Two-Step Sequence Alignment to Increase Thermostability of β-Glucosidase BgxA and D-Aminoacylase DA1

• Main Authors: Chang, Ting, 張庭
• Other Authors: Tseng, Ching-Ping
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/5mabtj

碩士 === 國立交通大學 === 分子醫學與生物工程研究所 === 104 === Nowadays, thermostable enzymes are widely utilized in foods, chemical and therapeutic drugs industry. Consensus method is one of the effective methods for improving enzymes’ thermostability by designing amino acids sequence. Nevertheless, it need mutate all residues at once and can’t tell us truly valid mutations for improving thermostability. Therefore, the new method was developed in this study using β-glucosidase/xylosidase (BgxA) as a monomeric model. Two amino acid sequence alignments of BgxA, based on its same family enzymes’ alignment and conserved domain alignment from NCBI, were used to select the suitable residues for mutations and substitutions, respectively. Upon selected 9 single mutants, Q290E and Q421L showed a 1.5 to 1.7-fold increase in half-life. It displayed that relative residues in the second alignment were all charged or hydrophobic. After adding the new screening requirement into original screening conditions, modified D-aminoacylases (DA1), S14R and S441V, showed separately the 2.1-fold and 2.6-fold increase in half-life. Thus, this new method is successful for effectively improving thermostability of protein in protein engineering.