Genetic engineering bacterial strain for expressing bacillus subtilis laccase in strain cell and method for realizing secreting and expressing laccase in bacterial strain

A technology of Bacillus subtilis and genetically engineered strains, applied in the directions of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of secondary environmental pollution, low degradability, high cost, and achieve good reference value, The effect is remarkable and the method is simple

Active Publication Date: 2015-05-06
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complex structure, high chemical stability, and low degradability of most dyes, the traditional physical and chemical degradation methods are costly and low

Method used

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  • Genetic engineering bacterial strain for expressing bacillus subtilis laccase in strain cell and method for realizing secreting and expressing laccase in bacterial strain
  • Genetic engineering bacterial strain for expressing bacillus subtilis laccase in strain cell and method for realizing secreting and expressing laccase in bacterial strain
  • Genetic engineering bacterial strain for expressing bacillus subtilis laccase in strain cell and method for realizing secreting and expressing laccase in bacterial strain

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specific Embodiment approach 1

[0027] Specific embodiment one: the method for constructing the genetically engineered bacterial strain expressing Bacillus subtilis laccase intracellularly in the present embodiment is carried out according to the following steps:

[0028] 1. Extraction of Bacillus genomic DNA and cloning of bacterial laccase gene:

[0029] The genomic DNA of Bacillus subtilis LS02 was extracted, using the genomic DNA as a template, using primers LS02-F and LS02-R for PCR amplification, introducing restriction sites NdeI and BamhI at both ends of the laccase gene sequence, and the amplified product was purified Afterwards, it was connected to the pEASY-Blunt cloning vector to obtain the recombinant vector pEASY / CotA. Using pEASY / CotA as a template, the point mutation primers LS02-Mut-F and LS02-Mut-R were used to amplify the whole plasmid by PCR, and the CotA DNA sequence was The 927th base T was mutated into base C. On the basis of not changing the amino acid sequence of the CotA protein, th...

specific Embodiment approach 2

[0046] Specific embodiment two: this embodiment realizes the method for inducing the secretion and expression of laccase by the bacterial strain described in specific embodiment one, and proceeds according to the following steps:

[0047] 1. Insert the recombinant expression strain PSD glycerol into LB medium containing 50mg / L Amp, and culture overnight at 37°C and 180rpm for 12-14h to obtain the bacterial solution;

[0048] 2. Inoculate the bacterial liquid obtained in step 1 into fresh LB medium containing 50mg / L Amp according to 1% inoculum amount, and cultivate to OD at 30°C and 180rpm 600 is 0.8, add 0.1mM IPTG and 0.1mM CuSO 4 , cultured at 120 rpm at 25°C for 4 hours, and then cultured statically at 30°C; from the time of adding the IPTG inducer, samples were taken every 4 hours to detect the laccase activity of the medium supernatant, intracellular, and periplasmic space, and simultaneously prepared Protein samples were used for SDS-PAGE analysis.

[0049] Laccase ac...

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Abstract

The invention discloses a genetic engineering bacterial strain for expressing bacillus subtilis laccase in a strain cell and a method for realizing secreting and expressing laccase in a bacterial strain, and relates to the genetic engineering bacteria strain for expressing bacillus the subtilis laccase in the strain cell and the method for realizing secreting and expressing laccase in the bacterial strain. A preparation method for the genetic engineering bacterial strain comprises the following steps: I, extracting DNA of a bacillus genome and cloning a bacterial laccase gene; and II, constructing a recombinant expression bacterial strain. An inducing method for realizing secreting and expressing recombinant laccase comprises the following steps: I, accessing the engineering bacterial strain into an LB (liquid-phase basicity) culture medium to culture until OD600 is 0.8; and II, adding 0.1 mM of IPTG (isopropyl-beta-d-thiogalactoside) and 0.1 mM of CuSO4 to culture for 4 hours at 25 DEG C under 120 rpm, and then performing static culture at 30 DEG C. The inducing method provided by the invention can remarkably improve the secretion effects of the recombinant laccase, is simple to operate, and has a high reference value in improving recombinant escherichia coli to secrete laccase.

Description

technical field [0001] The invention relates to a genetically engineered bacterial strain expressing Bacillus subtilis laccase in cells and an induction method for realizing the secretion and expression of the laccase from the bacterial strain. Background technique [0002] Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases, the largest subgroup of multi-copper oxidases. The enzyme utilizes O 2 As the electron acceptor catalytic substrate, water is the only by-product in the reaction process, which is a typical "green catalyst". Laccase has a wide range of substrates. So far, the application range of laccase has covered many fields such as environmental pollution control, lignocellulosic material improvement, biosynthesis, paper industry, dye wastewater degradation, and flavor food improvement. [0003] Laccase widely exists in fungi, insects, plants, and prokaryotes. At present, the research on fungal laccase is relatively in-depth. However, although fungal...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/66C12N9/02C12R1/125
CPCC12N9/0061C12Y110/03002
Inventor 王天女赵敏卢磊王靖瑶刘彤郑小芳
Owner NORTHEAST FORESTRY UNIVERSITY
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