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Immobilized lipase and preparation method thereof

A technology for immobilizing lipase and lipase, applied in biochemical equipment and methods, and enzymes fixed on/in organic carriers, can solve the problems of severe cross-linking reaction, activity damage, poor stability, etc., and achieve Effects of improved usage efficiency, high reuse rate, high thermal stability and operational stability

Inactive Publication Date: 2018-03-30
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immobilized enzyme obtained by sodium alginate immobilization alone has disadvantages such as poor stability and easy leakage, so it is necessary to use cross-linking methods to further strengthen the binding of the enzyme carrier.
In the existing lipase immobilization methods, almost all of them use glutaraldehyde as a cross-linking agent. After cross-linking, glutaraldehyde can make the enzyme molecule and the carrier more tightly combined, but at the same time, the cross-linking reaction between glutaraldehyde and protein is relatively difficult. Severe, easy to cause greater activity damage

Method used

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  • Immobilized lipase and preparation method thereof
  • Immobilized lipase and preparation method thereof
  • Immobilized lipase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Obtaining of immobilized lipase

[0021] 1.1 Effect of concentration of sodium alginate solution on lipase immobilization

[0022] The amount of enzyme given per milliliter of sodium alginate solution was set to be 800U, the concentration of calcium chloride solution was 3% by mass fraction, the fixed time was 30min, and under the condition of buffer solution pH 7.0, the concentration of sodium alginate solution (mass fraction 1.5, 2.0, 2.5, 3.0, 3.5%) on the activity of immobilized lipase.

[0023] The specific steps are:

[0024] S1. Weigh sodium alginate and add it to the phosphate buffer solution of pH 7.0, fully stir until the sodium alginate is completely dissolved, and prepare sodium alginate solutions with mass fractions of 1.5%, 2.0%, 2.5%, 3.0% and 3.5%; Lipase (Shenzhen Hengsheng Biotechnology Co., Ltd., article number: 9001-62-1) was dissolved in phosphate buffer solution of pH 7.0 to prepare 800U / mL lipase solution;

[0025] S2. Mix sodium ...

Embodiment 2

[0055] Example 2: Cross-linking of immobilized lipase

[0056] 2.1 The effect of cross-linking agent selection on the activity of immobilized lipase

[0057] The best preparation conditions obtained by Example 1: the concentration of sodium alginate solution is 2.5% by mass fraction, the amount of enzyme given is 400U / mL sodium alginate solution, the concentration of calcium chloride solution is 5% by mass fraction, and the fixed time is 20min (buffer solution at pH 7.0). Set the concentration of the cross-linking agent aqueous solution to 0.1% by volume, shake and cross-link for 1 hour at 25°C, investigate different cross-linking agents (glutaraldehyde, ethylene glycol glycidyl ether, neoglutaraldehyde glycidyl ether, polyethylene glycol Alcohol diglycidyl ether) on the activity of immobilized lipase after crosslinking.

[0058] The specific steps are:

[0059] S1. Weigh 2.5g sodium alginate and add it to 97.5g pH7.0 phosphate buffer solution, fully stir until the sodium a...

Embodiment 3

[0084] Example 3: Comparison of enzymatic properties between immobilized lipase and free lipase after crosslinking

[0085] According to the optimal condition that embodiment 1 and embodiment 2 obtain prepare immobilized lipase, concrete steps are:

[0086] S1. Weigh 2.5g sodium alginate and add it to 97.5g pH7.0 phosphate buffer solution, fully stir until the sodium alginate is completely dissolved to obtain a sodium alginate solution with a mass fraction of 2.5%; Co., Ltd., product number: 9001-62-1) was dissolved in the phosphate buffer solution of pH7.0, and the lipase liquid of 400U / mL was prepared;

[0087] S2. Mix and stir the sodium alginate solution with a mass fraction of 2.5% and the lipase solution of 400U / mL according to the volume ratio of 1:1 to obtain the sodium alginate / lipase mixed solution, and then mix the sodium alginate / lipase mixed solution Use a syringe (No. 5 needle) to add dropwise to a calcium chloride solution with a mass fraction of 5% to form smo...

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Abstract

The invention discloses immobilized lipase and a preparation method thereof. The preparation method comprises the following steps: dissolving sodium alginate and lipase in a buffer solution respectively to obtain a sodium alginate solution and lipase liquid; uniformly mixing the sodium alginate solution and the lipase liquid according to a ratio of (15-35)mg:(400-1,600)U between the sodium alginate and lipase, so as to obtain sodium alginate / lipase mixed liquid; dropwise adding the sodium alginate / lipase mixed liquid into 2-6% calcium chloride solution by mass; immobilizing the formed gel beads; filtering and adding the gel beads into a cross-linking agent aqueous solution; shaking and cross-linking for 0.5-3.5h at 20-50 DEG C; performing washing and suction filtration to obtain immobilized lipase. The enzyme activity of the immobilized lipase prepared in the invention reaches 84.9U / g; the immobilized lipase has relatively high thermal stability and operational stability and high recycle rate and meets the needs of industrial application.

Description

Technical field: [0001] The invention belongs to the fields of biochemical industry and biotechnology, and in particular relates to an immobilized lipase and a preparation method thereof. Background technique: [0002] Lipase (Lipase, EC3.1.1.3) is a general term for a class of acyl hydrolases that can catalyze fat hydrolysis. It can catalyze ester hydrolysis, alcoholysis, ester synthesis, transesterification, stereoisomer resolution, etc. at the oil-water interface. Organic synthesis reaction, which has a wide range of applications in food, tanning, washing, pharmaceutical and environmental treatment and other industrial fields. As an emerging renewable clean energy, biodiesel has always been a research hotspot. It can be prepared by lipase-catalyzed animal and vegetable oils and transesterification with short-chain fatty alcohols. When using lipase to catalyze the production of biodiesel, its cost is the main limiting factor, which seriously restricts the industrializatio...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04
CPCC12N9/20C12N11/04C12N11/10C12Y301/01003
Inventor 胡云峰徐珊李任强郑振华张云孙爱君
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI