High-efficiency and high-throughput screening method for high-yield strain of alpha-glucosidase
A screening method and high-throughput technology, applied in microorganism-based methods, biochemical equipment and methods, fungi, etc., can solve the problems of poor correlation, low pertinence, low efficiency, etc. The effect of screening
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Embodiment 1
[0076] Embodiment 1: Utilize nitrosoguanidine mutagenesis mode to carry out mutagenesis to Aspergillus niger bacterial strain CGMCC 3.795
[0077] Scrape the Aspergillus niger strain CGMCC 3.795 slant and 0.1% twenn80 solution cultivated for 3 days (30° C.) into an Erlenmeyer flask with glass beads, vibrate in a shaker for 30 minutes, filter with absorbent cotton and remove the hyphae to obtain a monospore suspension; Use 0.05mol / L Tris-maleic acid buffer solution, pH value 6.0, prepare a nitrosoguanidine solution with a concentration of 2.0mg / mL, and add equal amounts of the above nitrosoguanidine solution and spore suspension to sterile Mix well in the test tube, shake at room temperature for 30 minutes, centrifuge at 5,000 rpm for 5 minutes after the mutagenesis treatment to terminate the reaction, remove the supernatant, wash the precipitate with normal saline for 3 times, and resuspend to obtain the mutagenized bacterial solution;
Embodiment 2
[0078] Example 2: Screening of α-glucosidase enzyme high-yielding strains after mutagenesis-α-methylglucose (α-MG)-maltose-Bengal red screening plate method
[0079] (1) Selective plate primary screening
[0080] Appropriately dilute the mutagenized spore suspension and spread it on the α-methylglucose (α-MG)-maltose-Red Bengal screening plate medium, invert it, incubator temperature 30°C, and cultivate for 2 days, because, observe the mutagenesis The growth and growth speed of the strains on the screening plate were initially screened ( figure 1 )For example figure 1 The single colony pointed by the arrow has a larger diameter than other single colonies, a fuller shape, and a faster growth rate. It is speculated that the colony may be better at utilizing α-methylglucose, so it may have higher enzyme production capacity;
[0081] (2) 96-well plate high-throughput screening
[0082] Pick a single colony that can grow on the screening plate and has a larger diameter, fuller s...
Embodiment 3
[0088] Embodiment 3: Screening of α-glucosidase enzyme high-yielding bacterial strain after mutagenesis: α-methylglucose (α-MG)-Bengal red screening plate method
[0089] Selective plate primary screening: the spore suspension of the starting strain CGMCC 3.795 after mutagenesis was appropriately diluted and spread on the α-methylglucose (α-MG)-Bengal red screening plate respectively. The screening plate picked 67 strains of single colonies with larger colony diameter, plump shape and faster growth rate ( Figure 7 ). Streak the single colony picked from the α-methylglucose-Red Bengal screening plate onto a fresh PDA plate, culture it upside down at 30°C for 2 days, and then inoculate it directly into a 250mL shaker flask containing 50mL of liquid fermentation medium, at 30°C , 200r / min shaker after culture for 5 days, measure its enzyme activity, and the results of primary screening enzyme activity are as follows: Figure 5 shown.
[0090] Depend on Figure 5 It can be se...
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