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Optical selective screening process for amidase

A selective amidase, optically selective technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as no amidase screening model, and meet the requirements of high speed, strong versatility and equipment. low effect

Active Publication Date: 2006-08-30
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past few years, U.T.Borscheuer et al. reported a high-throughput screening model for high-activity and high-selectivity hydrolases such as esterase and lipase (Eng.Life.Sci.4: 539-542), so far, no See report on screening model for amidase

Method used

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  • Optical selective screening process for amidase
  • Optical selective screening process for amidase
  • Optical selective screening process for amidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Screening of microorganisms containing acetamide hydrolysis amidase activity After culturing each microorganism in the microbial library to be screened, centrifuge, wash with physiological saline, and evenly disperse in a phosphate buffer solution with pH=7.0 to prepare OD 600 =10.0 bacterial suspension. The enzyme-catalyzed reaction was carried out in a stoppered Erlenmeyer flask (50ml), and the reaction temperature was 30°C.

[0037] The composition of the reaction solution: (1) freshly prepared hydroxylamine hydrochloride solution, adjusted to pH 7 (5ml, 1M) with 10N NaOH before use; (2) disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (5ml, 100mM); (3 ) cells dispersed in buffer solution (5ml, OD 600=10.0); (4) Acetamide solution (5ml, 100mM). Sample 2ml every 5 minutes, add 4ml FeCl 3 HCl solution (355mM FeCl 3 Soluble in 0.65M HCl). The cells were removed by centrifugation, and the absorbance value of the filtrate was measure...

Embodiment 2

[0040] The strain Rhodococcus erythropolis A21 preserved in the laboratory is a known S-selective amidase strain consistent with the report in the literature (Appl Microbiol Biotechnol. (1997) 47:668-674). After the strain is cultivated, a bacterial suspension is made. The composition and detection method of the reaction solution are the same as in Example 1. The substrates were R-2-phenylpropanamide and S-2-phenylpropanamide (10 mM). These two catalytic reactions proceed simultaneously. React for 1h, take a sample, add FeCl 3 After the color development of the HCl solution, it was found that the conversion solution using R-2-phenylpropanamide as the substrate was light yellow, while the conversion solution using S-2-phenylpropanamide as the substrate was reddish brown, indicating that the amide The chiral selectivity of the enzyme is S type.

Embodiment 3

[0041] Embodiment 3: Contain the screening of R-amidase activity microorganism

[0042] The composition and detection method of the reaction solution are the same as in Example 1. Just replace the substrate with an amide with the α carbon atom as the chiral center, such as R-(-)-2,2-dimethylcyclopropylcarboxamide and S-(+)-2,2-dimethyl Cyclopropylcarboxamide (20 mM). These two catalytic reactions are carried out at the same time, and samples are taken every 10 minutes to detect the content of hydroxamic acid generated, and the initial reaction rate r can be calculated respectively 1 , r 2 , r 1 / r 2 The magnitude of the value reflects the stringency of amidase selectivity. Delftia sp. uses the above two enantiomers as substrates respectively, and the generation curve of hydroxamic acid in the reaction solution is as follows: figure 1 ;

[0043] From figure 1 It can be seen that the amount of generated R-hydroxamic acid is much greater than that of S-hydroxamic acid, i...

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Abstract

This invention provides an optical selecting amidase filtration method. The sample detected is dissolved in solution of pH 5 to 9; hydroxylamines are added and make its final concentration between 0.5 to 1M. Then chiral S-amide or R-amide is added as substrate, and make its concentration between 5 to 30mM. They react in water at 20 to 50 degree centigrade, and then the reacted transformation liquor is added solution with Fe<3+> to colored. If it is reddish brown, it reveals that the sample's optical selection is consistent with the substrate spatial configuration, or the amidase didn't have the optical selection. If it is faint yellow, it reveals that the sample's optical selection is not consistent with the substrate spatial configuration. The substrate is don't need derivation, the method can use simple colorimetry to quickly identify the amidase's activity and optical selectivity of the detected microorganism and its extract. Its filtration process is simple, fast, and its equipment request is low, versatility is strong, so it brings great convenience to the optical selecting amidase filtration.

Description

(1) Technical field [0001] The invention relates to an optical selective amidase screening method. (2) Background technology [0002] Amidase (EC 3.5.1.4) can catalyze the hydrolysis of amides to form the corresponding acid and ammonia, and in the presence of hydroxylamine, it can also catalyze the acyl transfer reaction to form hydroxamic acid. The two responses are as follows: [0003] <chemistry num="001"> <chem file="200510062182_cml001.xml" / > < / chemistry> [0004] <chemistry num="002"> <chem file="200510062182_cml002.xml" / > < / chemistry> [0005] R represents all possible and acyl groups (CONH 2 ) to form an amide group. [0006] Amidases were originally discovered in the dual enzymatic degradation of nitriles. However, in recent years, it has attracted increasing attention due to its ability to catalyze the synthesis of many optically pure pharmaceutical intermediates, especially D-amino acids. Although it has been found t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/25
Inventor 郑裕国郑仁朝沈寅初
Owner ZHEJIANG UNIV OF TECH
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