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Esterase free enzymes

A spherical particle, enzyme activity technology, applied to biochemical equipment and methods, fixed on/in organic carriers, enzymes, etc., can solve the problem of reducing esterase activity, 7-ACA quality and yield decline, incomplete And other issues

Inactive Publication Date: 2004-11-03
SANDOZ AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may cause a significant decrease in 7-ACA quality and yield
via acetone or CuSO 4 Conventional methods of treatment that reduce esterase activity in the presence of DAO do not completely reduce esterase activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] General procedure for obtaining mixtures with DAO activity substantially depleted of esterase activity

[0100] From the aqueous fermentation broth with esterase activity and DAO activity, the cell pellets can be harvested according to conventional methods (see for example Kublicek-Pranz, E.M., etc., Can.J.Microbiol., 1985, 31, pp624-628), and the obtained 10 g of cell pellets were resuspended in 50 ml of water and treated with caustic soda solution at pH 11 at room temperature for approximately 90 minutes to remove catalase activity. Phosphoric acid was added to adjust the pH of the mixture to approximately 7.

[0101] To the resulting mixture was added 500 l of an ethanol solution containing 10 mg of PMSF per ml of ethanol, and the resulting mixture was incubated at room temperature for about 3 hours. The resulting cell suspension can be used as such in reactions requiring DAO activity.

[0102] DAO activity of cell pellet: 530U oxidase

[0103] DAO activity after ...

Embodiment 2

[0106] The PMSF-treated cell suspension with DAO activity obtained as described in Example 1 was added to a solution containing 10 grams of cephalosporin C in 1000 ml of water, and the pH value of the obtained solution was adjusted to 7.5. The obtained mixture was stirred at room temperature for about 180 minutes under the introduction of oxygen into the mixture.

[0107] Gl-7-ACA yield: 91%

[0108] Total amount of deacetylated product at position 3 in the starting cephalosporin C solution: equivalent to 3% of cephalosporin C

[0109] The total amount of the 3-position deacetylated product in the obtained reaction solution: equivalent to 3% of the starting cephalosporin C.

[0110] Repeat Example 2, but use DAO activity without PMSF treatment

[0111] Gl-7-ACA yield: 88%

[0112] Total amount of deacetylated product at position 3 in the starting cephalosporin C solution: equivalent to 3% of cephalosporin C

[0113] The total amount of 3-position deacetylated products in t...

Embodiment 3

[0115] Before being treated with PMSF, the cell suspension obtained as described in Example 1 was homogenized (DAO activity in homogenized cells: 385U oxidase) and the homogenized cells obtained by PMSF were treated as described in Example 1 (homogenized cells) DAO activity in cells treated with PMSF: 372U oxidase, equivalent to 96% of the activity before treatment with PMSF). The resulting cell suspension was reacted with cephalosporin C as described in Example 2.

[0116] Gl-7-ACA yield: 93%

[0117] Amount of deacetylated product at position 3 in the starting cephalosporin C solution: equivalent to 3% of cephalosporin C

[0118] The amount of the 3-position deacetylated product in the obtained reaction solution: equivalent to 3% of the starting cephalosporin C.

[0119] Repeat Example 3, but use DAO activity without PMSF treatment

[0120] Gl-7-ACA yield: 89%

[0121] Amount of deacetylated product at position 3 in the starting cephalosporin C solution: equivalent to 3%...

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Abstract

A process for decreasing esterase activity present in the presence of D-amino acid oxidase activity or in the presence of glutarylacylase activity in a mixture having esterase activity and D-amino acid oxidase activity and / or having esterase activity and glutarylacylase activity by treatment with phenylmethylsulphonyl fluoride; immobilized biocatalysts in spherical particle form obtainable by treating microorganism cells having enzyme activity with a primary or secondary amine containing polymer, an organic solvent which is able to form a two-phase system with water and a bifunctional agent; and the use of that process and biocatalysts in the production of 7-aminocephalo-sporanic acid, 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid and cephalosporin antibiotics.

Description

field of invention [0001] The invention relates to a method for inhibiting esterase activity in an enzyme preparation, such as an enzyme preparation for enzymatically producing 7-aminocephalosporanic acid (7-ACA); also relates to a production method for an enzyme preparation, such as for producing 7-ACA and / or an enzyme preparation of 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid. Background technique [0002] 7-ACA is a key intermediate in the production (e.g. pharmaceutical) of active cephalosporin antibiotics, e.g. acylation of the 7-position amino group of the 7-ACA ring system, such as with known valuable in the production of cephalosporin antibiotics Or the acylation of valuable groups in intermediates for the production of these antibiotics; for example to obtain 7-acylated aminocephalosporins with acetoxymethyl at the 3-position of the ring system, such as cefotaxime, cefpodoxime (cefpodoxime oxime ester), cephalosporin; and for the production of other cephalos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/98C12N9/06C12N9/80C12N11/00C12N11/04C12P35/00
CPCC12N9/0024C12P35/00C12N11/04C12N11/00C12N9/80C12N9/0012
Inventor A·雷克特W·里索斯特F·克诺斯德N·帕尔马
Owner SANDOZ AG
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