Breeding Of High Production Strain With Xylanase By Genome Shuming

Aspergillus oryzae FST036being a starting strain was mutagenized by UV, UV-LiCl and microwaves. The best dosage of UV and microwaves was irradiated for210seconds and35seconds respectively, the concentration of LiCl was0.8%, Through the mutation, four mutant strains with high production xylanase, namely FU-41、FU-112、 FW-115、FW-17were breeded.The protoplast preparation and regeneration methods of Aspergillus oryzae were studied. The optimized conditions were as follows:the spore being cultured for6.5h; hydrolyzed for2.5h by mixed enzymes of1.0%cellulase+0.5%lysonzyme+0.5%lysonzyme under the temperature30℃;0.6mol/L NaCl and PDA medium being as osmotic stabilizer and regeneration medium respectively. Under the optimized conditions the rate of protoplast preparation and regeneration reached92.8%and28.6%respectively.The optimized conditions of protoplast fusion of Aspergillus oryzae were studied. The protoplast inactivation methods were determined to be UV irradiation for360s and microwaves irradiation for50s and fusion conditions were as follows:30%PEG,0.02%Ca2+, temperature30℃, pH7.0and fusion for12min. The last fusion rate could reach0.69%.A high production xylanase strain named C-516was obtained by three rounds of genome shuffling from parent strains namely the mutant strains FU-41、FU-112、 FW-115、FW-17. The fermentation level of the xylanase was increased50.7%more then that of FST036.The primary studies on fermentation performance of the C-516were carried out. The results of single factor test for medium component showed that the best carbon was bran and corncob, the total concentration was40g/L, the ratio of the bran to corncob was6:4; the optimum organic nitrogen was yeast extract, the concentration was lOg/L. Secondly a Plackett-Bunnan design was used to select3important factors from medium; they were bran, yeast extract, NaNO3, The response surface analysis method studies showed that three components (g/L) were bran19.41, yeast extrac9.92, NaNO31.76. Through the single factor test the optium fermentation conditions were as follow:temperature28℃, the amount of inoculation10%, liquid volume50ml culture in250ml shake flasks and fermentation time72h. After the optimization, the xylanase production of C-516was up to8092.5U/ml, which was31.79%higher than that of non-optimized fermentation.