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題名 | 隨機突變處理產生之突變Bacillus Macerans環狀糊精葡萄糖苷基轉移酶之性質=Properties of the Mutant Bacillus Macerans Cyclodextrin Glucanotransferase Generated by Random Mutagenesis |
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作者姓名(中文) | 陳貴凰; 蔣啟玲; | 書刊名 | 農林學報 |
卷期 | 49:3 2000.09[民89.09] |
頁次 | 頁15-23 |
分類號 | 463.37 |
關鍵詞 | 環狀糊精葡萄糖苷基轉移酶; 隨機突變; 突變株篩選; Cyclodextrin glucanotransferase; Random mutagenesis; Screening of mutants; |
語文 | 中文(Chinese) |
中文摘要 | 利用hydroxylamine對含Bacillus macerans環狀糊精葡萄糖?基轉移?(cyclodextrin glucanotransferase, CGTase)基因之重組質體(pUG)進行隨機突變,轉形至E. coli後,培養於固體篩檢培養基。自20,000單一菌落中,篩選出七株突變株,經DNA定序發現每株含一至二個定點突變,其中五株為改變一個胺基酸殘基。改變CGTase之環化及偶合活性測定結果顯示,不同突變株之間的酵素相對活性具有差異性(p<0.05)。同時,用偶合反應法分析最適作用pH值、溫度及貯存時間對於野生株與突變株之CGTase的影響。結果發現J-7 CGTase在低與高pH值下的相對活性較野生型及其它突變 CGTase高。而J-1 CGTase最適作用溫度在45℃,和野生型與其它突變酵素(50℃)不同。此外,J-4、J-6與J-7 CGTase 於4℃貯存20天,其酵素活性為原來21~38%,有明顯下降情形。 |
英文摘要 | The plasmin-pUG, containing the cloned Bacillus macerans cyclodextrin glucanotransferase (CGTase) gene. Was treated with hydroxylamine to perform random mutation. Then, the mutant plasmids were transformed into E. coli. Seven strains were selected by solid screening plates from 20,000 clones, and the mutations of cgt gene were identified by DNA sequencing. Among the seven mutant strains. Five strains showed one amino acid residue substitution, and two strains showed two amino acid residues alteration. All mutant CGTases still had ability for cyclization and coupling reaction. The relative activities for either reaction differed with the mutants (p<0.05). The effects of optimal pH, temperature and time of storage on the coupling reaction activities were determined for the wild-type and the seven mutant CGTases. The J-7 CGTase showed higher relative activities at both acidic and alkaline ranges of pH than the wild-type and other mutant CGTases. The J-1 CGTase had the optimal temperature at 45℃, and the wild-type and six mutant CGTases had the same maximum relative activity at 50℃. Furthermore, results showed that the activity of J-4, J-6 and J-7 CGTases were decreased dramatically after storage at 4℃ for 20 days, and the residual activity of these mutant CGTases ranged from 21 to 38%. |
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