Breeding of Fungal α-amylase High-producing Strain by Genome Shuffling
|School||Fujian Normal University|
|Keywords||α-amylase Aspergillus oryzae mutation Genome shuffling fermentation optimization|
Original strain Aspergillus oryzae FS-16 preserved in our laboratory was treated with ultraviolet and microwaves radiation. The mutation conditions were as follows, UV for 160s and microwaves for 25s. Prescreening method was starch plate hydrolysis spot method. Rescreening method was liquid fermentation. The mutant strains with high yield, namely FU-8、FU-26 were obtained by UV mutagenesis and the mutant strains named FW-111, FW-130 were obtained by microwave irradiationThrough single factor test, the optimized conditions for protoplast preparation and regeneration were as follows:the cells being cultured for 15 h, incubation at 28℃for 2.5h to allow cell wall lysis; 0.6 mol/L NaCl as osmotic stabilizer, and PDA plate with 0.6mol/L NaCl for regeneration. Through the response surface analysis method, the optimized enzyme composition for breaking cell wall was as follows:0.8% cellulose, 0.8% snailase and 0.5% lysozyme.The protoplast inactivation methods were determined to be ultraviolet irradiation for 180s and heat treatments for 12 min. The effects of the PEG concentration, the Ca2+ concentration, the fusion time and temperature on protoplast fusion were studied. The fusion was undergone in the fusion system of 30% PEG containing 0.02mol/L Ca2+ and processed for 10 min at 30℃The four strains we obtained from UV and microwaves mutation and FS-16 preserved in our lab were selected as parent strains for genome shuffling. After three cycles of recursive protoplast fusion, a shuffled strain named F3-542 was selected. The productionα-amylase activity of F3-542 was increased 61.5% more than that of FS-16. At the same time, the fermentation period was relatively shortened 24h. The results of continues culture and liquid fermentation showed the strains had stable hereditary property. On the whole the breeding purpose was achieved successfully.The primary studies on fermentation performance of the strain F3-542 was carried out.Through single factor test, the result showed that the best carbon was corn flour, the optimum nitrogen were beef extract and NaNO3; the best fermentation conditions were obtained based on the single factor test, it showed the optimal amount of inoculation was 10%, the optimum initial pH was 6 and the optimal liquid volume were 50mL culture in 250mL shake flasks.The uniform design study showed that optimal fermentation medium components (g/L) were corn flour 56,beef extract 26, NaNO3 4.5, K2HPO4 1.4, MgSO4 1.5, FeSO4 0.005. After the optimization, the activity of F3-542 was up to 5147.5U/mL, of which was increased 31.3% more than the activity before optimization.