Thermostable alkaline pectinase mutants and their coding genes and their applications

A technology encoding gene and pectinase, applied in the field of pectinase protein, can solve problems such as inability to withstand high temperature

Active Publication Date: 2018-01-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defect that the existing pectinase can not withstand high temperature, provide a kind of high temperature resistant pectinase protein and its encoding gene, in addition, also provide the gene containing the pectinase protein encoding Recombinant vectors, cells, and compositions containing the pectinase protein, and their applications

Method used

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  • Thermostable alkaline pectinase mutants and their coding genes and their applications
  • Thermostable alkaline pectinase mutants and their coding genes and their applications
  • Thermostable alkaline pectinase mutants and their coding genes and their applications

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Experimental program
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Effect test

Embodiment 1

[0034] Construction of pectinase mutant library by error-prone PCR method

[0035] Firstly, the PCR method was used (the upstream primer sequence of pelA is: 5'-GTGCGCTAGCATGGGACG-3', as shown in SEQ ID No: 9; the downstream primer sequence of pelA is 5'-GCGAAGCTTTCATCGATTTG-3', as shown in SEQ ID No: 10) from The pectinase Bsp165PelA gene pelA was amplified from the genome of alkalophilic Bacillus sp. N16-5 (CGMCC No.0369), and the recombinant plasmid pET28a-pelA was constructed. Using pET28a-pelA as a template, nucleotide mutations were introduced into pelA in vitro by using error-prone PCR technology. The sequences of random mutation primers are shown in SEQ ID No: 7 (MegaF: 5'-GCGGCAGCCATATGGCTAGCT-3) and SEQ ID No: 8 (MegaR: 5'-GCTCGAGTGCGGCCGCAAGCTTC-3).

[0036] The amplification system is:

[0037]

[0038] The PCR amplification conditions were: 94°C for 4min; 94°C for 45s, 55°C for 1min, 72°C for 1min, 30 cycles; 72°C for 10min.

[0039] The construction of the ...

Embodiment 2

[0044] Screening of mutants with improved thermostability

[0045] The mutant and wild-type strains were replicated in fresh LB (containing 50ng / μL kanamycin, 0.1mM IPTG) liquid medium with a sterilized 96-well plate needle replicator, and induced overnight. The obtained bacterial fluid was used for further screening.

[0046] 1. Preliminary screening of 96-well plate: Dilute the cultured bacterial solution by 10 times, incubate at 65°C for 10 minutes, and then place it on ice to cool, absorb 20 μL of bacterial solution and 80 μL of 0.2% polygalacturonic acid (PGA) (w / v ) Glycin-NaOH buffer solution (pH10.0), mix well, react at 55 °C for 5 min, then cool on ice, add 80 μL DNS (3,5-dinitrosalicylic acid) solution, and react at 98 °C for 10 min, Determination of OD 540 . After being incubated at the selected temperature for 10 minutes, the residual enzyme activity of the wild type is basically 0, so the color is lighter; if the color of the mutant is darker after being incuba...

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Abstract

The invention discloses a pectinase protein, wherein the amino acid sequence of the pectinase protein is shown in SEQ ID No: 5 or SEQ ID No: 6. In addition, the present invention also provides recombinant vectors and transgenic cells containing the coding gene, compositions containing the pectinase protein as an active ingredient, and the above-mentioned pectinase protein, coding gene, recombinant vector, and transgenic cells And the application of composition in hydrolyzed pectin. The pectinase protein provided by the invention has higher thermal stability and activity.

Description

technical field [0001] The present invention relates to a pectinase protein, the coding gene of the pectinase protein, a recombinant vector and a transgenic cell containing the coding gene, a composition containing the pectinase protein as an active ingredient, and the above-mentioned Application of pectinase protein, coding gene, recombinant vector, transgenic cell and composition in hydrolyzing pectin. Background technique [0002] Pectin is an acidic polysaccharide substance that widely exists in the plant kingdom, mainly in the primary cell wall and cell middle layer of plants, and is the support material of internal cells. The chemical structure of pectin is complex. The main chain of pectin is partially methylated galactan, and the branched chain changes according to the source of pectin. Deesterified pectin is known as pectic acid or polygalacturonic acid. Enzymes that hydrolyze such pectic substances are broadly known as pectinases. Endopolygalacturonate lyase (EC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21
Inventor 马延和叶金统周成薛燕芬
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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