臺灣博碩士論文加值系統

本研究自數種不同來源土壤中，分離出83株具生產植酸酉每能力之黴菌，其中以M46具最佳之酵素活性。利用誘突變劑NTG(N-methyl-N-nitro-N'-nitrosoguanidine)將M46及Asper-gillus ficuum NRRL3135行多次變異處理，並以含有植酸鈣之PSM agar為選擇性培養基；經進一步分析結果發現，Aficuum變異株A200413之胞外植酸酉每產量最高，故以此作為本實驗之研究菌株。於三角瓶振盪培養下，A.ficuum變異株A200413生產植酸鹽之最適培養基及培養條件如下：糖蜜12.93%，硝酸銨0.5%，KCl 0.05%，MgSO4．7H2O 0.05%，KH2PO4 0.002%，FeSO4．7H2O 0.001%及MnSO4．4H2O0.001%；起始pH4.0，接種量10(7)spores/50ml，培養溫度35℃，振盪速率150rpm。以該條件培養變異株A200413六天所得之最大植酸酉每生成量為5633.34U/ml。粗酵素之基本性質如下：最適作用溫度58∼60℃，最適作用pH5.5；於pH2.0及4.0之酵素穩定性最好；在熱穩定性方面，酵素於55℃下保溫10分鐘後，仍保有90%左右之活性；將粗酵素液貯存於-20℃60天後，植酸酉每只減少將近20%之活性；Ag(+)、Cu(2+)、Fe(3+)、Fe(2+)、Mn(2+)、Na(+)、Zn(2+)及oxalate會抑制粗植酸酉每之活性，Mg(2+)、citrate及EDTA則可促進植酸酉每活性，而Ca(2+)及K(+)之存在則對植酸酉每活性無任何影響。

Eighty three microorganisms containing phytase activity were isolated from different sources of soil. Strain M46 was found to have the highest enzyme activity among these organisms. For further improvement, Aspergillus ficuum NRRL 3135 and strain M46 were treated with the mutagenic agent NTG (N-methyl-N-nitro-N'-nitrosoguanidine) and plated onto PSM agar containing calcium phytate as a selective medium. One of the NTG-treated strains, A. ficuum mutant A200413, showed considerably higher phytase activity than the parent strain A. ficuum NRRL 3135 and was chosen for further investigation. This mutant strain A200413 was cultivated in shaking flasks and the optimal medium for the phytase production by this strain were as follows: molasses, 12.93%; ammonium nitrate, 0.5%; KCl; 0.05%; MgSO4．7H2O, 0.05%; KH2PO4, 0.002%; FeSO4． 7H2O, 0.001%; and MnSO4．4H2O, 0.001%. For cuitivations, initial pH at 4.0, inoculum size at 10(7) spores/50 ml, incubation temperature at 35℃, and shaking rate at 150 rpm were found to be the optimal conditions. The maximal phytase activity of 5633.34 U/ml was obtained after 6 days of incubation under the optional conditions. The optimal temperature and pH for the crude ezyme activity of mutant A200413 were 58-60℃ and 5.5, respectively. This enzyme was stable at pH 2.0 and pH 4.0 and maintained 90% of its activity after incubated at 55℃ for 10 min. After 60 days of storage at -20℃, only 20% loss of enzyme activity was found. The activity of the crude phytase was found to be inhibited by Ag+, Cu2+, Fe3+, Mn2+, Na+, Zn2+ and oxalate, whereas the enzyme activity was increased by the presence of Mg2+, citrate and EDTA. Ca2+ and K+ had no effect on the catalytic rate of the crude phytase from strain A200413.