CS198554B1 - Process for single stage preparing polyethylenimin isothiocyanate enzyme carrier - Google Patents

Description

SUMMARY OF THE INVENTION The present invention provides a novel process for preparing an enzyme support based on polyethyleneimine with substituted primary amino groups thiophosgene.

The prior art method for preparing isothiocyanate enzyme carriers based on polyethyleneimine is a two-step process. Kuniak, J. Zemek, Csl. pat. no. 159063), wherein in the first step the initially water-soluble polyethyleneimine is crosslinked with epicMorhydrin and in the subsequent step the primary amino groups are substituted with thiophosgene. The drawback of this process is that the substitution with thiophosgene already takes place on the insoluble cross-linked polyethyleneimine, i. as a result of which the distribution of the -NCS groups in the resulting product is not uniform.

This deficiency can be eliminated by a novel process based on the substitution of the primary amino groups of polyethyleneimine by thiophosgene in a homogeneous phase, i. directly in solution, wherein the appropriate cross-linking of the substituted polyethyleneimine macromolecules takes place at the level of the reaction of a portion of the resulting -NCS groups with free primary amino groups in the reaction mixture.

The polyethyleneimine base polymer is commercially manufactured for the major industries and is therefore more affordable and faster than other types of polymers. The intrinsic reaction with thiophosgene is very simple, and therefore, it is assumed that the resulting carrier will be substantially less expensive than the preparations made according to prior art (e.g. Kuniak, J. Zemek, US Pat. No. 159063; SA Barker, PJ Somers, EM). Crook et al., Res. 14, 287 (1970).

198 554

SUMMARY OF THE INVENTION The aqueous solution of commercial polyethyleneimine at a concentration of 1.0 to 40% is reacted with thiophosgene at a molar ratio of 1: 0.25 to 1: 2 with vigorous stirring at a temperature of from 10 to 40 ° C for 2 hours. up to 5 hours.

This results in polyethyleneimine isothiocyanate containing up to 150 eq. -NCS groups per gram of starting polyethyleneimine, which is a very suitable carrier for binding various enzymes and substances, and -SH groups.

Enzyme carrier preparation and protein binding can be expressed by the following reaction scheme:

B - NH ₂ + CSCl ₂ - R - NCS + 2 HCl (1)

R = NH _K

R - CS + H ₂ N - R - * C = S (2)

R = NH

R-NCS + HS-Protein - * ~ R-NH-C-S-Protein (3)

B S

R «Polyethylenimine

An advantage of the novel method of preparing a suitable enzyme carrier is that:

- enables the preparation of an enzyme carrier with an absolutely homogeneous distribution of -NCS groups,

- allows the preparation of both water-soluble and some organic solvents and insoluble preparations,

- allows higher efficiency of the main reaction of amino groups 9 with thiophosgene.

Example 1

1.8 g of a 50% aqueous solution of polyethyleneimine (0.02 mole) with an average molecular weight of 40,000 are dissolved in 18 ml of distilled water and neutralized with hydrochloric acid. 10 g of CaCO3 powder are added to the solution and 2.3 g of thiophosgene (0.02 mol) are added to the suspension under vigorous stirring on a magnetic stirrer. After stirring at 18 ° C for one hour, a new 2.3 g of thiophosgene was added to the reaction mixture. After stirring for a further 2 hours, excess CaCO 3 was filtered off from the reaction mixture. The filtrate was shaken in chloroform (25 mL) in a separatory funnel, the aqueous layer was diluted with distilled water (50 mL) and concentrated in vacuo at 40 ° C to a smaller volume to give an aqueous solution of the crude final product usable for impregnating suitable surfaces.

Example 2

The procedure of Example 1 was followed, except that the obtained aqueous solution of the crude product was evaporated to dryness in vacuo and extracted into chloroform (500 mL). By carefully distilling off chloroform ea, the desired product is obtained in the form of a brown, water-soluble paste which is converted into a water-insoluble form by the ethoxylation. The content of the -NCS groups was determined by titration with cysteine (U-C) to 114 wells. per g of starting polyethyleneimine.

Example 3

The process of Example 1, except that the aqueous polyethyleneimine solution is preceded by a water treatment step

198 554 does not neutralize by the addition of thiophosgene. A product with a sulfur content of 6.9% and an -NOS content of 82 baking groups is obtained. per g of starting polyethyleneimine.

The present invention has a wide range of applications for the preparation of binding enzymes, both for research purposes and applications in various sectors of the food industry.

Claims (1)

Process for the preparation of a polyethyleneimine isothiocyanate enzyme carrier, characterized in that an aqueous solution of commercial polyethyleneimine at a concentration of 1.0 to 40% is treated with thiophosgene in a molar ratio of 1: 0.025 to 1: 2 with vigorous stirring at a temperature of 10 to 40 ° C for 2 to 5 hours.