Preparation method of hyaluronic acid
The technology of hyaluronic acid and gene is applied in the field of preparation of hyaluronic acid, which can solve the problems of complex technological process of hyaluronic acid, low yield, difficulty in meeting industrial production, etc., and achieves safety, high molecular weight, and simple process requirements. Effect
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Embodiment 1
[0041] A whole-cell catalytic process for preparing hyaluronic acid is as follows:
[0042] (1) Seed culture: Inoculate the engineering bacteria E.coli pBPAB / BW25113 glycerol tube strain into a 500mL shake flask containing 100mL of sterilized LB medium according to 1% inoculum, add 100mg / L of Amp, and then culture at 35°C 3h, rotate at 200r / min, observe the growth of the bacteria, and transfer when the bacteria grow to the logarithmic growth phase.
[0043] (2) Bacterial fermentation: inoculate the shake flask seed solution into a 500mL shake flask equipped with 100mL of sterilized fermentation medium (casein 10.0g / L, yeast powder 5.0g / L, glycerin 1.0g) by 1% inoculum / L, NaCl 10.0g / L, urea 30mM), and then cultivated at 37°C at 200r / min for 3h until the cell OD was 0.6.
[0044] (3) Protease-induced expression: add 2.0 g / L inducer L-arabinose, lower the temperature to 35° C., rotate at 250 r / min, cultivate for 16 h, and collect the cells by centrifugation at 6000 r for 15 min...
Embodiment 2
[0048] (1) Seed culture: Inoculate the engineering bacteria E.coli pBPAB / BW25113 glycerol tube strain into a 500mL shake flask containing 100mL of sterilized LB medium according to the inoculum amount of 1%, add 100mg / L of Amp, and then culture at 37°C 4h, rotating at 300r / min, observe the growth of the bacteria, and transfer when the bacteria grow to the logarithmic growth phase.
[0049] (2) Bacterial fermentation: Inoculate the shake flask seed solution into a 500mL shake flask with 100mL of sterilized fermentation medium (casein 5.0g / L, yeast powder 3.0g / L, glycerin 5.0g) by 1% inoculum / L, NaCl 7.0g / L, ammonium nitrate 50mM), and then cultivated at 35°C with a rotation speed of 250r / min for 4h until the cell OD was 1.2.
[0050] (3) Protease-induced expression: add 1.0 g / L inducer L-arabinose, lower the temperature to 33° C., rotate at 200 r / min, cultivate for 18 h, and centrifuge at 6000 r for 15 min to collect the bacteria.
[0051] (4) Biotransformation: prepare trans...
Embodiment 3
[0054] (1) Seed culture: Inoculate the engineering bacteria E.coli pBPAB / BW25113 glycerol tube strain into a 500mL shake flask containing 100mL of sterilized LB medium according to 1% inoculum, add 100mg / L of Amp, and then culture at 40°C 2h, rotating at 250r / min, observe the growth of the bacteria, and transfer when the bacteria grow to the logarithmic growth phase.
[0055] (2) Bacterial fermentation: Inoculate the shake flask seed solution into a 500mL shake flask with 100mL of sterilized fermentation medium (casein 1.0g / L, yeast powder 1.0g / L, glycerin 8.0g) by 1% inoculum / L, NaCl 5.0g / L, ammonium sulfate 70mM), and then cultured at 30°C and 200r / min for 5h until the cell OD was 1.5.
[0056] (3) Protease-induced expression: Add 0.1 g / L inducer L-arabinose, lower the temperature to 27°C, rotate at 300r / min, cultivate for 20h, and centrifuge at 6000r for 15min to collect the bacteria.
[0057] (4) Biotransformation: prepare transformation medium according to the following...
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