Paecilomyces lilacinus中幾丁聚醣酶與葡萄糖胺苷水解酶之研究

Abstract

本研究係經由淡紫擬青黴 (Paecilomyces lilacinus) 菌種中，誘導出兩種不同催化型式的幾丁聚醣酵素 (Chitosanase)，經由陽離子管柱純化後，以蛋白質電泳測定其分子量分別為27 kDa的幾丁聚醣水解酵素 (Chitosanase) 及95 kDa 葡萄糖胺苷水解酵素(□-glucosaminidase)。幾丁聚醣水解酵素最適反應酸鹼值為6.0，最適反應溫度為37 ℃，酵素在pH 3.8~9.9穩定性良好；葡萄糖胺苷水解酵素之最適反應pH值為6.0，最適反應溫度為45 ℃，酵素在pH 6.0時穩定性佳。 幾丁聚醣水解酵素水解幾丁聚醣所得之產物以三、四及五糖為主，由產物分析可判斷為一內切型 (Endo-type) 酵素，經由質譜分析測定其分子量為22,711 Da，同時發現有醣類和酵素緊密結合，利用醣類甲基化反應與質譜分析，可得知此醣類的組成與排列方式，分別為GlcN-GlcN-GlcNAc與GlcNAc-GlcN-GlcN-GlcNAc。 葡萄糖胺苷水解酵素水解幾丁六糖 (Chitohexomers) 所得產物為單糖，由此判斷一外切型 (Exo-type) 酵素，具有轉醣功能 (Transglycosylation)。利用此功能將轉醣產物 (Methyl-□-D-glucosaminide)，以核磁共振光譜 (NMR) 判斷產物構形為β-型式，由此亦可證明酵素反應機制是屬於構形保留型 (Retention) ，此轉醣功能亦可用以合成Alkyl-□-D-glucosaminide (R-GlcN)，有利於簡化有機合成的複雜步驟。 由一系列的實驗成功的鑑定出，葡萄糖胺苷水解酵素可水解GlcN-GlcN與GlcN-GlcNAc之糖苷鍵，但無法水解GlcNAc-GlcNAc之糖苷鍵，代表酵素對於非還原端位置的醣基，有明顯的選擇性。

Paecilomyces lilacinus produces two types of chitosan degrading enzyme which were purified and characterized in this study. These purified enzyme were identified as chitosanase and β-Glucosaminidase with the molecular weights of 22,711 Da by ESI-MS analysis, and 95 kDa by SDS-PAGE, respectively. Chitosan and chitooligosaccharides are their substrates. Chitosanase exhibits endo-splitting activity with chitobiose, chitotriose, and chitotetraose as the end products, whereas β-Glucosaminidase exhibits exo-splitting activity to form glucosamine. The optimal temperature and pH of chitosanse are 37 ℃ and 6.0, and those of β-Glucosaminidase are 45 ℃ and 6.0 For hydrolysis of chitosans with various N-acetyl contents, both enzymes degrade higher degree of deacetylated chitosan more effectively. Interestingly, a monoacetylated chitotriose and a diacetylated chitotetraose can tightly bind to chitosanase. These chitooligosaccharide sequences are determined by chemical methylation, followed by the tandem mass spectrometry analysis. These sequences are identified as GlcN-GlcN-GlcNAc and GlcNAc-GlcN-GlcN-GlcNAc. Methyl-β-D-glucosaminide is the product of the transglycosylation reaction of chitooligosaccharides catalyzed by β-Glucosaminidase in the presence of methanol. Methyl-β-D-glucosaminide is then analyzed by 1H nuclear magnetic resonance spectroscopy to prove that the β-Glucosaminidase is a retaining enzyme. In a serial experiment of hydrolysis of Chitobiose, the enzyme appeared to be effective in cleaving glucosamine on GlcN-GlcN and GlcN-GlcNAc, but not GlcNAc-GlcNAc. Abbreviation：GlcN, glucosaminie；GlcNAc, N-acetylglucosamine