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Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability

A polygalactose, aldolase technology, applied in the field of genetic engineering, can solve problems such as instability

Inactive Publication Date: 2016-07-20
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the vast majority of commercial pectinases are produced by Aspergillus niger, and several endopolygalacturonases derived from this fungus have been reported. Guoetal., 2002.Liuetal., 2014.Singhetal., 2002), unstable when the pH is lower than 4.0 (Liuetal., 2014)

Method used

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  • Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability
  • Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability
  • Cloning, definition and application of internally tangent polygalacturonase gene with temperature resistance and acid stability

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Embodiment Construction

[0029] 1. Enzymes, Kits, Carriers and Reagents

[0030] LATaq enzyme, pMD TM 18-T vector cloning kit, Taq TM HotStartVersion kit, restriction enzymes SnaBI and AvrII, T4DNA ligase, RNAisoPlus for RNA extraction and protein molecular weight markers were purchased from TaKaRa Company (Liaoning, China); reverse transcription for cDNA first-strand synthesis The kit was purchased from TransGenBiotech (Beijing, China); the Bradford method protein concentration assay kit was purchased from GenerayBiotech (Shanghai, China); the expression vector pPIC9K was purchased from Invitrogen (California, USA); Aldehydic acid, polygalacturonic acid, orange peel pectin and apple peel pectin were all purchased from Sigma.

[0031] 2. Strains and Media

[0032] 1) strain

[0033] Penicillium oxalicum CZ1028 is a high-yielding polygalacturonase strain obtained by our laboratory through mutagenesis screening, and has been preserved in the China General Microorganism Culture Collection and Managem...

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Abstract

The invention discloses internally tangent polygalacturonase rEPG4 with temperature resistance and acid stability, a gene epg4 cloned by the internally tangent polygalacturonase rEPG4, a cloning vector, an expression vector, recombinant escherichia coli, a recombinant Pichia pastoris, and application of the gene epg4, the cloning vector, the expression vector, the recombinant escherichia coli and the recombinant Pichia pastoris to preparation of the internally tangent polygalacturonase rEPG4. After the internally tangent polygalacturonase gene epg4 is expressed and purified by the Pichia pastoris, the optimum pH value is 5.0, the optimum temperature is 65 DEG C, and the specific enzyme activity is as high as 12,038.1U / mg. After the rEPG4 is processed at 25 DEG C under the pH of 3.0-7.0 for 24h, 85.9% of enzyme activity is also maintained; after the rEPG4 is processed at 50 DEG C for 1h, the 100% enzyme activity is maintained; after the rEPG4 is processed at 55 DEG C for 1h, 61.2% of the enzyme activity is maintained. Therefore, enzymes have excellent acid stability and excellent heat stability.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the application of a temperature-resistant and acid-stable endopolygalacturonase rEPG4 capable of decomposing polygalacturonic acid or pectin. Background technique [0002] Pectin is a complex polysaccharide polymer, and its structure is mainly formed by the polymerization of partially methylated D-galacturonic acid. Pectin is an important component of the plant middle layer and cell wall, and plays a vital role in promoting plant tissue structure and stability (VanBurenetal., 1991). Due to the complexity of pectin structure, pectinase is a compound enzyme system. Based on the type of action of the enzyme and the preference for the reaction substrate, pectinase can be divided into protopectinase, pectin methylesterase, polygalacturonase and pectin lyase (Alkorta et al., 1998). Among these complex enzymes, endopolygalacturonase can randomly cleave the α-1,4 glycosid...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12P19/14
CPCC12N9/2402C12P19/14C12Y302/01015
Inventor 程忠陈东卢波陆琦黎贞崇王青艳黄日波
Owner GUANGXI ACAD OF SCI
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