Bacillus pumilus laccase gene as well as expression and application thereof

Abstract

The invention relates to a bacillus pumilus (Bacillus pumilus) L-9 laccase gene cotA as well as recombinant expression and application thereof, belonging to the field of bioengineering. The cotA is separated from the bacillus pumilus (Bacillus pumilus) L-9, an expression vector pColdII-cotA is constructed, and an escherichia coli expression strain BL21(DE3) is transformed to realize high-efficiency expression. The obtained recombinase laccase has the advantages of high alkaline environment resistance and high high-temperature resistance, has a good effect of discoloring azo dyes and anthraquinone dyes and has high application potential.

Description

technical field

[0001] The invention relates to a laccase gene, in particular to a Bacillus pumilus laccase gene and its expression and application, belonging to the technical field of bioengineering. Background technique

[0002] Laccase (Laccase, E.C.1.10.3.2) is a copper-containing polyphenol oxidase that can catalyze the redox reaction of phenolic substances and play an important role in the biodegradation of lignin and its precursor analogs. Laccase has a wide range of oxidation substrates, including phenols and their derivatives, aromatic amines and their derivatives, aromatic carboxylic acids and their derivatives, etc., so the application potential of laccase is huge. In the field of wood processing, laccase can replace chemical adhesives, which can not only improve product quality, but also reduce the harm to human health and environmental pollution; in the paper industry, laccase is used for paper biological bleaching and pulping, which can Reducing pollution in p...

Examples

Embodiment 1

[0033] Example 1 PCR amplification of B. pumilus L-9 laccase gene cotA, protein sequence functional region prediction

[0034] Bacterial Genomic DNA Extraction Kit (TaKaRa Company) was used to extract the total genomic DNA of B. pumilus L-9 bacteria according to its operation manual, its quality and purity were identified by agarose gel electrophoresis, and its concentration was determined by ultraviolet spectrophotometer. The upstream and downstream primers were designed according to the laccase gene sequences of other Bacillus pumilus strains published in the GenBank database: g3 (SEQ ID NO: 3, 5′-ATGAACCTAGAAAAATTTGTTGACGAGCTG-3′) and g4 (SEQ ID NO: 4, 5′- TTACTGGATGATATCCATCGGCCGCATC-3'), using the PCR method, using the above-mentioned B. pumilus L-9 genome as a template, and using the above-mentioned g3 and g4 as specific primers, the full-length coding frame sequence of the B. pumilus L-9 laccase gene was amplified ( 1533bp) (eg figure 1 Shown in A), then use the T-A c...

Embodiment 2

[0035] Example 2 Obtaining the Predicted Crystal Structure of B. pumilus L-9 Laccase Using "Homologous Modeling" Method

[0036] Submit the amino acid sequence of B. pumilus L-9 laccase to the SWISS-MODEL protein online modeling server (http: / / swissmodel.expasy.org / ) for homology modeling, and the optimal modeling template obtained by automatic analysis of the server is The crystal structure of B. subtilis MB24 laccase I494A mutant (PDB file: 2wsdA) was subsequently shown using the RasWin Molecular Graphics Program (RasMol, 156version2.7.2) software package (http: / / www.umass.edu / microbio / rasmol / ) The predicted structure of B.pumilus L-9 laccase crystal shown by Figure software is as follows figure 2 shown.

Embodiment 3B

[0037] Escherichia coli recombinant expression, purification, activity staining and immunoblotting identification of embodiment 3B.pumilus L-9 laccase

[0038] Since the laccase protein CotA of the wild strain of B. pumilus is only expressed on the exine wall of the spore and not secreted outside the cell, and the expression level is generally very low, which is far from meeting the requirements of industrial applications, the recombinant expression system of Escherichia coli is used to produce laccase It can greatly increase its output, making it easier to meet the requirements of industrial application. Design primer g5: (SEQ ID NO:5, 5'-TCAGGA GGTACC ATGAACCTAGAAAAATTTGTTG-3′) and g6: (SEQ ID NO:6, 5′-ACTGAG GAATTC TTACTGGATGATATCCATCG-3′), the 5′ and 3′ sides of the DNA coding frame of B. pumilus L-9 laccase were introduced into Kpn1 and EcoRI restriction enzyme sites (shown underlined) respectively by PCR. Ligation and other genetic operations Insert the laccase gene i...

technical field

[0001] The invention relates to a laccase gene, in particular to a Bacillus pumilus laccase gene and its expression and application, belonging to the technical field of bioengineering. Background technique

[0002] Laccase (Laccase, E.C.1.10.3.2) is a copper-containing polyphenol oxidase that can catalyze the redox reaction of phenolic substances and play an important role in the biodegradation of lignin and its precursor analogs. Laccase has a wide range of oxidation substrates, including phenols and their derivatives, aromatic amines and their derivatives, aromatic carboxylic acids and their derivatives, etc., so the application potential of laccase is huge. In the field of wood processing, laccase can replace chemical adhesives, which can not only improve product quality, but also reduce the harm to human health and environmental pollution; in the paper industry, laccase is used for paper biological bleaching and pulping, which can Reducing pollution in p...

Examples

Embodiment 1

[0033] Example 1 PCR amplification of B. pumilus L-9 laccase gene cotA, protein sequence functional region prediction

[0034] Bacterial Genomic DNA Extraction Kit (TaKaRa Company) was used to extract the total genomic DNA of B. pumilus L-9 bacteria according to its operation manual, its quality and purity were identified by agarose gel electrophoresis, and its concentration was determined by ultraviolet spectrophotometer. The upstream and downstream primers were designed according to the laccase gene sequences of other Bacillus pumilus strains published in the GenBank database: g3 (SEQ ID NO: 3, 5′-ATGAACCTAGAAAAATTTGTTGACGAGCTG-3′) and g4 (SEQ ID NO: 4, 5′- TTACTGGATGATATCCATCGGCCGCATC-3'), using the PCR method, using the above-mentioned B. pumilus L-9 genome as a template, and using the above-mentioned g3 and g4 as specific primers, the full-length coding frame sequence of the B. pumilus L-9 laccase gene was amplified ( 1533bp) (eg figure 1 Shown in A), then use the T-A c...

Embodiment 2

[0035] Example 2 Obtaining the Predicted Crystal Structure of B. pumilus L-9 Laccase Using "Homologous Modeling" Method

[0036] Submit the amino acid sequence of B. pumilus L-9 laccase to the SWISS-MODEL protein online modeling server (http: / / swissmodel.expasy.org / ) for homology modeling, and the optimal modeling template obtained by automatic analysis of the server is The crystal structure of B. subtilis MB24 laccase I494A mutant (PDB file: 2wsdA) was subsequently shown using the RasWin Molecular Graphics Program (RasMol, 156version2.7.2) software package (http: / / www.umass.edu / microbio / rasmol / ) The predicted structure of B.pumilus L-9 laccase crystal shown by Figure software is as follows figure 2 shown.

Embodiment 3B

[0037] Escherichia coli recombinant expression, purification, activity staining and immunoblotting identification of embodiment 3B.pumilus L-9 laccase

[0038] Since the laccase protein CotA of the wild strain of B. pumilus is only expressed on the exine wall of the spore and not secreted outside the cell, and the expression level is generally very low, which is far from meeting the requirements of industrial applications, the recombinant expression system of Escherichia coli is used to produce laccase It can greatly increase its output, making it easier to meet the requirements of industrial application. Design primer g5: (SEQ ID NO:5, 5'-TCAGGA GGTACC ATGAACCTAGAAAAATTTGTTG-3′) and g6: (SEQ ID NO:6, 5′-ACTGAG GAATTC TTACTGGATGATATCCATCG-3′), the 5′ and 3′ sides of the DNA coding frame of B. pumilus L-9 laccase were introduced into Kpn1 and EcoRI restriction enzyme sites (shown underlined) respectively by PCR. Ligation and other genetic operations Insert the laccase gene i...

Images