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Pectic lyase mutant [delta]Pel419 as well as coding gene, preparation method and application thereof

A technology of pectin lyase and mutants, which is applied in the field of molecular biology, can solve the problem of transforming pectin lyase with point mutations that have not been seen yet, and achieve the effect of improving enzyme activity and heat resistance

Active Publication Date: 2021-12-31
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] So far, there have been no reports on point mutation transformation of pectin lyase

Method used

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  • Pectic lyase mutant [delta]Pel419 as well as coding gene, preparation method and application thereof
  • Pectic lyase mutant [delta]Pel419 as well as coding gene, preparation method and application thereof
  • Pectic lyase mutant [delta]Pel419 as well as coding gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment one: the construction of recombinant plasmid

[0044] Cultivate D. dadantii DCE-01 to the logarithmic growth phase, take 1.5 mL of the bacterial liquid and centrifuge at 12,000 rpm for 1 min to collect the bacterial pellet; then extract genomic DNA according to the kit instructions.

[0045] According to the pectin lyase gene pel419 and the MCS segment of the vector pET28a, the restriction sites Nde I and Xho I were sequentially selected and the following primers were designed with the bioinformatics software SnapGene:

[0046]

[0047] The PCR reaction system is: genomic DNA, 1 μL; Primer F (10 μM), 0.75 μL; Primer R (10 μM), 0.75 μL; 2×UltraHiFi Mix (with dye) 12.5 μL; 2 O to make up to 25μL; mix well and perform PCR reaction.

[0048] The parameters are set to:

[0049] (1) Pre-denaturation at 94°C for 2min; (2) Denaturation at 98°C for 10s; (3) Refolding at 60°C for 30s; (4) Extension at 68°C for 15s; repeat steps (2)-(4) for 35 cycles; (5) ) at 68°C ...

Embodiment 2

[0051] Example 2: Site-directed mutagenesis

[0052] Principle of site-directed mutagenesis: the construction of the point mutation plasmid adopts the Dpn I method ( image 3 ).

[0053] According to the amino acid site to be mutated, PCR point mutation primers are designed as follows:

[0054]

[0055] Wherein, the underlined part represents the codon corresponding to the 52nd alanine encoded by the mutant gene.

[0056] Using the rapid site-directed mutagenesis kit and pET28a-pel419 recombinant plasmid as a template, the whole plasmid PCR was used to introduce the mutation site.

[0057] The PCR reaction system is: Primer F V52A (10 μM) 1 μL, Primer R V52A (10μM) 1μL, 5×FastAlteration Buffer 5μL, plasmid DNA 1μL, Fast Alteration DNA Polymerase 0.5μL, with ddH 2 O to make up to 25 μL.

[0058] The parameters are set to:

[0059] (1) Pre-denaturation at 95°C for 2 minutes; (2) Denaturation at 94°C for 20 seconds; (3) Refolding at 60°C for 10 seconds; (4) Extension at...

Embodiment 3

[0061] Example 3: Induced expression and SDS-PAGE analysis of wild enzyme and mutant enzyme

[0062] Genetically engineered bacteria pET28a-pel419 / BL21 and pET28a-pel419 V52A A single colony of / BL21 was inoculated in LB liquid medium containing 60mg / L Kan, cultured to OD at 37°C and 220r / min 600 To 0.6, add 0.5mmol / L IPTG, induce expression at 28°C, 120r / min for 12-15h.

[0063] Take 1mL of the induced maturation fermentation broth in a 1.5mL centrifuge tube, centrifuge at 10,000r / min for 5min, discard the supernatant, add 500 μL of normal saline, vortex, centrifuge, and wash twice. Sterilize ddH with 40 μL 2 O to suspend the bacterial pellet, add 10 μL of 5× protein loading buffer, boil for 5 minutes, cool naturally, and store at -20°C for later use (note: use a boiling water bath for 3 minutes before loading).

[0064] The prepared samples were analyzed by discontinuous SDS-PAGE (5% stacking gel and 12% separating gel) (such as Figure 4 shown), the pET28a / BL21 strain w...

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Abstract

The invention discloses a pectic lyase mutant [delta]Pel419 as well as a coding gene, preparation method and application thereof. The pectic lyase mutant [delta]Pel419 is obtained by mutating a 52nd amino acid in a rigid region of wild type pectic lyase Pel419 from branched-chain valine with a large molecular weight into alanine with a small molecular weight; and an amino acid sequence and a nucleotide sequence of the pectic lyase mutant [delta]Pel419 are SEQ ID NO. 1 and SEQ ID NO. 2 separately. The enzyme activity and heat resistance of the mutant enzyme provided by the invention are obviously improved under alkaline conditions, the problems of low catalytic activity and insufficient heat stability of the wild type pectic lyase under the alkaline conditions are solved, and good conditions are created for application of the enzyme in the industries of cotton and linen processing, pulping and papermaking, industrial wastewater treatment and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and more specifically relates to a pectin lyase mutant ΔPel419 and its coding gene, preparation method and application. Background technique [0002] Pectinase is an important class of industrial enzymes, which have been widely used in the paper industry, food processing, environmental protection and textile industries. [0003] According to the substrate and mode of action, pectinase can be divided into protopectinase, polygalacturonase, pectate lyase and pectin esterase. According to the optimum pH of the enzymatic hydrolysis reaction, it is divided into acid pectinase and alkaline pectinase; Pectinase is mainly used in cotton yarn biorefining and hemp biodegumming in textile processing. [0004] Compared with the traditional chemical method, the bio-enzyme method applied to hemp degumming has the advantages of energy saving, emission reduction and consumption reduction, and is favo...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C02F3/34C12R1/19C02F101/34
CPCC12N9/88C12N15/70C12Y402/02002C02F3/342C02F2101/34
Inventor 成莉凤彭源德段盛文冯湘沅杨琦郑科彭正红
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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