification from cereals or grains from corn or maize
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    • 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)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22002—Papain (3.4.22.2)

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Abstract

The invention relates to a magnetic graphene oxide immobilized enzyme and application thereof in wet corn soaking, wherein the magnetic graphene oxide immobilized enzyme can quickly separate corn starch from protein, has simple process steps, high corn starch yield and low protein content, and basically keeps the corn starch yield and the product quality after the magnetic graphene oxide immobilized enzyme is recycled for 10 times.

Description

Magnetic graphene oxide immobilized enzyme and application thereof in wet corn soaking
Technical Field
The invention belongs to the field of corn starch extraction, and particularly relates to a magnetic graphene oxide immobilized enzyme and application thereof in wet corn soaking.
Background
The traditional corn starch extraction process adopts old pulp and new acid (0.2 percent of sulfurous acid solution) to be soaked together in a soaking tank, the whole soaking process comprises the steps of adding the old pulp in a newly-installed tank, adding the new acid to the corn with long soaking time, and regularly circulating the tank and the tank. The circulation process can maintain a certain concentration difference between the soluble substances in the corn and the soaking solution, so as to soak out the soluble substances in the corn. However, sulfur dioxide generated in the traditional soaking process is a main component of acid rain, so that environmental pollution is caused, and meanwhile, sulfurous acid can corrode equipment and pipelines, so that the production cost is increased. In the final product, the residue of sulfur dioxide can affect the quality of the starch product and the utilization of byproducts, reducing economic benefits. Therefore, it is urgent to find a new soaking extraction process that can replace sulfurous acid.
Disclosure of Invention
The invention provides a magnetic graphene oxide immobilized enzyme, which is characterized in that the preparation method of the magnetic graphene oxide immobilized enzyme comprises the following steps:
(1) adding Graphene Oxide (GO) into 30-50% ethanol solution by volume fraction, ultrasonically dispersing for 0.5h, adding ferrous salt and ferric salt while stirring, continuously stirring for 30min, reacting at 50 ℃ to reflux temperature for 1-2 h, adding ammonia water to adjust pH to 11, continuously stirring for 1-2 h, and performing magnetic separation, water washing and drying to obtain GO/Fe3O4A composite magnetic material;
(2) the GO/Fe obtained in the step (1)3O4And (3) placing the composite magnetic material and papain in a phosphate buffer solution, carrying out ultrasonic treatment for 30-45min, and then carrying out magnetic separation, water washing and drying to obtain the magnetic graphene oxide immobilized enzyme.
The ferrous salt in the step (1) is preferably one or more of ferrous chloride, ferrous sulfate, ferrous nitrate or hydrates thereof, and the ferric salt is preferably one or more of ferric chloride, ferric sulfate, ferric nitrate or hydrates thereof; 40-60mL of ethanol solution is used for every gram of Graphene Oxide (GO), and 1.5-2.5mmol of ferrous salt is used; wherein the molar ratio of ferrous salt to ferric salt is 1: 2;
GO/Fe in step (2)3O4The mass ratio of the composite magnetic material to the papain is 10:0.5-0.8, and the phosphoric acidThe pH of the salt buffer solution is preferably 6-7, per gram GO/Fe3O480-100mL of phosphate buffer solution is used as the composite magnetic material;
the frequency of the ultrasound in the steps (1) and (2) is preferably 30-40 KHz.
Another embodiment of the present invention provides a preparation method of the magnetic graphene oxide immobilized enzyme, including the above steps.
Another embodiment of the present invention provides a process for extracting corn starch by wet steeping, which is characterized by comprising the following steps:
crushing the degermed corn to 20-40 meshes, adding water and the magnetic graphene oxide immobilized enzyme, stirring and soaking for 6-10 hours at 30-50 ℃, recovering the magnetic graphene oxide immobilized enzyme through magnetic separation, transferring the rest soaked mixture into a crusher for fine crushing, sieving by a 200-mesh sieve, standing the filtrate overnight, performing centrifugal separation, and drying to obtain the corn starch.
The degerming corn can be commercially available degerming corn or can be degerming by a corn degerming machine;
the using amount of the water is 3-5 times of the mass of the degerming corn, and the using amount of the magnetic graphene oxide immobilized enzyme is 0.1-0.2% of the mass of the degerming corn.
Another embodiment of the invention provides an application of the magnetic graphene oxide immobilized enzyme in corn starch extraction.
Compared with the prior art: the method disclosed by the invention has the advantages that the corn starch is extracted by using the magnetic graphene oxide immobilized enzyme, so that the defects in the traditional process are overcome, the corn starch yield is high, the protein content is low, the using amount of the magnetic graphene oxide immobilized enzyme is small, and the corn starch yield and the product quality are basically unchanged after the magnetic graphene oxide immobilized enzyme is recycled for 10 times.
Detailed Description
In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.
Example 1
(1) Adding 10g of Graphene Oxide (GO) into 600mL of 30% ethanol solution, ultrasonically dispersing for 0.5h at 30KHz, adding ferrous chloride (15mmol) and ferric chloride (30mmol) while stirring, continuously stirring for 30min, reacting at 50 ℃ for 2 h, adding ammonia water to adjust pH to 11, continuously stirring for 1 h, and obtaining GO/Fe through magnetic separation, water washing and drying3O4A composite magnetic material;
(2) taking GO/Fe obtained in the step (1)3O4The composite magnetic material (1.0g) and papain (50mg) are placed in 80mL of phosphate buffer solution with the pH value of 6.0, and after 30KHz ultrasonic treatment is carried out for 45min, the magnetic graphene oxide immobilized enzyme (hereinafter referred to as product A, the immobilization rate of the enzyme is 82.1%) is obtained through magnetic separation, water washing and drying.
Example 2
(1) Adding 10g of Graphene Oxide (GO) into 400mL of ethanol solution with volume fraction of 50%, ultrasonically dispersing for 0.5h at 40KHz, adding ferrous sulfate (25mmol) and ferric sulfate (50mmol) while stirring, continuously stirring for 30min, reacting for 1 h at reflux temperature, adding ammonia water to adjust pH to 11, continuously stirring for 2 h, and obtaining GO/Fe through magnetic separation, water washing and drying3O4A composite magnetic material;
(2) taking GO/Fe obtained in the step (1)3O4The composite magnetic material (1.0g) and papain (80mg) are placed in 100mL phosphate buffer solution with pH of 7.0, and after 40KHz ultrasonic treatment for 30min, the magnetic graphene oxide immobilized enzyme (hereinafter referred to as product B, with the enzyme immobilization rate of 80.5%) is obtained through magnetic separation, water washing and drying.
Example 3
Crushing commercially available degerming corn (100kg) to 20-40 meshes, adding water (300kg) and a product A (100g), stirring and soaking at 50 ℃ for 6 hours, recovering magnetic graphene oxide immobilized enzyme through magnetic separation (the recovery rate is 99.2%), transferring the rest soaked mixture into a crusher for fine crushing, sieving with a 200-mesh sieve, standing the filtrate overnight, performing centrifugal separation, and drying to obtain 71.3kg of corn starch (the protein is less than or equal to 0.4%).
Example 4
Crushing the degerming corn (100kg) to 20-40 meshes, adding water (500kg) and a product B (200g), stirring and soaking for 10 hours at 30 ℃, recovering the magnetic graphene oxide immobilized enzyme (the recovery rate is 98.9%) through magnetic separation, transferring the rest soaked mixture into a crusher for fine crushing, filtering through a 200-mesh sieve, standing the filtrate overnight, centrifugally separating, and drying to obtain 71.8kg of corn starch (the protein is less than or equal to 0.4%).
Example 5
The method comprises the steps of crushing the degerming corn (100kg) to 20-40 meshes, adding water (300kg) and papain (10g), stirring and soaking for 6 hours at 50 ℃, then transferring into a crusher to be finely crushed, sieving by a 200-mesh sieve, standing filtrate overnight, centrifugally separating, and drying to obtain a product (corn starch) containing a large amount of protein (about 3.8-4.0%), wherein the protein cannot be fully destroyed by the papain, the starch cannot be completely separated out, and the yield and the purity of the starch are greatly reduced.
Example 6
Crushing commercially available degerming corn (10kg) to 20-40 meshes, adding water (30kg) and a product A (10g) which is recovered and used for 10 times, stirring and soaking for 6 hours at 50 ℃, recovering magnetic graphene oxide immobilized enzyme through magnetic separation, transferring the rest soaked mixture into a crusher for fine crushing, sieving by a 200-mesh sieve, standing the filtrate overnight, performing centrifugal separation, and drying to obtain 7.1kg of corn starch (the protein is less than or equal to 0.4%).
In conclusion, the magnetic graphene oxide immobilized enzyme is used, so that the corn starch and the protein can be quickly separated, the process steps are simple, the corn starch yield is high, the protein content is low, and the corn starch yield and the product quality are basically unchanged after the magnetic graphene oxide immobilized enzyme is recycled for 10 times.

Claims (2)

1. An application of a magnetic graphene oxide immobilized enzyme in corn starch extraction; the preparation method of the magnetic graphene oxide immobilized enzyme comprises the following steps:
(1) adding Graphene Oxide (GO) into 30-50% ethanol solution by volume fraction, ultrasonically dispersing for 0.5h, adding ferrous salt and ferric salt while stirring, continuously stirring for 30min, reacting at 50 ℃ to reflux temperature for 1-2 h, adding ammonia water to adjust pH to 11, and adjusting pH to 11Stirring for 1-2 hr, magnetically separating, washing with water, and drying to obtain GO/Fe3O4A composite magnetic material;
(2) the GO/Fe obtained in the step (1)3O4Placing the composite magnetic material and papain in a phosphate buffer solution, carrying out ultrasonic treatment for 30-45min, and then carrying out magnetic separation, water washing and drying to obtain the magnetic graphene oxide immobilized enzyme;
the ferrous salt in the step (1) is selected from one or more of ferrous chloride, ferrous sulfate, ferrous nitrate or hydrates thereof, and the ferric salt is selected from one or more of ferric chloride, ferric sulfate, ferric nitrate or hydrates thereof; 40-60mL of ethanol solution is used for every gram of Graphene Oxide (GO), and 1.5-2.5mmol of ferrous salt is used; wherein the molar ratio of ferrous salt to ferric salt is 1: 2;
GO/Fe in step (2)3O4The mass ratio of the composite magnetic material to the papain is 10:0.5-0.8, the pH of a phosphate buffer solution is 6-7, and GO/Fe/g3O480-100mL of phosphate buffer solution is used as the composite magnetic material;
the ultrasonic frequency in the steps (1) and (2) is 30-40 KHz.
2. A process for extracting corn starch by wet soaking is characterized by comprising the following steps:
crushing the degermed corn to 20-40 meshes, adding water and magnetic graphene oxide immobilized enzyme, stirring and soaking for 6-10 hours at 30-50 ℃, recovering the magnetic graphene oxide immobilized enzyme through magnetic separation, transferring the rest soaking mixture into a crusher for fine crushing, sieving by a 200-mesh sieve, standing the filtrate overnight, performing centrifugal separation, and drying to obtain corn starch; the using amount of water is 3-5 times of the mass of the degerming corn, and the using amount of the magnetic graphene oxide immobilized enzyme is 0.1-0.2% of the mass of the degerming corn;
the preparation method of the magnetic graphene oxide immobilized enzyme comprises the following steps:
(1) adding Graphene Oxide (GO) into 30-50% ethanol solution by volume fraction, ultrasonically dispersing for 0.5h, adding ferrous salt and ferric salt while stirring, continuously stirring for 30min, and reacting at 50 ℃ to reflux temperatureAfter 1-2 hours, adding ammonia water to adjust the pH value to 11, continuously stirring for 1-2 hours, and then obtaining GO/Fe through magnetic separation, water washing and drying3O4A composite magnetic material;
(2) the GO/Fe obtained in the step (1)3O4Placing the composite magnetic material and papain in a phosphate buffer solution, carrying out ultrasonic treatment for 30-45min, and then carrying out magnetic separation, water washing and drying to obtain the magnetic graphene oxide immobilized enzyme;
the ferrous salt in the step (1) is selected from one or more of ferrous chloride, ferrous sulfate, ferrous nitrate or hydrates thereof, and the ferric salt is selected from one or more of ferric chloride, ferric sulfate, ferric nitrate or hydrates thereof; 40-60mL of ethanol solution is used for every gram of Graphene Oxide (GO), and 1.5-2.5mmol of ferrous salt is used; wherein the molar ratio of ferrous salt to ferric salt is 1: 2;
GO/Fe in step (2)3O4The mass ratio of the composite magnetic material to the papain is 10:0.5-0.8, the pH of a phosphate buffer solution is 6-7, and GO/Fe/g3O480-100mL of phosphate buffer solution is used as the composite magnetic material;
the ultrasonic frequency in the steps (1) and (2) is 30-40 KHz.
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Publication number Priority date Publication date Assignee Title
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CN105154429A (en) * 2015-10-29 2015-12-16 齐齐哈尔大学 Preparation method of magnetic graphite oxide compound carrier for fixing lipase and method for fixing lipase
CN105441419A (en) * 2015-12-22 2016-03-30 陕西师范大学 Magnetic-graphene-oxide-immobilized CPO (chloroperoxidase), and preparation method and application thereof
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Publication number Priority date Publication date Assignee Title
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CN105154429A (en) * 2015-10-29 2015-12-16 齐齐哈尔大学 Preparation method of magnetic graphite oxide compound carrier for fixing lipase and method for fixing lipase
CN105441419A (en) * 2015-12-22 2016-03-30 陕西师范大学 Magnetic-graphene-oxide-immobilized CPO (chloroperoxidase), and preparation method and application thereof
CN105801712A (en) * 2016-05-19 2016-07-27 重庆市农业科学院 Corn soaking method in waxy corn starch production

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