靈芝之主要生理活性成份是一群具有β-1,3鍵結之聚葡萄糖主鏈之高分子多醣體。本研究主要是以靈芝屬中之松杉靈芝Ganoderma tsugae建立起β-葡聚醣酶之分離、純化方法並探討其物化特性。
松杉靈芝中之β-1,3葡聚醣酶利用硫酸銨分劃、Sephadex G-50及DEAE Spharose CL-6B管柱層析等步驟純化後，其回收率為4.2％，β-1,3葡聚醣酶分別被純化15.1倍。其最適作用pH值為5.0，此酵素在pH值6.0時最為穩定。β-1,3葡聚醣酶之最適作用溫度為50℃，而當溫度在60℃或60℃以上時，此酵素會因變性而失去活性。在受質特異性方面，當以laminarin作為受質時，可得到最大之酵素活性，而以curdlan、lichenan及zymosan為受質時，其相對水解活性分別為16.1、43.7及26.3%。在金屬離子方面，10 mM Cu+2與酵素作用之後，其活性完全受到抑制，而當以Fe+2, Zn+2, Mn+2, Mg+2, Na+2及Ca+2與酵素作用之後，其抑制率介於30.0-85.2%，而Co+2對β-1,3葡聚醣酶之活性影響最小。在抑制劑方面，常見之抑制劑皆能對β-1,3葡聚醣酶活性加以抑制，當L-抗壞血酸濃度達10 mM時，幾乎能完全的抑制β-1,3葡聚醣酶活性。在酵素動力學方面，當以laminarin最為受質時所求得之Km值為5.99 mg/ml，而Vmax則為129.87 OD/min.ml。
β-1,6葡聚醣酶利用硫酸銨分劃、Sephadex G-50及DEAE Spharose CL-6B管柱層析等步驟純化後，其回收率為3.2％，β-1,3葡聚醣酶分別被純化15.6倍。β-1,6葡聚醣酶顯示其具有較廣範圍之最適作用pH值；在pH 5.0時其活性最大，而此酵素在pH 6.0時最為穩定。其最適作用溫度為60℃，但當此酵素在50或50℃以上的溫度下時則很容易快速變性失活。在受質特異性方面，當以pustulan作為受質時，其具有最大之酵素活性，而zymosan也有48.3%的相對活性。在金屬離子方面，當以10 mM Ca+2與酵素作用之後，其活性完全受到抑制，而當以Fe+2, Zn+2, Mn+2, Mg+2, Na+2及Cu+2與酵素作用之後，其抑制率介於41.0- 84.6%。而Co+2對β-1,6葡聚醣酶之活性影響不大。在抑制劑方面，常見之抑制劑皆能對β-1,3葡聚醣酶活性加以抑制，當sodium metabisulfite濃度達10 mM時幾乎能完全的抑制β-1,6葡聚醣酶活性。在酵素動力學方面，當以pustulan最為受質時所求得之Km值為1.88 mg/ml，而Vmax則為75.19 OD/min.ml。
綜合以上結果可知，β-1,3葡聚醣酶及β-1,6葡聚醣酶在受質特異性方面，僅對β-1,3及β-1,6鍵結之多醣體具有活性，對其他方式鍵結之多醣體沒有任何活性存在。因此，利用酵素分析方法來測定市售靈芝產品中之多醣體，應具有其之可行性。

The bioactive carcinostatic substance in Lingzhi is high molecular weight polysaccharides linked byβ-(1,3)-D-glucosidic bond. In this project, the β-glucanases from Ganoderma tsugae were isolated, purified and characterized.
Purification of the β-1,3 glucanase from Ganoderma tsugae with a recovery of 4.2% and purity increase of 15.1 fold was achieved by ammonium sulfate fractionation, Sephadex G-50 and DEAE Sepharose CL-6B chromatography. The optimum pH forβ-1,3 glucanase was 5.0. The enzyme was stable at pH 6.0. The optimum temperature for β-1,3 glucanase was 50℃. The enzyme was rapidly denatured at temperature of 60℃ and above. Substrate specificity studies indicated this enzyme has maximum activity toward laminarin. The relative rate of hydrolysis of curdlan, lichenan and zymosan to that of laminarin was 16.1, 43.7 and 26.3%, respectively. Almost complete inhibition was observed with 10 mM Cu+2, while moderate inhibition was seen with Fe+2, Zn+2, Mn+2, Mg+2, Na+2及Ca+2. Activity was unaffected by 10 mM Co+2. All the common inhibitors tested inhibitedβ-1,3 glucanase activity to various extents. The enzyme was completely inhibited by 10 mM L-asccorbic acid. The Km value of β-1,3 glucanase toward laminarin was 5.99 mg/ml, while Vmax was 129.87 OD/min. ml.
Purification of the β-1,6 glucanase from Ganoderma tsugae with a recovery of 3.2% and purity increase of 15.6 fold was achieved by ammonium sulfate fractionation, Sephadex G-50 and DEAE Sepharose CL-6B chromatography. β-1,6 glucanase displayed relatively broad pH optimum. The enzyme was stable at pH 6.0. The optimum temperature for β-1,6 glucanase was 60℃. The enzyme was rapidly denatured at temperature of 50℃ and above. Substrate specificity studies indicated this enzyme has maximum activity toward pustulan. The relative rate of hydrolysis of zymosan to that of pustulan was 48.3%. Almost complete inhibition was observed with 10 mM Ca+2, while moderate inhibition was seen with Fe+2, Zn+2, Mn+2, Mg+2, Na+2及Cu+2. Activity was unaffected by 10 mM Co+2. All the common inhibitors tested inhibitedβ-1,6 glucanase activity to various extents. The enzyme was completely inhibited by 10 mM Sodium metabisulfite. The Km value ofβ-1,6 glucanase of toward pustulan was 1.88 mg/ml, while Vmax was 75.19 OD/min. ml.
This study displayed thatβ-1,3 glucanase andβ-1,6 glucanase from Ganoderma tsugae only hydrolyze specific 1→3 and 1→6 linkage types of β-glucans, respectively. Therefore, substrate specific β-1,3 glucanase andβ-1,6 glucanase could be employed in the enzymatic assay to identify the authenticity of Lingzhi products on the market.

封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 ix
目錄 x
圖目錄 xiv
表目錄 xvi
第一章 緒論 1
1.1研究背景 1
1.2研究動機 1
1.3研究大綱 2
1.4研究重要性 2
第二章 文獻回顧 4
2.1靈芝之簡介 4
2.1.1靈芝之分類 5
2.1.2靈芝之多醣體 6
2.1.3靈芝之保存方法 11
2.1.4多醣體之測定方法 12
2.2β-葡聚醣酶 14
2.2.1β-葡聚醣酶之存在 15
2.2.2β-葡聚醣酶之分類 16
(1)外切β-葡聚醣酶 18
(2)內切β-葡聚醣酶 18
(3)外切-內切β-葡聚醣酶 19
2.2.3β-葡聚醣酶之純化 20
2.2.4β-葡聚醣酶之物化特性 25
2.2.4.1分子量 25
2.2.4.2 pH值與等電點 29
2.2.4.3最適溫度 30
2.2.4.4專一性 30
2.2.5β-葡聚醣酶活性之測定 32
2.2.6β-葡聚醣酶之應用 33
第三章 材料與方法 37
3.1材料 37
3.1.1菌株 37
3.2化學藥品 37
3.3儀器與裝置 39
3.4實驗方法 41
一、靈芝之β-葡聚醣酶之萃取、分離及純化方法之建立
(1)純種菌種保存方法 41
(2)液態培養 41
(3)菌絲體乾重 42
(4)殘糖 42
(5)β-葡聚醣酶之純化 43
(6)酵素活性之測定 45
(7)蛋白質含量測定 46
二、靈芝中β-葡聚醣酶生化特性探討
(1)原態分子量 47
(2)受質專一性 47
(3)最適 pH 值 48
(4)pH 穩定性 48
(5)最適溫度 48
(6)熱穩定性 48
(7)各種可能之化學抑制劑及重金屬離子之影響 49
(8)酵素動力學測定 49
第四章 結果與討論 51
一、靈芝之β-葡聚醣酶之萃取、分離及純化方法之建立
(一)靈芝屬Ganoderma tsugae培養期間菌絲乾重、pH值及殘糖之變化 51
(二)硫酸銨飽和沈澱活性劃分 53
(三)Sephadex G-50管柱層析 56
(四)DEAE Sepharose CL-6B離子交換管柱層析 56
(五)純化總表 59
二、靈芝中β-葡聚醣酶生化特性探討
(一)原態分子量及次單元分子量 64
(二)最適pH及pH穩定性 67
(三)最適作用溫度及熱穩定性 70
(四)受質特異性 73
(五)金屬離子 76
(六)抑制劑 79
(七)酵素動力學 82
第五章 結論 87
參考文獻 90
圖目錄
圖一、具有抗腫瘤活性之多醣體結構 7
圖二、多醣體抗腫瘤之作用機制 9
圖三、酵素回收純化之流程 21
圖四、Ganoderma tsugae培養天數對菌絲乾重、pH值及殘糖之變化 52
圖五、靈芝屬β-1,3葡聚醣酶之硫酸銨飽和沉澱活性劃分 54
圖六、靈芝屬β-1,6葡聚醣酶之硫酸銨飽和沉澱活性劃分 55
圖七、β-1,3葡聚醣酶之Sephadex G-50管柱層析圖
57
圖八、β-1,6葡聚醣酶之Sephadex G-50管柱層析圖
58
圖九、β-1,3葡聚醣酶之DEAE Sepharose CL-6B管柱層析圖 60
圖十、β-1,6葡聚醣酶之DEAE Sepharose CL-6B管柱層析圖 61
圖十一、以膠體過濾層析測定β-1,3葡聚醣酶之原態分子量 65
圖十二、以膠體過濾層析測定β-1,6葡聚醣酶之原態分子量 66
圖十三、β-1,3葡聚醣酶之最適pH及pH穩定性 68
圖十四、β-1,6葡聚醣酶之最適pH及pH穩定性 69
圖十五、β-1,3葡聚醣酶之最適作用溫度及熱穩定性
71
圖十六、β-1,6葡聚醣酶之最適作用溫度及熱穩定性
72
圖十七、β-1,3葡聚醣酶作用於不同濃度受質之雙倒數圖 83
圖十八、β-1,6葡聚醣酶作用於不同濃度受質之雙倒數圖 84
表目錄
表一、β-glucan分解酵素之命名及作用機制 17
表二、各種鹽析及沉澱法之比較 22
表三、常用生物技術產物之液相層析分離技術 23
表四、真菌中β-葡聚醣酶之物化性質 26
表五、不同基質之鍵結形式 31
表六、胞外β-1,3葡聚醣酶之純化總表 62
表七、胞外β-1,6葡聚醣酶之純化總表 63
表八、純化後β-1,3葡聚醣酶對不同基質作用之相對活性 74
表九、純化後β-1,6葡聚醣酶對不同基質作用之相對活性 75
表十、金屬離子對β-1,3葡聚醣酶活性之影響 77
表十一、金屬離子對β-1,6葡聚醣酶活性之影響 78
表十二、抑制劑對β-1,3葡聚醣酶活性之影響 80
表十三、抑制劑對β-1,6葡聚醣酶活性之影響 81
表十四、β-葡聚醣酶與不同濃度受質之動力學參數 85

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