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Method of cultivating microorganisms having nitrile hydratase activity

A technology of nitrile hydratase and cultivation methods, applied in biochemical equipment and methods, adding compounds to stimulate growth, enzymes, etc., can solve problems such as low enzyme production rate, low microbial growth rate, and few microorganisms

Active Publication Date: 2017-08-18
BASF AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Known fermentation methods for cultivating microorganisms producing nitrile hydratase face several different difficulties, for example, high concentrations of carbon or nitrogen sources suitable for growth may inhibit enzyme production, so-called "catabolite inhibition" (Leonova, Applied Biochem Biotechnol (2000), 88:231-241) or inhibition of metal ion cofactors (EP-B11283256; Pei, Biotechnology letters (2013), 35:1419-1424)
These difficulties result in a low rate of nitrile hydratase activity in a given period of time, either due to a low rate of enzyme production (i.e. the microorganism primarily grows but still exhibits a low rate of enzyme production), or due to a low rate of microbial growth (i.e. show high production rates, but still have too few microorganisms)

Method used

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  • Method of cultivating microorganisms having nitrile hydratase activity
  • Method of cultivating microorganisms having nitrile hydratase activity
  • Method of cultivating microorganisms having nitrile hydratase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Culture of Rhodococcus

[0047] In a microfermentation system, different sugar / organic acid ratios were evaluated against different Rhodococcus strains, Rhodococcus rhodochrous "CIBA" (NCIMB 41164) and Rhodococcus rhodochrous "J1" (FERM BP-1478)).

[0048] Basal medium containing yeast extract (Biospringer) (1.25g / l), KH 2 PO 4 (10g / l), K 2 HPO 4 (10g / l), (NH 4 ) 2 SO 4 (1g / l), MgSO 4 *7H 2 O(0.375g / l), CaCl 2 *2H 2 O (25mg / l), trace element solution (2.5g / l) (see below), Co(NO 3 ) 2 (31.2mg / l), defoamer P2000 (BASF) (62.5mg / l), urea (7g / l) and ammonium glutamate (2.5g / l), in H 2 O middle.

[0049]Trace element solution: citric acid monohydrate (40g / l), ZnSO 4 *7H 2 O(11g / l), (NH 4 ) 2 Fe(SO 4 ) 2 *6H 2 O(8.5g / l), MnSO 4 *H 2 O (3g / l) and CuSO 4 *5H 2 O(0.8g / l), in H 2 O middle.

[0050] The medium was then supplemented with 10 g / l of a carbon source (sugar, organic acid or a mixture of both, as indicated in the table below). Use H 3 PO 4 or...

Embodiment 2

[0061] Determination of nitrile hydratase activity

[0062] Take a sample from the culture medium of embodiment 1, and with 50mmol / l KH 2 PO 4 Buffer (pH7.0) was diluted 1:10 (v / v). The obtained solution was treated with 50mmol / l KH 2 PO 4 Buffer (pH 7.0) was further diluted 1:20 (v / v) to achieve a final dilution factor of 1:200 for the medium.

[0063] reaction:

[0064] 875 μl KH 2 PO 4 Buffer (pH 7.0) was pipetted into 2ml Eppendorf tubes. Add 100 μl of diluted medium and pre-incubate the mixture in a thermostatic mixer at 500 rpm for 5 min at 25 °C. After pre-incubation, the reaction was started by adding 25 μl of acrylonitrile. The reaction was carried out at 25°C and 1,000 rpm for 10 minutes.

[0065] Sample Preparation:

[0066] After a reaction time of 10 minutes, the reaction was quenched by transferring 300 μl of the reaction solution to a 1.5 ml Eppendorf tube containing 300 μl of 1.4% (m / v) hydrochloric acid. The mixture was vortexed briefly and cent...

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Abstract

The present invention relates to methods for cultivating a nitrile hydratase producing microorganism, compositions for cultivating a nitrile hydratase producing microorganism, and use of compositions comprising a saccharide and an organic acid for cultivating a nitrile hydratase producing microorganism. The composition provided in and to be employed in context with the present invention is particularly suitable for inducing both, growth and nitrile hydratase production of corresponding microorganisms.

Description

technical field [0001] The present invention relates to a method for cultivating microorganisms producing nitrile hydratase, a composition for cultivating microorganisms producing nitrile hydratase, and the use of a composition containing sugars and organic acids for cultivating microorganisms producing nitrile hydratase. The compositions provided in the context of and used in the present invention are particularly suitable for inducing the growth and production of nitrile hydratase of the corresponding microorganisms. Background technique [0002] Polyacrylamides are widely used as flocculants, as thickeners in the paper industry, as additives in tertiary oil recovery, and in many other fields. The raw material of polyacrylamide is usually its monomer acrylamide. In principle, two different methods exist for the industrial-scale production of acrylamide: chemical synthesis and biosynthesis, with the biosynthetic method on the rise due to milder reaction conditions and inhe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/88
CPCC12N1/20C12N9/88C12Y402/01084C12N1/38
Inventor K·哈格C·施瓦尔布P·斯普洛特P·奥德曼K-U·鲍尔代纽斯B·恩斯特G·斯泰格尔曼S·费赖尔C·博尔施韦勒M·G·布劳恩J·道韦尔
Owner BASF AG
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