Amidase xam and its encoding gene and application

A technology encoding a gene and amidase, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of low amidase expression, by-product environmental pollution, separation and purification difficulties, etc., and achieve high enzyme activity and wide substrate Spectrum, effect of broad substrate spectrum

Active Publication Date: 2022-05-31
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • 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 its encoding gene and application
  • Amidase xam and its encoding gene and application
  • Amidase xam and its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Separation, identification and preservation of aromatic fungus DLY26

[0037] 1. Separation

[0038] Take about 1ml of activated sludge sample (taken from the petrochemical refining sewage treatment system) and dilute it with sterile distilled water to 10 times the volume, 20 times the volume, 50 times the volume, 100 times the volume and 1000 times the volume. 100 μl of the diluted samples were spread on the surface of the solid medium by the coating plate method, cultured at 30°C, and the growth of the surface colonies was observed every day. Pick colonies with different colors and shapes, purify and cultivate by the three-zone streak method, until a single colony with the same shape, size and other characteristics can be observed growing on the surface of the medium.

[0039] 2. Identification

[0040] The purified strains were inoculated on TSA plates, cultured at 30°C for 24 hours, and then the morphology, size and other characteristics of the cells we...

Embodiment 2

[0057] Example 2. Preparation of amidase (XAM protein)

[0058] After extensive sequence analysis, alignment and functional verification, a new protein was discovered from Aromatica spp. DLY26, which was named XAM protein, as shown in Sequence 1 of the sequence listing. The gene encoding XAM protein in Aromatia sinensis DLY26 is named as XAM gene, and its coding frame is shown in sequence 2 of the sequence listing.

[0059] 1. Construction of recombinant plasmids

[0060] 1. Using the genomic DNA of Aromatia sinensis DLY26 as a template, using a primer pair composed of am-F and am-R to carry out PCR amplification, and recover the PCR amplification product.

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

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

[0063] Restriction sites KpnI and HindIII (indicated by underline) were added to the upstream and downstream primers, respectively. Using the genomic DNA of Xinfangfangia sp. DLY26 as the template and am-F and am-R as the ...

Embodiment 3

[0078] Example 3. Enzymatic properties of amidase (XAM protein)

[0079] Tris-HCl buffer (50 mM, pH 7.4): Weigh 6.06 g of Tris, dissolve 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 in Tris-HCl buffer, and make up to 100 mL.

[0081] Substrate 2 solution (5.0M hydroxylamine hydrochloride): Weigh 34.75 g of hydroxylamine hydrochloride, dissolve it in Tris-HCl buffer, make up 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 make up to 200 mL with ultrapure water.

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

[0084] The effect of pH on amidase activity

[0085] 1. Optimum pH

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

[0087] Detecti...

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Abstract

The invention discloses amidase XAM and its coding gene and application. The protein provided by the present invention is derived from Xinfangfangia sp., is an amidase tag family amidase, named XAM protein, and is a protein composed of the amino acid sequence shown in Sequence 1 in the sequence listing. The invention also protects the use of XAM protein as amidase. The invention also protects the use of XAM protein in the production of hydroxamic acid. The present invention has great application prospects for related medical fields, chemical industry fields and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to amidase XAM and its encoding gene and application. Background technique [0002] Amidase (EC 3.5.1.X) is an important hydrolase that catalyzes the hydrolysis of amide compounds to the corresponding carboxylic acids and ammonia. In addition to hydrolytic activity, amidases can also catalyze the acyl transfer of different amides, acids, esters and hydrazines to the corresponding hydroxamic acids. At present, amidases are mainly used in the biosynthesis of optically pure pharmaceuticals, chemical intermediates and the synthesis of hydroxamic acids. 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, nicotinic hydroxamic acid is used as an anti-HIV and anti-tumor drug. ...

Claims

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Application Information

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Patent Type & Authority Patents(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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