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Laccase mutant and encoding gene and application thereof

A coding and gene technology, applied in the field of genetic engineering, can solve the problems of poor stability, low specific activity, and expensive price, and achieve the effect of high specific activity and wide range of pH tolerance

Active Publication Date: 2014-04-09
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to its low specific activity, poor stability, and high price, it has not been widely used.

Method used

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  • Laccase mutant and encoding gene and application thereof
  • Laccase mutant and encoding gene and application thereof
  • Laccase mutant and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the cloning of wild-type laccase gene and the construction of recombinant engineering bacteria

[0044] 1. Cloning of POXA1b gene of laccase from Pleurotus florida ACCC No.50035

[0045] Specific steps for amplifying the laccase POXA1c gene from Pleurotus florida ACCC No.50035:

[0046]1. The total RNA of Pleurotus florida ACCC No.50035 was extracted with TRIZOL Total RNA Extraction Kit (Invitrogen).

[0047] 2. Take oligo-(dT) 15 (Promega) was used as a primer, and cDNA was synthesized by reverse transcription according to conventional methods.

[0048] 3. Use the following primers to amplify the POXa1b gene of laccase.

[0049] poxa1c-F:5'-CG GAATTC AGCATTGGGCCCCGCGGAACGCTG-3' (the underlined part is the recognition sequence of EcoR I, and the following sequence is the 1-24th position of sequence 8);

[0050] poxa1c-R: 5'-ATAGTTTA GCGGCCGC TCATGCTTTCAATGGCGCAGGCAG-3' (the underlined part is the recognition sequence of Not I, and the following seq...

Embodiment 2

[0060] Embodiment 2, the acquisition of laccase mutant gene and the construction of recombinant engineering bacteria

[0061] 1. Acquisition of laccase mutant POXA1c R5V coding gene and construction of recombinant expression vector

[0062] Using the recombinant expression vector pPICZαA-POXA1c obtained in Example 1 as a template, PCR amplification was performed using primers poxa1c R5V-F and poxa1c R5V-R.

[0063] poxa1c R5V-F: 5'-ATTGGGCCC GTT GGAACGCTGAACATCGCGAAC-3' (position 4-36 of sequence 6, the underlined part is the mutation site compared with sequence 8);

[0064] poxa1c R5V-R:5'-TTCC AAC GGGCCCAATGCT GAATTC -3' (the underlined part in italics is the recognition sequence of EcoR Ⅰ, the sequence before it is the reverse complementary sequence of the 1-19 position of sequence 6, and the underlined part is the mutation site compared with sequence 8).

[0065] The obtained PCR product was transformed into Escherichia coli competent DH5α, and the PCR product circu...

Embodiment 3

[0085] Embodiment 3, wild-type laccase and enzyme activity determination of different laccase mutants

[0086] 1. Fermentation of laccase-producing engineering strains, acquisition and purification of laccase

[0087] 1. Fermentation of laccase-producing engineering strains

[0088] The engineering bacteria X33 / pPICZαA-POXA1c expressing wild-type laccase POXA1c obtained in Example 1, the engineering bacteria X33 / pPICZαA-POXA1c R5V, X33 / pPICZαA-POXA1cΔ13-R5V and X33 expressing laccase mutants obtained in Example 2 / pPICZαA-POXA1cΔ13 were respectively cultured with fermentation medium, and after 24 hours of culture, anhydrous methanol was added to the fermentation system to induce the expression of laccase; the method of adding anhydrous methanol was once every 12 hours (the first The time point of the first addition is the 24th hour of cultivation), and a total of 5 additions were added, and anhydrous methanol was added each time to make the initial concentration of anhydrous ...

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Abstract

The invention discloses a laccase mutant and an encoding gene and an application thereof. The laccase mutant disclosed by the invention is any one of the following protein in a) to d): a) protein as shown in a sequence 1; b) protein as shown in a sequence 3; c) protein as shown in a sequence 5; d) protein with laccase activity and obtained by substituting and / or deleting and / or adding one or multiple amino acid residues in the amino acid sequences of the proteins limited by any one of a) to c). As shown in experiments, compared with wild laccase, the laccase mutant disclosed by the invention has the advantages of high activity, wide pH tolerance range and the like. The laccase mutant disclosed by the invention is expected to be applied in such fields as wastewater treatment in pulp bleaching and dyeing and printing industry, biological detection in medicine field, food industry, organic synthesis, organic pesticide degradation, etc.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a laccase mutant, its coding gene and its application. Background technique [0002] Laccase (EC.1.10.3.2) belongs to the blue multi-copper oxidase family, which can oxidize a variety of phenols and other organic compounds, so it is widely used in industrial fields such as dye degradation, pulp bleaching, organic synthesis and biodegradation. However, due to its low specific activity, poor stability and high price, it has not been widely used. Laccase is widely distributed in fungi, bacteria, plants and so on. Among them, laccases in fungi are the most extensively studied, and the laccases expressed in fungi have higher specific activities, such as Trametes versicolor, Phalomyces wood-rot, Pleurotus spp., and so on. But the laccases they express often contain several isozymes. The specific activity of some isozymes is very low, so it is meaningful to find laccases with high spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12R1/84
CPCC12N9/0061C12Y110/03002
Inventor 胡美荣周雪陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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