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Culture medium for high-efficient production of acetonic acid oxidase by using recombinant escherichia coli

A technology of pyruvate oxidase and Escherichia coli, applied in the field of bioengineering, can solve the problem that the yield of pyruvate oxidase is not high enough

Inactive Publication Date: 2008-05-21
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the method for expressing pyruvate oxidase by recombinant method has appeared in the prior art, the recombinant strain adopted is such as Escherichia coli DH5α / pSMLPyOD; However, the yield of pyruvate oxidase produced by the current recombinant method is not high enough, Some aspects of production conditions still need to be optimized

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Construction of E.coli DH5α / pSMLPyOD

[0084] The pyruvate oxidase gene was amplified from the ATCC 10400 genome of Aerococcus viridans.

[0085] Upstream primer (SEQ ID NO: 1):

[0086] 5'CATG CCATGGG AATGCATCACCATCACCATCACTCAGATAACAAAATTAACATC-3' (the underlined part is the NcoI restriction site, and the nucleotide sequence corresponding to 9 amino acid residues MGMHHHHHH (SEQ ID NO: 3) is added at the same time, which can be used for Ni affinity chromatography);

[0087] Downstream primer (SEQ ID NO: 2):

[0088] 5'CGC GGATCC TTATCATTTGATGTATTTAGATTCTAAGCCTTCAGC-3' (the underlined part is the restriction site of BamHI).

[0089] Using the genome of Aerococcus viridans ATCC 10400 as a template, the AvPyOD gene was amplified by PCR using the above primer pairs, and the PCR amplification product was identified. After identification, the AvPyOD amplification product with the correct sequence was obtained. The PCR amplification product is purified by conventional me...

Embodiment 2

[0093] Preparation of culture medium

[0094] Several formulations of the medium are shown in Table 3.

[0095] table 3

[0096] Medium 1

Medium 2

Medium 3

Medium 4

Glycerin (ml / L)

10

12

10

15

Peptone (g / L)

15

15

17

13

Yeast extract (g / L)

12

12

10

12

Ammonium sulfate (g / L)

5

5

4

6

Magnesium sulfate (g / L)

2

2

3

2

Compound phosphate (g / L)

5

7

4.5

6.5

Trace element mother solution (ml / L)

1.25

1.25

1.7

1.2

NaCl(g / L)

10

10

11

8.5

[0097] Among them, the composition of trace element mother liquor is: FeSO 4 ·7H 2 O 12.8g / L; ZnSO 4 ·7H 2 O 2.84g / L; CuSO 4 ·5H 2 O 1.6g / L; MnSO 4 ·H 2 O 3.2g / L; CaCl 2 0.28g / L; CoCl 2 ·6H 2 O 1.6g / L; H 3...

Embodiment 3

[0100] Cultivation of Pyruvate Oxidase in Fermentation Medium 2 Shake Flasks

[0101] Fermentation medium 1: the formula is as medium 1 in Example 2, pH 7.

[0102] Mix other components except magnesium sulfate according to the stated concentration, put 30ml / bottle into a 250ml shake flask, sterilize at 121°C for 20 minutes, and cool. Magnesium sulfate was sterilized separately (sterilized by filter sterilization), and added according to the concentration before inoculation.

[0103] E.coli DH5α / pSMLPyOD stored in glycerol tubes was inserted into a 500ml shake flask filled with 100ml LB medium (containing 50mg / L tetracycline) at 1% inoculum volume, cultured overnight at 37°C and 220RPM, and then inoculated at 10% inoculum volume Shake flasks of the above-mentioned fermentation medium, culture at 37°C and 220RPM for 12 hours, then lower the fermentation temperature to 30°C, and continue to cultivate until the end of fermentation for about 16 hours. After testing, the activity ...

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Abstract

The invention belongs to the field of bioengineering and provides a culture medium mainly using glycerol as a carbon source. The invention also provides a method for producing pyruvate oxidase by using the culture medium. The present invention discloses for the first time that glycerol is used as a carbon source in the production of pyruvate oxidase, which enables recombinant Escherichia coli to continuously express pyruvate oxidase during the whole fermentation process, greatly improving the yield of pyruvate oxidase; and, adopting the present invention The culture medium is fermented, and the pH value is stable throughout the fermentation process, and the fermentation process is greatly simplified.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically, the invention relates to a fermentation medium for expressing pyruvate oxidase efficiently in recombinant Escherichia coli and a method for producing pyruvate oxidase using the medium. Background technique [0002] Pyruvate oxidase (Pyruvate oxidase, EC 1.2.3.3) is a very important enzyme for clinical diagnosis, and its most important application is for alanine aminotransferase (ALT) and aspartate aminotransferase in blood Enzyme (AST) activity determination; in addition, it can also be used for pyruvate determination, pyruvate kinase activity determination, etc. [0003] Currently commercially produced pyruvate oxidase is mainly derived from wild-type Lactobacillus plantarum, and the enzyme production is low, only about 120U / L. Although there have been methods for expressing pyruvate oxidase by recombinant methods in the prior art, the recombinant strains adopted are such as...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/04C12N1/32C12R1/19
Inventor 张嗣良赵劼王永红储炬庄英萍袁中一
Owner EAST CHINA UNIV OF SCI & TECH
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