Preparation of valuable sialinic acid from cheap sodium lactate by multi-step coupling bio-conversion

[0081] Example 1: Cultivation of lactate oxidase-containing strains

[0082] 1. Culture of Pseudomonas sp. ATCC11452

[0083] (1) Lactate oxidase strain: select Pseudomonas sp. ATCC11452;

[0084] (2) Slant culture: Pseudomonas sp. ATCC11452 was inoculated on a solid slant basic medium containing 1.5% mass / volume agar and 1% mass / volume sodium L-lactate at 30°C cultured for 20 hours;

[0085] (3) Primary seed culture: the strains cultivated in step (2) were inoculated into 30 mL liquid basic medium BLM containing L-type sodium lactate with a mass-to-volume ratio of 1% under aseptic conditions, at 30° C. Under the condition of shaking culture on a shaker for 10 hours, first-class seeds were obtained;

[0086] (4) Expanded culture: with an inoculation amount of 5% by volume, the first-class seeds were placed in 500 mL of BLM containing 2% by volume of L-type sodium lactate, and under the condition of 30° C., shaken and cultured on a shaking table for 16 hours to prepare seco...

[0104] Example 2: Construction of engineering strain E. coli HB101 (pBV220-ALD)

[0105] Construction of sialyl aldolase strain: Escherichia coli engineering strain, namely E.coli HB101 (pBV220-ALD), was constructed by conventional methods:

[0106]Construction of recombinant engineering strains: Genomic DNA of E.coli K12 strains was prepared by conventional methods. This process can be referred to the method of small-scale preparation of bacterial genomes in the "Guidelines for Molecular Biology" published by Science Press to extract E.coli K12 strains. Genomic DNA of the coli K12 bacterial strain; use synthetic primers to amplify the sialic acid aldolase gene from the genomic DNA of the E.coli K12 bacterial strain; The sialic acid aldolase gene was connected to obtain the recombinant plasmid pBV220-ALD; then the recombinant plasmid pBV220-ALD was transformed into the vector E.coli HB101 to obtain the engineering strain E.coli HB101 (pBV220-ALD);

[0107] Wherein the host is...

[0108] Embodiment 3: the cultivation of engineering strain Escherichia coli E.coli HB101 (pBV220-ALD)

[0109] (1) Plate culture: Streak the engineering strain Escherichia coli HB101 (pBV220-ALD) on an ampicillin LB plate containing 100 μg / mL of 1.5% agar in mass volume ratio, and culture at 30° C. for 12 hours;

[0110] (2) First-class seeds: under sterile conditions, use a sterile toothpick to pick a single colony on the plate of step (1), and then inoculate it into 5 mL of liquid medium containing 100 μg / mL ampicillin, for 30 Cultivate with shaking in a shaker for 12 hours;

[0111] (3) Secondary seeds: under aseptic conditions, take the culture solution cultivated in step (2) with a volume ratio of 2% inoculum, inoculate into 240 mL of LB liquid medium containing 100 μg / mL of ampicillin, 30°C shaker culture for 6 hours;

[0112] (4) fermentor culture: under aseptic conditions, get the nutrient solution obtained in step (3) to inoculate the improved M9 liquid medium of 3L...