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Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector

Active Publication Date: 2011-02-09
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the immobilization carriers of enzymes mainly include inorganic carrier materials (such as silica, activated carbon, porous glass, etc.), organic synthetic polymer materials (such as polyacrylamide, polyethylene, polystyrene, polyurethane, etc.) and natural polymer materials. materials (such as structural proteins, chitosan, sodium alginate, etc.), but there is no report on using bacterial cellulose beads as a carrier to immobilize enzymes

Method used

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  • Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector
  • Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector
  • Method for preparing immobilized enzyme by taking bacterial cellulose bead/membrane as vector

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Pick 1 ring single colony from the acetic acid bacteria slant and inoculate in 100mL sterile seed medium, described seed medium comprises (mass percentage): glucose 2.0%, yeast powder 0.5%, tryptone 0.5%, Na 2 HPO 4 12H 2 O0.27%, 0.115% citric acid, pH5.0, cultured at 30°C, 130rpm for 12h. The seed solution was inserted into a 500mL Erlenmeyer flask containing 300mL sterile fermentation medium with a 6% inoculum amount. Described fermentation medium comprises (mass percentage): glucose 2.0%, yeast powder 0.5%, tryptone 0.3%, Na 2 HPO 4 12H 2 O 0.27%, citric acid 0.115%, pH 5.0; 30°C, 110rpm after culturing for 72 hours, many bacterial cellulose pellets were formed in the medium. After decanting the culture medium, take out the cellulose pellets, rinse them repeatedly with deionized water to remove the culture medium and impurities on the surface of the pellets, then soak the pellets in 0.1% NaOH solution, and treat them at 80°C for 60 minutes to remove the cellulos...

Embodiment 2

[0077] Preparation of Bacterial Cellulose Membrane Block Carrier

[0078] Pick 1 ring single colony from the activated slant strain and inoculate it in 100mL sterile seed culture medium. After cultivating for 12h under the condition of 100-200rpm at 30°C, the described seed culture medium comprises (mass percentage): Mannitol 2.5%, yeast powder 0.5%, tryptone 0.5%, pH5.0. The liquid seed is transferred to the fermentation medium with 4-10% inoculation amount, and the fermentation medium comprises (mass percentage): mannitol 2.5%, yeast powder 0.5%, tryptone 0.3%, pH5.0; 25 -30°C, culture statically for 5-14 days, decant, and collect the bacterial cellulose membrane. Treat the bacterial cellulose membrane collected by fermentation with 0.1% NaOH in a water bath at 80°C until the membrane is milky white and translucent, rinse with deionized water for 3-5 times, collect, and neutralize the remaining residue in the membrane with 0.1% acetic acid Lye, let it stand overnight, rins...

Embodiment 3

[0081] Preparation of immobilized laccase (simple adsorption immobilization)

[0082] Add pH 5.0, enzyme activity unit, 10mL laccase enzyme solution of 1000U / L to a 50mL beaker, add 0.2g freeze-dried bacterial cellulose membrane block and make it submerged, shake slightly for 3-5min, at 4°C After 24 hours of adsorption, the bacterial cellulose membrane was taken out, rinsed 1-2 times with 200 mM acetic acid-sodium acetate buffer solution, pH 5.2, and the moisture on the surface of the membrane was blotted, and stored in a refrigerator at 4°C for later use.

[0083] Compared with the free enzyme, the immobilized enzyme has the following characteristics: from Figure 6-10 The change of relative enzyme activity (in the same group of experiments, the highest enzyme activity is 100%, compared with the activities of other immobilized enzymes or free enzymes, expressed as a percentage) shows that the optimum reaction temperature of immobilized enzymes is 60°C, which is 10°C higher t...

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Abstract

The invention relates to a method for preparing immobilized enzyme by taking a bacterial cellulose bead / membrane as a vector. The method comprises the following steps of: fermenting to prepare the bacterial cellulose bead or the bacterial cellulose membrane; treating the bacterial cellulose bead or the bacterial cellulose membrane in a water bath with 0.1 percent NaOH at the temperature of between 80 and 90 DEG C for 30 to 120 minutes; rinsing the mixture with deionized water; collecting the solution; neutralizing residual alkali liquor with 0.1 percent acetic acid; standing the solution; rinsing the mixture with deionized water; taking the bead or the membrane out; absorbing surface moisture; freezing the bead or the membrane; performing freeze drying on the bead or the membrane for layer use; and preparing the immobilized enzyme by a physical adsorption method or an adsorption crosslinking method. The bacterial cellulose bead / membrane can efficiently adsorb biological enzyme, maintains the activity of the enzyme to the greatest extent and is safe and environmental friendly; and the method is simple, is easy to operate and has high controllability.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzymes, in particular to a method for preparing immobilized enzymes with bacterial cellulose pellets / membrane blocks as carriers. Background technique [0002] Because the enzyme has better substrate specificity, it has important application value in many fields. However, the free enzyme protein is susceptible to denaturation and inactivation under the influence of environmental conditions in the actual application process, and it is not easy to separate from the reaction system to achieve the purpose of repeated use, which limits the industrial application of enzymes to a certain extent. Enzyme immobilization technology is an effective means to achieve repeated and continuous use of enzymes and improve their stability. At present, the immobilization carriers of enzymes mainly include inorganic carrier materials (such as silica, activated carbon, porous glass, etc.), organic synthetic polyme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12R1/38C12R1/02C12R1/41C12P19/04C12N11/12C12R1/01
Inventor 洪枫杨雪霞杨光曹张军
Owner DONGHUA UNIV
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