- 學位論文
以結晶結構理性設計提升黑麴菌甘露聚醣酶 ManBK 與棘孢麴黴菌纖維素酶 FI-CMCase 之活性
Activity enhancement through crystal structure-based rational design for Aspergillus niger mannanase ManBK and Aspergillus aculeatus cellulase FI-CMCase
摘要
內切性甘露聚醣酶 (β-mannanase) 為分解植物細胞壁中大量存在的甘露聚醣的主要酵素之一。而從黑麴菌 (Aspergillus niger) 當中分離出來的內切甘露聚醣酶 (ManBK) 是一種具高耐熱性的酵素。ManBK 可以有效地表達於工業上常用的畢赤酵母 (Pichia pastoris) 表達系統，且此酵素相較於現今工業甘露聚醣酶有著高耐熱性的優勢，因而具有潛在的產業應用價值。此研究中，藉由 X 光蛋白質結晶學技術，解析了高解析度 (1.57 Å) ManBK 甘露聚醣酶的蛋白質三級結構。此蛋白質結構呈現出典型的糖苷水解酶家族 5 的 (β/α)8 蛋白質摺疊。為了更進一步了解甘露聚醣酶與受質之間的作用力及機制，我們利用分析軟體將受質與 ManBK 結構做重疊比對。接著藉由結構的分析找出關鍵性的胺基酸進行改造，以增進此酶蛋白的作用活性。結果顯示，將甘露聚醣酶胺基酸序列上第216個胺基酸由酪胺酸突變為色胺酸 (Y216W) 後可以增加此甘露聚醣酶的活性，相較於原始蛋白質，Y216W 突變蛋白增加了18 ± 2.7%的酵素活性。此外，Y216W 突變蛋白質的 kcat 以及 Km 值皆高於原始蛋白質，推測其具有較快的催化效率。 纖維素多醣是植物細胞壁的主要組成，它同時也是地球上最充裕的再生物質。多年來纖維素多醣的水解也已經被利用在許多工業應用上。內切性葡聚醣酶 (endoglucanase) 為水解纖維素多醣的主要酵素之一，此種酶可藉由水解β-1,4-糖苷鍵結而隨機地將纖維素多醣分解成較小片段。而從棘孢麴黴菌 (Aspergillus aculeatus) 當中分離出來的內切纖維素酶 (FI-CMCase) 被歸類於糖苷水解酶家族 12 的一員，且此纖維素酶已被證明可以有效地表達於工業上常用的畢赤酵母中。在此論文中，利用畢赤酵母表達及純化FI-CMCase蛋白，並藉由 X 光蛋白質結晶學技術解析了 FI-CMCase 纖維素酶的蛋白質三級結構。此蛋白質結構呈現出典型的糖苷水解酶家族 12 的 β-jelly roll 蛋白質摺疊，其主要具備兩個反向平行的 β-sheet 形成一個活性區裂縫以供受質進入與其結合。為了更進一步了解纖維素酶與受質之間辨識的作用力及機制，在此論文中也解析了 FI-CMCase 纖維素酶與其受質結合的複合體結構。並藉由結構的分析找出可能具關鍵性的胺基酸來進行改造及分析。 本論文藉由單點突變將 ManBK 之酪胺酸216突變為色胺酸可顯著增加酵素活性，改造後的甘露聚醣酶有較佳的工業應用性，亦可做為未來在其它酵素改造的參考。另一方面，此論文也提供了纖維素酶 FI-CMCase 與其受質結合的複合體結構，其改造及分析的結果可做為後續其它纖維素酶改造的依據。
並列摘要
β-1,4-Mannanase (β-mannanase) is a key enzyme involved in the decomposition of mannans, which are the most abundant components of hemicelluloses in the plant cell wall. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK). ManBK can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilization. ManBK holds great potential in biotechnological applications owing to its high thermostability. Here, we present the 1.57 Å crystal structure of ManBK. The protein adopts a typical (β/α)8 fold that resembles that of other glycoside hydrolase family 5 (GH 5) members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. On the basis of structure, ManBK was rationally designed to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18 ± 2.7% increase in specific activity compared with the wild-type enzyme. Kinetic studies showed that Y216W has higher kcat values than those of the wild-type enzyme, suggesting a faster turnover rate of catalysis. Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in various industries. Endo-1,4-β-D-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of β-1,4-glycosidic bonds to cleave cellulose into smaller fragments, is a key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase family 12 (GH 12), has been demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, FI-CMCase was expressed and purified in P. pastoris, and the crystal structure was determined through X-ray crystallography. Its structure shows a β-jelly roll protein fold that is typical of the GH 12 enzymes with a curved surface and the concave face forms an extended ligand-binding cleft. To further understand the catalytic machinery, the crystal structures of FI-CMCase in complex with oligosaccharides were determined. On the basis of the structural analysis, several active site residues of FI-CMCase were modified, and their enzymatic activities were examined. To summarize, the enzymatic activity of ManBK was improved through molecular engineering, and we identified that the Y216W mutant shows great potential for use in industrial applications. Furthermore, the structural information of FI-CMCase in complex with its ligands provided fundamental information for further enhancing the enzymatic properties of GH 12 cellulases through rational molecular engineering.
參考文獻
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