杏鮑菇漆酶之功能性表達與特性分析

Functional Expression and Characterization of Laccases from

薛桂青(Kuei-Ching Hsueh)

指導教授 ： 徐志宏 ； 陳幼光

國立屏東科技大學/農學院/生物科技研究所/碩士(2010年)

https://doi.org/10.6346/NPUST.2010.00250

摘要

木質纖維素是自然界中最豐富的碳源，其由纖維素、半纖維素和木質素所組成，其中以木質素的結構最為複雜，也較難分解。白腐真菌 (white-rot fungus) 在自然界中扮演重要的木質素分解角色，目前已知白腐真菌之木質素分解酶主要由三種酵素所組成，分別為漆氧化酶 (laccase, EC 1.10.3.2)、含錳過氧化酶 (manganese peroxidase, EC 1.11.1.13) 及木質素氧化酶 (lignin peroxidase, EC 1.11.1.14) 等。杏鮑菇 (Pleurotus eryngii) 屬於側耳屬的白腐真菌，已知具有分解木質素的能力，因此本研究將以探討杏鮑菇生產漆氧化酶之最佳誘導條件以及其漆氧化酶基因之異源性表達方式為方向。研究將漆氧化酶基因 (pel3 和pel4) 分別選殖至含 pET28a 和 pET29a 載體之大腸桿菌系統中表現，結果顯示 pel3 基因在 pET28a 和 pET29a 載體上皆可表達，其分子量大小分別約為 56 和 53 kDa；pel4 基因則在 pET28a 系統可得到較佳的表達，分子量大小約為 58 kDa，漆氧化酶會隨誘導時間增加而表現量增加，在漆氧化酶可溶性試驗的部分，發現蛋白質幾乎呈現不可溶之狀態，試用不同濃度之IPTG進行漆氧化酶誘導，發現不同濃度之IPTG對蛋白質表現量並沒有顯著之影響。在漆氧化酶之酵素活性分析試驗，結果確認在原核表現系統之蛋白，並不具漆氧化酶之活性。使用酵母菌之真核表達系統可表現出具酵素活性之PEL4，進行生化特性分析得知最適反應溫度為60℃，最適酸鹼值為在Citric acid -Na2HPO4 buffer的pH 3.0，酵素在 pH 5.0~10.0 範圍之間具穩定性，熱穩定性分析的結果顯示酵素在溫度 10~60℃ 範圍內仍具有漆氧化酶活性。

關鍵字

漆氧化酶 ； 杏鮑菇 ； 蛋白質異源性表達

並列摘要

Lignocellulosics are the most abundant carbon resource in nature. They are composed of celluloses, hemicelluloses, and lignins. Lignin is the hardest composition to be degraded for its complex constitutions. White-rot fungus plays an important role for degrading lignin in nature that degrades lignin by secreting extracellular oxidase-lignin degradable enzymes. Three kinds of enzymes are able to degrade lignins: laccase (EC 1.10.3.2), manganese peroxidase (EC 1.11.1.13), and lignin peroxidase (EC 1.11.1.14). Pleurotus eryngii is a white rot fungus which has the ability to degrade lignins. The aims of this study were to investigate the optimal induction condition of laccase production by P. eryngii and the heterologous expression of laccase genes. Preliminary results showed that higher laccase activity was obtained when p. eryngii were grown in liquid medium with the addition of copper sulfate. In addition, two laccase genes (pel3 and pel4) were cloned into pET28a and pET29a vectors and transformed to E. coli for expression, respectively. The results showed that PEL3 proteins were both expressed in pET28a and pET29a systems by E. coli. The molecular weight of which were approximately 56 and 53 KDa, respectively. PEL4 proteins could be expressed in the pET28a system with a molecular weight of about 58 kDa. Expression of laccase PEL3 and PEL4 was found that the laccases expression increased with time, different IPTG concentration was used for the induction of PEL3 and PEL4. However no soluble proteins were found in supernatant, and they were insoluble proteins without enzymatic activity. Thus laccase genes were subsequently cloned into Pichia pastoris for the production of laccases. The crude PEL4 have enzymatic activity. The optimum temperature was 60°C and the optimum pH was 3.0 in citric acid- Na2HPO4 buffer. The crude laccase was stable in a pH ranging from 5.0~10.0. Crude laccases exhibited thermostability in a range of 10~60℃.

並列關鍵字

Laccase ； Pleurotus eryngii ； Heterologous expression

參考文獻

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