靈芝屬漆氧化酶基因選殖、分類與異源表現

Abstract

纖維素、半纖維素、木質素是自然界中存量最多的大分子物質， 其中木質素由於結構複雜最難以分解。已知有三種酵素具有分解木質 素之功能分別為漆氧化酶（laccase, 1.10.3.2）、含錳過氧化酶(manganese peroxidase, 1.11.1.13)和木質素氧化酶( lignin peroxidase, 1.11.1.14)。 靈芝屬真菌屬於白腐型真菌，具有能夠分解木質素的能力。本文 將探討靈芝屬真菌中漆氧化酶基因之種類、特性與異源表現之結果。 以漆氧化酶基因之保守性序列設計引子，對十一株靈芝屬真菌進行聚 合酶鍊鎖反應，將產物定序，共得到26 條漆氧化酶部分基因序列，比 對顯示，靈芝屬真菌普遍具有兩條以上的漆氧化酶基因，將選殖之序 列與其他物種之漆氧化酶基因序列進行演化計算，發現靈芝屬真菌之 漆氧化酶可分為三種類型，其中第一類型與第二種類型下可分別再分 為兩群，序列比對與演化分析結果顯示，這三類基因有屬於自己的 intron，可能有獨立的演化來源，其中第三類的漆氧化酶基因可能在靈 芝屬與雲芝屬的分類形成前就已出現。 自靈芝屬真菌Ganoderma lucidum RZ、G. tsuage 1109、G. fornicatum 0814 中，分別選殖出漆氧化酶基因RZ.lac4 、0814.lac1 、1109.lac1，基 因全長依序為2121 bp、2019 bp、2110bp，皆具有9 個intron，其蛋白 質各由520、521、521 個胺基酸組成，其中前21 個胺基酸為signal peptide，在保守性Cysteine 之後第十個胺基酸為Phenylalanine，顯示 三者皆屬具有高還原電位之第三類漆氧化酶。 針對G. lucidum 之RZ.lac4 基因進行5’端Genome walking 所得上 游啟動子區域中含有真核生物常見的啟動子TATA、CAAT 序列之外， 還有MRE（ Mental-responsive element ）、STRE （ Stress-responsive promoter element）序列，顯示此基因之表現可能受到金屬離子之調控。 使用AOX1 起動子、pPICZ 載體，將所選殖之三條基因轉殖入嗜 甲醇酵母Pichia pastoris KM71 進行異源表現，所得之重組蛋白質皆具 有漆氧化酶活性，漆氧化酶轉型株reRZ.lac4、re0814.lac1、re1109.lac1 胞外上清液之最適反應pH 值為3.0，最適反應溫度分別為55、55-75、 65℃。以BMMHY 為誘導培養基，以30℃、0.5% 甲醇、250rpm 之條 件誘導七天，以ABTS 為活性測定基質，在最適反應條件之下測得轉 型株reRZ.lac4、re0814.lac1、re1109.lac1 之胞外上清液活性依序為 1.13U/ml、5.9U/ml、6.6U/ml，比活性依序為 32U/mg、90.2U/mg、 81.7U/mg。reRZ.lac4、re0814.lac1、re1109.lac1 之胞外上清液在30℃環 境下放置24 小時後，皆保有90%以上的活性，而re1109.lac1 在55℃ 環境下放置24 小時後，仍保有將近100%的活性，顯示具有良好之耐 熱性。而pH 穩定性實驗顯示re0814.lac1 之上清液置於不同pH 緩衝液 中，在室溫放置24 小時後，皆仍保有90%以上之活性，顯示其重組蛋 白可能具有良好的pH 穩定性。

Cellulose, hemicellulose, and lignin are most abundant macromolecules in nature. Lignin is hardest to be degraded for its complex constitutions. There are three enzymes have the ability to degrade lignin: laccase (1.10.3.2), manganese peroxidase (1.11.1.13), and lignin peroxidase (1.11.1.14). Whitr-rot fungi, such as Ganoderma spp., can degrade lignin. In this study, gene family, characteristics, and heterologous expression of laccase genes from Ganoderma spp. are discussed. The specific primers according to laccase conserved copper-binding regions used to amplify the laccase genes in the eleven strains of Ganoderma spp. There are 26 partial laccase genes were cloned in this study. The result shows that there are at least two laccase genes in each strain. The phylogenetic relationship shows that laccases from G. spp can be divided into three groups and each group has their own introns which might suggest that these three groups have different evolutionary relationship. The third group might exist before the speciation for G. spp and Trametes. The full length laccase gene of RZ.lac4 、0814.lac1 、1109.lac1 from G. lucidum RZ、G. tsugae 1109、G. fornicatum 0814 were 2121bp, 2019bp, and 2110bp, and there are 9 introns in each sequence. Laccase cDNA of RZ.lac4 、0814.lac1 、1109.lac1 were cloned and encodes for proteins with 520, 521, and 521 amino acids, including a 21-residue secrection signal peptide for each protein. Phenylalnine in additional residue 10 amino acids downstream of the conserved cysteine of each encoding protein shows that these proteins belong to class 3 laccase and may have high redox potential of the cupric ion. 5’ Genome walking of RZ.lac4 from G. lucidum RZ shows that TATA box, CAAT box, The Mental-responsive elements (MREs) and stress-responsive promoter element (STRE) exist in the promoter region of RZ.lac4. The Mental-responsive elements and stress-responsive promoter element found in the promoter of RZ.lac4 suggest that RZ.lac4 might be regulated by mentals. The cloned cDNA were subcloned to pPICZ vectors and expressed in Pichia pastoris KM71 under the control of the AOX1 promoter. The transformants were found to secrete active recombinant enzymes after induction with methanol. The optimal temperature of the recombinant proteins, reRZ.lac4, re0814.lac1, re1109.lac1, are 55, 55-75, and 65 . The ℃ optimal pH for the recombinant proteins is 3.0. The transformants were incubated in BMMHY medium at 30℃ with 0.5% MeOH at 250rpm each day. The laccase activities for reRZ.lac4, re0814.lac1, re1109.lac1 were 1.13U/ml、5.9U/ml、6.6U/ml，and the specific activities were 32U/mg、 90.2U/mg、81.7U/mg measured at optimal pH and temperature with ABTS as substrate. These three recombinant proteins were incubated in 30℃ for 24 hours, and all of them retained activities above 90%. Re1109.lac1 was incubated in 55℃and still retained almost 100% activity which shows that the protein has good thermostability. The culture medium of Re0814.lac1 was added to different pH buffer at room temperature for 24h, all of them still retained above 90% activity. This infers that re0814.lac1 might have good pH stability.