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Agarase immobilization method

A technology of agarase, chitosan, applied in isotropic immobilization on/in an organic carrier

Inactive Publication Date: 2013-08-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Since the immobilization of agarase has not been studied at home and abroad, the use of cheap chitosan as an immobilization carrier to immobilize agarase has great theoretical and practical significance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Effect of pH value on enzyme activity

[0022] Get prepared chitosan immobilized enzyme 0.5g and corresponding free enzyme in parallel, add substrate and free enzyme and immobilized enzyme respectively under different pH (4.0-9.0) conditions, measure its enzyme activity respectively, the result is as follows figure 1 shown.

[0023] figure 1 It shows that the optimal pH value of the free enzyme is 8.0, and the enzyme activity decreases with the increase of the pH value; for the immobilized enzyme, under the experimental conditions, the enzyme activity reaches the maximum when the pH value reaches 8.5, and the immobilized enzyme The optimal pH of the enzyme is increased by 0.5 units to the alkaline side, which is in line with the basic law after the combination of the enzyme and the carrier.

Embodiment 2

[0024] Example 2: Effect of Temperature on Enzyme Activity

[0025] Take the prepared chitosan-immobilized enzyme 0.5g and the corresponding free enzyme in parallel, add the substrate to react with the free enzyme and the immobilized enzyme respectively under different temperature (30°C-80°C) conditions, and measure the enzyme activity respectively. The result is as figure 2 shown.

[0026] Depend on figure 2 It can be seen that the free agarase exhibits the highest activity at 40°C, and as the temperature rises, the activity drops sharply, and the enzyme begins to inactivate, but for the immobilized enzyme, there is a large enzyme activity in the range of 40-60°C, The enzyme activity reaches the maximum at 50°C, and the heat resistance of the immobilized enzyme is significantly improved.

Embodiment 3

[0027] Example 3: Thermostability of free and immobilized enzymes

[0028] Take the prepared chitosan-immobilized enzyme 0.5g and the corresponding free enzyme in parallel, incubate at different temperatures (20°C-80°C) for 0.5h, then add the substrate to interact with it, and measure the enzyme activity respectively. The results are as follows: image 3 shown.

[0029] Depend on image 3 It can be seen that short-term incubation of the enzyme solution below 40°C has little effect on the enzyme activity, and when the temperature is further raised to 50°C, the effect on the activity of the free enzyme increases, with a loss of about 27.36%, while the effect on the activity of the immobilized enzyme is still small. At 60°C, the activity of the free enzyme is very low, while the activity of the immobilized enzyme is about 50%. At 70°C, the enzyme has basically no activity at all, indicating that the thermal stability of the immobilized enzyme is higher than that of the free enzy...

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Abstract

The invention belongs to the field of enzyme engineering research and development technology. The invention relates to an agarase immobilization method research. The invention comprises the following steps: preparing a 1:20 (W / V) of chitosan solution with 1.5-2% (V / V) of acetate for fully dissolving; dropping the dissolved chitosan solution drop by drop into a 1 mol / L sodium hydroxide solution, and filtering and collecting a chitosan bead; washing to neutral with distilled water, and then immersing with a 0.2 mol / L phosphate buffer solution of pH7.6 overnight; fetching the immersed chitosan bead carrier 2.5-5 g, and adding 2.5% of glutaraldehyde 20-25 ml, carrying out water-bath oscillation for 0.5 hours and room temperature crosslinking for 5-6 hours, and washing the chitosan bead cross-linked carrier with a phosphoric acid buffer; adding diluted agarase liquid 10-15 ml into the chitosan bead cross-linked carrier, stirring uniformly, fixing at 4 DEG C for 2-3 hours, washing with the phosphoric acid buffer; obtaining the immobilized agarase by vacuum-pumping. The agarase immobilization method provided by the invention has the advantages of cheap and easily available carrier, simple technology, mild condition and small loss of enzyme activity, and the enzyme activity recovery rate reaches to 77.6%.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering research and development. The invention relates to the research on the method of chitosan microspheres immobilizing agarase, which is suitable for industrial production. Background technique [0002] Agar is an important seaweed polysaccharide, mainly composed of agarose and sulfur agar. Agarase degrades agarose. According to its different modes of action, agarase can be divided into two categories: α-agarase and β-agarase. α-agarase cleaves the α-1,3-glucosidic bond of agarose to generate β-D-galactose as the non-reducing end and 3,6-internal ether-α-L-galactose as the reducing end Agar oligosaccharide column at the end; β-agarase cleaves the β-1,4-glucosidic bond of agarose to generate β-D-galactose as the reducing end and 3,6-endether-α-L - A new series of agar-oligosaccharides with galactose as the non-reducing end. The agarase degradation process has the advantages of high yiel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10
Inventor 朱启忠张瑞张扬
Owner SHANDONG UNIV
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