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
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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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