Thermophilic alkaline recombination manganese-containing catalase as well as expression carrier and engineering bacteria thereof

A technology of manganese catalase and expression vector, applied in the fields of thermophilic alkaline recombinant manganese-containing catalase and its expression vector and engineering bacteria

Active Publication Date: 2014-04-16
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the research on this type of thermophilic MnCAT is still in its infancy. The highest fermentation enzyme activity of this type of MnCAT reported in foreign literature is only about 20U / ml, and there is no related report in China (Non-heme manganese catalase–the'other 'catalase, Archives of biochemistry and biophysics, 2012, 525:111-120.)

Method used

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  • Thermophilic alkaline recombination manganese-containing catalase as well as expression carrier and engineering bacteria thereof
  • Thermophilic alkaline recombination manganese-containing catalase as well as expression carrier and engineering bacteria thereof
  • Thermophilic alkaline recombination manganese-containing catalase as well as expression carrier and engineering bacteria thereof

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Embodiment 1

[0061] The construction of embodiment 1 manganese catalase MnCAT prokaryotic expression genetic engineering bacteria

[0062] 1.1 MnCAT gene designed based on codon usage preference optimization in Escherichia coli

[0063] According to the preference of codon usage in Escherichia coli ( http: / / gcua.schoedl.de / seqoverall_v2.html ) optimization design, the resulting codon-optimized MnCAT gene has a nucleotide sequence as shown in SEQ ID No:1.

[0064] 1.2 Construction of MnCAT prokaryotic expression vector pET28a-MnCAT

[0065] At the 5'-end and 3'-end of the MnCAT gene, respectively design the restriction endonuclease Nco I and Hind III enzyme cutting sites that do not have in the gene but have on the multiple cloning site of the vector pET28a (+) (due to direct Adding the endonuclease Nco I nucleotide sequence CCATGG at the N-terminus of the gene will cause a frameshift mutation during the translation of the MnCAT gene. Therefore, in order to prevent the frameshift mutation...

Embodiment 2

[0068] Induced expression of embodiment 2 manganese catalase MnCAT

[0069] 2.1 Seed culture

[0070]Put the genetically engineered bacteria PB-01 into the LB medium containing 50μg / ml kanamycin, put 50ml of the medium into a 250ml culture bottle, at 37°C, the rotation speed is 200rpm, and the culture time is 10-12 hours. After cultivation, the OD600 absorbance value was between 4 and 5.

[0071] Note: The components of LB medium (g / L) are: 10 peptone, 5 yeast powder, and 10 sodium chloride.

[0072] 2.2 Shake flask fermentation culture:

[0073] Transfer the seed culture solution to TB medium containing 50mg / L kanamycin (Kan) at 1%, when the recombinant bacteria E.coli PB-01 grows to OD 600 At 0.7-0.8, add a final concentration of 0.2mmol / L IPTG, and at the same time add a final concentration of 14mmol / L MnCl 2 , induced and cultured at 42°C for 2 hours, collected the bacteria, broken the cells, and analyzed the supernatant by SDS-PAGE. It was found that there was an obvi...

Embodiment 3

[0075] Example 3 Construction of Manganese Catalase MnCAT and Mnt H Co-expression Genetic Engineering Bacteria

[0076] 3.1 Construction of prokaryotic expression vector pACYCDuet-Mnt H of manganese ion transporter Mnt H

[0077] Using the genome of E.coli W3110 (E.coli Genetic Stock Center, Yale University) as a template, primers were designed to amplify the gene DNA fragment of manganese ion transporter Mnt H by PCR. The upstream primers used were:

[0078] P1: 5'- CATATG ACGAACTATCGCGTTGAGAGTAGC-3' (underlined Nde I restriction site).

[0079] Downstream primers are:

[0080] P2: 5'- CTCGAG CTACAAATCCCAGCGCCGTC-3' (underlined Xho I restriction site).

[0081] The PCR product was recovered by gel and digested with PstI / PvuII, and then cloned into the intracellular expression vector pACYCDuet-1 after the same digestion to construct the recombinant expression plasmid pACYCDuet-Mnt H, which was verified by sequencing and stored in Escherichia coli DH5α ( Figure 4 , ...

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Abstract

The invention relates to the technical field of biology, and in particular relates to thermophilic alkaline recombination manganese-containing catalase as well as an expression carrier and engineering bacteria thereof. The invention provides a polynucleotide sequence of recombination manganese-containing catalase, which is expressed as SEQID NO.1. According to the colibacillus codon application preference, codon optimization is carried out on a manganese catalase gene sequence sourced from (i) thermusthermophilus(/i)HB27, when the optimized manganese catalase is expressed in the colibacillus, the maximal enzyme activity of the catalase detected in shake flask fermentation liquid is 120U/mL at most; when the manganese catalase is co-expressed with Mn ion transportprotein MntH, the enzyme activity of the catalase fermentation enzyme in the shake flask fermentation liquid can reach 1300U/mL.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a thermophilic alkaline recombinant manganese-containing catalase, its expression vector and engineering bacteria. Background technique [0002] Catalase (CAT) catalyzes the decomposition of hydrogen peroxide into water and oxygen. CAT is widely used in food, textile, paper and pharmaceutical industries, among which in the textile industry it is mainly used for residual H after bleaching of cloth 2 o 2 of elimination. Compared with water washing or chemical additives in traditional processes, CAT has the advantages of reducing pollution and improving the quality of subsequent printing and dyeing; but it is worth noting that the printing and dyeing process is usually in a high temperature (T>70°C) alkaline (pH>9) environment This requires CAT to have the application characteristics of thermobasophilic. Although CAT is rich in sources and exists in almost all aerobic microorg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/08C12N15/70C12N1/21C12R1/19
Inventor 赵志军史吉平孙俊孙何晓娟
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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