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Amidase XAM, and encoding gene and application thereof

An amidase and gene technology, which is applied to amidase XAM and its encoding gene and application fields, can solve the problems of low amidase expression, environmental pollution by by-products, difficult separation and purification, etc., and achieves high enzyme activity and broad substrate spectrum. , the effect of a broad substrate spectrum

Active Publication Date: 2020-11-17
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, hydroxamic acid is mainly synthesized by chemical methods, which usually require strong acid or strong alkali conditions, and must be refluxed at high temperature, and is often accompanied by the formation of a large number of salts, which brings great difficulties to separation and purification, and the by-products will also cause certain environmental pollution. Pollution
The bioenzyme synthesis process of hydroxamic acid can effectively solve the problems existing in the chemical synthesis method, but amidase has problems such as low expression, poor stability, and narrow substrate spectrum in industrial applications

Method used

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  • Amidase XAM, and encoding gene and application thereof
  • Amidase XAM, and encoding gene and application thereof
  • Amidase XAM, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, Isolation, Identification and Preservation of Xingfangfangbacterium DLY26

[0037] 1. Separation

[0038] Take about 1ml of activated sludge sample (taken from the petrochemical refinery sewage treatment system), and dilute to 10 times volume, 20 times volume, 50 times volume, 100 times volume and 1000 times volume in turn with sterile distilled water. Spread 100 μl of the diluted sample on the surface of the solid medium by the coating plate method, incubate at 30°C, and observe the growth of the colonies on the surface every day. Pick colonies with different colors and shapes, and purify and culture them by the three-section line method until a single colony with the same shape and size can be observed on the surface of the culture medium.

[0039] 2. Identification

[0040] The purified bacterial strains were inoculated on TSA plates, cultivated at 30°C for 24h, and then observed the morphology, size and other characteristics of the cells using a transmi...

Embodiment 2

[0057] Embodiment 2, the preparation of amidase (XAM protein)

[0058] After a large number of sequence analysis, alignment and functional verification, a new protein was found from Xingfangfang bacteria DLY26, which was named XAM protein, as shown in sequence 1 of the sequence table. The gene encoding the XAM protein in Xingfangxiang bacteria DLY26 is named XAM gene, and its coding frame is shown in sequence 2 of the sequence list.

[0059] 1. Construction of recombinant plasmids

[0060] 1. Using the genomic DNA of A. syringae DLY26 as a template, a primer pair composed of am-F and am-R is used for PCR amplification, and the PCR amplification product is recovered.

[0061] am-F:5'-CGG GGTACC ATGGCTGAATTCCTG-3';

[0062] am-R:5'-CC AAGCTT CTAGCGCGAAACCG-3'.

[0063] Restriction sites KpnI and HindIII (underlined) were added to the upstream and downstream primers, respectively. The genomic DNA of Xinfangfangia sp.DLY26 was used as a template and am-F and am-R were used...

Embodiment 3

[0078] Embodiment 3, the enzymatic property of amidase (XAM protein)

[0079] Tris-HCl buffer solution (50mM, pH 7.4): Weigh 6.06g Tris, dissolve it in ultrapure water, and adjust the pH to 7.5 with HCl.

[0080] Substrate 1 solution (1.0 M butanamide): Weigh 8.7 g of butanamide, dissolve it in Tris-HCl buffer, and dilute to 100 mL.

[0081] Substrate 2 solution (5.0 M hydroxylamine hydrochloride): Weigh 34.75 g of hydroxylamine hydrochloride, dissolve it in Tris-HCl buffer, and adjust the volume to 100 mL, and adjust the pH to 7.5 with NaOH.

[0082] HCl solution (0.65M): Measure 11 mL of hydrochloric acid (36%), and dilute to 200 mL with ultrapure water.

[0083] FeCl 3 Solution (356mM): Weigh 5.78g of ferric chloride and dilute to 100mL with 0.65M HCl.

[0084] 1. Effect of pH on amidase activity

[0085] 1. Optimal pH

[0086] Take the XAM protein solution prepared in Example 2, dilute it to 2 times the volume with buffer, and use the diluted solution as the test solu...

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Abstract

The invention discloses amidase XAM, and an encoding gene and application thereof. The protein provided by the invention is from Xinfangfangia sp., is an amidase tag family amidase, is named XAM protein and is the protein having an amino acid sequence disclosed by a sequence 1 in a sequence table. The invention also provides application of the XAM protein as the amidase. The invention also provides application of the XAM protein in hydroxamic acid production. The invention has a great application prospect in relevant medical care fields, chemical industry fields and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to amidase XAM and its coding gene and application. Background technique [0002] Amidase (EC 3.5.1.X) is an important hydrolase that catalyzes the hydrolysis of amides to generate corresponding carboxylic acids and ammonia. In addition to their hydrolytic activity, amidases catalyze the acyl transfer of various amides, acids, esters, and hydrazines to form the corresponding hydroxamic acids. At present, amidase is mainly used in optically pure medicine, biosynthesis of chemical intermediates and synthesis of hydroxamic acid. Hydroxamic acid is a very important metal complexing agent, which has great applications in medicine, agriculture, bioremediation, food additives, antibiotics, antifungal drugs, tumor suppressor siderophores, enzyme inhibitors and bioremediation. Among them, nicotine hydroxamic acid is used as an anti-HIV and anti-tumor drug. It is also used for the t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12P13/02
CPCC12N9/80C12P13/02
Inventor 刘建国郗丽君谭雯斐李子一
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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