本研究自白色腐生真菌 Cerrena sp. RSD1 中分離出高活性漆氧化酵素 DLac。經酵素動力學分析結果判定 DLac 屬於擴散限制型酵素。高解析蛋白質晶體結構分析指出 DLac 整體結構與其他真菌漆氧化酵素的蛋白單體具高度同源性，唯獨 DLac 的基質結合部位具有特異性。經與不同真菌漆氧化酵素比對基質結合部位後，顯示 DLac 的基質結合環狀結構是由較小側鏈的胺基酸組成，此外，第 IV 號基質結合環狀區段的也相對較短。綜合上述兩點，DLac 於結構上具有較寬大的基質結合入口，有利於促進基質進入酵素基質結合腔室。有別於大多數高度醣基化的真菌漆氧化酵素，DLac 的醣基化程度較低，即便如此，DLac 仍具備高度保守的醣基化位點 N432；同時有一獨特的醣基化點位於 N468。結構分析指出，N-聚醣穩定底物結合環狀區段和蛋白質結構。生化實驗亦證實，胺基酸上第一個N-乙醯葡糖胺對此酵素催化效率至關重要。另，本研究亦研發出能增進5倍酵素活性產量的浸置培養條件，作為工業量產應用的基礎。此外，在酵素產業應用上，高機溶劑耐受性的漆氧化酵素已被公認為具備高商業價值的生物催化劑。但在高濃度有機溶劑當中，已知大多數漆氧化酵素蛋白結構會被破壞，進而失去酵素活性。本研究發現，事先混合有機溶劑與真菌漆氧化酵素進行前置培養，可有效提高1.5倍至4.0倍的酵素活性，而且此方法具備可逆性。我們測試的有機溶劑包含丙酮、甲醇、乙醇、二甲基亞碸和二甲基甲醯胺，對於本研究中四種不同真菌來源的漆氧化酵素皆有增強酵素活性的作用。本研究也選用酚類和非酚類基質進行實驗，證實此酵素活性的增強並非基質特異性造成的結果。雖然事先混合有機溶劑與漆氧化酵素後，會導致酵素對於高溫較為敏感，但在常溫25°C下則是更有利於長時間的儲存。觀察事先混合丙酮與漆氧化酵素DLac的蛋白質結構，也證實DLac的蛋白質結構在高濃度有機溶劑中保持完整性及穩定性。此外，事先混合有機溶劑與漆氧化酵素的做法，也提高了單位時間內每莫耳酵素所能催化的基質數，其中又以DLac的活性增強表現最好。我們的研究提供在高濃度有機溶劑中真菌漆氧化酵素應用的高度潛能，並延伸了生物修復、脫色以及有機合成中運用的機會。

Fungal laccase is a blue, glycosylated four-copper oxidase that catalyzes the oxidation of phenolic units in lignin as well as a number of phenolic compounds and aromatic amines. A high-efficiency laccase, DLac, was secreted by an indigenous fungal strain Cerrena sp. RSD1 isolated from rice straw compost, it is identified using 18S rDNA and internal transcribed spacer sequencing analysis. A procedure of submerged culture using rice straw as a feedstock was carried out to produce DLac with high catalytic efficiency of 1.5×109 s-1 M-1 show the enzyme to be diffusion-limited. So far only a few study focus on structural properties of the diffusion-limited enzyme, it is important to investigate the crucial structural features that govern the enzyme kinetics. The crystal structure of DLac was determined at 1.38 Å resolution. DLac displays the typical fungal laccase architecture consisting of three domains, and each domain is folded into the greek key β-barrel topology. Four copper atoms were coordinated with His and Cys residues to create the catalytic site. The crystal structure was determined to atomic resolution and its overall structure was found to be closely homologous to the monomeric laccases. However, DLac displays some unique substrate-binding loops different from those in other laccases. The substrate-binding residues with small side-chains and the short substrate-binding loop IV widen the substrate-binding cavity and this may facilitate access by larger substrates. DLac is not as highly-glycosylated as other fungal laccases and contains one highly-conserved glycosylation site at N432 and another unique site at N468. The N-glycans stabilize the substrate-binding loops and the protein structure and the first N-acetylglucosamine is crucial for catalytic efficiency. A submerged culture method useful for industrial application allows a five-fold increase of protein yield to be achieved. Laccases that are tolerant to organic solvents are powerful bio-catalysts with broad applications in biotechnology, most frequently carried out at high concentration of solvent, during which process the proteins can be unfolded and enzyme activity can be lost. In this study it will be shown that pre-incubation of fungal laccases with organic solvents can result in an effective (and reversible) 1.5 to 4.0 fold enhancement of enzyme activity. Several organic solvents, including acetone, methanol, ethanol, DMSO, and DMF were effective in this specific enhancement in all the laccases studied. The enhancement was not substrate-specific and could be observed in both phenolic and non-phenolic substrates. Although laccase pre-incubated with organic solvents was sensitive to high temperature, it remained stable at 25°C, which was an advantage for long term storage. The 3-D structure of DLac, pre-incubated with acetone, was determined and it was confirmed that the protein structure remained intact and stable at high concentrations of organic solvent. Furthermore, the turnover rate of fungal laccases was improved by pre-incubation in an organic-solvent and DLac showed the highest enhancement among the fungal laccases examined. This investigation has shed light on improving fungal laccase usage under extreme conditions and has extended the opportunities for bioremediation, decolorization, and organic synthesis.

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