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Stabilization of enzymes

一种脂肪酶、酶结构的技术，应用在酶稳定、水解酶等方向，能够解决活性位点没有暴露、不易控制、不易控制颗粒尺寸等问题

Inactive Publication Date: 2007-02-14
CSIR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both CLEC and CLEA are limited due to the fact that some active sites of the enzyme are not exposed, so in order to exert special functions, methods utilizing CLEC or CLEA require excess enzyme catalyst (and consequently increased cost) to overcome the above problems
In addition, these methods do not readily control particle size and morphology over a wide range of particle sizes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 (not optimized) Preparation of cross-linked or stabilized lipase spheres (structure) from water-in-oil emulsions

[0043] 1 g of lipase Amano AK was added to 195 g of phosphate buffered saline (PBS) solution (pH 7.8) and 5 g of mineral oil (Castrol). Homogenize the mixture using a Silverson L4R laboratory rotor-stator homogenizer at 6000 rpm for 5 minutes. 1.5 g of hexamethylene diisocyanate (Merck Schuchardt) were added to the emulsion, and the emulsion was stirred at room temperature for 2 hours. Subsequently, the cross-linked enzyme structure was recovered by filtration with 0.45 μm filter paper, and washed 5 times with PBS, each time using 50 ml of PBS (250 ml of PBS in total). figure 1 Typical stable enzyme spheres or structures obtained according to the method are shown. Using laser light scattering (Malvern Mastersizer 2000) to measure the particle size, the average diameter of the sand (Sautermean diameter) obtained is 49.4 μm (see figure 2 ).

[...

Embodiment 2

[0046] Example 2 Preparation of cross-linked or stabilized lipase spheres (structure) from water-in-oil emulsions

[0047] A lipase solution was prepared by resuspending Candida rugosa lipase (Candida rugosa lipase) (Altus Biologics, Inc) in 100 mM Tris-Cl [tris(hydroxymethyl)aminomethane] buffer (pH 8.0) to a final concentration of 100mg / ml. The enzyme was dialyzed in an Amicon ultrafiltration unit equipped with a 10K polyethersulfone membrane (Microsep (Pty) Ltd, PO Box 391647, Bramley 2018, South Africa) using 3 volumes of 100 mM Tris-Cl buffer (pH 8.0) sample.

[0048] Lipase spheres were prepared using the following volumes of the following reagents: 200 μl Candida rugosa lipase solution (prepared above); 50 μl nonoxynol-4; 50 μl tributyrin; 5 ml mineral oil. The mixture was stirred at 1500 rpm for 1 minute to emulsify. To the above solution was added 40 µl of gluteraldehyde (25% in water) and further stirred for 10 minutes. The emulsion was left at 4°C for 12 hours. ...

Embodiment 3

[0052] Example 3 Dextran aldehyde (dextran aldehyde) as a crosslinking agent

[0053] Repeat Example 2, except that the cross-linking agent used is activated dextran (dextran aldehyde) with an average molecular weight of 20 kDa from leuconostoc species, the oil phase is vegetable oil, lipase solution and Tris Example 2 was repeated except that the buffer ratio was 1:1 and no surfactant was used. Such as Hong, T., Guo, W., Yuan, H., Li, J., Liu, Y., Ma, L., Bai, Y., & Li, T. (2004) "Periodate oxidation of nanoscaled magneticdextran composites", Journal of Magnetism and Magnetic Materials, 269, 95-100, dextran aldehyde is prepared by reacting dextran with excess sodium metaperiodate. An activity of 7.5% (for p-nitrophenyl palmitate) was obtained relative to the activity of the initial free enzyme in aqueous solution.

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Abstract

A process for producing enzyme structures includes providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase. The one liquid phase is a hydrophilic phase, while the other liquid phase is a hydrophobic phase which is immiscible with the hydrophilic phase. Enzyme molecules are located at or within interfacial boundaries of the droplets and the second liquid phase. The enzyme molecules of the respective droplets are cross-linked so that individual enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets.

Description

technical field [0001] The present invention relates to the stabilization of enzymes. More specifically, the present invention relates to methods of producing stable enzyme structures, said stable enzyme structures and uses of said stable enzyme structures. Background technique [0002] Enzymes are commonly used as catalysts in many industries such as chemical, pharmaceutical and cosmetic industries. However, unlike chemical catalysts, enzymes have limited applications and shelf life due to their instability. Enzymes are extremely temperature and pH dependent, making their application difficult in many processes. In addition, soluble enzymes are not easy to recover from the aqueous phase, and enzyme activity usually decreases during storage or processing, thus limiting the application of enzymes as catalysts in chemical processes. [0003] The commercial application of enzymes as catalysts can be enhanced by immobilization of enzymes, which offers a double advantage: maki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N9/20
CPCC12N9/96
Inventor 弗朗西斯·肖恩·穆尔曼迪安·布雷迪阿瓦西里·沙姆帕克西·塞韦尔海丁·罗尔夫斯贾斯廷·霍尔达纳
Owner CSIR
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