Ultra-Mn2<+>-resistant bacterial laccase, recombinant vector, recombinant bacteria, enzymic preparation, compound enzyme system and preparation method and application thereof

A recombinant vector, recombinant bacteria technology, applied in the biological field, can solve the problems of reduced enzyme activity, low yield, remaining 20%, etc.

Inactive Publication Date: 2017-07-14
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Before the 1980s, laccase was mainly directly isolated from plants or fungi, with low yield and high cost
Ye Mao et al. isolated an alkaline laccase from mangroves using metagenomic methods and carried out directed evolution of the enzyme, but the enzyme was restricted by Mn 2+ Inhibition, low concentrations of Mn 2+ That is, the enzyme activity can be reduced to 70%; the optimal reaction temperature is 55°C with guaiacol as the substrate, and only 20% of the activity remains after treatment at this temperature for 30 minutes
[0005] The Chinese invention patent with publication number CN106434707A discloses a bacterial laccase gene derived from Bacillus subtilis ZNXH4, bacterial laccase and its application, but it can only tolerate 10mM Mn 2+

Method used

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  • Ultra-Mn2&lt;+&gt;-resistant bacterial laccase, recombinant vector, recombinant bacteria, enzymic preparation, compound enzyme system and preparation method and application thereof
  • Ultra-Mn2&lt;+&gt;-resistant bacterial laccase, recombinant vector, recombinant bacteria, enzymic preparation, compound enzyme system and preparation method and application thereof
  • Ultra-Mn2&lt;+&gt;-resistant bacterial laccase, recombinant vector, recombinant bacteria, enzymic preparation, compound enzyme system and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The acquisition of bacterial laccase gene in the present embodiment comprises the following steps:

[0071] (1) Acquisition of metagenomic DNA in soil samples: The samples were collected from the sewage outfall sludge of a paper mill in Xuchang City, Henan Province. Add DNAextraction buffer 18mL (containing 100mM EDTA, 100mM sodium phosphate, 1.5M NaCl, 1% CTAB and 100mM Tris-Hcl, pH 8.0), vortex to mix, shake at 220rpm, 37℃ for 30min, then add 2mL to each tube 20% SDS to make the final concentration 2% (w / v). Then put it in a water bath at 65°C for 2 hours, and gently invert it up and down several times every 15 minutes to mix well. Centrifuge at 6,000g at room temperature for 10min. After collecting the supernatant, transfer it to a clean centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1, V / V), and gently invert up and down to mix. Then centrifuge at 11,000g for 20min at 4°C to collect the supernatant, add 0.6 times the volume of isopropanol a...

Embodiment 2

[0080] The acquisition of bacterial laccase recombinant vector in the present embodiment comprises the following steps:

[0081] 1. The positive clones obtained in Example 1 were inoculated in 5 mL of LB medium (100 μg / mL Amp), cultured overnight at 37° C. on a shaker at 220 rpm, and plasmids were extracted.

[0082] 2. Design primers LacF and LacR, respectively introduce EcoR I and Hind III restriction sites that can be inserted into the Escherichia coli expression vector pet28a at both ends of the primers, and remove the stop codon.

[0083] The nucleotide sequence of primer LacF (upstream primer) is shown in SEQ ID NO.3;

[0084] The nucleotide sequence of the primer LacR (downstream primer) is shown in SEQ ID NO.4.

[0085] 3. Using the extracted plasmid as a template, use primers LacF and LacR for PCR amplification: the total volume of the PCR system is 30 μL, including 15 μL of Taq mix (Tiangen Company), 1 μL of each of the two primers (10 μM), 12 μL of sterilized disti...

Embodiment 3

[0088]The acquisition of recombinant bacteria in this example includes: transforming the host bacteria Escherichia coli BL21 (DE3) with the recombinant vector obtained in Example 2, coating the LB medium plate containing 100 μg / mL kanamycin (Kam) with the transformation solution, 37 Cultivate overnight at ℃, extract positive colony plasmids, and use primers LacF and LacR for PCR identification. The correct recombinant bacteria obtained contain the above-mentioned bacterial laccase gene.

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Abstract

The invention relates to ultra-Mn2<+>-resistant bacterial laccase, a recombinant vector, recombinant bacteria, an enzymic preparation, a compound enzyme system and a preparation method and an application thereof and belongs to the biotechnical field. The ultra-Mn2<+>-resistant bacterial laccase is originated from bacterial laccase lac1542 of metagenome, and the gene nucleotide sequence of the laccase is as shown in SEQ ID NO.1. The optimum pH of the bacterial laccase taking ABTS as a primer is 4.0; the optimum reaction temperature is 75 DEG C and the bacterial laccase is highly stable at a high temperature; 100mmol/L Mn2<+> still can activate the enzyme. Bacterial laccase, manganese peroxidase and coprinus cinereus peroxidase form the compound enzyme system which can degrade 71.5% of lignin at a low concentration; the decolourization ratio of the bacterial laccase and coprinus cinereus peroxidase compound enzyme system on methyl orange can reach 54.8%. The bacterial laccase provided by the invention can be used in the field of papermaking, dyeing and weaving and environmental protection as a novel enzyme source and has a wide industrial application prospect.

Description

technical field [0001] The invention relates to a super resistant Mn 2+ The bacterial laccase, the recombinant vector, the recombinant bacteria, the enzyme preparation, the compound enzyme system and the preparation method and application thereof belong to the field of biotechnology. Background technique [0002] Laccase is a copper-containing polyphenol oxidase, which can oxidize polyphenols, toxic aromatic amines, decolorize dyes and decompose lignin, etc. It has always been a research hotspot in the fields of biology, chemistry and environmental science; It has been widely researched and applied in the fields of lignin degradation, pulp bleaching, and organic pollutant treatment. Before the 1980s, laccase was mainly isolated directly from plants or fungi, with low yield and high cost. With the development of molecular biology technology, it is possible to clone laccase gene to achieve heterologous expression and industrial production. In 1988, Fronhman et al first clon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N9/08D21C5/00C02F3/34C02F101/38
CPCC02F3/342C02F2101/308C02F2101/40C12N9/0061C12N9/0065C12Y110/03001C12Y111/01013D21C5/005
Inventor 董冰雪张磊张伟夏敏李鹏蔡心清吴倩倩
Owner NANYANG NORMAL UNIV
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