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A thermophilic alkaline recombinant manganese-containing catalase and its Pichia pastoris expression vector and engineering bacteria

A technology of manganese catalase and expression vector, which is applied in the field of thermophilic alkaline recombinant manganese-containing catalase and its expression vector of Pichia pastoris and engineering bacteria to achieve high-efficiency expression

Active Publication Date: 2016-01-20
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

Escherichia coli and Pichia pastoris expression systems are currently the most mature genetic engineering expression systems in industrial production, and there is no report on the expression of this enzyme in Pichia pastoris

Method used

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  • A thermophilic alkaline recombinant manganese-containing catalase and its Pichia pastoris expression vector and engineering bacteria
  • A thermophilic alkaline recombinant manganese-containing catalase and its Pichia pastoris expression vector and engineering bacteria
  • A thermophilic alkaline recombinant manganese-containing catalase and its Pichia pastoris expression vector and engineering bacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 manganese catalase MnCAT yeast expression genetic engineering bacteria

[0045] 1.1 MnCAT gene designed based on Pichia pastoris codon usage preference optimization

[0046] According to the preference of codon usage in Pichia pastoris ( 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.

[0047] 1.2 Construction of MnCAT Yeast Expression Vector pPIC3.5K-MnCAT

[0048] At the 5'-end and 3'-end of the MnCAT gene, the restriction endonuclease SnaBI and NotI restriction endonuclease sites that are not available in the gene but are available in the multiple cloning site of the vector pPIC3.5K were designed by Shanghai Sangon Biotechnology Directly synthesized by Engineering Technology Service Co., Ltd., the MnCAT gene fragment was digested by SnaBI and NotI, and then T4-ligated with the expression vector pPIC3.5K after the same digest...

Embodiment 2

[0053] Shake flask fermentation culture of embodiment 2 recombinant bacteria KM71 / pPIC3.5K-MnCAT

[0054] 2.1 Shake flask seed culture

[0055] Inoculate the recombinant strain KM71 / pPIC3.5K-MnCAT into 50mL of LYPD medium, and culture at 30°C and 200rpm / min for 19-22h;

[0056] 2.2 Shake flask fermentation culture:

[0057] Transfer the seed culture solution to 50mL BMGY medium according to the inoculum amount of 10%, culture at 30°C, 200r / min for 19-22h; collect the bacteria by centrifugation at 4°C, 6000r / min, and use 25mL containing the final concentration of 1mmol / LMnCl 2 Resuspended bacteria in BMMY medium, cultivated at 30°C and 200rpm / min, added 100% methanol to the medium every 20h to a final concentration of 0.25% (V / V) to induce expression of Mn-CAT, and cultured for 120h , collected the bacteria by centrifugation, broken the cells, and analyzed the supernatant by SDS-PAGE. It was found that there was an obvious protein band at 33Kda, which was consistent with the ...

Embodiment 3

[0062] 5L fermenter culture of embodiment 3 recombinant bacteria KM71 / pPIC3.5K-MnCAT

[0063] 3.1 Seed culture in fermenter

[0064] The recombinant strain KM71 / pPIC3.5K-MnCAT was inoculated into 50mL seed medium, and cultured at 30℃, 200rpm / min for 19-22h.

[0065] The components of the fermenter seed medium are: peptone 15g / L, yeast extract 15g / L, glycerol 10g / L, biotin 4×10-4g / L, potassium phosphate buffer 0.1mol / L (pH6.0).

[0066] 3.2 Fermentation tank fermentation

[0067] The seed culture solution was transferred to the BSM medium of the fermenter with a liquid content of 2L according to the inoculum amount of 10%. -20%; when the glycerol in the initial medium is exhausted, start to feed the feed medium, and control the final concentration of glycerol in the culture medium to be less than 10g / l; when the recombinant bacteria grow to OD 600 When the temperature is 100-120, start to add methanol continuously, control the final volume concentration of methanol in the cult...

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Abstract

The invention relates to the field of biotechnology, and particularly relates to thermophilic alkaline recombined manganese-containing catalase as well as a pichia pastoris expression vector and engineering bacteria thereof. A nucleotide sequence of the recombined manganese-containing catalase is as shown in SEQ ID No:1. According to preference in application of pichia pastoris codons, a gene sequence of manganese catalase derived from Thermus thermophilus HB27 is subjected to codon optimization, and when the optimized manganese catalase is expressed in the pichia pastoris, about 12000U / mL of enzyme activity of the catalase can be detected in 5L of culture solution in a fermentation tank.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a thermophilic alkaline recombinant manganese-containing catalase, an expression vector of Pichia pastoris 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 al...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/08C12N15/81C12N1/19C12R1/84
Inventor 赵志军史吉平姜标孙俊松何晓娟
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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