Abstract 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 30oC 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 35oC. 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.
摘要 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?(protease)、脂解?(lipase)及其他胞外?進行定性分析。以營養培養基添加gelatin及Tween 20培養基培養真菌，於30oC下恆溫培養七天後，所產生之?水解環直徑遠較培養於20、25及35oC溫度下真菌產生?水解環直徑來的大；除35oC外，真菌在其他三種溫度培養下，菌體外皆分泌蛋白?及脂解?。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?酸?(β-glucuronidase)、α-半乳糖??(α-galactosidase)、α-葡萄糖??(α-glucosidase)及甘露糖??(α-mannosidase)；蠟蚧輪枝菌未偵測到β-葡萄糖?酸?，但有微量的纈胺酸?胜分解?(valine aminopeptidase)、半胱氨酸?胜分解?(cysteine aminopeptidase)、胰蛋白?(trypsin)、α-半乳糖??、α-葡萄糖??及甘露糖??活性(￡ 1等級)；另三株白殭菌供試菌株（35502、35517及H0212）有較高酯解?(esterase)、酯解脂解?(esterase lipase)、脂解?(lipase)、酸性磷酸脢(acid phosphatase)、磷酸醯胺?(phosphoamidase)、β-葡萄糖??及β-氨基葡萄糖??(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??(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?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。
| Key words: enzymes, Beauveria bassiana, Verticillium lecanii, API ZYM | | 關鍵詞: 白殭菌、蠟蚧輪枝菌、酶、API ZYM |
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