A solid state matrix, process of preparation thereof, and process of preparation of theaflavins

一种茶黄素、基材的技术，应用在生物化学设备和方法、固定在有机载体上/在其中的、酵素等方向，能够解决靶分子失活、丧失生物学活性、不适合用作生物反应器或反复使用的材料等问题

Active Publication Date: 2009-05-27
COUNCIL OF SCI & IND RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1. Long coupling time
[0009] 2. Non-physiological pH leads to inactivation of target molecules
[0010] 3. The product contaminates the activated or deactivated polymer
[0011] 4. Polymer type or by-product toxicity
[0012] 5. Limited application in aqueous solution
[0013] 6. Activated polymer constructs are unstable
[0014] 7. The cyanuric chloride method (refer to Abuchowski et al. (1977a), Journal of Biological Chemistry, 252, 3582-3586) and the carbonyldiimidazole method (refer to Beauchamp et al. (1983), Analytical Biochemistry, 131, 25-33) are common And chloroformic acid phenyl ester (can refer to Veronese et al. (1985), Appl.Biochem.Biotechnol., 11,141-152) and succinimidyl (succinimedyl) active ester method (can refer to Shadle et al.; Katre et al. (1987) , Proc.Natl.Acad.Sci.USA, 84,1487-1491) in the substantial loss of biological activity often seen
The present inventors found the previously described methods to be unsuitable because of the high level of product binding during the reaction of the active enzyme:polymer adduct with the substrate, resulting in substrate poisoning and partial or complete loss of biological activity and the inability to extract from the active substrate The product is recovered so it is not suitable for use in bioreactors or as reusable material

Method used

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  • A solid state matrix, process of preparation thereof, and process of preparation of theaflavins
  • A solid state matrix, process of preparation thereof, and process of preparation of theaflavins
  • A solid state matrix, process of preparation thereof, and process of preparation of theaflavins

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specific Embodiment approach

[0088] 1. The method as described above, comprising, in step (c), reacting the polymer activated by carbonyldiimidazole with a target moiety at a predetermined molar ratio and at a predetermined molar concentration, thereby producing a mixture of the polymer and the target moiety :1 polymer:target adduct, and recover the adduct according to step (d).

[0089] 2. A method as described above, comprising, in step (c), reacting an activated polymer with a target moiety having a plurality of reactive groups at a predetermined molar ratio and molar concentration, thereby producing such a polymer and target Adducts of Adducts: In which a preselected proportion of reactive groups have reacted and attached to a polymer molecule, especially for macromolecular targets. The preselected proportion may be, for example, 1-100%, preferably 10-90%, such as 10, 20, 30, 40, 50, 60, 70, 80 or 90%.

[0090] 3. The method as described above, comprising, in step (c), reacting the polymer activated ...

Embodiment I

[0130] In a plastic beaker, 0.3 g of substrate (CDI-activated acrylic (ester) polymer resin) was resuspended in 3 ml of pH 6.2 phosphate buffer, and 2.0 ml of crude polyphenol oxidase (1.66 mg ×2-enzyme protein); at 25°C, incubate the whole mixed system in a shaking incubator at 100r.p.m. for 1 hour; after incubation, filter the mixture through glass wool, and detect the enzyme in the filtrate with tea source substrate Activity, it was found that there was no enzyme activity at all, indicating that all enzyme proteins were bound to the substrate; then the immobilized enzyme bound to the substrate was washed with 10ml of water, and the remaining enzyme activity in the filtrate was detected again, no enzyme activity was detected in the filtrate, indicating that the enzyme Covalently bond with the substrate; add 0.04g tea source substrate dissolved in 4.0ml water to the above substrate in a plastic beaker, then incubate in a shaking incubator at 37.5°C, 100r.p.m. for half an hour,...

Embodiment -II

[0135] In a plastic beaker, 0.5 g of the base material (CDI-activated acrylate polymer resin) was suspended in 5 ml of phosphate buffer at pH 6.2, and 5.0 ml of crude lychee (litchi chinensis) bud extracted Polyphenol oxidase (1.15 mg × 2-enzyme protein); 20 ° C, the whole mixed system was incubated in a shaking incubator at 100 r.p.m. for 2 hours; The enzyme activity in the filtrate was detected by the substrate, and it was found that there was no enzyme activity at all, indicating that all enzyme proteins were combined with the substrate; then the immobilized enzyme bound to the substrate was washed with 10ml of water, and the residual enzyme activity in the filtrate was detected again, and in the filtrate No detection of enzyme activity indicates that the enzyme is covalently bound to the substrate; add 0.05 g of lychee substrate dissolved in 5.0 ml of water to the above substrate in a plastic beaker, and then incubate in a shaking incubator at 25 ° C and 100 r.p.m. After i...

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Abstract

The present invention relates to a process for the development of a highly efficient solid state matrix by the activation of acrylate based polymer resin having specialized functional groups with 1,1-Carbonyl diimidazole for immobilizing biologically active macromolecules such as oxidases, in particular plant oxidases and the most preferred being tea polyphenol oxidase through indirect covalent bonding / cross linking on such activated polymer resin support, are thermally stable, gives very high number of turnovers in vitro ('n' times); with tea substrate forming exclusive product Theaflavins without any loss of biological activity and leaving the product remaining in vitro without adherence to matrix rendering the matrix safe towards product poisoning and subsequent partial or complete loss of biological activity of the matrix bound enzyme system and thus well adapted to and well suited bioreactors based on such systems. It is unique with respect to its recyclability of otherwise uneconomical tea substrates such as seed and flower substrates into theaflavins both with respect to crude substrate or purified ones.

Description

field of invention [0001] In particular, the invention relates to a solid-phase base material, a preparation method thereof and a preparation method of theaflavin (theaflavin). [0002] More specifically, the present invention relates to the development of efficient solid phase substrates for the immobilization of soluble and bound forms of plant oxidases, in particular tea polyphenol oxidase (EC 1.10.3.1), enabling continuous batch production and Complete conversion of tea-derived substrates into theaflavins from purified tea-derived substrates and crude tea-derived substrates, wherein the base material can completely convert all available substrates into theaflavins, thus providing a A method for preparing theaflavins (w / w) in a medium proportional to the substrate available. Background of the invention [0003] Theaflavins are a group of polyphenolic compounds with the general structure shown in Table 2, which are utilized during black tea production by native tea polyph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F220/18C08F8/30C12N11/08
CPCC12P17/06C08F220/18C08F8/30C12N11/08C12N11/087C08F20/00
Inventor H·P·辛格K·沙尔玛
Owner COUNCIL OF SCI & IND RES
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