Aspergillus niger strain bfa010-7 with high production of L-lactone hydrolase and its application in the preparation of d-pantolactone
The technology of pantolactone and Aspergillus niger strain is applied in the field of bioengineering, and can solve the problems of undisclosed mycelial cell immobilization method, difficulty in separation and recovery of immobilized cells, and high viscosity of reaction liquid, and achieves simple and feasible technology. , shorten the production cycle, the effect of high fermentation yield
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Embodiment 1
[0041] Example 1 Isolation, screening and identification of Aspergillus niger strain BFA010
[0042] More than 200 soil samples were collected in different locations and environments in Shanghai, Jiangsu, Zhejiang, Shandong, Guangxi and other provinces and cities. When collecting soil samples, the surface soil was shoveled, and deep soil samples were collected. The collected soil samples were used within 10 days. Screening of D-pantolactone-producing strains.
[0043] About 100 mg of soil samples were taken, added to 3 ml of enriched medium, incubated at 30°C, 200 rpm, and shaken for 3 days, and the enriched culture solution was detected by liquid chromatography for the enzymatic hydrolysis product pantothenic acid. The enriched culture solution with significant pantoic acid generation was taken and 100 μl of the bacterial solution was spread on a PDA solid medium plate containing calcium carbonate and pantoic acid lactone, and cultured at 30 °C for 3 days to isolate the Asper...
Embodiment 2
[0050] Example 2 Molecular identification of Aspergillus niger species
[0051] Genomic DNA of Aspergillus niger BFA010 was extracted by secondary precipitation method. The Aspergillus niger BFA010 obtained in Example 1 was inoculated into a liquid rich medium and cultured for 2 days, the mycelium was collected by centrifugation, washed twice with deionized water, and the water was sucked dry. Transfer 1 g of mycelium to a pre-cooled mortar, add liquid nitrogen to freeze, and grind it into a fine powder with a pestle. The mycelium powder was quickly transferred to a 20 ml centrifuge tube, 10 ml of SDS-EB buffer (10 mM Tris-HCl, 0.1 mM EDTA, 4% SDS, pH 8.0) was added, and the mixture was vortexed to mix well. The mixture was incubated in a water bath at 65°C for 20 min, and centrifuged at 10,000 rpm for 15 min. Transfer 8ml of supernatant to a new centrifuge tube, add 0.5ml of potassium acetate buffer (8M, pH 4.2), mix well, ice bath for 45min, centrifuge at 10000rpm for 20mi...
Embodiment 3
[0056] Example 3 Plasma mutation breeding of Aspergillus niger BFA010
[0057] Aspergillus niger BFA010 was inoculated on the slant of solid medium and cultured at 30°C for 48h. Add 1 ml of sterile water to the slanted solid medium, and scrape the spores of Aspergillus niger into the sterile water with an inoculating loop. Dilute the spore suspension to about 10 7 Spores / ml suspension, placed in a plasma box, and mutagenesis was performed. The working gas is high-purity helium, the irradiation power is 40W, the irradiation distance is 4mm, the plasma temperature is less than 30°C, the gas flow rate is 10L / min, the bacterial solution is 10μL, and the treatment time is 40s.
[0058] The mutagenized spore suspension was spread on a solid rich medium plate containing 20 g / L pantolactone and 5 g / L calcium carbonate, and cultured at 30° C. for 48 h, and colonies with obvious clear circles were selected. According to the method described in Example 1, the isolated Aspergillus nige...
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