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Alkaline pectinase mutant with improved heat stability as well as encoding gene and application of alkaline pectinase mutant

A technology that encodes genes and pectinases, which can be used in applications, genetic engineering, plant genetic improvement, etc., can solve problems affecting the effect and decline of pectinases

Active Publication Date: 2017-12-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the temperature rises above 45°C, the enzyme activity of pectinase will decrease sharply with the increase of temperature. After 0.5h of water bath treatment at 60°C, about 40% of the enzyme activity remains, which seriously affects the effect of pectinase Effect

Method used

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  • Alkaline pectinase mutant with improved heat stability as well as encoding gene and application of alkaline pectinase mutant
  • Alkaline pectinase mutant with improved heat stability as well as encoding gene and application of alkaline pectinase mutant
  • Alkaline pectinase mutant with improved heat stability as well as encoding gene and application of alkaline pectinase mutant

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Experimental program
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Effect test

Embodiment 1

[0054] Embodiment 1, the gene sequence preparation of alkaline pectinase PelN mutant

[0055] One, the preparation of the gene sequence of alkaline pectinase PelN single mutant

[0056] The wild-type alkaline pectinase PelN gene shown in sequence 1 is mutated to obtain the alkaline pectinase PelN mutant gene. The specific steps are as follows: the mutated nucleotides are introduced into the PelN gene by the method of segmented PCR amplification; the forward primer used in the front sequence PCR is the PelN universal primer PelN-F (the underlined part is the restriction site), and the reverse primer is the specific primer containing the mutation site (the part in bold is the mutated nucleotide), and the template is the PelN gene. The forward primer used in the subsequent sequence PCR is a specific primer containing a mutation site (the bold part is the mutated nucleotide), the reverse primer is the PelN general primer PelN-R (the underlined part is the restriction site), and t...

Embodiment 2

[0064] Embodiment 2, the preparation of alkaline pectinase PelN mutant

[0065] 1. Construction of expression vector

[0066] Insert the gene sequence of the wild-type alkaline pectinase PelN shown in sequence 1 between the Nde1 and Xho1 restriction sites of the expression vector pET-22b(+), and keep other sequences of the expression vector pET-22b(+) from The expression vector PelN is obtained; the expression vector PelN expresses the wild-type alkaline pectinase PelN with the HIS tag at the C-terminus, and the amino acid sequence of the wild-type alkaline pectinase PelN is sequence 6.

[0067] Insert the gene sequence of the alkaline pectinase PelN single mutant K93I shown in sequence 2 between the Nde1 and Xho1 restriction sites of the expression vector pET-22b(+), and keep the other parts of the expression vector pET-22b(+) The sequence remains unchanged, and the expression vector K93I is obtained; the expression vector K93I expresses the alkaline pectinase PelN mutant K9...

Embodiment 3

[0081] Embodiment 3, the thermostability comparison of wild-type alkaline pectinase PelN and mutant

[0082] The present invention compares wild-type alkaline pectinase PelN (PelN-WT, referred to as PelN or wild) and alkaline pectinase PelN single mutant K93I (PelN-K93I, referred to as K93I), alkaline pectinase PelN single mutant G241A (PelN-G241A, referred to as G241A), alkaline pectinase PelN single mutant G241V (PelN-G241V, referred to as G241V) and alkaline pectinase PelN double mutant K93I / G241A (PelN-K93I / G241A, referred to as K93I / G241A), wherein the wild-type alkaline pectinase PelN is Paenibacillus sp.0602 (GenBank accession number: KC351190) derived alkaline pectinase PelN-WT (amino acid sequence is sequence 6, which encodes The gene sequence is sequence 1).

[0083] 1. Comparison of remaining enzyme activity, half-life and specific activity of wild-type alkaline pectinase PelN and mutants after treatment at 60°C for different times

[0084] 1. Residual enzyme acti...

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Abstract

The invention discloses an alkaline pectinase mutant with improved heat stability as well as an encoding gene and application of the alkaline pectinase mutant. By carrying out site-specific mutagenesis at two sites of wild alkaline pectinase, the alkaline pectinase mutant with the improved enzyme activity and heat stability and the decreased optimal action temperature is obtained; the optimal enzyme activity temperature of a double mutant pelN-K93I / G241A is decreased by 5 DEG C than that before mutation, Tm is increased by 1.671 DEG C, the half-life period is prolonged by 15.86 minutes at 60 DEG C, Km is decreased from 2.59 g / L to 1.1 g / L, and the optimal enzyme activity temperature is decreased from 67.5 DEG C to 60 DEG C. The alkaline pectinase mutant has relatively high heat stability and relatively high ramie degumming rate, so that the alkaline pectinase mutant is more suitable for degumming processes and has relatively good application potentials in the textile industry.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alkaline pectinase mutant with improved thermostability, its coding gene and its application. Background technique [0002] In the traditional cotton and linen refining process, the desized cotton and linen fabric is generally treated with a high concentration of strong alkali solution to remove the non-fibrous components on the plant surface. In this process, not only the fiber components of cotton and linen will be damaged, affecting the quality of downstream products, but also a large number of chemical components are introduced in the process, which increases the difficulty of wastewater treatment and the production cost of products. In recent years, biological enzyme treatment methods have attracted more and more attention, among which alkaline pectinase is the main one. Enzyme refining can significantly reduce energy consumption, simplify wastewater treatment, a...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70D01C1/02
CPCC12N9/88C12N15/70C12Y402/0201D01C1/02
Inventor 周站平刘阳宋江宁马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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