利用API ZYM系統分析白殭菌及蠟蚧輪枝菌之胞外酶分泌種類及時程

Enzymatic Characterization of Two Entomopathogenic Fungi with the API ZYM System and Their Secretion Time Series

簡巧治(Chiao-Chih Chien) ； 蕭文鳳(Wen-Feng Hsiao) ； 翁秉霖(Ping-Lin Ong)

《台灣昆蟲》 26卷4期 (2006/12) Pp. ap2+319-328

https://doi.org/10.6661/TESFE.2006028

摘要

本實驗利用固體培養基及API ZYM系統對蟲生真菌白殭菌（Beauveria bassiana）及蠟蚧輪枝菌（Verticillium lecanii）的蛋白酶（protease）、脂解酶（lipase）及其他胞外酶進行定性分析。以營養培養基添加gelatin及Tween20培養基培養真菌，於30℃下恆溫培養七天後，所產生之酶水解環直徑遠較培養於20、25及35℃溫度下真菌產生酶水解環直徑來的大；除35℃外，真菌在其他三種溫度培養下，菌體外皆分泌蛋白酶及脂解酶。另以API ZYM系統分析真菌體外分泌酵素，結果顯示白殭菌並未偵測到β-葡萄糖苷酸酶（β-glucuronidase）、α-半乳糖苷酶（α-galactosidase）、α-葡萄糖苷酶（α-glucosidase）及甘露糖苷酶（α-mannosidase）；蠟蚧輪枝菌未偵測到β-葡萄糖苷酸酶，但有微量的纈胺酸肽胜分解酶（valine aminopeptidase）、半胱氨酸肽胜分解酶（cysteine aminopeptidase）、胰蛋白酶（trypsin）、α-半乳糖苷酶、α-葡萄糖苷酶及甘露糖苷酶活性（≤1等級）；另三株白殭菌供試菌株（35502、35517及H0212）有較高酯解酶（esterase）、酯解脂解酶（esterase lipase）、脂解酶（lipase）、酸性磷酸脢（acid phosphatase）、磷酸醯胺酶（phosphoamidase）、β-葡萄糖苷酶及β-氨基葡萄糖苷酶（β-glucosaminidase）等活性（2～3等級）。而另二株蠟蚧幹枝孢菌供試菌株（35548與35556）亦具有acid phosphatase、phosphoamidase、β-galactosidase、β-glucosidase、β-glucosaminidase及α-岩藻糖苷酶（α-fucosidase）等水解活性（等級2～3）。此外較特殊的是白殭菌35502菌株，較其他兩株白殭菌菌株具較高alkaline phosphatase、acid phosphatase、phosphoamidase、β-glucosidase及α-fucosidase酵素活性（等級4）。以上的結果顯示此二蟲生真菌在孢子發芽階段能產生esterase、lipase、acid phosphatase、phosphoamidase及chitinase等胞外酵素。API ZYM測試系統可提供快速半定量真菌細胞外酶活性測試，分析蟲生真菌入侵寄主昆蟲可能之水解酵素產生種類。本實驗發現白殭菌於培養第二天即產生可能參與昆蟲體表脂質水解的esterase及lipase酵素，及可能分解蟲體幾丁質的glucosidase及glucosaminidase酵素，及與蛋白質修飾有關的acid phosphatase及phosphoamidase酵素，上述大量分泌體外酵素在蟲生真菌侵入蟲體過程中扮演著特定角色。

關鍵字

白殭菌 ； 蠟蚧輪枝菌 ； 酶 ； API ZYM

並列摘要

The enzymatic profiles and enzyme secretion time series of protease and lipase of two entomopathogenic fungi, Beauveria bassiana and Verticillium lecanii were studied using an API ZYM system and culture media. The diameter of the hydrolytic zone when both fungi were cultured with two media below 30°C was larger than these of fungi cultured at the remaining temperatures7 days later. In the in vitro test, these two fungi were able to produce protease and lipase at the tested temperatures, except at 35°C. None of the isolates showed any reaction to substrates with β-glucuronidase. All isolates showed little activity or were completely without activity to substrates with valine aminopeptidase, cysteine aminopeptidaes, trypsin, α-galactosidase, α-glucosidase, and α-mannosidase. Three isolates of B. bassiana (35502, 35517, and HO212) showed moderate activity to substrates of esterase, esterase lipase, lipase, acid phosphatase, phosphoamidase, β-glucosidase, and β-glucosaminidase. Two isolates of V. lecanii (35548 and 35556) showed moderate activity to substrates of alkaline phosphatase, acid phosphatase, phosphoamidase, β-glucosidase, and α-fucosidase. Among them, B. bassiana isolate 35502 showed stronger reactions to the substrates of alkaline phosphatase, acid phosphatase, phosphoamidase, β-glucosidase and α-fucosidase than did the other two B. bassiana isolates that reached a category of four. Results suggest that these two entomopathogenic fungi produce extracellular enzymes, e.g., esterase, lipase, acid phosphatase, phosphoamidase, and chitinasem, during their spore germination period. In the enzyme secretion series conducted with B. bassiana, glucosidase and glucosaminidase appeared on day 2 to digest the carbohydrate. Meanwhile, phosphatase and phosphoamidase appeared on day 2 to digest the protein. The results of these studies indicate that the API ZYM technique provides a rapid and reproducible semiquantitative method for revealing the enzymatic activity and secretion time series.

並列關鍵字

enzymes ； Beauveria bassiana ； Verticillium lecanii ； API ZYM