Study of a secondary metabolite PR acid by penicillium roqueforti

• Main Authors: Lei, Wen-Yee, 雷文宜
• Other Authors: Chang, Shenq-Chyi
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/58095941189438557962

碩士 === 國立陽明大學 === 生物化學研究所 === 84 === PRtoxin與PRacid，是藍酪黴菌(Penicillium roqueforti)所產生之次級代謝產物。經由time course研究，我們發現不管採用液體培養基(YESC)或固態培養基(喬麥)都能觀察到隨著PR toxin產量下降相對會有PR acid產生之現象，而且PR acid會維持一穩定之量。同時利用液體培養所產生之PR toxin及PR acid的產量大於固態培養，但以固體培養方式時，此二次級代謝產物出現時間較早。此外，發現最適合菌體產生PR toxin及PR acid之液體培養基pH僅在4.0左右。 為了觀察在不同Penicillium菌種中PR toxin及PR acid產生之差異，我們針對十二株不同species之Penicillium及四株不同strains之Penicillium roqueforti,研究其產生PR toxin及PR acid之情形。發現在相同培養條件下，除了Penicillium roqueforti 48936及Penicillium roqueforti 48779可產生此早次級代謝產物外，其它兩株不同strains之Penicillium roqueforti及十二株不同species之Penicillium 則無此相關產物出現。 近來，我們發現了一種新的氧化酵素(PR oxidase)，其主要存在液體培養液中，且能促使PR toxin轉換成PR acid。我們收集這些培養液，藉由超過濾濃縮、80%硫酸銨鹽析後再進一步以陰離子交換層析、強疏水性層析柱、HPLC膠體層析柱加以純化，得到最終產物之純度提高6.4倍，比活性達998U/mg蛋白質，回收率為10.19%，而且我們發現此氧化酵素需有cofactor參與反應。 PR氧化酵素經由7.5%SDS-膠體電泳分析後，得知其分子量約為88kDa左右，除此之外，經由PAS染色後得知其為一種醣蛋白。經由endoglycosidase F作用後，發現醣鏈約佔PR氧化酵素分子量之23.9%。 PR氧化酵素之pI值為4.50，其最適反應之pH值為pH4-5，在pH4-6之範圍相當穩定，但在偏鹼範圍則易尖沽。最適反應溫度為50℃，對於溫度相當穩定，但超過60℃左右則活性大幅降低。 基於PR toxin及PR acid兩者結構相似且二者存在具相關性，再加上存在一氧化酵素能將PR toxin轉換為PR acid，顯示著PR toxin為PR acid之前驅物。至於PR氧化酵素可將PR toxin轉換為PR acid，此途徑即類似EC氧化酵素可將erernofortin C(EC)轉換為PR toxin一般。綜合以上結果，可畫出藍酪黴菌毒素之生合成及代謝途徑: EC oxidase PR oxidase EC---------------->PR toxin------------------>PR acid Both PR toxin and PR acid are the secondary metabolites produced by Perncillium roqueforti . In time course study, PR acid, was produced by association with the disappearance of PR toxin in the liquid (YESC) or the solid culture (buckwheat) of Penicillium roqueforti, but the production of PR toxin and PR acid in the liquid culture were much greater than those in the solid culture, and the optimal pH for the production of these two compounds were around pH4.0. After examining 12 species of Penicillium and 4 strains of P. roqueforti, only two strains of P. roqueforti , ATCC 48936 and 48779, could produce these metabolites, but others could not. Recently, a new enzyme (PR oxidase) was discovered which was almost exclusively present in the culture broth of the strains and involved in the conversion of PR toxin into PR acid. The culture broth was collected and concentrated by ultrafiltration, precipitated with 80% ammonium sulfate and further purified by Q-sepharose, hydrophobic (phenyl) and gel filtration column. The purity of PR oxidase was increased 6.4 fold and its specific activity reached to 998U/mg, and the recovery of the purified enzyme was 10.19%. Moreover, the enzyme needed a cofactor(s) to catalyze the enzymatic reaction. The PR oxidase was found to be a monomer with a molecular weight of approximately 88 kDa as determined by 7.5 % SDS-polyacrylamide gel electrophoresis. In addition, the enzyme was estimated to be a glycoprotein determined by PAS staining. The sugar residues account for 23.9% of the mass of PR oxidase. The pI value was around 4.50 and the optimal pH of PR oxidase was around pH 4-5, and the enzyme was stable between pH 4-6, but the enzyme activity was quickly lost in the basic condition. The PR oxidase was quite stable toward heat. The optimal temperature was 50 ℃. However, the enzyme activity was lost above 60℃. Based on these data, we strongly suggested that PR toxin was oxidized to PR add by the PR oxidase, just like the model of erernofortin C (EC) which is transformed to PR toxin by EC oxidase. We could estimate the degradation and synthesis pathway of PR toxin as following : EC oxidase PR oxidase EC---------> PR toxin -------------> PR acid