綠竹筍懸浮細胞幾丁質降解酵素之探討

Abstract

研究顯示植物幾丁質降解酵素之主要功能為防禦病原菌的入侵，並且可應用於製備真菌原生質體、單細胞蛋白質生產、生物防治劑及製備高經濟價值之 N-乙醯幾丁寡醣及幾丁寡醣。本實驗室先前結果顯示，竹筍外殼及可食部皆可偵測到幾丁質酶、幾丁聚醣酶及幾丁糊精酶 (chitodextrinase) 的活性。本論文首先以綠竹筍懸浮培養細胞為研究材料，探討幾丁質降解酵素的生化性質。藉由含有 4-MU-(GlcNAc)3 之 SDS-PAGE 及幾丁糊精酶活性染色，和含有乙二醇幾丁質之 SDS-PAGE 及幾丁質酶活性染色與活性測定，可知綠竹筍懸浮培養細胞有多種幾丁質降解酵素存在。其中幾丁糊精酶由純化結果顯示是比較偏疏水性的結構，活性染色分析顯示雙硫鍵的存在對於幾丁糊精酶活性的維持是個必要的因子，而在二維電泳分析指出此酵素是 pI 大約在 5 左右的酸性蛋白質。以 4-MU-(GlcNAc)3 為基質，幾丁糊精酶之 Km 值為 4.07 μM，以 4-MU-(GlcNAc)2 為基質，其Km值為9.04 μM，推論幾丁糊精酶對 4-MU-(GlcNAc)3 之親和力較 4-MU-(GlcNAc)2 大。由基質專一性分析顯示幾丁糊精酶具有水解寡醣但不水解高分子幾丁質的特性，其水解作用模式可能為內切型或任意型，其水解 4-MU-(GlcNAc)3 之最適反應溫度為 70℃，最適反應 pH 值為 3，在 pH 5 的環境下有最高活性，隨著 pH 值提高則逐漸下降；而幾丁糊精酶之活化能為 13.21 kJ/mol，比其它物種幾丁質酶之活化能較低。由膠體過濾法及 SDS-PAGE 活性染色測得幾丁糊精酶之分子量皆為 90.5 kDa，故屬單元體酵素。大多數金屬離子對酵素活性無影響，只有 Hg2+ 會有明顯的抑制作用。貯存安定性分析顯示此酵素可存放在 50 mM 磷酸緩衝液 (pH 7.4) 或 10 mM Tris-acetate (pH 7.4) 緩衝液於 4℃ 環境下達 14 個月，其活性甚為安定。 另外為了獲得足夠的蛋白質以進行各項研究探討，本研究將先前從綠竹筍懸浮細胞cDNA 庫篩選到的第三型幾丁質酶 cDNA (BoChi3-1a)，送入酵母菌 (Pichia pastoris) 中進行重組蛋白之表現與檢定。收取菌體培養外泌液經由硫酸銨劃分 (0-100%飽和度)、Phenyl-Sepharose 疏水性層析、Con A-Sepharose 親和性層析等連續純化步驟，可得到分子量分別為 28.3 kDa 與 35.7 kDa 的幾丁質降解酵素，並由 N 端定序及西方墨點法進一步確認所表現的蛋白質均為重組第三型幾丁質酶 (BoCHI3-1a)。疏水性管柱層析法可分別將 28.3 kDa-、35.7 kDa-BoCHI3-1a 與菌株內生性幾丁質酶分離開來，其中 35.7 kDa-BoCHI3-1a 的親水性遠大於 28.3 kDa-BoCHI3-1a。由 Con A-Sepharose 親和性層析與醣蛋白質染色法 (PAS staining) 也說明，原態分子量為 28.3 kDa 的重組第三型幾丁質酶在酵母菌體表現會有醣基化現象，造成分子量變大、親水性增強與生化性質的改變。EGC基質水解特性顯示，35.7 kDa -與 28.3 kDa-BoCHI3-1a 之最適 pH 值分別為 3 與4；最適溫度為 80 與 70℃；Km 值為 1.35 與 0.65 mg/mL；活化能為 13.4 與 15.5 kJ/mol；比活性為 289 與 117 mU/mg；兩者之熱穩定性與 pH 穩定性皆為 70℃ 與 3。35.7 kDa -與 28.3 kDa-BoCHI3-1a 的酵素混合液對於分離自綠竹空心稈腐生菌及十字花科蔬菜黑斑病菌均有抑制生長作用。貯存安定性分析顯示此兩者酵素可存放在 50 mM 磷酸緩衝液 (pH 7.4) 於 4℃ 環境下達 1 年，其活性甚為安定。

Chitinase was expressed in suspension-cultured cells of bamboo (Bambusa oldhamii) in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and secreted into the medium during cultivation. A novel chitinase, designated chitodextrinase, was purified from the medium of the suspension-cultured cells by 40-80% saturation of ammonium sulfate fractionation, hydrophobic chromatography, DEAE-Sephacel ion-exchange chromatograph, and preparative polyacrylamide gel electrophoresis. The purified chitodextrinase was active toward chitin oligomer substrates but almost inactive toward chitin polymer. The optimal pH for hydrolysis of 4-methylumbelliferyll-β-D-N, N’, N”-triacetylchitotrioside (4-MU-GlcNAc3) was 3, the optimal temperature was 70°C and the Km was 4.07 μM. The molecular mass was 90.5 kDa, which was estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 5. The chitodextrinase was thermally stable, as it retained almost all of its activity after incubation at 60°C for 30 min or storage at 4°C for a year. Mercuric ion (0.5 mM) significantly inhibited the activity of the enzyme. The end products of N-acetylglucosamine oligomers (GlcNAcn, n = 3~6) hydrolysis catalyzed by the enzyme were GlcNAc1、GlcNAc2 or GlcNA3, as analyzed using thin-layer chromatography. The smallest chitin oligomer substrate for the enzyme action was a chitin trimer.

A class III chitinase cDNA (BoChi3-1a) cloned from a cDNA library of bamboo suspension-cultured cells was transformed into yeast (Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 kDa and 35.7 kDa, respectively, were purified from the yeast’s culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography and Con A-Sepharose chromatography. N-terminal sequencing and western analysis revealed that both recombinant chitinases were encoded by BoChi3-1a, while SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1a) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1a) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pHs were 3 and 4 for 35.7 kDa and 28.3 kDa BoCHI3-1a, respectively; the optimal temperatures were 80°C and 70°C, and the Km values were 1.35 and 0.65 mg/mL, respectively. The purified 35.7 kDa-BoCHI3-1a hydrolyzed EGC more efficiently than 28.3 kDa isoform, as revealed from their specific activity and activation energy. Both recombinant BoCHI3-1a isoforms not only showed antifungal activity against Scolecobasidium longiphorum and Alternaria brassicicola but also displayed remarkable stability.