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|標題:||Bacillus macerans 環狀糊精葡萄糖基轉移結構與生產之研究|
Studies on the Structure and Production of Bacillus macerans Cyclodextrin Glucanotransferase
|摘要:||本研究主要目的除了偵測突變處理產生之突變 Bacillus macerans 環狀糊精葡萄糖基轉移（cyclodextrin glucanotransferase, CGTase）之性質，以了解酵素結構與功能相關性。同時亦探討以 B. subtilis (pHG) CGTase 生產環狀糊精（cyclodextrin, CD）。
含 B. macerans 環狀糊精葡萄糖基轉移基因之重組質體（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 7 株突變株，經 DNA 定序發現每株含一至二個定點突變殘基，其中 5 株為改變一個胺基酸殘基。突變 CGTase 進行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比活性具有差異性（p<0.05）。其中，J-8（L304F）CGTase 之γ-CD 生成比活性僅佔野生型的 41 %。而 J-11（A125R）CGTase 經環化或偶合反應法測定，雖然比活性皆較野生型 CGTase 高，但貯存於 4℃ 下，酵素容易失活。將 J-11 CGTase 置於含 4 mM CaCl2 或 NaCl 之 10 mM Tris-HCl buffer（pH 7.2）中，在 4℃ 下，貯存 40 天，酵素活性依舊維持 95 %，此結果或可應用於改善有利用價值之突變酵素的安定性，以提高它們在工業上的應用。而 Trp101 及 Leu304 位於 B. macerans CGTase 活性部位凹袋附近，為暸解其上殘基在酵素催化活性上所扮演的角色，乃在 cgt 基因這兩個殘基所在之位置上分別進行定點突變後，發現 W101L、L304Y、L304W、W101F/L304W 及 W101L/L304W CGTase 進行 CD 生產時，反應初期產物以β-CD 為主，這和野生型 CGTase 初期產物以α-CD 為主，有明顯差異。同時亦發現 W101F/L304W 及 W101L/L304W CGTase 於反應前 8 小時之α-CD/β-CD 比例小於 1，即β-CD 產量大於α-CD，且 W101L/L304W CGTase 於作用 48 小時內，並未被偵測到γ-CD，推測 Trp101 及 Leu304 與 CGTase 之產物特異性有關。
另一方面，以 500 mL 溝槽的三角錐形瓶盛 100 mL medium B（4 % 麥麩、0.5 % (NH4)2SO4、0.5 % CaCO3、0.075 % K2HPO4、0.05 % NaHPO4·12H2O 及 0.03 % MgSO4·7H2O）來生產 B. subtilis (pHG) CGTase，於 37℃ 下，震盪培養（120 rpm）72 小時，粗酵素液經過β-CD 親和性管柱層析及 DEAE Sepharose CL-6B 管柱層析等純化後，酵素產量為 100 ~ 110 mg/L，為酵素來源之菌株 B. macerans 在相同條件下產量的 10 倍，且可不須添加抗生素 tetracycline。經純化之 B. subtilis (pHG) CGTase 之生化性質分析結果與原菌株相似。繼之利用反應曲面法探討基質與酵素濃度對生產 CD 之影響，結果顯示在所設定範圍內，總 CD、α-CD 及β-CD 產率受到基質與酵素濃度顯著的影響（p<0.05）。在低濃度澱粉溶液中、添加 CGTase 量越高，總 CD 及β-CD 產率越高，而α-CD 產率則越低。若以高的總 CD 與β-CD 產率為生產目標時，則在澱粉濃度為 2.8 %，B. subtilis (pHG) CGTase 為 0.014 % 時，於 37℃、100 rpm 下，作用 2 小時，可得總 CD 產率約為 50 %，α-CD 產率為 19 %，β-CD 產率為 26 %。於相同作用條件下，若以等量之酵素欲得較高之 CD 產量，則澱粉濃度為 5.2 %，可得總 CD 產率為 45 %，其中α-CD 及β-CD 產率分別是 16 % 及 22 %。依不同設定條件所得之實驗值與模式所得估計值並無顯著差異，顯示所建立模式是適當的。|
The study was aimed to investigate the structure and functional relationships the mutant Bacillus macerans cyclodextrin glucanotransferases (CGTase) generated by mutagenesis. And the investigate production of cyclodextrin (CD) with B. subtilis (pHG) CGTase. The plasmid - pUG containing the cloned B. macerans cgt gene was mutated with random mutagenesis. Mutated plasmids were transformed into E. coli. Seven mutant strains were selected on solid screening plates from 20,000 clones, and DNA sequencing identified mutations of the cgt gene. Among the seven strains, five showed one-amino-acid substitution, and two showed two-amino-acid alteration. All mutant CGTases still had the activities for cyclization and coupling reactions. The relative specific activities for either reaction differed with the mutants(p<0.05). The relative specific activity γ-CD formation by J-8 (L304F) CGTase was 41 % of than the wild-type. In cyclization and coupling assay, the J-11 (A125R) CGTase showed higher specific activity than wild-type, but it was found that the activity was decreased dramatically after storage at 4℃ for 40 days. In order to stabilize J-11 CGTase, the mutant was stored in 10 mM Tris-HCl buffer (pH 7.2) containing either 4 mM CaCl2 or NaCl. Ninety-five percent of the enzymatic activity was maintained after storage at 4℃ for 40 days. It revealed that we could ameliorate the stability of the unstable mutant enzyme to improve their industrial application. Furthermore, Trp101 and Leu304 are located near the active site pocket of CGTase from B. macerans. The roles of Trp101 and Leu304 of CGTase in the structure and function were investigated. The pUG, was used for the construction of mutants via site-directed mutagenesis. Acting on soluble starch, the wild-type CGTase mainly produced α-CD in the early stage. However, W101L, L304Y, L304W, W101F/L304W and W101L/L304W CGTases produced mainly β-CD. A clear change in CD product ratio was observed in W101F/L304W and W101L/L304W CGTases. They produced less 1 in α-CD/β-CD product ratio than wild-type CGTase for 8 hours. And W101L/L304W CGTase almost lost the ability to produced γ-CD at all incubation periods (48 hours). These results indicated that the amino acid residues, Trp101 and Leu304 in CGTase, were related to the product specificity. Besides, as Bacillus subtilis (pHG) was cultivated in medium B containing 4 % wheat bran, 0.5 % (NH4)2SO4, 0.5 % CaCO3, 0.075 % K2HPO4, 0.05 % NaHPO4·12H2O and 0.03 % MgSO4·7H2O for CGTase production. The optimum condition for B. subtilis (pHG) CGTase production in 500 mL hinton flask was found as following : cultural volume, 100 mL; incubation temperature, 37℃; shaking speed, 120 rpm and incubation time, 72 hours. The CGTase was purified by a procedure including β-CD coupling affinity and DEAE Sepharose CL-6B chromatographs. The cgt gene from B. macerans was expressed in B. subtilis (pHG) and 100 ~ 110 mg/L of the enzyme was produced, which is about 10 times high as that of the parental strain. And the host could produce CGTase without adding any antibiotics (eg. tetracycline). The biochemical properties of B. subtilis (pHG) CGTase were similar with parental strain CGTase. Furthermore, effect of the substrate and enzyme concentration on the production of CD using response surface methodology. Results was applied to each variable to determine the productivity of total CD, α-CD and β-CD, the substrate and enzyme concentration were found to be significantly related (p<0.05). When a low concentration starch solution, was employed, it was found that the productivity of total CD and β-CD increased as the enzyme concentration increased. However, the productivity of α-CD was increased with decreasing CGTase concentration. The conditions for high productivity of total CD and β-CD were as follows : starch, 2.8 % ; B. subtilis (pHG) CGTase, 0.014 % ; agitation, 100 rpm ; incubation time, 2 hour and incubation temperature, 37℃. The productivity of total CD, α-CD and β-CD was 50 %, 19 % and 26 %, respectively. After the same amount of CGTase, was applied, a higher CD yield in same reaction volume could be obtained at a starch concentration of 5.2 %. Under this condition, the productivity of total CD was 45 % ; that of α-CD was 16 %; and that of β-CD was 22 %. No significant differences were observed between the experimental data obtained under the various conditions and the predicted values, which indicated that the regression equation established in this study was acceptable.
|Appears in Collections:||食品暨應用生物科技學系|
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