Breeding of Fungal -amylase High-producing Strain by Genome Shuffling

Author :LiuYuanHui
Tutor:ZhengYi
School :Fujian Normal University
CLC :TQ925
TYPE :Masters thesis
Download the PDF Full Text:http://www.topresearch.org/showinfo-153-665310-0.html
Year:2011
Abstract:
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 28for 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 30The 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 production-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.