Culture medium and method for producing laccase by Ascheronia placenta fermentation
A technology of Ascospora and culture medium, which is applied in the field of medium for fermenting and producing laccase from Ascosporidium, achieving the effects of high laccase yield, easy control and mild conditions
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Embodiment 1
[0022] Single factor experiment to determine the optimal composition of culture medium
[0023] (1) Preparation of seed medium and basal medium
[0024] (a) Seed medium: 1L of 20% potato juice, 2% glucose, 1% yeast extract, natural pH. 1.01MPa, sterilize for 20min.
[0025] (b) Basal medium: 1L of 20% potato juice, 2% glucose, 1% yeast extract, natural pH. 1.01MPa, sterilize for 20min.
[0026] (2) Production of fermented seeds: inoculate Sophora platinospores on potato agar medium, culture at 25°C for 5 days, and activate after spore production; use the inoculation needle to inoculate the spore powder to the seeds of the activated strain The culture medium was cultured at 25°C for 24 hours at a rotational speed of 160 r / min, which was the fermentation broth.
[0027] (3) Preparation of enzyme liquid: take the fermentation liquid in step (2) and centrifuge at 5000 r / min for 15 min at 4°C, and the supernatant is the crude enzyme liquid for use. The absorbance was measured ...
Embodiment 2
[0045] Orthogonal experiment to determine optimal fermentation conditions
[0046] (1) Orthogonal experiment to determine the best medium
[0047] On the basis of the culture medium determined by the single factor experiment, the solubility and pH value of each component were changed, and different culture media were configured, and the method was the same as that of steps (1) and (2) in Example 1. See Table 1. Inoculate 250mL of the same amount of inoculum (10%) into 250mL of culture solution, and culture at 25°C with a rotation speed of 160 r / min for 3 days. Then measure the enzyme activity according to the steps (3) and (4) of Example 1. Select the experimental group number with the optimal result of the orthogonal experiment and the highest enzyme production, and carry out the verification experiment. In order to avoid being cumbersome, the preparation of the culture medium, the preparation of the crude enzyme solution, and the enzyme activity assay method in the embodim...
Embodiment 3
[0065] Fungal Laccase Fermentation Method
[0066] (1) Preparation of fermented seeds
[0067] (A) Strain and culture medium
[0068] Bacteria: Cyclospora
[0069] Slant medium: PDA medium
[0070] Liquid seed medium: 1L of 20% potato juice, 2% glucose, 1% yeast extract, natural pH.
[0071] (B) Preparation of seed solution
[0072] Transfer the pure Septoria platina on the slant to several 250mL Erlenmeyer flasks filled with 100mL of culture medium, and then cultivate them at 25°C for 3 days on a reciprocating shaker at a speed of 160 r / min. When the silk grows vigorously and the bacterial liquid is viscous, it means that the seeds have grown well.
[0073] (2) Fermentation culture
[0074] Fermentation medium: According to the results of the orthogonal experiment of medium optimization, that is, containing 0.5w% glucose, (NH 4 ) 2 SO 4 4w%, Zn 2+ 0.5×10 -4 mol / L, V B4 5×10 -4 w %, the initial pH of the medium is 4.4, and it is prepared.
[0075] With the Se...
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