Activity enhancement through crystal structure-based rational design for Aspergillus niger mannanase ManBK and Aspergillus aculeatus cellulase FI-CMCase

• Main Authors: Jian-Wen Huang, 黃建文
• Other Authors: 劉嚞睿
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/59900965852796055867

博士 === 國立臺灣大學 === 生物科技研究所 === 104 === 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.