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Catalase and high-yielding genetic engineering strains thereof

A technology of catalase and genetically engineered bacteria, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of many by-products, difficulty in obtaining mutant strains, and heavy workload.

Active Publication Date: 2016-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of wild strains to produce catalase has defects such as more by-products and lower yields. Although wild strains can be screened through breeding techniques, the workload is large and it is difficult to obtain ideal mutant strains.

Method used

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  • Catalase and high-yielding genetic engineering strains thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Construction and Transformation of Recombinant Plasmid pET-28a-KatX2

[0021] [1] Genomic DNA of Bacillus pumilus was used as a template.

[0022] [2] According to the catalase gene sequence of Bacillus pumilus (KatX2, GenBank Accession No. CP011150.1) and the restriction site on the pET-28a or pMA5 plasmid, the KatX2 gene primers were designed.

[0023] P1: 5'-ACGCGTCGACAAATGACAAATTCAAATCAT-3' (Sal I)

[0024] P2: 5'-CCGCTCGAGTTATTTCATGCTTCCTTG-3'(Xho I)

[0025] [3] Using the DNA of Bacillus pumilus as a template to do PCR amplification to obtain the gene. PCR amplification system: template 2 μL, upstream and downstream primers P1 and P2 0.5 μL each, dNTP Mix 4 μL, 10×Ex Taq Buffer 5 μL, sterilized ddH 2 O 37 μL, Ex Taq DNA polymerase 1 μL. PCR reaction conditions: 94°C pre-denaturation, 5min, one cycle; 94°C denaturation, 1min, 58°C annealing, 1min, 72°C extension, 1min 30s, 30 cycles; 72°C, 10min, one cycle; 15°C, 10min, a cycle. The PCR product was...

Embodiment 2

[0028] Example 2: Preparation of Escherichia coli Competent and Transformation of Plasmid

[0029][1] Preparation of competent Escherichia coli. Activate the monoclonal Escherichia coli in 10ml LB medium, then transfer to 37°C shaking culture to OD 600 0.35 to prepare the competent state; put the cultured bacterial solution in ice water, shake gently to cool the bacterial solution for about 10 minutes; prepare several 1.5ml centrifuge tubes for sterilization, and divide the bacterial solution into the tubes, The amount of bacteria in the tube is 1.2ml, put the centrifuge tube in ice; centrifuge the bacteria solution at 8000r / min for 10-20s, let it stand in ice water for 2min, discard the supernatant, add pre-cooled 0.1M CaCl 2 400μL, gently blow the suspension, put it in ice for 15min (repeat this step 2-3 times); finally, add pre-cooled 0.1M CaCl 2 80 μL, gently pipette the suspension and place it on ice.

[0030] [2] Transformation of plasmids. Take the competent cell...

Embodiment 3

[0031] Embodiment 3: Construction and transformation of recombinant plasmid pET-28a-KatX2

[0032] [1] Genomic DNA of Bacillus pumilus was used as a template.

[0033] [2] According to the catalase gene sequence of Bacillus pumilus (KatX2, GenBank Accession No. CP011150.1) and the restriction site on the pMA5 plasmid, KatX2 gene primers were designed.

[0034] P3: 5'-CGGGATCCATGACAAATTCAAATCATAAAAAT-3'(BamH I)

[0035] P4: 5'-CGACGCGTTTATTCATGCTTCCTTG-3'(MluI)

[0036] [3] Using the DNA of Bacillus pumilus as a template to do PCR amplification to obtain the gene. PCR amplification system: template 2 μL, upstream and downstream primers P3 and P4 0.5 μL each, dNTP Mix 4 μL, 10×Ex Taq Buffer 5 μL, sterilized ddH 2 O 37 μL, Ex Taq DNA polymerase 1 μL. PCR reaction conditions: 94°C pre-denaturation, 5min, one cycle; 94°C denaturation, 1min, 58°C annealing, 1min, 72°C extension, 1min 30s, 30 cycles; 72°C, 10min, one cycle; 15°C, 10min, a cycle. The PCR product was purified and...

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Abstract

The invention relates to a novel-originated catalase and the construction of recombinant engineering strains of the catalase. The construction is characterized in that primers are designed according to a catalase gene of bacillus pumilus for the first time, and recombinant expression vectors are constructed; the vectors are converted to genetic engineering strains, i.e., Escherichia coli and Bacillus subtilis for heterologous expression; the recombinant engineering strains are subjected to fermented cultivation through a 5L fermentation tank by using a fermentation culture medium, wherein catalase of recombinant Escherichia coli is mainly subjected to intracellular expression and has the enzyme activity of 6645U / mL, catalase of recombinant B. subtilis 168 has the total enzyme activity of 23,263U / mL and the extracellular enzyme activity of 15,368U / mL, catalase of B. subtilis WB600 has the total enzyme activity of 26,635U / mL and the extracellular enzyme activity of 20,128U / mL, the extracellular synthesis of the catalase is better facilitated by using the B. subtilis WB600 as a host, and thus the B. subtilis WB600 has an important industrial application value. The invention achieves the maximum yield compared with currently-reported methods which are used for producing the catalase by using microbes, so that a practical and effective policy for industrial production of the catalase is provided.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a novel catalase gene derived from Bacillus pumilus, which is connected to pET-28a or pMA5 plasmid and expressed in Escherichia coli or Bacillus subtilis respectively , using recombinant Escherichia coli or recombinant Bacillus subtilis to ferment a method for high-production catalase. Background technique [0002] All oxygen-dependent organisms in nature can produce reactive oxygen species such as O 2- , H 2 o 2 And hydroxyl radicals, and if this type of active oxygen is excessive, it will have a toxic effect on cells and cause the oxidation of nucleic acids, proteins and lipids. Catalase (Catalase, EC 1.11.1.6), as the core enzyme for removing active oxygen radicals, widely exists in a large part of oxygen-dependent organisms, and it can catalyze H 2 o 2 Oxygen and water are produced to maintain the redox balance in living organisms. Ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N1/21C12N15/70C12N15/75C12R1/19C12R1/125
CPCC12N9/0065C12Y111/01006
Inventor 饶志明杨套伟周俊平张显徐美娟菲利波
Owner JIANGNAN UNIV
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