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A method for directional immobilization of lipase by nano-iron ferric oxide

A technique for immobilizing lipase and ferric oxide, which is applied to biochemical equipment and methods, and enzymes immobilized on or in inorganic carriers, can solve the problem of reduced enzyme activity, loss of immobilized enzyme activity, and poor performance. Improve enzyme performance and other issues

Active Publication Date: 2021-06-15
浙江容锐科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immobilization of enzymes does not improve the performance of enzymes very well
It has been reported that many inappropriate immobilization processes lead to the impairment of enzyme performance, such as loss of activity ( C., et al. Immobilized heterologous Rhizopus oryzae lipase: A feasible biocatalyst for the production of human milk fat substitutes. Biochemical Engineering Journal, 2012, 67: 104-110; RedekerS.E., et al. Protein engineering for directed immunojugization, Biochemical Engineering. 2013, 24(11): 1761-77.)
The decrease in enzyme activity during immobilization may be due to conformational changes of the enzyme during immobilization or mass transfer limitations of the immobilized enzyme in the catalytic reaction.
For lipases, inappropriate immobilization procedures often lead to loss of immobilized enzyme activity, since most immobilization is random, resulting in the lipase "lid" being masked

Method used

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  • A method for directional immobilization of lipase by nano-iron ferric oxide
  • A method for directional immobilization of lipase by nano-iron ferric oxide
  • A method for directional immobilization of lipase by nano-iron ferric oxide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Nanometer Fe 3 o 4 Synthesis

[0065] 11.75g ​​FeCl 3 ·6H 2 O, 5.97g FeSO 4 ·7H 2 O was dissolved in 250ml of deionized water, heated to 80°C, and 14ml of ammonia water was quickly added under rapid stirring, and kept under nitrogen protection for 2h. The precipitate was washed with deionized water until the aqueous solution was neutral to obtain Fe 3 o 4 . Its transmission electron microscope as figure 1 (a) shown.

[0066] will get Fe 3 o 4 Add 500ml of 80% ethanol, slowly add 20ml of APTES (3-aminopropyl-triethoxysilane) at 30°C, react at room temperature for 12h, wash with deionized water and ethanol three times to obtain aminated Fe 3 o 4 . Aminated Fe 3 o 4 Add it to 100ml of 2% glutaraldehyde solution, react at 30°C for 2h, wash and freeze-dry to get about 5g of Fe with aldehyde groups on the surface 3 o 4 . Its transmission electron microscope as figure 1 (b) shown.

[0067] Depend on figure 1 (a) and figure 1 (b) It can be seen ...

Embodiment 11～18

[0075] Effects of different pH values ​​of Examples 11 to 18 on the activity of immobilized lipase

[0076] Get 1g Rhizopus oryzae lipase enzyme powder (protein content is 10.5%) and be dissolved in 25ml pH respectively in the phosphate buffer saline (100mM) of 4,5,6,6.5,7,7.5,8 and 8.5, add final concentration 1% (w / v) sucrose ester SE-11, stirred to dissolve the enzyme powder, then added 2 g of the activated ferric iron tetroxide prepared in Example 1, stirred and reacted at 20°C for 4 hours, and added a final concentration of 1 % (v / v) glutaraldehyde, continue to react for 2h. The obtained immobilized lipase is separated with a strong magnet, and washed with buffer solution for several times to wash away unbound protein and interfacial activator to obtain ferric oxide immobilized lipase.

[0077] Measure the relative activity of protein recovery rate, protein binding amount and ferric iron tetroxide immobilized lipase in each embodiment (defining the highest relative activ...

Embodiment 19～26

[0079] Examples 19-26 Effects of different enzyme protein concentrations on the activity of immobilized lipase

[0080] Get different quality Rhizopus oryzae lipase enzyme powder (protein content is 10.5%) and be dissolved in 25ml pH and be respectively in the phosphate buffer saline (100mM) of 6.5, make the protein concentration in the solution be respectively 1.1,2.2,3.2,4.2, 5.3, 6, 8.4, 10.5mg / ml, add the sucrose ester SE-11 that final concentration is 1% (w / v), stir to make enzyme powder dissolve, then add the activated iron ferric oxide prepared in 2g embodiment 1, After stirring and reacting at 20° C. for 4 h, glutaraldehyde with a final concentration of 1% (v / v) was added, and the reaction was continued for 2 h. The obtained immobilized lipase is separated with a strong magnet, and washed with buffer solution for several times to wash away unbound protein and interfacial activator to obtain ferric oxide immobilized lipase.

[0081] Determination of the relative activi...

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Abstract

The invention discloses a method for directional immobilization of lipase with nanometer ferric oxide, comprising the following steps: (1) synthesizing nanometer ferric oxide by hydrothermal method, using silane coupling agent and glutaraldehyde to synthesize the nanometer ferric oxide Ferric iron tetroxide is modified; (2) lipase and interfacial activator are dissolved in the buffer solution to obtain immobilization solution, and the modified nano iron ferric oxide is added to the immobilization solution as a carrier to carry out immobilization reaction; The interfacial activator includes at least one of Tween, sucrose ester, n-hexanol, n-hexane, Triton X-100 and sodium lauryl sulfate; (3) after the immobilization reaction finishes, add to the immobilization solution Glutaraldehyde is used for cross-linking, and after the cross-linking is completed, the lipase is separated, washed and dried to obtain immobilized lipase. The immobilized lipase prepared by the invention has the advantages of high efficiency, stability, good dispersibility, easy recovery and the like.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to a method for directionally immobilizing lipase with nanometer ferric oxide. Background technique [0002] Lipase (triacylglycerol acyl hydrolase, E.C. 3.1.1.3) is an enzyme that hydrolyzes triacylglyceride bonds ubiquitously in nature. Lipase is a biocatalyst widely used in food, medicine and other chemical industries. Lipase has a "lid" structure, and its conformational changes are closely related to lipase activation. In the closed conformer of the enzyme, the active site is completely buried under the "lid" of the alpha helix. The opening of the "lid" of lipase leads to the exposure of its active center, showing catalytic activity, which is called "interface activation". [0003] The use of lipases in the biocatalytic industry has grown significantly in recent years, especially in the food industry. However, the high cost and low operational stability of commercial lipa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14
CPCC12N9/20C12N11/14C12Y301/01003
Inventor 杨立荣吴绵斌赵炯烽
Owner 浙江容锐科技有限公司
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