Engineering of Agrobacterium radiobacter N-Carbamoyl D-Amino Acid Amidohydrolase to Increase Thermostability by a Structure-based Approach

• Main Authors: Ji-Yu You, 游季禹
• Other Authors: Wen-Ching Wang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/46365063934575806202

碩士 === 國立清華大學 === 生命科學系 === 91 === The structure of the N-carbamoyl D-amino acid amidohydrolase (D-NCAase) enzyme from Agrobacterium radiobacter (CCRC 14924) has been determined to 1.95 Å. The crystal structure reveals a tetramer with 222 symmetry. The industrial application of this enzyme is to produce hydroxyphenylglycine (HPG). Since the dissolution of the reactant D,L-p-hydroxyphenylhydantoin (D,L-p-HPH) requires high temperature, a thermostable enzyme is acquired. The purpose of this study is to design heat-resistant mutant for industrial application. The potential D-NCAase mutants with additional disulfide bonds are predicted based on the MODIP software by Balaram et .al.. Five residues (V44C, P178C, A222C, P295C-F304C, A302C) are mutated by site-directed mutagenesis. The V44C mutant is designed for the formation of intramolecular disulfide bond, the others are predicted to form additional intermolecular linkages. The relative activity of the V44C, P295C-F3204C and A302C remains about 50% as compared with the wild-type enzyme. The circular dichorism analysis of Tm for the intermolecular mutants are 74.8, 72.3 and 77.4℃, respectively. V44C, P295C-F304C and A302C could increase the thermostability of the enzyme, but neither P178C nor A222C had increase in Tm. Crystal structure of A222C showed no disulfide bond. On the other hand, a covalent disulfide bond was seen in A302C structure. Despite higher Tm, V44C and P295C-F304C showed limited improvement in the enzyme stability at 50℃. A double mutant V44C/A302C containing the intra and inter molecular disulfide linkages, nevertheless showed significant stability at 50℃. In addition, circular dichroism analysis demonstrated a 9.2℃ increase in Tm. Our results demonstrate that this is a robust approach to engineer an enzyme with enhanced thermostability.