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Laccase gene, engineering bacteria and application

A technology of genetically engineered bacteria and engineered bacteria, applied in the field of environmental biology, can solve the problems of discovering endogenous laccase and achieve the effect of improving expression activity

Active Publication Date: 2013-09-18
ZHEJIANG SHUANGLIANG SUNDA ENVIRONMENTAL PROTECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The fungi that secrete laccase mainly include Basidiomycetes, Polypores, Deuteromycetes, Ascomycetes, Neurospora, Podosporium and Aspergillus, and most of them are distributed in Basidiomycetes, followed by Ascomycetes. Most of them are white rot fungi of Basidiomycotina, but no endogenous laccase has been found in yeast

Method used

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  • Laccase gene, engineering bacteria and application
  • Laccase gene, engineering bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Optimization of the codon of laccase gene

[0022] According to the cotA gene sequence of Bacillus subtilis (GENE ID: 936023), combined with the codon preference expressed by Pichia pastoris, codon optimization was carried out on the cotA gene sequence, and a restriction site was introduced in the upstream and downstream of the full-length gene: EcoRI and NotI. At the same time, in order to prevent the optimized gene sequence from containing the EcoRI restriction site and the subsequent DNA linearization Bgl II restriction site, and adjust the GC content of the optimized gene, the 855th base A was mutated to G, and the 1098th base was mutated. A base T is mutated to a C. The full length of the gene is 1542 bases, encoding 514 amino acids. The optimized laccase gene was named Tcot.

[0023] Design restriction sites and protective bases at both ends of the gene, and design primers as follows:

[0024] P1: ACGGAATTCATGACTTTGGAAAAGTTTGTTGATGCTTTGCCAATTCCAGATACTTTGAAGCCA...

Embodiment 2

[0056] Expression of Laccase Gene in Yeast Cells

[0057] 1. Construction of expression vector

[0058] The codon-optimized PCR product was digested with EcoRI / NotⅠ, the fragment was recovered, and inserted into the Pichia pastoris constitutive expression vector pGAPZaA (Invitrogen, USA) with the correct reading frame, ligated and transformed E. coli DH5α, the recombinant plasmid pGAPZaA-Tcot was obtained. The recombinant plasmid was verified by EcoRI / NotI double enzyme digestion.

[0059] 2. Preparation of competent cells for electroporation

[0060] In a 50 ml centrifuge tube containing 5 ml YPD, culture yeast cells for 30 overnight; take 30~50 ul of the overnight culture, inoculate a 250 ml shake flask containing 50 ml of fresh medium, grow overnight until OD600=1.3~1.5 ;Collect the cells by centrifugation at 1500 g at 4°C for 5 min, and suspend the cells with 50 ml of pre-cooled sterile water; centrifuge as above, and suspend the cells with 25 ml of pre-cooled sterile...

Embodiment 3

[0076] Study on the Properties of Laccase

[0077] 1. Effect of pH value on laccase activity

[0078] Laccase was added to the buffer system with different pH values ​​to measure the enzyme activity, the pH was set from 2.0 to 10.0, and points were taken every 0.5 for measurement. The enzyme activity assay reaction was carried out at 25°C, and the reaction time was 15 min. The result is as figure 1 It was shown that the optimum pH value of laccase is 4.5~5.5, and in the range of pH4.0 to pH6.5, the enzyme activity of laccase can maintain more than 50%.

[0079] 2. Effect of temperature on laccase activity

[0080] The optimal reaction temperature of laccase was determined at the optimal pH value, the reaction time was 15 min, the temperature was set from 15 °C to 75 °C, and the measurement was taken every 5 °C. The result is as figure 2 It was shown that the optimum temperature of laccase was 25-35°C, and the enzyme activity could maintain more than 60% at 20-45°C. ...

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Abstract

The invention relates to a field of environmental biology, and discloses a laccase gene, an engineering bacteria of the laccase gene, and an application thereof in treatment of papermaking waste water by microbial hydrolysis. The laccase gene has a nucleotide sequence as shown in a SEQIDNO:2, and codes a protein having an amino acid sequence as shown in a SEQIDNO:1. According to the invention, the laccase gene optimized according to preferred codons of pichia yeast can be constitutively expressed in high efficiency in yeast, and an expression vitality of the laccase can be improved, thereby being of great significance for promoting the application of the laccase in biological treatment of the papermaking waste water.

Description

technical field [0001] The invention relates to the field of environmental biology, in particular to a laccase gene, a laccase gene engineering bacterium and its application in microbial hydrolysis treatment of papermaking wastewater. Background technique [0002] Laccase is a protein that binds multiple copper atoms and belongs to the group of ceruloid oxidases. Laccase has a wide range of substrates and can treat various pollutants such as various phenolic dyes, substituted phenols, chlorophenols, thiophenols, bisphenols, and aromatic amines. Laccase is the most potential biological enzyme discovered in the paper industry in recent years, which has the ability to catalyze the oxidation of lignin. Laccase can selectively catalyze the degradation of lignin without producing toxic substances, and the production is carried out under mild conditions of normal temperature and normal pressure, saving equipment and energy consumption. Therefore, laccase has a very great applicat...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/10C12N1/19C02F3/34C12R1/125C12R1/84C02F103/28
Inventor 郑展望杨瑾
Owner ZHEJIANG SHUANGLIANG SUNDA ENVIRONMENTAL PROTECTION CO LTD
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