微生物幾丁聚醣酵素之探討

Abstract

近年來，數種可分解幾丁聚醣 (chitosan) 之菌種於本實驗室中被篩出。此論文針對其中三株分別為仙人掌桿菌 (Bacillus cereus)，淡紫擬青霉菌(Paecilomyces lilacinus) 與煙麴菌 (Aspergillus fumigatus) 菌種之幾丁聚醣酵素 (chitosanase) 做進一步之研究。 仙人掌桿菌中胞外幾丁聚醣酵素之生產，在添加適量之幾丁聚醣於培養液後能提高10 ~ 30% 產量。經純化後得知其為一分子量約42 kDa之內切幾丁聚醣酵素，等電點為5.0，最適反應條件為60℃及pH 5.7，最終水解產物為2、3、4醣。其催化過程以核磁共振光譜鑑定為位向反轉的機制 (inversion) ，與其所屬的醣類水解酵素第8家族者一致。在酵素回收再利用的兩項研究中， 酵素固定於矽膠 (silica gel) 時，在為期24小時之批次反應中，連續使用7次以上仍可維持99%之活性。此外，直接以溶解態酵素 (soluble enzyme)進行水解反應後，利用陰離子交換樹脂可有效回收反應中所使用之酵素達99%。 另外，由淡紫擬青霉菌中，可以利用單一離子交換樹脂從誘導醱酵液中純化取得達90%以上純度之胞外內切幾丁聚醣酵素。分子量以分子篩、SDS-電泳與質譜儀分別鑑定為24 kDa、25k Da與22,711 Da，即說明此酵素為一單體 (monomeric)。部分N端氨基酸序列已被鑑定出為“xQLPANLxxIYD”，因為與黑殭菌 (Metarhizium anisopliae) 的序列幾近相同，故將其定位為第75家族。另，有兩種特定的寡醣 (Gln-Gln-GlnAc與GlnAc-Gln-Gln-GlnAc) 即使經多次純化步驟，仍與此酵素緊密結合，這種現象在其他相關研究中並未曾被發現過。 煙麴菌是一具有強大幾丁聚醣酵素活性之墨綠色黴菌。可使用1 %的幾丁聚醣從菌絲誘導生產大量的幾丁聚醣酵素。培養液經濾膜處理、濃縮及離子交換樹脂處理後可得到26 kDa酵素，已可應用於生產公斤級幾丁寡醣之生物製程反應。 此外，配合cDNA library與inverse PCR的技術，可獲知全長866個鹼基對的煙麴菌幾丁聚醣酵素之染色體基因，其中包含717個鹼基的開放讀架 (ORF) 以及兩段插入序列 (intron)。爾後可轉譯成238個胺基酸之原酵素 (pro-protein)，其中包含一段17個胺基酸之訊息月生月太。經序列比對得知隸屬於第75家族。在大腸桿菌進行基因表達時，會以包涵體 (inclusion body) 的型式大量生成於菌體內。以5 M尿素 (urea) 處理後，可將此重組酵素純化至90%，並回復 30% 的酵素活性。此過表達系統 (over-expression) 與簡便純化的方法，可將酵素進一步應用在甲殼寡醣的大量生產上。 在酵素催化機制的研究上，以核磁共振光譜鑑定為位向反轉的機制 (inversion)。此外，比較 Family 75 的五個成員，發現十個高度保留的 (conserved) 胺基酸，預期藉由定點突變，以釐清何者為此酵素催化機制中的必需胺基酸 (essential groups)。初步實驗數據顯示，D96N、D175N與D178N突變後尚有不錯的活性，初步排除為必需胺基酸之可能。

Three different chitosanases independently purified from Bacillus cereus, Paecilomyces lilacinus, and Aspergillus fumigatus were investigated for their biochemical properties. All of these microorganisms were screened in our laboratory and identified by Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands). A 42 kDa extra-cellular chitosanase from B.cereus is purified. This enzyme is a monomeric protein with pI 5.0 and an optimal pH and temperature of 5.7 and 60°C, respectively. The major catalytic products are DP2 and DP3 chitooligo- saccharides. A time-course NMR study confirms that the catalytic mechanism of Bacillus chitosanase follows an inversion of anomeric configuration, which is consistent with the Family 8 glycohydrolase. A few enzyme recovery systems designed were tested. Among them, the batch enzymatic reaction combining with a post-Q-column separation can effectively isolate both products and enzyme in one step. Most of all, the activity of the recycled chitosanase is fully recovered. Another extracellular chitosanase from P. lilacinus is purified and characterized. Gel filtration analysis reveals the molecular weight of enzyme about 24 kDa, which corresponds with the estimated value of 25 kDa by SDS-PAGE. ESI-MS gives a more precise measurement with molecular weight of 22,711 Da. Clearly, the native Paecilomyces chitosanase is a monomeric protein and likely without any posttranslational modifications, such as glycosylation. It is classified to Family 75 according to its partial N-terminal amino acid sequences—“xQLPANLxxIYD”(x: indefinite), which is nearly identical to that of Metarhizium anisopliae in Family 75. It is also an endo-splitting enzyme mainly cleaving chitosan as chitobiose, chitotriose and chitotetrose. Interestingly, a monoacetylated chitotriose and a diacetylated chitotetraose, which are determined to be Gln-Gln-GlnAc and GlnAc-Gln-Gln-GlnAc, respectively, can tightly bind to the Paecilomyces chitosanase. However, this feature was not observed in Aspergillus chitosanase. An extremely powerful and highly stable chitosanase was found and purified from A. fumigatus culture containing chitosan as inducer. The purified enzyme has been successfully applied for a 275 g-scale of chitooligomers preparation with the products of DP2 to DP12. The corresponding gene has been cloned from the cDNA library and over-expressed in E. coli. By combining the manipulation of cDNA library and inverse PCR technology, the 866-base complete genome containing 717-base ORF, which includes a 17-amino-acid signal peptide and a 221-amino-acid mature chitosanase, are accomplished. When the recombinant DNA is expressed in E. coli., inclusion bodies are formed. At least 30% of enzymatic activity can be rescued by treating the inclusion body with 5 M urea without further dialysis. Based on the multialignment of chitosanases in Family 75, ten conserved amino acid residues, which should contain the catalytic essential groups, are found. Research on the identification of the catalytic essential groups by site-directed mutagenesis is in progress. The preliminary results show that D96N, D175N and D178N are unlikely to be the essential group.